Effects of preoperative aspirin on perioperative platelet activation and dysfunction in patients undergoing off-pump coronary artery bypass graft surgery: A prospective randomized study.

PubMed

Lee, Jiwon; Jung, Chul-Woo; Jeon, Yunseok; Kim, Tae Kyong; Cho, Youn Joung; Koo, Chang-Hoon; Choi, Yoon Hyeong; Kim, Ki-Bong; Hwang, Ho Young; Kim, Hang-Rae; Park, Ji-Young

2017-01-01

The benefit of aspirin use after coronary artery bypass graft surgery has been well proven. However, the effect of preoperative aspirin use in patients undergoing off-pump coronary artery bypass graft surgery (OPCAB) has not been evaluated sufficiently. To evaluate platelet function changes during OPCAB due to preoperative aspirin use, we conducted a randomized controlled trial using flow cytometry and the Multiplate® analyzer. Forty-eight patients scheduled for elective OPCAB were randomized to the aspirin continuation (100 mg/day until operative day) and discontinuation (4 days before the operative day) groups. Platelet function was measured using the platelet activation markers CD62P, CD63, and PAC-1 by flow cytometry, and platelet aggregation was measured using the Multiplate® analyzer, after the induction of anesthesia (baseline), at the end of the operation, and 24 and 48 h postoperatively. Findings of conventional coagulation assays, thromboelastography by ROTEM® assays, and postoperative bleeding-related clinical outcomes were compared between groups. No significant change in CD62P, CD63, or PAC-1 was observed at the end of the operation or 24 or 48 h postoperatively compared with baseline in either group. The area under the curve for arachidonic acid-stimulated platelet aggregation, measured by the Multiplate® analyzer, was significantly smaller in the aspirin continuation group (P < 0.01). However, chest tube drainage and intraoperative and postoperative transfusion requirements did not differ between groups. Our study showed that preoperative use of aspirin for OPCAB did not affect perioperative platelet activation, but it impaired platelet aggregation, which did not affect postoperative bleeding, by arachidonic acid.

Evaluation of the effect of ketoprofen and carprofen on platelet function in dogs studied by PFA-100 point-of-care analyser.

PubMed

Gaál, T; Halmay, Dóra; Kocsis, R; Abonyi-Tóth, Z

2007-09-01

The effect of two nonsteroidal anti-inflammatory drugs (carprofen and ketoprofen) on platelet adhesion and aggregation functions was evaluated by the PFA-100 analyser (Dade-Behring, CA, U.S.A.) using its collagen-adenosine diphosphate (ADP) and collagen-epinephrine (EPI) cartridges. The function of platelets was evaluated in 55 healthy dogs, in 7 dogs treated with ketoprofen and in 31 dogs treated with carprofen in a therapeutic dose for minimum 5 days. The therapeutic doses of carprofen had no effect on the closure time of PFA-100 (which is the marker of platelet function) but ketoprofen caused a significant increase when using collagen-EPI stimulation The closure times for both the healthy (control) and the treated dogs using EPI cartridges were often longer than the upper default cut-off point (300 sec) of the device. The PFA-100 analyser with collagen-ADP cartridges could be a useful tool for veterinary applications including the evaluation of platelet aggregation in dogs treated with NSAIDs. The upper cut-off point of PFA-100 might be extended.

Platelet dysfunction detected at high shear in patients with heart valve disease.

PubMed

Francis, J L

2000-05-01

Whether patients with valvular heart disease have a defect of platelet function has been unclear. Despite evidence that these individuals have an abnormality detectable only under conditions of high shear stress, no methods have been widely available to adequately assess platelet function under such conditions. The Platelet Function Analyzer (PFA)-100 measures platelet function in a high shear environment and is well suited to the detection of platelet dysfunction in the clinical laboratory. The instrument records the time for platelets to occlude a membrane coated with collagen and either epinephrine (CEPI) or ADP (CADP). We studied the PFA-100 in 398 patients before open heart surgery; 308 for coronary artery bypass grafting (CABG) and 90 for aortic or mitral valve replacement (VR). Patients were classified as normal (CEPI < or = 153 s); 'aspirin effect' (CEPI > 153 s but CADP < or = 109 s) or abnormal (CEPI > 153 s and CADP > 109 s). In the CABG group, 41.2% were classified as normal, 43.2% as 'aspirin effect' and 15.6% as abnormal. In contrast, in patients undergoing VR, these values were 6.7, 11.1 and 82.4%, respectively. Patients with valvular disease had significantly longer closure times for both CEPI and CADP tests (P < 0.001). In addition, the valvular disease group had a significantly higher proportion of patients with markedly prolonged (> 150 s) closure times in the CADP cartridge (43.3 vs. 3.6%, respectively). Only one (0.3%) patient in the CABG group had non-closure (> 300 s) in the CADP test compared to seven (7.8%) in the valvular disease group. Three of six patients in the latter group bled excessively during surgery. We conclude that abnormal CADP closure is much more frequent among patients with aortic or mitral valve disease compared to those with coronary artery disease. This may reflect pre-existing high-shear damage to platelets that renders them refractory to subsequent shear activation and aggregation in the PFA-100 system. Further studies are needed to more precisely define the platelet defect in these patients. Markedly prolonged CADP closure in patients with valvular disease may indicate an increased likelihood of intra-operative bleeding, although an appropriately designed prospective study is needed to adequately address this hypothesis.

Failure of platelet parameters and biomarkers to correlate platelet function to severity and etiology of heart failure in patients enrolled in the EPCOT trial. With special reference to the Hemodyne hemostatic analyzer. Whole Blood Impedance Aggregometry for the Assessment of Platelet Function in Patients with Congestive Heart Failure.

PubMed

Serebruany, Victor L; McKenzie, Marcus E; Meister, Andrew F; Fuzaylov, Sergey Y; Gurbel, Paul A; Atar, Dan; Gattis, Wendy A; O'Connor, Christopher M

2002-01-01

Data from small studies have suggested the presence of platelet abnormalities in patients with congestive heart failure (CHF). We sought to characterize the diagnostic utility of different platelet parameters and platelet-endothelial biomarkers in a random outpatient CHF population investigated in the EPCOT ('Whole Blood Impedance Aggregometry for the Assessment of Platelet Function in Patients with Congestive Heart Failure') Trial. Blood samples were obtained for measurement of platelet contractile force (PCF), whole blood aggregation, shear-induced closure time, expression of glycoprotein (GP) IIb/IIIa, and P-selectin in 100 consecutive patients with CHF. Substantial interindividual variability of platelet characteristics exists in patients with CHF. There were no statistically significant differences when patients were grouped according to incidence of vascular events, emergency revascularization needs, survival, or etiology of heart failure. Aspirin use did not affect instrument readings either. PCF correlates very poorly with whole blood aggregometry (r(2) = 0.023), closure time (r(2) = 0.028), platelet GP IIb/IIIa (r(2) = 0.0028), and P-selectin (r(2) = 0.002) expression. Furthermore, there was no correlation with brain natriuretic peptide concentrations, a marker of severity and prognosis in heart failure reflecting the neurohumoral status. Patients with heart failure enrolled in the EPCOT Trial exhibited a marginal, sometimes oppositely directed change in platelet function, challenging the diagnostic utility of these platelet parameters and biomarkers to serve as useful tools for the identification of platelet abnormalities, for predicting clinical outcomes, or for monitoring antiplatelet strategies in this population. The usefulness of these measurements for assessing platelets in the different clinical settings remains to be explored. Taken together, opposite to our expectations, major clinical characteristics of heart failure did not correlate well with the platelet characteristics investigated in this study. Copyright 2002 S. Karger AG, Basel

Comparative evaluation of Plateletworks, Multiplate analyzer and Platelet function analyzer-200 in cardiology patients.

PubMed

Kim, Jeeyong; Cho, Chi Hyun; Jung, Bo Kyeung; Nam, Jeonghun; Seo, Hong Seog; Shin, Sehyun; Lim, Chae Seung

2018-04-14

The objective of this study was to comparatively evaluate three commercial whole-blood platelet function analyzer systems: Platelet Function Analyzer-200 (PFA; Siemens Canada, Mississauga, Ontario, Canada), Multiplate analyzer (MP; Roche Diagnostics International Ltd., Rotkreuz, Switzerland), and Plateletworks Combo-25 kit (PLW; Helena Laboratories, Beaumont, TX, USA). Venipuncture was performed on 160 patients who visited a department of cardiology. Pairwise agreement among the three platelet function assays was assessed using Cohen's kappa coefficient and percent agreement within the reference limit. Kappa values with the same agonists were poor between PFA-collagen (COL; agonist)/adenosine diphosphate (ADP) and MP-ADP (-0.147), PFA-COL/ADP and PLW-ADP (0.089), MP-ADP and PLW-ADP (0.039), PFA-COL/ADP and MP-COL (-0.039), and between PFA-COL/ADP and PLW-COL (-0.067). Nonetheless, kappa values for the same assay principle with a different agonist were slightly higher between PFA-COL/ADP and PFA-COL/EPI (0.352), MP-ADP and MP-COL (0.235), and between PLW-ADP and PLW-COL (0.247). The range of percent agreement values was 38.7% to 73.8%. Therefore, various measurements of platelet function by more than one method were needed to obtain a reliable interpretation of platelet function considering low kappa coefficient and modest percent agreement rates among 3 different platelet function tests.

A detailed examination of platelet function inhibition by nitric oxide in platelet-rich plasma and whole blood.

PubMed

Zimmermann, Robert; Krueger, Julia; Filipović, Milos R; Ivanović-Burmazović, Ivana; Calatzis, Andreas; Weiss, Dominik R; Eckstein, Reinhold

2013-01-01

The question of whether novel instruments such as multiple electrode aggregometry (MEA) can be used for measurement of the effects of nitric oxide (NO) on platelets (PLTs) has not been examined. Therefore, we compared the effects of NO concentrations (1, 10, and 100 microM) on the PLT aggregation response to ADP, arachidonic acid (AA), collagen, ristocetin, and thrombin receptor-activating peptide 6 (TRAP6) using light transmission aggregometry (LTA) and multiple electrode aggregometry (MEA) and examined the effects of NO using the platelet function analyzer (PFA)-100. The response of PLTs to ADP and AA was strongly inhibited by all NO concentrations in LTA and MEA. The inhibition of the responses to ristocetin and collagen was detectable in MEA at lower NO concentrations than in LTA. However, the typically increasing lag phase between collagen addition and the aggregation response in the presence of NO was more obvious in LTA. TRAP caused a reproducible early response in the presence of NO in LTA which was followed by rapid PLT disaggregation, whereas even 100 microM NO did not inhibit the response to TRAP in MEA. Finally, NO prolonged the in-vitro bleeding time remarkably more in the PFA-100 collagen-epinephrin cartridge than in the collagen-ADP cartridge. Whole blood versus PLT rich plasma, citrate versus hirudin, and high versus low shear influenced the effects of NO. This shows that a careful selection of models and potentially a combination of different methods is appropriate for a differentiated evaluation of pharmacological or physiological mechanisms of NO-donors or of NO-inhibitors.

Acute effects of 30 minutes of exposure to a smartphone call on in vitro platelet function

PubMed Central

Lippi, Giuseppe; Danese, Elisa; Brocco, Giorgio; Gelati, Matteo; Salvagno, Gian Luca; Montagnana, Martina

2017-01-01

Background Significant concerns are now regularly raised about the safety of excessive mobile phone use. This study was aimed to assess the acute effects of radiofrequency waves emitted by a commercial smartphone on platelet function. Materials and methods Two sequential citrated blood samples were collected from 16 healthy volunteers recruited from laboratory staff. The first sample was placed in a plastic rack, 1 cm distant from a commercial smartphone receiving a 30-min call and emitting 900 MHz radiofrequency waves. The second sample was placed in another plastic rack, isolated from radiofrequency wave sources, for the same period. The platelet count and the mean platelet volume were then assessed in all blood samples, whereas platelet function was evaluated using the platelet function analyser-100 (PFA-100). Results A 30-min exposure of citrated blood to smartphone radiofrequency waves induced significant prolongation of collagen-epinephrine aggregation (median increase, 10%) and a considerable increase of mean platelet volume (median increase, 5%), whereas collagen-adenosine diphosphate aggregation and platelet count remained unchanged. Discussion This study demonstrates that smartphone radiofrequency waves induce significant perturbation of platelet structure and function, thus providing further support to concerns regarding excessive use of mobile phones. Caution should also be taken with regards to blood products containing platelets, which should be kept far away from mobile phones and smartphones throughout the production pipeline and storage period. PMID:27177410

Acute effects of 30 minutes of exposure to a smartphone call on in vitro platelet function.

PubMed

Lippi, Giuseppe; Danese, Elisa; Brocco, Giorgio; Gelati, Matteo; Salvagno, Gian Luca; Montagnana, Martina

2017-05-01

Significant concerns are now regularly raised about the safety of excessive mobile phone use. This study was aimed to assess the acute effects of radiofrequency waves emitted by a commercial smartphone on platelet function. Two sequential citrated blood samples were collected from 16 healthy volunteers recruited from laboratory staff. The first sample was placed in a plastic rack, 1 cm distant from a commercial smartphone receiving a 30-min call and emitting 900 MHz radiofrequency waves. The second sample was placed in another plastic rack, isolated from radiofrequency wave sources, for the same period. The platelet count and the mean platelet volume were then assessed in all blood samples, whereas platelet function was evaluated using the platelet function analyser-100 (PFA-100). A 30-min exposure of citrated blood to smartphone radiofrequency waves induced significant prolongation of collagen-epinephrine aggregation (median increase, 10%) and a considerable increase of mean platelet volume (median increase, 5%), whereas collagen-adenosine diphosphate aggregation and platelet count remained unchanged. This study demonstrates that smartphone radiofrequency waves induce significant perturbation of platelet structure and function, thus providing further support to concerns regarding excessive use of mobile phones. Caution should also be taken with regards to blood products containing platelets, which should be kept far away from mobile phones and smartphones throughout the production pipeline and storage period.

[Effect of endogenous H2S on platelet L-Arg transport].

PubMed

Duan, Wen-zhuo; Wang, Yi-peng; Gong, Hai-min

2010-05-01

To observe the effect of novel air neuromodulator H2S on platelet function of L-Arg transport for discussing H2S of effect on platelet function. Saturate H2S solution as donate made rat rich platelet plasma and pre-incubation rat platelet with different density of H2S. To measure the velocity of L-Arg transport in platelet by radioactivity technique. At different concentrations of H2S (6.25, 12.5, 25, 50, 100 micromol/L), the velocity of L-Arg transport was lower than that in control. H2S reduced rapidly the Vmax and velocity of L-Arg transport in platelet (P < 0.05) and this effect had no effect to Km. H2S can affect platelet function by changing rapidly platelet L-Arg transport system function.

High incidence of defective high-shear platelet function among platelet donors.

PubMed

Harrison, Paul; Segal, Helen; Furtado, Charlene; Verjee, Salim; Sukhu, Kampta; Murphy, Michael F

2004-05-01

Because single-donor plateletpheresis concentrates now account for a large percentage of PLT concentrates, the PLT quality of individual donations is increasingly important. There has been little previous work on PLT function in blood donors. The aim of this study was to investigate the prevalence of defective PLT function among 100 healthy UK PLT donors on 3 consecutive donation days. Citrated blood samples were taken before plateletpheresis. High-shear PLT function testing was performed by a PLT function analyzer (PFA-100, Dade Behring), within both collagen-ADP (CADP) and collagen-epinephrine (CEPI) cartridges. Sixteen percent of PLT donors (mainly over the 3 donating days) had prolonged CEPI closure times (CTs) only, indicative of an aspirin-like pattern; 9 percent had a severe defect with prolonged CEPI CTs between 200 and 300 seconds, and 4 percent had maximal CEPI CTs of greater than 300 seconds. The majority of prolonged CEPI CTs appeared transient in nature. PLT dysfunction as detected by the PFA-100 is surprisingly common within a voluntary PLT donor population. The majority of CEPI defects appeared transient in nature and are indicative of surreptitious intake of cyclooxygenase inhibitors (e.g., aspirin). Identification of defective PLT function within donors would provide a simple means to eradicate defective PLTs from being clinically utilized.

Comparative Analysis of Various Aspects of Plateletpheresis on the Fenwal Amicus and Fresenius COM.TEC Cell Separator Instruments.

PubMed

Philip, Joseph; Biswas, Amit Kumar; Chatterjee, Tathagata; Mallhi, Rajiv Singh

2014-01-01

To compare the Fenwal Amicus and the Fresenius COM.TEC apheresis instruments regarding donor peripheral blood parameters, operational variables of the instruments, and quality control parameters of the product obtained. We performed 100 platelet collections from 100 voluntary donors using the 2 studied devices. We measured platelet count using an automated analyzer and analyzed the activation statuses using a flow cytometer. The median time needed to perform the procedures was significantly longer with the COM.TEC. However, the product we obtained using the Amicus instrument showed higher degrees of platelet-activation. All products we obtained with both instruments had white blood cell counts of less than 5 × 10(6) per bag. We observed no statistical difference regarding collection efficiency and collection rates between the devices. Both instruments collected platelets efficiently, with minimal donor discomfort. Compared with the COM.TEC instrument, the Amicus reached the platelet target yield more quickly; however, it displayed an increase in platelet activation. Copyright© by the American Society for Clinical Pathology (ASCP).

Aspirin administered to women at 100 mg every other day produces less platelet inhibition than aspirin administered at 81 mg per day: implications for interpreting the women's health study.

PubMed

Swaim, Lisa; Hillman, Robert S

2009-07-01

We aimed to determine the relative level of platelet inhibition achieved with low-dose aspirin (81 mg daily) compared with a very low-dose (100 mg every other day). The Womens Health Study (WHS) found that a dose of 100 mg every other day of aspirin provided protection against stroke as primary prophylaxis, but not myocardial infarction. In the United States, the most commonly prescribed dose of aspirin for primary prophylaxis is 81 mg per day. As a result, it is important to know whether these doses are equivalent before extrapolating the results of the WHS to women in the U.S. To achieve this goal, we have studied the effects of these two dosing regimens on platelet function in healthy women meeting the WHS inclusion criteria using a randomized design. We enrolled 49 healthy female volunteers and used a sequential, crossover design to compare the two regimens. The participants received a 17-day course of each aspirin-dosing regimen separated by a 7-day washout period. The degree of platelet inhibition was measured on days 14-17 of each dosing regimen using a point-of-care platelet function assay utilizing arachidonic acid to activate platelets (VerifyNow-Aspirin). Participants platelet response, expressed as Aspirin Response Unit (ARU) attained a significantly greater level of platelet inhibition on days 14-17 while taking aspirin 81 mg daily compared to aspirin 100 mg every other day (31.3% vs. 12.7%, P < 0.0001) with mean +/- SD ARU values of 445 +/- 50 and 570 +/- 68, P < 0.0001. Significantly more daily readings in participants were >or=550 ARU, a value correlated with clinical outcomes in several studies, with the 100 mg every other day regimen (72.0% vs. 6.4% with 81 mg daily, P < 0.0001), and this alternate-day regimen also resulted in more day-to-day variability in platelet function (P = 0.0002). We found significantly less inhibition of platelet function with the dose used in the WHS than the usual U.S. dose. We observed that the degree of platelet inhibition was significantly less with aspirin 100 mg every other day compared with aspirin 81 mg daily, suggesting that results of the Women's Health Study may have underestimated both the efficacy and toxicity of aspirin as it is commonly administered. These data need to be considered when developing recommendations about the use of aspirin in the primary prevention of cardiovascular disease in women.

Dogs with heart diseases causing turbulent high-velocity blood flow have changes in platelet function and von Willebrand factor multimer distribution.

PubMed

Tarnow, Inge; Kristensen, Annemarie T; Olsen, Lisbeth H; Falk, Torkel; Haubro, Lotte; Pedersen, Lotte G; Pedersen, Henrik D

2005-01-01

The purpose of this prospective study was to investigate platelet function using in vitro tests based on both high and low shear rates and von Willebrand factor (vWf) multimeric composition in dogs with cardiac disease and turbulent high-velocity blood flow. Client-owned asymptomatic, untreated dogs were divided into 4 groups: 14 Cavalier King Charles Spaniels (Cavaliers) with mitral valve prolapse (MVP) and no or minimal mitral regurgitation (MR), 17 Cavaliers with MVP and moderate to severe MR, 14 control dogs, and 10 dogs with subaortic stenosis (SAS). Clinical examinations and echocardiography were performed in all dogs. PFA100 closure times (the ability of platelets to occlude a hole in a membrane at high shear rates), platelet activation markers (plasma thromboxane B2 concentration, platelet surface P-selectin expression), platelet aggregation (in whole blood and platelet-rich plasma with 3 different agonists), and vWf multimers were analyzed. Cavaliers with moderate to severe MR and dogs with SAS had longer closure times and a lower percentage of the largest vWf multimers than did controls. Maximal aggregation responses were unchanged in dogs with SAS but enhanced in Cavaliers with MVP (regardless of MR status) compared with control dogs. No significant difference in platelet activation markers was found among groups. The data suggest that a form of platelet dysfunction detected at high shear rates was present in dogs with MR and SAS, possibly associated with a qualitative vWf defect. Aggregation results suggest increased platelet reactivity in Cavaliers, but the platelets did not appear to circulate in a preactivated state in either disease.

Platelet impedance adhesiometry: A novel technique for the measurement of platelet adhesion and spreading.

PubMed

Polgár, L; Soós, P; Lajkó, E; Láng, O; Merkely, B; Kőhidai, L

2018-06-01

Thrombogenesis plays an important role in today's morbidity and mortality. Antithrombotics are among the most frequently prescribed drugs. Thorough knowledge of platelet function is needed for optimal clinical care. Platelet adhesion is a separate subprocess of platelet thrombus formation; still, no well-standardized technique for the isolated measurement of platelet adhesion exists. Impedimetry is one of the most reliable, state-of-art techniques to analyze cell adhesion, proliferation, viability, and cytotoxicity. We propose impedimetry as a feasible novel method for the isolated measurement of 2 significant platelet functions: adhesion and spreading. Laboratory reference platelet agonists (epinephrine, ADP, and collagen) were applied to characterize platelet functions by impedimetry using the xCELLigence SP system. Platelet samples were obtained from 20 healthy patients under no drug therapy. Standard laboratory parameters and clinical patient history were also analyzed. Epinephrine and ADP increased platelet adhesion in a concentration-dependent manner, while collagen tended to have a negative effect. Serum sodium and calcium levels and age had a negative correlation with platelet adhesion induced by epinephrine and ADP, while increased immunoreactivity connected with allergic diseases was associated with increased platelet adhesion induced by epinephrine and ADP. ADP increased platelet spreading in a concentration-dependent manner. Impedimetry proved to be a useful and sensitive method for the qualitative and quantitated measurement of platelet adhesion, even differentiating between subgroups of a healthy population. This novel technique is offered as an important method in the further investigation of platelet function. © 2018 John Wiley & Sons Ltd.

In vitro properties of concentrated canine platelets stored in two additive solutions: a comparative study.

PubMed

Hlavac, N; Lasta, C S; Dalmolin, M L; Lacerda, L A; de Korte, D; Marcondes, N A; Terra, S R; Fernandes, F B; González, F H D

2017-11-15

Platelet transfusion therapy poses many challenges in veterinary clinical practice. Lack of readily available blood donors, short shelf-life, and inability to administer a sufficient number of platelets to meet a dog's transfusion need are the major difficulties encountered. Platelet additive solutions are already in use at American and European human blood banks, showing to be a realistic alternative. This study compares the in vitro platelet function in plasma, Composol, or SSP+ during storage for 13 days. Platelet rich plasma-platelet concentrate with 35% plasma and 65% platelet additive solutions (Composol or SSP+) and a control group (100% plasma) were prepared. Swirling, platelet count, blood gases, metabolic variables, platelet activation markers, and apoptosis markers were analyzed on days 1, 5, 9 and 13. Swirling was well preserved and pH was acceptable (> 6.2) during storage for all platelet additive solutions units until day 9. SSP + units showed more stable pH and metabolic variables until day 13. Platelets in plasma showed higher glucose consumption than in Composol or in SSP+. The platelet additive solutions units showed better platelet metabolism maintenance, reduced glucose consumption and lactate production. The apoptotic markers were still low for 9 days in platelet concentrates with platelet additive solutions, suggesting the possibility to extend the shelf life with the use of SSP+ or Composol. Our findings suggest that the uses of Composol and SSP+ in canine platelet concentrates are potential alternatives in veterinary blood banks.

Comparison of the effects of a balanced crystalloid-based and a saline-based tetrastarch solution on canine whole blood coagulation and platelet function.

PubMed

Reuteler, Annina; Axiak-Flammer, Shannon; Howard, Judith; Adamik, Katja-Nicole

2017-01-01

To evaluate the effects of a 6% hydroxyethyl starch (130/0.42) solution in either a buffered, electrolyte-balanced (HES-BAL), or a saline (HES-SAL) carrier solution on canine platelet function and whole blood coagulation. Prospective, randomized study. University teaching hospital. Thirty-seven client-owned dogs undergoing general anesthesia for arthroscopy or imaging studies. Dogs received a 15 mL/kg intravenous bolus of HES-SAL (n = 13), HES-BAL (n = 14), or a modified Ringer's solution (n = 10) over 30-40 minutes. Coagulation was analyzed using a Platelet Function Analyzer-100 (closure time [Ct PFA ]), and whole blood thromboelastometry (ROTEM) with extrinsically (ex-tem and fib-tem) and intrinsically (in-tem) activated assays, which assessed clotting time (CT), clot formation time (CFT), maximal clot firmness (MCF), and lysis index (LI). Coagulation samples were assayed prior to fluid administration (T0), and 5 minutes (T1), and 3 hours (T2) following fluid bolus administration, respectively. Both HES solutions resulted in impaired platelet function as indicated by a significant prolongation of Ct PFA at T1 as compared to T0, but which resolved by T2. An IV bolus of Ringer's solution did not alter platelet function. In both HES groups, whole blood coagulation was significantly impaired at T1 as indicated by a significant increase in in-tem CFT, and a significant decrease in ex-tem, in-tem, and fib-tem MCF compared to T0. Furthermore, a significant increase in ex-tem CFT at T1 compared to T0 was found in the HES-SAL group. With the exception of in-tem MCF after HES-BAL, these effects were not present at T2. No significant differences were found in Ct PFA or any ROTEM variable at any time point between HES-SAL and HES-BAL. Administration of a single bolus of 15 mL/kg 6% HES 130/0.42 results in significant but short-lived impairment of canine platelet function and whole blood coagulation, regardless of carrier solution. © Veterinary Emergency and Critical Care Society 2016.

Assessment of platelet function in acute ischemic stroke patients previously treated with aspirin.

PubMed

Lago, Aida; Parkhutik, Vera; Tembl, Jose Ignacio; Vallés, Juana; Santos, Maria Teresa; Moscardó, Antonio

2014-01-01

Platelet inhibition measured by platelet function tests could be critical to understand the reasons for early recurrence and to guide therapeutic recommendations. We assess the platelet function during the acute phase of ischemic stroke in patients pretreated with aspirin who continue their treatment with aspirin only, are started on clopidogrel only, or add clopidogrel to aspirin. Sixty-four patients were taking aspirin before the stroke. Depending on the administered antiplatelet, 3 groups were defined: ASA: patients who continued on aspirin orally or intravenous acetylsalicylate of lysine, n = 30; CLO: patients who discontinued aspirin and were started on clopidogrel, n = 16; and ASA + CLO: patients who were prescribed both aspirin and clopidogrel, n = 10. Collagen-induced thromboxane A2 (TXA2) synthesis, ADP (adenosine diphosphate)-induced aggregation, and occlusion time (PF-100) were measured. CLO group only had a marked elevation of TXA2 (17.44 ± 15.62 ng/mL, P = .000) and a shortening of the platelet function analyzer (PFA)-100 closure time (157.13 ± 88 seconds, P = .047) compared with the other 2 groups (ASA: TXA2, .62 ± 1.59 ng/mL; ASA + CLO: TXA2 1.79 ± 4.59 ng/mL). They achieved a small (13%) but significant reduction of ADP-induced aggregation (87.00 ± 23.06 mm, P = .008) compared with the ASA group (102.82 ± 22.38 seconds). Stopping aspirin intake within the first 72 hours of the acute stroke drastically increases TXA2 synthesis. During the same time window, the freshly prescribed clopidogrel manages to reduce the ADP-induced aggregation only slightly (13%). This study offers analytic proof that the common practice of replacing aspirin with clopidogrel does not leave stroke patients fully protected during the first days after an ischemic stroke. Possible solutions could be to preserve aspirin during a few days or to use loading doses of clopidogrel at hospital admission. Copyright © 2014 National Stroke Association. Published by Elsevier Inc. All rights reserved.

Comparison between a new platelet count drop method PL-11, light transmission aggregometry, VerifyNow aspirin system and thromboelastography for monitoring short-term aspirin effects in healthy individuals.

PubMed

Guan, Jie; Cong, Yulong; Ren, Junwei; Zhu, Yuan; Li, Li; Deng, Xinli; Bai, Jie

2015-01-01

Platelet function has been described by many laboratory assays, and PL-11 is a new point-of-care platelet function analyzer based on platelet count drop method, which counts platelet before and after the addition of agonists in the citrated whole blood samples. The present study sought to compare PL-11 with other three major more established assays, light transmission aggregometry (LTA), VerifyNow™ aspirin system and thromboelastography (TEG), for monitoring the short-term aspirin responses in healthy individuals. Ten healthy young men took 100 mg/d aspirin for 3-day treatment. Platelet function was measured via PL-11, LTA, VerifyNow and TEG, respectively. The blood samples were collected at baseline, 2 hour, 1 day during the aspirin treatment and 1 day, 5 ± 1 days, 8 ± 1 days after the aspirin withdrawal. Moreover, 90 additional healthy subjects were recruited to establish a reference range for PL-11. Platelet function of healthy subjects decreased significantly 2 hours after 100 mg/d aspirin intake and began to recover during 4-6 days after the aspirin withdrawal. Correlations between methods were PL-11 vs. LTA (r = 0.614, p < 0.01); PL-11 vs. VerifyNow (r = 0.829, p < 0.01); PL-11 vs. TEG (r = 0.697, p < 0.001). There was no significant bias between PL-11 and LTA at baseline (bias = 1.94%, p = 0.804) using Bland-Altman analysis, while the data of PL-11 were significantly higher than LTA (bias = 24.02%, p < 0.001) during the aspirin therapy. The reference range for PL-11 in healthy young individuals was from 66.8 to 90.5% (95%CI). When aspirin low-responsiveness was defined as LTA > 20%, the cut-off values for each method were, respectively: PL-11 > 50%, VerifyNow > 533 ARU, TEG > 60.2%. The results of different platelet function assays were uninterchangeable for monitoring aspirin response and correlations among them were also varied. Correlations among PL-11 and other three major assays suggested the ability of PL-11 to assess the treatment effects of aspirin. But a large cohort study is needed to confirm the cut-off value of aspirin response detected by PL-11.

Multiple electrode whole blood aggregometry, PFA-100, and in vivo bleeding time for the point-of-care assessment of aspirin-induced platelet dysfunction in the preoperative setting.

PubMed

Jámbor, Csilla; von Pape, Klaus-Werner; Spannagl, Michael; Dietrich, Wulf; Giebl, Andreas; Weisser, Heike

2011-07-01

Acquired platelet dysfunction due to aspirin ingestion may increase bleeding tendency during surgery. Thus, we examined the diagnostic accuracy of in vivo bleeding time (BT) and 2 platelet function assays for the preoperative assessment of a residual antiplatelet effect in patients treated with aspirin. Consecutive patients scheduled for surgery were prospectively enrolled in this study. The patients' last aspirin ingestion had occurred within the previous 48 hours before blood sampling in the "full aspirin effect" group, between 48 and 96 hours before in the "variable aspirin effect" group, and >96 hours before in the "recovered aspirin effect" group. The control group had not taken any aspirin. Multiple electrode aggregometry, platelet function analyzer (PFA)-100, and in vivo BT were performed to assess the effects of aspirin. One-way analysis of variance on ranks with a post hoc multiple-comparison procedure (Dunn) was used to detect differences among the groups. Categorical data were compared using the z test. Receiver operating characteristic (ROC) curves were created to determine the diagnostic accuracy of the platelet function assays investigated. The area under the ROC curve (AUC), sensitivity, and specificity of the assays were calculated. The level of statistical significance was set at P < 0.05. Three hundred ninety-four patients were included in the analysis (133 control and 261 aspirin-treated patients). All 3 methods were able to detect the antiplatelet effect of aspirin in the full aspirin effect group. Furthermore, no difference in the measurement values between the recovered aspirin effect and control group was found, irrespective of the assay performed. Measurement values in the variable aspirin effect group were different from those of the control group in the ASPItest using multiple electrode aggregometry and COL-EPI using PFA-100 but not in BT. ROC analysis showed the highest diagnostic accuracy in excluding the residual aspirin effect in the ASPItest (AUC 0.81, P < 0.001), followed by COL-EPI (AUC 0.78, P < 0.001) and BT (AUC 0.56, P = 0.05). The cutoff value of 53 U in the ASPItest excluded the effect of aspirin with a sensitivity of 88% and specificity of 71%. The full therapeutic antiplatelet effects of aspirin can be expected within 48 hours of the patient's last aspirin ingestion. Platelet function recovered in our study if aspirin cessation occurred >96 hours (4 days) before; thus, in these patients, preoperative platelet function testing is not useful. To quantify any residual aspirin effect in patients who ceased their intake of aspirin between 48 and 96 hours before surgery, the ASPItest might have the highest diagnostic accuracy.

Antiplatelet properties of escitalopram in patients with the metabolic syndrome: a dose-ranging in vitro study.

PubMed

Atar, Dan; Malinin, Alex; Pokov, Alex; van Zyl, Louis; Frasure-Smith, Nancy; Lesperance, Francois; Serebruany, Victor L

2007-11-01

There is an increasing body of evidence suggesting that selective serotonin reuptake inhibitors exhibit clinical benefit beyond treating depression, by simultaneously inhibiting platelet activity. We recently demonstrated that escitalopram (ESC), but not its major metabolites, inhibits multiple platelet biomarkers in healthy volunteers. Considering that the metabolic syndrome represents one of the major risk factors for vascular disease, we here determined how ESC affects platelet activity in such patients. We assessed the in vitro effects of preincubation with escalating (50-200 nM/l) concentrations of ESC on platelet aggregation, expression of major surface receptors by flow cytometry, and quantitatively by platelet function analyzers. Blood samples were obtained from 20 aspirin-naïve patients with documented metabolic syndrome. Pretreatment of blood samples with medium (150 nM/l), or high (200 nM/l) doses of ESC resulted in a significant inhibition of platelet aggregation induced by ADP (p=0.007) and by collagen (p=0.004). Surface platelet expression of GPIb (CD42, p=0.03), LAMP-3 (CD63, p=0.04), and GP37 (CD165, p=0.03) was decreased in the ESC-pretreated samples. Closure time by the PFA-100 analyzer was prolonged after the 200 nM/l dose (p=0.02), indicating platelet inhibition under high shear conditions. On the other hand, the lowest tested concentration of ESC (50 nM/l) did not affect platelet activity in these patients. The in vitro antiplatelet characteristics of ESC in patients with the metabolic syndrome are similar to those in healthy volunteers. However, higher ESC doses are required to induce equally potent platelet inhibition. These data justify prospective ex vivo studies with the highest therapeutic dose to determine the potential clinical advantage of ESC in high-risk patients with vascular disease.

Effects of aspirin, carprofen, deracoxib, and meloxicam on platelet function and systemic prostaglandin concentrations in healthy dogs.

PubMed

Blois, Shauna L; Allen, Dana G; Wood, R Darren; Conlon, Peter D

2010-03-01

To determine effects of therapeutic dosages of aspirin, carprofen, deracoxib, and meloxicam on platelet function and systemic prostaglandin concentrations in healthy dogs. 10 hound-crossbred dogs. Aspirin (10 mg/kg, PO, q 12 h), carprofen (4.4 mg/kg, PO, q 24 h), deracoxib (2 mg/kg, PO, q 24 h), meloxicam (0.1 mg/kg, PO, q 24 h), and a placebo were administered for 7 days in a random order to each of 10 healthy dogs; there was a 21-day washout period between subsequent treatments. One-stage prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen concentration, and plasma concentrations of thromboxane (TX)B(2) and 6-keto prostaglandin (PG)F(1alpha) were measured before and after treatment administration. Platelet function was assessed by use of a platelet-function analyzer and aggregation. Aspirin, carprofen, and meloxicam did not significantly affect platelet function. Deracoxib caused a mild decrease in platelet aggregation induced by 50microM ADP. Platelet number, Hct, PT, aPTT, and plasma TXB(2) and 6-keto PGF(1alpha) concentrations were unchanged after NSAID administration. Meloxicam administration resulted in a significant decrease in fibrinogen concentration, but results remained within the laboratory reference interval. Oral administration of commonly used NSAIDs at therapeutic dosages in healthy dogs did not alter plasma TXB(2) and 6-keto PGF(1alpha) concentrations. Deracoxib administration resulted in a minor abnormality in platelet aggregation. Anti-inflammatory doses of aspirin did not affect platelet function as measured by use of optical aggregometry and a platelet-function analyzer. Further evaluation of the effects of aspirin and cyclooxygenase-2-selective inhibitors on hemostasis should be performed.

Platelet closure time in anesthetized Greyhounds with hemorrhagic shock treated with hydroxyethyl starch 130/0.4 or 0.9% sodium chloride infusions.

PubMed

McBride, Duana; Hosgood, Giselle; Raisis, Anthea; Smart, Lisa

2016-07-01

To measure platelet closure time (PCT) in dogs during controlled hemorrhagic shock and after fluid resuscitation with hydroxyethyl starch (HES) 130/0.4 or 0.9% sodium chloride. Experimental interventional study. University veterinary teaching hospital. Eleven healthy Greyhounds. Dogs were anesthetized and had 48 mL/kg of blood removed to induce hemorrhagic shock. Dogs received 20 mL/kg of HES 130/0.4 (n = 6) or 80 mL/kg of 0.9% sodium chloride (NaCl; n = 5) intravenously over 20 minutes. PCT was measured using the Platelet Function Analyzer-100 with collagen and adenosine-diphosphate cartridges at: T0 = 60 minutes after induction of anesthesia prior to hemorrhage, T1 = during hemorrhagic shock, and T2 = 40 minutes after completion of fluid bolus. Packed cell volume and platelet count were concurrently measured. Hemorrhagic shock did not significantly change PCT, with no difference between T0 and T1. Both the HES 130/0.4 and 0.9% NaCl group had a significantly increased mean PCT at T2 of 91.4 seconds (95% CI 69.3-113.4) and 95.5 seconds (95% CI 78.2-112.8), respectively, compared to T1. The magnitude of change was significantly greater for the 0.9% NaCl group than the HES 130/0.4 group. There was no difference in the magnitude of change in PCV and platelet count between the 2 groups. The PCV and platelet count were >25% and >100,000/μL, respectively, in all dogs, except for dogs in the HES 130/0.4 group at T2 where platelet counts were <100,000/μL. Controlled hemorrhagic shock in Greyhounds under anesthesia did not cause a significant change in PCT. Both HES 130/0.4 and 0.9% NaCl administration after induction of shock increased PCT. These results do not support that HES 130/0.4 causes relevant platelet dysfunction beyond hemodilution. © Veterinary Emergency and Critical Care Society 2016.

Generation of Platelet Microparticles after Cryopreservation of Apheresis Platelet Concentrates Contributes to Hemostatic Activity.

PubMed

Eker, İbrahim; Yılmaz, Soner; Çetinkaya, Rıza Aytaç; Pekel, Aysel; Ünlü, Aytekin; Gürsel, Orhan; Yılmaz, Sebahattin; Avcu, Ferit; Muşabak, Uğur; Pekoğlu, Ahmet; Ertaş, Zerrin; Açıkel, Cengizhan; Zeybek, Nazif; Kürekçi, Ahmet Emin; Avcı, İsmail Yaşar

2017-03-01

In the last decade, substantial evidence has accumulated about the use of cryopreserved platelet concentrates, especially in trauma. However, little reference has been made in these studies to the morphological and functional changes of platelets. Recently platelets have been shown to be activated by cryopreservation processes and to undergo procoagulant membrane changes resulting in the generation of platelet-derived microparticles (PMPs), platelet degranulation, and release of platelet-derived growth factors (PDGFs). We assessed the viabilities and the PMP and PDGF levels of cryopreserved platelets, and their relation with thrombin generation. Apheresis platelet concentrates (APCs) from 20 donors were stored for 1 day and cryopreserved with 6% dimethyl sulfoxide. Cryopreserved APCs were kept at -80 °C for 1 day. Thawed APCs (100 mL) were diluted with 20 mL of autologous plasma and specimens were analyzed for viabilities and PMPs by flow cytometry, for thrombin generation by calibrated automated thrombogram, and for PDGFs by enzyme-linked immunosorbent assay testing. The mean PMP and PDGF levels in freeze-thawed APCs were significantly higher (2763±399.4/µL vs. 319.9±80.5/µL, p<0.001 and 550.9±73.6 pg/mL vs. 96.5±49 pg/mL, p<0.001, respectively), but the viability rates were significantly lower (68.2±13.7% vs. 94±7.5%, p<.001) than those of fresh APCs. The mean endogenous thrombin potential (ETP) of freeze-thawed APCs was significantly higher than that of the fresh APCs (3406.1±430.4 nM.min vs. 2757.6±485.7 nM.min, p<0.001). Moreover, there was a significant positive poor correlation between ETP levels and PMP levels (r=0.192, p=0.014). Our results showed that, after cryopreservation, while levels of PMPs were increasing, significantly higher and earlier thrombin formation was occurring in the samples analyzed despite the significant decrease in viability. Considering the damage caused by the freezing process and the scarcity of evidence for their in vivo superiority, frozen platelets should be considered for use in austere environments, reserving fresh platelets for prophylactic use in blood banks.

[Dose-response of aspirin on platelet function in very elderly patients].

PubMed

Feng, X R; Liu, M L; Liu, F; Fan, Y; Tian, Q P

2016-10-18

To assess the consequences of switching aspirin dosage from 100 mg/d to 40 mg/d on cardiovascular benefit, bleeding risk and platelet aggregation in very elderly patients. Arachidonic acid induced platelet aggregation(AA-Ag) was measured in 537 patients aged 80 or older treated with aspirin (100 mg/d). In the study, 100 patients with low on-treatment platelet aggregation and at high risk of bleeding and low risk of cardiovascular events, were switched to aspirin (40 mg/d) and their platelet aggregation was measured again 7 days later.Their bleeding and upper gastrointestinal symptoms were also recorded in following 3 months. The study observed a heterogeneous distributed aspirin 100 mg/d AA-Ag (range: 0.42% to 28.78%)in the 537 very elderly patients.Aspirin 100 mg/d AA-Ag before the switch in aspirin 40 mg/d group was 5.00%±2.32% and the rate of the patients with low on-treatment platelet aggregation was 71.00%. The rates of melena or occult blood positive, other minimal bleeding,upper gastrointestinal symptoms and a history of gastrointestinal bleeding in 40 mg/d group were higher than those in 100 mg/d group. On a regimen of aspirin 40 mg/d, AA-Ag increased to 11.21%±4.95%(range: 2.12% to 28.84%) with 95.00%of the patients with AA-Ag<20% and the rate of the patients with low on-treatment platelet aggregation was 15.00%. Multiple variable analysis revealed that aspirin 40 mg/d AA-Ag was significantly influenced by aspirin 100 mg/d AA-Ag, BMI and platelet counts. The rate of gastrointestinal bleeding decreased from 12.00% to 5.00%,and upper gastrointestinal symptoms decreased from 59.00% to 21.00% after the switch in 40 mg/d group. Switching aspirin dosage from 100 mg/d to 40 mg/d reduces the bleeding events and improves upper gastrointestinal symptoms, thus inhibiting platelet aggregation effectively in very elderly patients.

Influence of a cyclic combination chemotherapeutic protocol on primary haemostasis in dogs suffering from malignant lymphoma.

PubMed

Eberle, N; Mischke, R

2010-03-01

The purpose of this study was to examine the influence of cyclic combination chemotherapy on primary haemostasis in dogs with malignant lymphoma. Seventeen dogs receiving cytostatic treatment for high-grade lymphoma were included in the study. The dogs were treated with a Madison-Wisconsin derived protocol, which included asparaginase, vincristine, doxorubicin and prednisolone. At different time points during the first 4 weeks of induction, platelet count, capillary bleeding time, analysis of the platelet function using the platelet function analyser PFA-100, and platelet aggregation by the Born-method were measured. The most obvious changes were found for median values of the platelet count, which increased significantly from 210,000/microL before induction to 349,000/microL during the second week of induction (P=0.0010). Median platelet count subsequently decreased by the fourth week of treatment (Friedman-test: P<0.0001). None of the parameters of platelet function (capillary bleeding time, automatic platelet function analysis, aggregation maximum) showed significant changes with time (P>0.05, Friedman-test). The results did not suggest that significant platelet dysfunction was induced by the chemotherapeutic protocol used in the study. 2009 Elsevier Ltd. All rights reserved.

In vitro analysis of platelet function in acute aneurysmal subarachnoid haemorrhage.

PubMed

von der Brelie, Christian; Subai, Alexander; Limperger, Verena; Rohde, Veit; Dempfle, Astrid; Boström, Azize

2018-04-01

Platelet function might play an essential role in the pathogenesis of delayed cerebral ischemia (DCI) after aneurysmal subarachnoid haemorrhage (SAH). Thus, impaired platelet function and disturbed primary haemostasis induced by intake of acetylsalicylic acid (ASA) might influence the rate of DCI. Primary haemostasis and platelet function can be measured with in vitro diagnosis (platelet function analyser test, PFA 100). The aim of this study is to evaluate the rate of DCI, haemorrhagic complications and the neurological outcome. Two groups were compared (patients with regular platelet function versus patients with impaired platelet function). This is a retrospective observational study. An initial cohort of 787 patients with SAH has been treated from January 2005 to September 2012. Seventy-nine patients (10%) with aneurysmal SAH, a history of ASA medication and PFA testing within the first 24 h after aneurysm rupture have been included. The overall rate of DCI in the present study was 43%. In vitro platelet function testing showed pathological primary haemostasis in 69.6%. The DCI rate was higher in patients with regular tested primary haemostasis (p = 0.02, OR = 3.16, 95%CI = [1.19; 8.83]). However, outcome assessment by mGOS did not show a significant difference between the groups. Patients with impaired primary haemostasis did not display a higher rate of haemorrhagic complications. Impairment of primary haemostasis resulting from an impairment of platelet function at an early stage after SAH might lead to a lower rate of DCI. In vitro testing of platelet function might be useful to predict the occurrence of DCI in the course.

In vitro anti-platelet effects of simple plant-derived phenolic compounds are only found at high, non-physiological concentrations.

PubMed

Ostertag, Luisa M; O'Kennedy, Niamh; Horgan, Graham W; Kroon, Paul A; Duthie, Garry G; de Roos, Baukje

2011-11-01

Bioactive polyphenols from fruits, vegetables, and beverages have anti-platelet effects and may thus affect the development of cardiovascular disease. We screened the effects of 26 low molecular weight phenolic compounds on two in vitro measures of human platelet function. After platelets had been incubated with one of 26 low molecular weight phenolic compounds in vitro, collagen-induced human platelet aggregation and in vitro TRAP-induced P-selectin expression (as marker of platelet activation) were assessed. Incubation of platelet-rich plasma from healthy volunteers with 100 μmol/L hippuric acid, pyrogallol, catechol, or resorcinol significantly inhibited collagen-induced platelet aggregation (all p<0.05; n≥15). Incubation of whole blood with concentrations of 100 μmol/L salicylic acid, p-coumaric acid, caffeic acid, ferulic acid, 4-hydroxyphenylpropionyl glycine, 5-methoxysalicylic acid, and catechol significantly inhibited TRAP-induced surface P-selectin expression (all p<0.05; n=10). Incubation with lower concentrations of phenolics affected neither platelet aggregation nor activation. As concentrations of 100 μmol/L are unlikely to be reached in the circulation, it is doubtful whether consumption of dietary phenolics in nutritionally attainable amounts plays a major role in inhibition of platelet activation and aggregation in humans. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pharmacogenetic testing for clopidogrel using the rapid INFINITI analyzer: a dose-escalation study.

PubMed

Gladding, Patrick; White, Harvey; Voss, Jamie; Ormiston, John; Stewart, Jim; Ruygrok, Peter; Bvaldivia, Badi; Baak, Ruth; White, Catherine; Webster, Mark

2009-11-01

Our aim was to assess whether a higher clopidogrel maintenance dose has a greater antiplatelet effect in CYP2C19*2 allele carriers compared with noncarriers. Clopidogrel is a prodrug that is biotransformed by the cytochrome P450 enzymes CYP2C19, 2C9, and 3A4, 2B6, 1A2. The CYPC219*2 loss of function variant has been associated with a reduced antiplatelet response to clopidogrel and a 3-fold risk of stent thrombosis. Forty patients on standard maintenance dosage clopidogrel (75 mg), for 9.4 +/- 9.2 weeks, were enrolled into a dose escalation study. Platelet function was assessed at baseline and after 1 week of 150 mg once daily using the VerifyNow platelet function analyzer (Accumetrics Ltd., San Diego, California). Genomic DNA was hybridized to a BioFilmChip microarray on the INFINITI analyzer (AutoGenomics Inc., Carlsbad, California) and analyzed for the CYP19*2, *4, *17, and CYP2C9*2, *3 polymorphisms. Platelet inhibition increased over 1 week, mean +8.6 +/- 13.5% (p = 0.0003). Carriers of the CYP2C19*2 allele had significantly reduced platelet inhibition at baseline (median 18%, range 0% to 72%) compared with wildtype (wt) (median 59%, range 11% to 95%, p = 0.01) and at 1 week (p = 0.03). CYP2C19*2 allele carriers had an increase in platelet inhibition of (mean +9 +/- 11%, p = 0.03) and reduction in platelet reactivity (mean -26 +/- 38 platelet response unit, p = 0.04) with a higher dose. Together CYP2C19*2 and CYP2C9*3 loss of function carriers had a greater change in platelet inhibition with 150 mg daily than wt/wt (+10.9% vs. +0.7%, p = 0.04). Increasing the dose of clopidogrel in patients with nonresponder polymorphisms can increase antiplatelet response. Personalizing clopidogrel dosing using pharmacogenomics may be an effective method of optimizing treatment.

In vitro effects of 3% hypertonic saline and 20% mannitol on canine whole blood coagulation and platelet function.

PubMed

Adamik, Katja-Nicole; Butty, Emmanuelle; Howard, Judith

2015-09-24

Hyperosmolar therapy, using either mannitol or hypertonic saline (HTS), is considered the treatment of choice for intracranial hypertension. However, hyperosmolar agents may impair coagulation and platelet function, limiting their use in patients at risk for hemorrhage. Despite this, studies evaluating the effects of mannitol compared to other hyperosmolar agents in dogs are largely lacking. The aim of this study was to compare the in vitro effects on global hemostasis and platelet function of 20% mannitol and 3% HTS on canine blood. Citrated whole blood from 15 healthy dogs was diluted with 0.9% saline, 20% mannitol and 3% HTS in ratios of 1:16 and 1:8. Rotational thromboelastometry (ROTEM) was used to assess clotting time (CT), clot formation time (CFT) and maximal clot firmness (MCF) following extrinsic activation (Ex-tem) and after platelet inhibition (Fib-tem). A platelet function analyzer (PFA-100) was used to assess closure time (Ct(PFA)). No significant differences were observed between untreated whole blood and samples diluted with saline. Samples diluted with both mannitol and HTS were hypocoagulable compared to untreated whole blood samples. At a dilution of 1:16, no significant differences were found between any measured parameter in samples diluted with saline compared to mannitol or HTS. At a 1:8 dilution, Ct(PFA) was prolonged in samples diluted with mannitol and HTS compared to saline, and Ct(PFA) was prolonged more with mannitol than HTS. Ex-tem CT was increased at a 1:8 dilution with mannitol compared to HTS. Ex-tem CFT was prolonged at a 1:8 dilution with both agents compared to saline, and was prolonged more with mannitol than HTS. Ex-tem MCF was reduced at a 1:8 dilution with both agents compared to saline. Data in this study indicate that both mannitol and HTS affect canine platelet function and whole blood coagulation in vitro in a dose-dependent fashion. The most pronounced effects were observed after high dilutions with mannitol, which impaired platelet aggregation, clot formation time, clot strength, and fibrin formation significantly more than HTS. Further in vivo studies are necessary before recommendations can be made.

Comparison of increased aspirin dose versus combined aspirin plus clopidogrel therapy in patients with diabetes mellitus and coronary heart disease and impaired antiplatelet response to low-dose aspirin.

PubMed

Duzenli, Mehmet Akif; Ozdemir, Kurtulus; Aygul, Nazif; Soylu, Ahmet; Tokac, Mehmet

2008-08-15

The effects of therapy with aspirin 300 mg/day and with combined aspirin 100 mg/day plus clopidogrel 75 mg/day on platelet function were compared in patients with diabetes mellitus and coronary artery disease and impaired antiplatelet responses to aspirin 100 mg/day. The study population consisted of 151 outpatients with type II diabetes mellitus and coronary artery disease who were taking aspirin 100 mg/day. Of the 151 patients, a subgroup of subjects with impaired aspirin response were selected on the basis of the results of platelet aggregometry. Nonresponsiveness to aspirin was defined as mean aggregation > or =69% with 3 micromol/L adenosine diphosphate and mean aggregation > or =70% with 2 micromol/L collagen. Aspirin semiresponders were defined as meeting 1 but not both of these criteria. Nonresponders and semiresponders were randomized equally to aspirin 300 mg/day and aspirin 100 mg/day plus clopidogrel 75 mg/day, and aggregation tests were repeated after 2 weeks. Sixty of the 151 patients with diabetes (40%) were found to respond to aspirin inadequately. Platelet aggregation induced by adenosine diphosphate and collagen decreased significantly after aspirin 300 mg/day or combined therapy. Combined treatment was found to have a stronger inhibitory effect on platelet aggregation induced by adenosine diphosphate than aspirin 300 mg/day (p = 0.002). Impaired aspirin response was resolved by increasing the aspirin dose or adding clopidogrel to aspirin (p <0.0001 for each). However, desired platelet inhibition was achieved in significantly more patients by combined treatment than by aspirin 300 mg/day (p <0.05). In conclusion, aspirin 100 mg/day does not inhibit platelet function adequately in a significant number of patients with diabetes mellitus and coronary artery disease. Increasing the aspirin dose to 300 mg/day or adding clopidogrel to aspirin can provide adequate platelet inhibition in a significant number of those patients with impaired responses to low-dose aspirin.

Platelet function in whole-blood donors is impaired: the effects of painkillers.

PubMed

Curvers, Joyce; Dielis, Arne W J H; Heeremans, Judith; van Wersch, Jan W J

2007-01-01

Aspirin (ASA) or non-aspirin-like nonsteroidal anti-inflammatory drugs (NSAIDs) influence platelet (PLT) function by inhibiting cyclooxygenase enzymes. In this study, the aim was to address the use of ASA or NSAIDs before donation and the effect on PLT function. Donors were asked questions about recent use of ASA or NSAIDs. Furthermore, PLT function was evaluated by measurement of the closure time (CT) in a PLT function analyzer (PFA-100, Dade Behring) and by aggregometry (response to ADP or arachidonic acid [AA]). Of 100 questioned donors, 22 percent had used ASA (n = 4), NSAIDs (n = 6), or paracetamol (n = 12) before donation. Upon assessment of the PLT function in the PFA-100, 27 donors showed values of greater than 180 seconds, indicative of impaired PLT function. Of these, only 7 had used pain killers before donation. Furthermore, 15 of 22 users had normal CTs. Aggregation after stimulation with AA was absent in 33 PLT-rich samples. Again only 8 had reported use of ASA (3), NSAIDs (1), or paracetamol (4). Of the 22 users, 14 had normal AA aggregation responses. All donor samples showed ADP-induced aggregation, indicating PLT integrity. There was no difference between the group of donors who reported the intake of ASA or NSAIDs and the group of donors who did not with respect to the tested PLT function assays. It is concluded that there is a considerable group of donors that use PLT-influencing medication before donation. A relation between the reported use and impaired PLT function in blood donors could not be established, however. Impaired PLT function as tested may have other causes than intake of ASA or NSAIDs.

Development of a New Method for Platelet Function Test and Its Shearing Condition in Microfludic System

NASA Astrophysics Data System (ADS)

Lee, Hoyoon; Kim, Gyehyu; Choi, Seawhan; Shin, Sehyun; Korea University Department of Mechanical Engineering Team

2015-11-01

Platelet is a crucial blood cell on hemostasis. As platelet exposed to high shear stress, it can be activated showing morphological and functional changes to stop bleeding. When platelet is abnormal, there is high risk of cardiovascular diseases. Thus, quick and precise assay for platelet function is important in clinical treatment. In this study, we design a microfluidic system, which can test platelet function exposed with the stimulation of shear and agonists. The microfluidic system consists of three parts: 1) a shear mechanism with rotating stirrer; 2) multiple microchannels to flow samples and to stop; 3) camera-interfaced migration distance(MD) analyzing system. When sheared blood is driven by pressure through the microchannel, shear-activated platelets adhere to a collagen-coated surface, causing blood flow to significantly slow and eventually stop. As the micro-stirrer speed increases, MD decreases exponentially at first, but it increases beyond a critical rpm after all. These results are coincident with data measured by FACS flowcytometry. These results imply that the present system could quantitatively measure the degree of activation, aggregation and adhesion of platelets and that blood MD is potent index for measuring the shear-dependence of platelet function.

Effects of carprofen, meloxicam and deracoxib on platelet function in dogs.

PubMed

Mullins, Kathleen B; Thomason, John M; Lunsford, Kari V; Pinchuk, Lesya M; Langston, Vernon C; Wills, Robert W; McLaughlin, Ronald M; Mackin, Andrew J

2012-03-01

To determine effects of anti-inflammatory doses of COX-2 selective NSAIDs carprofen, meloxicam, and deracoxib on platelet function in dogs and urine 11-dehydro-thromboxane B2. Randomized, blocked, crossover design with a 14-day washout period. Healthy intact female Walker Hounds aged 1-6 years and weighing 20.5-24.2 kg. Dogs were given NSAIDs for 7 days at recommended doses: carprofen (2.2 mg kg(-1), PO, every 12 hours), carprofen (4.4 mg kg(-1), PO, every 24 hours), meloxicam (0.2 mg kg(-1), PO, on the 1st day then 0.1 mg kg(-1), PO, every 24 hours), and deracoxib (2 mg kg(-1), PO, every 24 hours). Collagen/epinephrine and collagen/ADP PFA-100 cartridges were used to evaluate platelet function before and during and every other day after administration of each drug. Urine 11-dehydro-thromboxane B(2) was also measured before and during administration of each drug. All NSAIDs significantly prolonged PFA-100 closure times when measured with collagen/epinephrine cartridges, but not with collagen/ADP cartridges. The average duration from drug cessation until return of closure times (collagen/epinephrine cartridges) to baseline values was 11.6, 10.6, 11 and 10.6 days for carprofen (2.2 mg kg(-1) every 12 hours), carprofen (4.4 mg kg(-1) every 24 hours), meloxicam and deracoxib, respectively. Oral administration of some COX-2 selective NSAIDs causes detectable alterations in platelet function in dogs. As in humans, PFA-100 collagen/ADP cartridges do not reliably detect COX-mediated platelet dysfunction in dogs. Individual assessment of platelet function is advised when administering these drugs prior to surgery, particularly in the presence of other risk factors for bleeding. © 2011 The Authors. Veterinary Anaesthesia and Analgesia. © 2011 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists.

Evaluation of Dried Storage of Platelets and RBC for Transfusion: Lyophilization and other Dehydration Techniques.

DTIC Science & Technology

1992-11-02

linking agents. Water replacement molecules, such as trehalose or sucrose at concentrations up to 1 M, did not suffice to stabilize platelets for...concentration of albumin (5% w/v) or trehalose (250 mM - I M) is required in the final suspension prior to freeze-drying to ensure platelet yield and...structurally or functionally intact platelets post- rehydration; the presence of albumin or trehalose raises this yield to 90-100%. Lvophilization and Storage

Monitoring survival and function of transfused platelets in Bernard-Soulier syndrome by flow cytometry and a cone and plate(let) analyzer (Impact-R).

PubMed

Panzer, Simon; Eichelberger, Beate; Koren, Daniela; Kaufmann, Karin; Male, Christoph

2007-01-01

Bernard-Soulier syndrome (BSS) patients may repeatedly require transfusion of platelets (PLTs). The hemostatic competence of transfused PLTs requires monitoring. Flow cytometry and a cone and plate(let) analyzer (Impact-R, DiaMed) were used to monitor survival and function of transfused PLTs in a 7-year-old girl with BSS undergoing surgery. Flow cytometry was applied to differentiate autologous PLTs from transfused PLTs by staining for CD42b. The Impact, which measures PLT adhesion and aggregation in response to high shear stress, was used to evaluate PLT function. Transfused PLTs were detectable by flow cytometry for 1 week after transfusion. While the patient's PLTs did not respond to high shear stress before transfusion, a normal response was documented by the Impact on the day after transfusion and 1 week thereafter. Transfused PLTs were detectable by flow cytometry, and their functional activity was demonstrated by the Impact.

Postoperative Decrease in Platelet Counts Is Associated with Delayed Liver Function Recovery and Complications after Partial Hepatectomy.

PubMed

Takahashi, Kazuhiro; Kurokawa, Tomohiro; Oshiro, Yukio; Fukunaga, Kiyoshi; Sakashita, Shingo; Ohkohchi, Nobuhiro

2016-05-01

Peripheral platelet counts decrease after partial hepatectomy; however, the implications of this phenomenon are unclear. We assessed if the observed decrease in platelet counts was associated with postoperative liver function and morbidity (complications grade ≤ II according to the Clavien-Dindo classification). We enrolled 216 consecutive patients who underwent partial hepatectomy for primary liver cancers, metastatic liver cancers, benign tumors, and donor hepatectomy. We classified patients as either low or high platelet percentage (postoperative platelet count/preoperative platelet count) using the optimal cutoff value calculated by a receiver operating characteristic (ROC) curve analysis, and analyzed risk factors for delayed liver functional recovery and morbidity after hepatectomy. Delayed liver function recovery and morbidity were significantly correlated with the lowest value of platelet percentage based on ROC analysis. Using a cutoff value of 60% acquired by ROC analysis, univariate and multivariate analysis determined that postoperative lowest platelet percentage ≤ 60% was identified as an independent risk factor of delayed liver function recovery (odds ratio (OR) 6.85; P < 0.01) and morbidity (OR, 4.90; P < 0.01). Furthermore, patients with the lowest platelet percentage ≤ 60% had decreased postoperative prothrombin time ratio and serum albumin level and increased serum bilirubin level when compared with patients with platelet percentage ≥ 61%. A greater than 40% decrease in platelet count after partial hepatectomy was an independent risk factor for delayed liver function recovery and postoperative morbidity. In conclusion, the decrease in platelet counts is an early marker to predict the liver function recovery and complications after hepatectomy.

High content evaluation of shear dependent platelet function in a microfluidic flow assay

PubMed Central

Hansen, Ryan R.; Wufsus, Adam R.; Barton, Steven T.; Onasoga, Abimbola A.; Johnson-Paben, Rebecca M.; Neeves, Keith B.

2012-01-01

The high blood volume requirements and low throughput of conventional flow assays for measuring platelet function are unsuitable for drug screening and clinical applications. In this study, we describe a microfluidic flow assay that uses 50 μL of whole blood to measure platelet function on ~300 micropatterned spots of collagen over a range of physiologic shear rates (50–920 s−1). Patterning of collagen thin films (CTF) was achieved using a novel hydrated microcontact stamping method. CTF spots of 20, 50, and 100 μm were defined on glass substrates and consisted of a dense mat of nanoscale collagen fibers (3.74 ± 0.75 nm). We found that a spot size of greater than 20 μm was necessary to support platelet adhesion under flow, suggesting a threshold injury is necessary for stable platelet adhesion. Integrating 50 μm CTF microspots into a multishear microfluidic device yielded a high content assay from which we extracted platelet accumulation metrics (lag time, growth rate, total accumulation) on the spots using Hoffman modulation contrast microscopy. This method has potential broad application in identifying platelet function defects and screening, monitoring and dosing antiplatelet agents. PMID:23001359

Edge-Selectively Functionalized Graphene-Like Platelets as a Co-curing Agent and a Nanoscale Additive to Epoxy Resin

DTIC Science & Technology

2012-08-12

21 For mass production , the Report Documentation Page Form ApprovedOMB No. 0704-0188 Public reporting burden for the collection of information is...analyzer. The field emission scanning electron microscope ( FE -SEM) used in this work was a NanoSem 230 (FEI, USA). High-resolution transmission...WAXD) powder patterns were recorded with a Rigaku RU-200 diffractometer using Ni-filtered Cu K radiation (40 kV, 100 mA,  = 0.15418 nm). Dynamic

Dark chocolate inhibits platelet aggregation in healthy volunteers.

PubMed

Innes, Andrew J; Kennedy, Gwen; McLaren, Margaret; Bancroft, Anne J; Belch, Jill J F

2003-08-01

Cardiovascular disease is a leading cause of death in the UK. The flavonoids found in cocoa may produce a cardio-protective role for chocolate with a high cocoa content. Thirty healthy volunteers were randomised to receive 100 g of white, milk or dark chocolate, and assessments of platelet function were undertaken on venous blood samples before and after chocolate consumption. White and milk chocolate had no significant effect on platelets. However dark chocolate inhibited collagen-induced platelet aggregation in platelet rich plasma. In the future dark chocolate may have a role in prevention of cardiovascular and thromboembolic diseases.

Low angle light scattering analysis: a novel quantitative method for functional characterization of human and murine platelet receptors.

PubMed

Mindukshev, Igor; Gambaryan, Stepan; Kehrer, Linda; Schuetz, Claudia; Kobsar, Anna; Rukoyatkina, Natalia; Nikolaev, Viacheslav O; Krivchenko, Alexander; Watson, Steve P; Walter, Ulrich; Geiger, Joerg

2012-07-01

Determinations of platelet receptor functions are indispensable diagnostic indicators of cardiovascular and hemostatic diseases including hereditary and acquired receptor defects and receptor responses to drugs. However, presently available techniques for assessing platelet function have some disadvantages, such as low sensitivity and the requirement of large sample sizes and unphysiologically high agonist concentrations. Our goal was to develop and initially characterize a new technique designed to quantitatively analyze platelet receptor activation and platelet function on the basis of measuring changes in low angle light scattering. We developed a novel technique based on low angle light scattering registering changes in light scattering at a range of different angles in platelet suspensions during activation. The method proved to be highly sensitive for simultaneous real time detection of changes in size and shape of platelets during activation. Unlike commonly-used methods, the light scattering method could detect platelet shape change and aggregation in response to nanomolar concentrations of extracellular nucleotides. Furthermore, our results demonstrate that the advantages of the light scattering method make it a choice method for platelet receptor monitoring and for investigation of both murine and human platelets in disease models. Our data demonstrate the suitability and superiority of this new low angle light scattering method for comprehensive analyses of platelet receptors and functions. This highly sensitive, quantitative, and online detection of essential physiological, pathophysiological and pharmacological-response properties of human and mouse platelets is a significant improvement over conventional techniques.

Mice Lacking the SLAM Family Member CD84 Display Unaltered Platelet Function in Hemostasis and Thrombosis

PubMed Central

Hofmann, Sebastian; Braun, Attila; Pozgaj, Rastislav; Morowski, Martina; Vögtle, Timo; Nieswandt, Bernhard

2014-01-01

Background Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity by forming thrombi at sites of vascular injury. Although the early events of thrombus formation—platelet adhesion and aggregation—have been intensively studied, less is known about the mechanisms and receptors that stabilize platelet-platelet interactions once a thrombus has formed. One receptor that has been implicated in this process is the signaling lymphocyte activation molecule (SLAM) family member CD84, which can undergo homophilic interactions and becomes phosphorylated upon platelet aggregation. Objective The role of CD84 in platelet physiology and thrombus formation was investigated in CD84-deficient mice. Methods and Results We generated CD84-deficient mice and analyzed their platelets in vitro and in vivo. Cd84−/− platelets exhibited normal activation and aggregation responses to classical platelet agonists. Furthermore, CD84 deficiency did not affect integrin-mediated clot retraction and spreading of activated platelets on fibrinogen. Notably, also the formation of stable three-dimensional thrombi on collagen-coated surfaces under flow ex vivo was unaltered in the blood of Cd84−/− mice. In vivo, Cd84−/− mice exhibited unaltered hemostatic function and arterial thrombus formation. Conclusion These results show that CD84 is dispensable for thrombus formation and stabilization, indicating that its deficiency may be functionally compensated by other receptors or that it may be important for platelet functions different from platelet-platelet interactions. PMID:25551754

NF-E2 p45 Is Important for Establishing Normal Function of Platelets

PubMed Central

Fujita, Rie; Takayama-Tsujimoto, Mariko; Satoh, Hironori; Gutiérrez, Laura; Aburatani, Hiroyuki; Fujii, Satoshi; Sarai, Akinori; Bresnick, Emery H.

2013-01-01

NF-E2 is a heterodimeric transcription factor consisting of p45 and small Maf subunits. Since p45−/− mice display severe thrombocytopenia, p45 is recognized as a critical regulator of platelet production from megakaryocytes. To identify direct p45 target genes in megakaryocytes, we used chromatin immunoprecipitation (ChIP) sequencing to analyze the genome-wide chromatin occupancy of p45 in primary megakaryocytes. p45 target gene candidates obtained from the analysis are implicated in the production and function of platelets. Two of these genes, Selp and Myl9, were verified as direct p45 targets through multiple approaches. Since P-selectin, encoded by Selp, plays a critical role in platelet function during thrombogenesis, we tested whether p45 determines the intrinsic reactivity and potency of platelets generated from megakaryocytes. Mice expressing a hypomorphic p45 mutant instead of wild-type p45 in megakaryocytes (p45−/−:ΔNTD-Tg mice) displayed platelet hypofunction accompanied by mild thrombocytopenia. Furthermore, lung metastasis of melanoma cells, which requires platelet activation, was repressed in p45−/−:ΔNTD-Tg mice compared to control mice, validating the impaired function of platelets produced from p45−/−:ΔNTD-Tg megakaryocytes. By activating genes in megakaryocytes that mediate platelet production and function, p45 determines the quantity and quality of platelets. PMID:23648484

Safety and efficacy of a ginkgo biloba-containing dietary supplement on cognitive function, quality of life, and platelet function in healthy, cognitively intact older adults.

PubMed

Carlson, Joseph J; Farquhar, John W; DiNucci, Ellen; Ausserer, Laurie; Zehnder, James; Miller, Donald; Berra, Kathy; Hagerty, Lisa; Haskell, William L

2007-03-01

To determine if a ginkgo biloba-containing supplement improves cognitive function and quality of life, alters primary hemostasis, and is safe in healthy, cognitively intact older adults. Four-month, randomized, double-blind, placebo-controlled parallel design. Ninety men and women (age range 65 to 84 years) were recruited to a university clinic. Eligibility included those without dementia or depression, not taking psychoactive medications or medications or supplements that alter hemostasis. Ninety subjects were randomly assigned to placebo or a ginkgo biloba-based supplement containing 160 mg ginkgo biloba, 68 mg gotu kola, and 180 mg decosahexaenoic acid per day for 4 months. Assessments included: six standardized cognitive function tests, the SF-36 Quality of Life questionnaire, the Platelet Function Analyzer-100 (Dade Behring, Eschbom, Germany), and the monitoring of adverse events. Baseline characteristics and study hypotheses were tested using analysis of covariance. Tests were two-tailed with a 0.05 significance level. Seventy-eight subjects (87%) completed both baseline and 4-month testing (n=36 in placebo group, n=42 in ginkgo biloba group). At baseline, the participants' cognitive function was above average. One of six cognitive tests indicated significant protocol differences at 4 months (P=0.03), favoring the placebo. There were no significant differences in quality of life, platelet function, or adverse events. These finding do not support the use of a ginkgo biloba-containing supplement for improving cognitive function or quality of life in cognitively intact, older, healthy adults. However, high baseline scores may have contributed to the null findings. The ginkgo biloba product seems safe and did not alter platelet function, though additional studies are needed to evaluate the interaction of varying doses of ginkgo biloba and ginkgo biloba-containing supplements with medications and supplements that alter hemostasis.

Detection of microbial contamination in platelets

NASA Astrophysics Data System (ADS)

Berg, Tracy L.; Leparc, German; Huffman, Debra E.; Gennaccaro, Angela L.; Garcia-Lopez, Alicia; Klungness, Greta; Stephans, Christie; Garcia-Rubio, Luis H.

2005-03-01

In the United States, approximately 100 patients develop fatal sepsis associated with platelet transfusions every year. Current culture methods take 24-48 hours to acquire results, which in turn decrease the shelf life of platelets. Many of the microorganisms that contaminate platelets can replicate easily at room temperature, which is the necessary storage temperature to keep platelets functional. Therefore, there is a need for in-situ quality control assessment of the platelet quality. For this purpose, a real time spectrophotometric technique has been developed. The Spectral Acquisition Processing Detection (SAPD) method, comprised of a UV-vis spectrophotometer and modeling algorithms, is a rapid method that can be performed prior to platelet transfusion to decrease the risk of bacterial infection to patients. The SAPD method has been used to determine changes in cell suspensions, based on size, shape, chemical composition and internal structure. Changes in these cell characteristics can in turn be used to determine microbial contamination, platelet aging and other physiologic changes. Detection limits of this method for platelet suspensions seeded with bacterial contaminants were identified to be less than 100 cfu/ml of sample. Bacterial counts below 1000 cfu/ml are not considered clinically significant. The SAPD method can provide real-time identification of bacterial contamination of platelets affording patients an increased level of safety without causing undue strain on laboratory budgets or personnel while increasing the time frame that platelets can be used by dramatically shortening contaminant detection time.

Women with Red Hair Report A Slightly Increased Rate of Bruising, but Have Normal Coagulation Tests

PubMed Central

Liem, Edwin B.; Hollensead, Sandra C.; Joiner, Teresa V.

2005-01-01

There is an anecdotal impression that redheads experience more perioperative bleeding complications than those with other hair colors. We, therefore, tested the hypothesis that perceived problems with hemostasis could be detected with commonly used coagulation tests. Se studied healthy female Caucasian volunteers, 18 to 40 years, comparable in terms of height, weight, and age, with natural bright red (n = 25) or black or dark brown (n = 26) hair. Volunteers were questioned about their bleeding history and the following tests were performed: complete blood count, prothrombin time/international normalized ratio, activated partial thromboplastin time, platelet function analysis (PFA-100), and platelet aggregation using standard turbidimetric methodology. Agonists for aggregation were adenosine diphosphate, arachidonic acid, collagen, epinephrine, and two concentrations of ristocetin. The red-haired volunteers reported significantly more bruising, but there were no significant differences between the red- and dark-haired groups in hemoglobin concentration, platelet numbers, prothrombin time/international normalized ratio, or activated partial thromboplastin time. Furthermore, no significant differences in platelet function, as measured with the PFA-100 or with platelet aggregometry, were observed. We conclude that if redheads have hemostasis abnormalities, they are subtle. PMID:16368849

Platelet Function Analyzer 100 and Brain Natriuretic Peptide as Biomarkers in Obstructive Hypertrophic Cardiomyopathy.

PubMed

Blackshear, Joseph L; Safford, Robert E; Thomas, Colleen S; Bos, J Martijn; Ackerman, Michael J; Geske, Jeffrey B; Ommen, Steve R; Shapiro, Brian P; Johns, Gretchen S

2018-03-15

To test dual blood biomarkers compared with electrocardiogram (ECG) for hypertrophic cardiomyopathy (HC) screening, we performed 3 analyses and cut-point assessments. First, we measured platelet function analyzer (PFA)-100 (n = 99) and normalized B-type natriuretic peptide (BNP) or NT-proBNP (BNP/upper limit of normal [ULN], n = 92) in 64 patients with HC and 29 normal controls (NCs). Second, from the regression equation between PFA and gradient (r = 0.77), we derived estimated PFA in a population of 189 patients with functional class I HC in whom measured BNP/ULN and ECG were available, and calculated single and dual biomarker sensitivity and specificity compared with ECG. Finally, we compared BNP/ULN in class I patients based on mutation and familial history status. In 42 patients with obstructive HC versus NCs, there was a slight overlap of PFA and BNP/ULN, but for the product of PFA × BNP/ULN, there was near-complete separation of values. Among patients with class I obstructive HC, estimated PFA × BNP/ULN had a sensitivity of 93% and a specificity of 100%; in latent and nonobstructive HC, sensitivity dropped to 61% and 72%; for ECG in obstructive, latent, and nonobstructive HC, sensitivity was 71%, 34%, and 67%. Functional class I patients with positive (n = 28) and negative (n = 36) sarcomere mutations and a positive (n = 71) or a negative (n = 109) family history had significant elevations of BNP/ULN versus NC, with no between-group differences. In conclusion, PFA and BNP were highly associated with obstructive HC and could potentially be used for screening; BNP was not uniquely elevated in patients with familial versus nonfamilial or mutation-positive versus mutation-negative HC. Copyright © 2017 Elsevier Inc. All rights reserved.

Rapid Evaluation of Platelet Function With T2 Magnetic Resonance

PubMed Central

Cuker, Adam; Husseinzadeh, Holleh; Lebedeva, Tatiana; Marturano, Joseph E.; Massefski, Walter; Lowery, Thomas J.; Lambert, Michele P.; Abrams, Charles S.; Weisel, John W.

2016-01-01

Objectives: The clinical diagnosis of qualitative platelet disorders (QPDs) based on light transmission aggregometry (LTA) requires significant blood volume, time, and expertise, all of which can be barriers to utilization in some populations and settings. Our objective was to develop a more rapid assay of platelet function by measuring platelet-mediated clot contraction in small volumes (35 µL) of whole blood using T2 magnetic resonance (T2MR). Methods: We established normal ranges for platelet-mediated clot contraction using T2MR, used these ranges to study patients with known platelet dysfunction, and then evaluated agreement between T2MR and LTA with arachidonic acid, adenosine diphosphate, epinephrine, and thrombin receptor activator peptide. Results: Blood from 21 healthy donors was studied. T2MR showed 100% agreement with LTA with each of the four agonists and their cognate inhibitors tested. T2MR successfully detected abnormalities in each of seven patients with known QPDs, with the exception of one patient with a novel mutation leading to Hermansky-Pudlak syndrome. T2MR appeared to detect platelet function at similar or lower platelet counts than LTA. Conclusions: T2MR may provide a clinically useful approach to diagnose QPDs using small volumes of whole blood, while also providing new insight into platelet biology not evident using plasma-based platelet aggregation tests. PMID:28028118

In vitro effect of sodium nitrite on platelet aggregation in human platelet rich plasma--preliminary report.

PubMed

Kadan, M; Doğanci, S; Yildirim, V; Özgür, G; Erol, G; Karabacak, K; Avcu, F

2015-10-01

The role of nitrates and nitric oxide on platelet functions has obtained an increasing attention with respect to their potential effects on cardiovascular disorders. In this study we aimed to analyze the effect of sodium nitrite on platelet functions in human platelets. This in vitro study was designed to show the effect of sodium nitrite on platelet functions in seven healthy volunteers. Blood samples were centrifuged to prepare platelet rich plasma and platelet poor plasma. Platelet rich plasma was diluted with the platelet poor plasma to have a final count of 300,000 ± 25,000 platelets. Platelet rich plasma was incubated with six different increasing doses (from 10 μM to 5 mM) of sodium nitrite for 1 hour at 37°C. Then stimulating agents including collagen (3 μg ml-1), adenosine diphosphate (10 μM), and epinephrine (10 μM) were added to the cuvette. Changes in light transmission were observed for 10 minutes. In addition spontaneous aggregation were performed in control group with all aggregating agents separately. Effect of sodium nitrite on agonist-induced platelet aggregation depends on the concentration of sodium nitrite. Compared with control group, agonist-induced platelet aggregations were significantly suppressed by sodium nitrite at the concentration of 5, 1.0 and 0.5 mM. Our results suggested that sodium nitrite has inhibitory effects in vitro on platelet aggregation in a dose-dependent manner.

Effect of Hawthorn (Crataegus aronia syn. Azarolus (L)) on platelet function in albino Wistar rats.

PubMed

Shatoor, Abdullah S; Soliman, Hesham; Al-Hashem, Fahaid; Gamal, Basiouny El-; Othman, Adel; El-Menshawy, Nadia

2012-07-01

This study was designed to investigate the possible antiplatelet effect of aqueous whole-plant C. aronia syn: Azarolus (L) extract using Wistar albino rats as a model. Forty-two male albino Wistar rats weighing 200 to 250 g were divided into seven groups with six rats in each group. Group 1 served as the control and received equal volumes of distilled water. Groups 2-6 served as the experimental groups and were given C. aronia extract at doses of 100, 200, 500, 1,000, and 2,000 mg/kg, while group 7 served as a positive control and was given aspirin (25mg/kg). All the doses were administered orally once a day and the treatment was continued for seven days. In all groups, at the end of the experimental procedure, blood samples were obtained for platelet function measurements, including PFA-100, thromboxane B2 levels, platelet count, and haematocrit. The bleeding time was determined using a modified tail cutting method described previously. The aqueous C. aronia syn. Azarolus (L) extract significantly altered the bleeding time and the closure time, as determined by the PFA-100 and thromboxane B2 levels, suggesting significant platelet function inhibition. These effects were observed with C. aronia doses between 100 - 500 mg/kg, which yielded thromboxane B2 levels of 1,000 mg/kg, whereas the higher dose (2,000 mg/kg) produced opposite effects on these parameters. C. aronia syn. Azarolus (L) aqueous extract has antiplatelet effects in Wistar albino rats. Copyright © 2012 Elsevier Ltd. All rights reserved.

Patients with metabolic syndrome exhibit higher platelet activity than those with conventional risk factors for vascular disease.

PubMed

Serebruany, Victor L; Malinin, Alex; Ong, Stephen; Atar, Dan

2008-04-01

The metabolic syndrome is a matter of ongoing debate with regard to its existence, classification, clinical meaningfulness, and associated risks for vessel occlusion. Considering that persistent platelet activation is a cornerstone for the development of acute vascular events, and that patients with type 2 diabetes consistently exhibit high platelet activity, these characteristics may be critical for distinguishing and triageing specific features of metabolic syndrome among established risk factors for vascular disease. We assessed the platelet activity by conventional aggregation, expression of major surface receptors by flow cytometry, and quantitatively by rapid bedside analyzers in 20 aspirin-naïve patients with documented metabolic syndrome, and compared these with 20 untreated subjects with multiple cardiovascular risk factors. Closure time by the PFA-100 analyzer was significantly (P = 0.002) shorter in patients with metabolic syndrome indicating platelet inhibition under high shear conditions. Ultegra analyzer readings revealed increased fibrinogen binding (P = 0.0003) what in combination with the increased expression of PAC-1 (P = 0.32) strongly suggest activation of platelet glycoprotein IIb/IIIa receptor. Surface expression of CD107a (P = 0.014), and SPAN-12 (P = 0.003) were also higher in patients with metabolic syndrome. In contrast, platelet aggregation induced by collagen or ADP, CD31, CD41, CD42b, CD51/61, CD62p, CD63, CD154, CD165, so as formation of platelet-monocyte aggregates, PAR-1 thrombin receptor, and thrombospondin did not differ between groups. Patients with metabolic syndrome exhibited a higher degree of platelet activation than subjects with conventional risk factors for vascular disease. Conceptually, applying adequate antiplatelet strategies may reduce the risk of acute thrombotic events in these patients. Further prospective studies exploring this notion are encouraged.

The administration of a loading dose of aspirin to patients presenting with acute myocardial infarction while receiving chronic aspirin treatment reduces thromboxane A2-dependent platelet reactivity.

PubMed

Santos, Maria Teresa; Madrid, Isabel; Moscardo, Antonio; Latorre, Ana M; Bonastre, Juan; Ruano, Miguel; Valles, Juana

2014-01-01

Abstract The optimal dose of aspirin for patients presenting with acute myocardial infarction (AMI) while receiving chronic aspirin therapy has not been clearly established. We evaluated whether continued treatment with 100 mg of aspirin or a loading dose (200-500 mg) influences thromboxane A2 (TX) suppression or platelet reactivity. Sixty-four consecutive patients with AMI and 98 healthy subjects (82 aspirin-free and 16 receiving 100 mg daily for a week) were evaluated. Treatment was at the discretion of the attending physician. Collagen (1 µg/ml)-induced TX synthesis, (14)C-serotonin-release, platelet aggregation, and the PFA-100 assay were evaluated. The platelet TX synthesis of patients receiving a loading dose of aspirin was sixfold lower than that of patients receiving 100 mg of aspirin (p<0.005). This was associated with marked reductions in (14)C-serotonin-release and arachidonic-acid-induced aggregation and an increase in the PFA-100 closure time (p<0.01). Categorization of patients according to their TX synthesis (<95% or ≥ 95% inhibition vs. healthy aspirin-free subjects) revealed that 8% of the patients treated with loading doses had a poor response (<95% inhibition) vs. 53% of those treated with 100 mg (p<0.001). Patients with lower TX inhibition had higher serum NT-Pro-BNP (p<0.005), a marker of poor left ventricular systolic function. Administration of a loading dose of aspirin to patients with AMI during existing chronic aspirin treatment induced greater reductions in platelet TX synthesis and TX-dependent platelet reactivity than the continued treatment alone.

Comparative lipidomics and proteomics analysis of platelet lipid rafts using different detergents.

PubMed

Rabani, Vahideh; Davani, Siamak; Gambert-Nicot, Ségolène; Meneveau, Nicolas; Montange, Damien

2016-11-01

Lipid rafts play a pivotal role in physiological functions of platelets. Their isolation using nonionic mild detergents is considered as the gold standard method, but there is no consensual detergent for lipid raft studies. We aimed to investigate which detergent is the most suitable for lipid raft isolation from platelet membrane, based on lipidomics and proteomics analysis. Platelets were obtained from healthy donors. Twelve sucrose fractions were extracted by three different detergents, namely Brij 35, Lubrol WX, and Triton X100, at 0.05% and 1%. After lipidomics analysis and determination of fractions enriched in cholesterol (Ch) and sphingomyelin (SM), proteomics analysis was performed. Lipid rafts were mainly observed in 1-4 fractions, and non-rafts were distributed on 5-12 fractions. Considering the concentration of Ch and SM, Lubrol WX 1% and Triton X100 1% were more suitable detergents as they were able to isolate lipid raft fractions that were more enriched than non-raft fractions. By proteomics analysis, overall, 822 proteins were identified in platelet membrane. Lipid raft fractions isolated with Lubrol WX 0.05% and Triton X100 1% contained mainly plasma membrane proteins. However, only Lubrol WX 0.05 and 1% and Triton X100 1% were able to extract non-denaturing proteins with more than 10 transmembrane domains. Our results suggest that Triton X100 1% is the most suitable detergent for global lipid and protein studies on platelet plasma membrane. However, the detergent should be adapted if investigation of an association between specific proteins and lipid rafts is planned.

Von Willebrand factor antigen predicts response to double dose of aspirin and clopidogrel by PFA-100 in patients undergoing primary angioplasty for ST elevation myocardial infarction.

PubMed

Gianetti, Jacopo; Parri, Maria Serena; Della Pina, Francesca; Marchi, Federica; Koni, Endrin; De Caterina, Alberto; Maffei, Stefano; Berti, Sergio

2013-01-01

Von Willebrand factor (VWF) is an emerging risk factor in acute coronary syndromes. Platelet Function Analyzer (PFA-100) with Collagen/Epinephrine (CEPI) is sensitive to functional alterations of VWF and also identifies patients with high on-treatment platelet reactivity (HPR). The objective of this study was to verify the effect of double dose (DD) of aspirin and clopidogrel on HPR detected by PFA-100 and its relation to VWF and to its regulatory metalloprotease ADAMTS-13. Between 2009 and 2011 we enrolled 116 consecutive patients with ST elevation myocardial infarction undergoing primary PCI with HPR at day 5 after PCI. Patients recruited were then randomized between a standard dose (SD, n = 58) or DD of aspirin and clopidogrel (DD, n = 58), maintained for 6 months follow-up. Blood samples for PFA-100, light transmittance aggregometry, and VWF/ADAMTS-13 analysis were collected after 5, 30, and 180 days (Times 0, 1, and 2). At Times 1 and 2 we observed a significantly higher CEPI closure times (CT) in DD as compared to SD (P < 0.001). Delta of CEPI-CT (T1 - T0) was significantly related to VWF (P < 0.001) and inversely related to ADAMTS-13 (0.01). Responders had a significantly higher level of VWF at T0. Finally, in a multivariate model analysis, VWF and ADAMTS-13 in resulted significant predictors of CEPI-CT response (P = 0.02). HRP detected by PFA-100 in acute myocardial infarction is reversible by DD of aspirin and clopidogrel; the response is predicted by basal levels of VWF and ADAMTS-13. PFA-100 may be a useful tool to risk stratification in acute coronary syndromes given its sensitivity to VWF.

Advances and controversies in neonatal ICU platelet transfusion practice.

PubMed

Christensen, Robert D

2008-01-01

Some of the platelet transfusions currently given to NICU patients are unnecessary and convey no benefits. Although ordered with good intentions, unnecessary platelet transfusions carry known and unknown risks. Identifying and eliminating any unnecessary platelet transfusions in NICUs would be a step toward better care, lower costs, and more careful preservation of blood component resources. A renewed interest in platelet transfusion studies is needed, if essential data is to be gathered to improve NICU platelet transfusion practice. Retrospective studies can be of value: for instance, seeking associations between bleeding events and platelet counts can suggest the possibility of cause and effect relationships. Such studies might identify approximate platelet count levels that convey high hemorrhagic risk and might help focus future prospective trials. Prospective indirect studies also can be of value, for instance, measuring the template bleeding time and the PFA-100 closure time as a function of platelet count and perhaps as a function of circulating platelet mass, and would provide new information with relevance to platelet transfusion benefits. Such studies might give a better awareness of how low the platelet count can fall before platelet plug formation is impaired. It seems inescapable, however, that new, multicentered, randomized, prospective studies are needed, where NICU patients are assigned different platelet transfusion triggers and then carefully tracked for bleeding events and long-term neurodevelopmental outcomes. Only that type of study is likely to generate the evidence base needed for widespread implementation of improvements in NICU platelet transfusion practice.

Novel whole blood assay for phenotyping platelet reactivity in mice identifies ICAM-1 as a mediator of platelet-monocyte interaction

PubMed Central

Kirkby, Nicholas S.; Chan, Melissa V.; Finsterbusch, Michaela; Hogg, Nancy; Nourshargh, Sussan; Warner, Timothy D.

2015-01-01

Testing of platelet function is central to the cardiovascular phenotyping of genetically modified mice. Traditional platelet function tests have been developed primarily for testing human samples and the volumes required make them highly unsuitable for the testing of mouse platelets. This limits research in this area. To address this problem, we have developed a miniaturized whole blood aggregometry assay, based on a readily accessible 96-well plate format coupled with quantification of single platelet depletion by flow cytometric analysis. Using this approach, we observed a concentration-dependent loss of single platelets in blood exposed to arachidonic acid, collagen, U46619 or protease activated receptor 4 activating peptide. This loss was sensitive to well-established antiplatelet agents and genetic manipulation of platelet activation pathways. Observations were more deeply analyzed by flow cytometric imaging, confocal imaging, and measurement of platelet releasates. Phenotypic analysis of the reactivity of platelets taken from mice lacking intercellular adhesion molecule (ICAM)-1 identified a marked decrease in fibrinogen-dependent platelet-monocyte interactions, especially under inflammatory conditions. Such findings exemplify the value of screening platelet phenotypes of genetically modified mice and shed further light upon the roles and interactions of platelets in inflammation. PMID:26215112

Platelet Function Analyzed by Light Transmission Aggregometry.

PubMed

Hvas, Anne-Mette; Favaloro, Emmanuel J

2017-01-01

Analysis of platelet function is widely used for diagnostic work-up in patients with increased bleeding tendency. During the last decades, platelet function testing has also been introduced for evaluation of antiplatelet therapy, but this is still recommended for research purposes only. Platelet function can also be assessed for hyper-aggregability, but this is less often evaluated. Light transmission aggregometry (LTA) was introduced in the early 1960s and has since been considered the gold standard. This optical detection system is based on changes in turbidity measured as a change in light transmission, which is proportional to the extent of platelet aggregation induced by addition of an agonist. LTA is a flexible method, as different agonists can be used in varying concentrations, but performance of the test requires large blood volumes and experienced laboratory technicians as well as specialized personal to interpret results. In the present chapter, a protocol for LTA is described including all steps from pre-analytical preparation to interpretation of results.

Dual Therapy with Aspirin and Cilostazol May Improve Platelet Aggregation in Noncardioembolic Stroke Patients: A Pilot Study.

PubMed

Ohnuki, Yoichi; Ohnuki, Yuko; Kohara, Saori; Shimizu, Mie; Takizawa, Shunya

2017-01-01

Objective Some previous studies have found clinical benefit of dual antiplatelet therapy with aspirin and cilostazol for prevention of secondary stroke, but the physiological mechanism involved remains unknown. We aimed to clarify the effects of aspirin/cilostazol therapy on the platelet and endothelial functions of patients with acute noncardioembolic ischemic stroke, in comparison to patients who were treated with aspirin alone. Methods The present randomized prospective pilot study enrolled 24 patients within a week after the onset of noncardioembolic ischemic stroke. The patients were randomly allocated to receive aspirin (100 mg/day) (A group; 11 patients) or cilostazol (200 mg/day) plus aspirin (100 mg/day) (CA group; 13 patients). We measured platelet aggregation, platelet activation, and the thrombomodulin (TM), highly sensitive C-reactive protein (hs-CRP), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and von Willebrand (vWF) antigen levels and vWF activity over a 4-week period after enrollment. Results There was no significant difference in the platelet functions of the A and CA groups. However, the platelet aggregation induced by adenosine diphosphate (ADP) was decreased at 2 and 4 weeks (p<0.05) after treatment in comparison to the pre-treatment values in the CA group, but not in the A group. Platelet activation, and the hs-CRP, TM, ICAM-1, VCAM-1 and vWF values did not significantly decrease after treatment in either group. Conclusion Although there were no significant differences in platelet aggregation, platelet activation or the endothelial biomarker levels of the A and CA groups, dual therapy with aspirin and cilostazol inhibited platelet aggregation in comparison to the pre-treatment values, similarly to patients who received aspirin alone. This may suggest the clinical usefulness of dual therapy with aspirin and cilostazol in the treatment of patients with noncardioembolic ischemic stroke.

The Effect of Regular Intake of Dry-Cured Ham Rich in Bioactive Peptides on Inflammation, Platelet and Monocyte Activation Markers in Humans

PubMed Central

Martínez-Sánchez, Sara María; Minguela, Alfredo; Prieto-Merino, David; Zafrilla-Rentero, María Pilar; Abellán-Alemán, José; Montoro-García, Silvia

2017-01-01

Background and aims: Dietary studies have shown that active biopeptides provide protective health benefits, although the mediating pathways are somewhat uncertain. To throw light on this situation, we studied the effects of consuming Spanish dry-cured ham on platelet function, monocyte activation markers and the inflammatory status of healthy humans with pre-hypertension. Methods: Thirty-eight healthy volunteers with systolic blood pressure of >125 mmHg were enrolled in a two-arm crossover randomized controlled trial. Participants received 80 g/day dry-cured pork ham of >11 months proteolysis or 100 g/day cooked ham (control product) for 4 weeks followed by a 2-week washout before “crossing over” to the other treatment for 4 more weeks. Soluble markers and cytokines were analyzed by ELISA. Platelet function was assessed by measuring P-selectin expression and PAC-1 binding after ADP (adenosine diphosphate) stimulation using whole blood flow cytometry. Monocyte markers of the pathological status (adhesion, inflammatory and scavenging receptors) were also measured by flow cytometry in the three monocyte subsets after the interventional period. Results: The mean differences between dry-cured ham and cooked ham followed by a time period adjustment for plasmatic P-selectin and interleukin 6 proteins slightly failed (p = 0.062 and p = 0.049, respectively), notably increased for MCP-1 levels (p = 0.023) while VCAM-1 was not affected. Platelet function also decreased after ADP stimulation. The expression of adhesion and scavenging markers (ICAM1R, CXCR4 and TLR4) in the three subsets of monocytes was significantly higher (all p < 0.05). Conclusions: The regular consumption of biopeptides contained in the dry-cured ham but absent in cooked ham impaired platelet and monocyte activation and the levels of plasmatic P-selectin, MCP-1 and interleukin 6 in healthy subjects. This study strongly suggests the existence of a mechanism that links dietary biopeptides and beneficial health effects. PMID:28333093

Platelet activation and function in response to high intensity interval exercise and moderate continuous exercise in CABG and PCI patients.

PubMed

Ahmadizad, Sajad; Nouri-Habashi, Akbar; Rahmani, Hiwa; Maleki, Majid; Naderi, Nasim; Lotfian, Sara; Salimian, Morteza

2016-01-01

The effects of high intensity interval training (HIIT) on inflammatory markers and endothelial function have been extensively shown. However, the acute effect of HIIT on platelet activation and function in patients with recent revascularization is unclear. The purpose of present study was to compare the responses of platelet activation (CD62P) and function (platelet aggregation) to high intensity interval exercise (HIIE) and moderate continuous exercise (MCE) in coronary artery bypass grafting (CABG) and percutaneous coronary interventions (PCI) patients. Thirty patients who had CABG or PCI were randomly divided into HIIE, MCE and control groups. After determining the VO2peak, subjects in the MCE group carried out 30 min of continuous exercise at 60% of VO2peak, whereas, the subjects in HIIE group performed an interval protocol consisted of 8 repetitions of 2 min activity (running on treadmill) at 90% of VO2peak interspersed by 2 min of active recovery between repetitions at 30% of VO2peak .  Subjects in control group were seated and had no activity for the same period of time. Two blood samples were collected before and immediately after exercise and were analyzed for markers of platelet activation and function. Data analyzes revealed that increases in platelet aggregation induced by ADP and corrected for increases in platelet count in response to MCE trial was significantly lower than HIIE group (P < 0.05). In addition, responses of CD62P to MCE trial was significantly lower compared to HIIE group (P < 0.05). Changes in plateletcrit and platelet distribution width were significantly different among the three trials where the PCT and PDW following the HIIE were higher than MCE. Platelet count increased significantly (P < 0.05) by 13% following HIIE trial. Based on the findings of the present study it could be concluded that the risk of exercise-induced thrombosis is higher during HIIE than MCE in patients with recent revascularization.

Suspension properties of whole blood and its components under glucose influence studied in patients with acute coronary syndrome

NASA Astrophysics Data System (ADS)

Malinova, Lidia I.; Simonenko, Georgy V.; Denisova, Tatyana P.; Dovgalevsky, Pavel Y.; Tuchin, Valery V.

2004-05-01

The protocol of our study includes men with acute myocardial infarction, stable angina pectoris of II and III functional classes and unstable angina pectoris. Patients with arterial hypertension, disorders in carbohydrate metabolism were excluded from the study. Blood samples taken under standardized conditions, were stabilized with citrate sodium 3,8% (1:9). Erythrocytes and platelets aggregation activity under glucose influence (in vitro) was studied by means of computer aided microphotometer -- a visual analyzer. Erythrocyte and platelets were united in special subsystem of whole blood. Temporal and functional characteristics of their aggregation were analyzed by creation of phase patterns fragments. The received data testify to interrelation of erythrocytes and platelets processes of aggregation under conditions of increasing of glucose concentration of the incubatory environment, which temporal and functional characteristics may be used for diagnostics and the prognosis of destabilization coronary blood flow at an acute coronary syndrome.

The Effects of 2′-O-Methoxyethyl Containing Antisense Oligonucleotides on Platelets in Human Clinical Trials

PubMed Central

Baker, Brenda F.; Witztum, Joseph L.; Kwoh, T. Jesse; Pham, Nguyen C.; Salgado, Nelson; McEvoy, Bradley W.; Cheng, Wei; Hughes, Steven G.; Bhanot, Sanjay; Geary, Richard S.

2017-01-01

A thorough analysis of clinical trial data in the Ionis integrated safety database (ISDB) was performed to determine if there is a class effect on platelet numbers and function in subjects treated with 2′-O-methoxyethyl (2′MOE)-modified antisense oligonucleotides (ASOs). The Ionis ISDB includes over 2,600 human subjects treated with 16 different 2′MOE ASOs in placebo-controlled and open-label clinical trials over a range of doses up to 624 mg/week and treatment durations as long as 4.6 years. This analysis showed that there is no class generic effect on platelet numbers and no incidence of confirmed platelet levels below 50 K/μL in subjects treated with 2′MOE ASOs. Only 7 of 2,638 (0.3%) subjects treated with a 2′MOE ASO experienced a confirmed postbaseline (BSLN) platelet count between 100 and 50 K/μL. Three of sixteen 2′MOE ASOs had >10% incidence of platelet decreases >30% from BSLN, suggesting that certain sequences may associate with clinically insignificant platelet declines. Further to these results, we found no evidence that 2′MOE ASOs alter platelet function, as measured by the lack of clinically relevant bleeding in the presence or absence of other drugs that alter platelet function and/or number and by the results from trials conducted with the factor XI (FXI) ASO. PMID:28145801

Ambient hemolysis and activation of coagulation is different between HeartMate II and HeartWare left ventricular assist devices.

PubMed

Birschmann, Ingvild; Dittrich, Marcus; Eller, Thomas; Wiegmann, Bettina; Reininger, Armin J; Budde, Ulrich; Strüber, Martin

2014-01-01

Thromboembolic and bleeding events in patients with a left ventricular assist device (LVAD) are still a major cause of complications. Therefore, the balance between anti-coagulant and pro-coagulant factors needs to be tightly controlled. The principle hypothesis of this study is that different pump designs may have an effect on hemolysis and activation of the coagulation system. Referring to this, the HeartMate II (HMII; Thoratec Corp, Pleasanton, CA) and the HeartWare HVAD (HeartWare International Inc, Framingham, MA) were investigated. For 20 patients with LVAD support (n = 10 each), plasma coagulation, full blood count, and clinical chemistry parameters were measured. Platelet function was monitored using platelet aggregometry, platelet function analyzer-100 system ( Siemens, Marburg, Germany), vasodilator-stimulated phosphoprotein phosphorylation assay, immature platelet fraction, platelet-derived microparticles, and von Willebrand diagnostic. Acquired von Willebrand syndrome could be detected in all patients. Signs of hemolysis, as measured by lactate dehydrogenase levels (mean, 470 U/liter HMII, 250 U/liter HVAD; p < 0.001), were more pronounced in the HMII patients. In contrast, D-dimer analysis indicated a significantly higher activation of the coagulation system in HVAD patients (mean, 0.94 mg/liter HMII, 2.01 mg/liter HVAD; p < 0.01). The efficacy of anti-platelet therapy using clopidogrel was not sufficient in more than 50% of the patients. Our results support the finding that all patients with rotary blood pumps suffered from von Willebrand syndrome. In addition, a distinct footprint of effects on hemolysis and the coagulation system can be attributed to different devices. As a consequence, the individual status of the coagulation system needs to be controlled in long-term patients. © 2013 Published by International Society for the Heart and Lung Transplantation on behalf of International Society for Heart and Lung Transplantation.

Effects of immunosuppressive agents on the hemostatic system in normal dogs.

PubMed

Thomason, John M; Archer, Todd M; Wills, Robert W; Mackin, Andrew J

2018-05-10

In dogs, the effects of immunosuppressive medications on hemostasis are not well known. The objective was to determine the effects of immunosuppressive medications on primary and secondary hemostasis. Our hypothesis was that cyclosporine and prednisone would increase markers of hypercoagulability and thromboxane synthesis, while azathioprine, mycophenolate mofetil, and leflunomide would have minimal effects on hemostasis. Eight healthy dogs. A randomized, cross-over study used aggregometry, the PFA-100 platelet function analyzer, viscoelastometry, platelet count, and prothrombin and activated partial thromboplastin times to evaluate hemostasis during the administration of prednisone, azathioprine, cyclosporine, mycophenolate mofetil, and leflunomide for 1 week each at standard oral doses. Urine 11-dehydro-thromboxane-B 2 (11-dTXB 2 ) and 6-keto-prostaglandin-F 1α (6-keto-PGF 1α ) concentrations, normalized to urine creatinine concentration, were measured. The aggregometry amplitude decreased from 51 ± 21 to 27 ± 14 (P = .002) during leflunomide treatment (ADP activation), but there were no differences in amplitude (P = .240) for any medications when platelets were activated with collagen. For all medications, there were no significant differences in viscoelastometry indices (ACT, P = .666; ClotRate, P = .340; and platelet function, P = .411) and platelet count (P = .552). Compared with pretreatment values, urinary 11-dTXB 2 -to-creatinine ratio increased (P = .001) after drug administration (from 3.7 ± 0.6 to 5.6 ± 1.1). Cyclosporine was associated with an increase (P < .001) in the 6-keto-PGF 1α -to-creatinine ratio (from 10.3 ± 4.6 to 22.1 ± 5.3). Most immunosuppressive drugs do not enhance platelet function or coagulation in healthy dogs, suggesting that these medications might not predispose hypercoagulable dogs to thromboembolism. The results of our study need to be correlated with the clinical outcomes of hypercoagulable dogs. Copyright © 2018 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

Comparison platelet indices in diabetic patients with and without diabetic foot ulcer

NASA Astrophysics Data System (ADS)

Mardia, A. I.; Gatot, D.; Lindarto, D.

2018-03-01

Diabetes Mellitus is a group of metabolic disease which incidence increases every year. Some diabetic patients have diabetic foot ulcer as acomplication. The occurrence of ulcers in diabetic patients can be caused by the presence of thrombosis due to increased platelet function. Therefore, a cross-sectional study on 40 diabetic patients was performed at RSUP Adam Malik Medan to see whether there were differences in platelet indices between diabetic patients with and without diabetic foot ulcers. Platelets indices were examined and looked for differences in diabetic patients with and without diabetic foot ulcers. Data were analyzed using Chi-Square and Mann-Whitney U test with 95% CI. P-value<0.05 was considered statistically significant. There were differences in hemostasis function (prothrombin time, thrombin time, INR, aPTT, and fibrinogen) between the two groups with p values of 0.001; 0.004; 0.015; 0.021; 0.009, respectively. From the platelet indices examination, there were differences in the number of platelets, PDW and PCT with p values of 0.041; 0.027; 0.007, respectively, whereas there was no difference for MPV value (p=0,05). Platelet indices were found to increase in diabetic patients with diabetic foot ulcers indicating more reactive and aggregatable platelet function.

Cyclooxygenase Expression and Platelet Function in Healthy Dogs Receiving Low Dose Aspirin

PubMed Central

Dudley, Alicia; Thomason, John; Fritz, Sara; Grady, Jesse; Stokes, John; Wills, Robert; Pinchuk, Lesya; Mackin, Andrew; Lunsford, Kari

2014-01-01

Background Low dose aspirin is used to prevent thromboembolic complications in dogs, but some animals are non-responsive to the anti-platelet effects of aspirin (‘aspirin resistance’). Hypothesis/Objectives That low dose aspirin would inhibit platelet function, decrease thromboxane synthesis, and alter platelet cyclooxygenase (COX) expression. Animals Twenty-four healthy dogs Methods A repeated measures study. Platelet function (PFA-100® closure time, collagen/epinephrine), platelet COX-1 and COX-2 expression, and urine 11-dehydro-thromboxane B2 (11-dTXB2) was evaluated prior to and during aspirin administration (1 mg/kg Q24 hours PO, 10 days). Based on prolongation of closure times after aspirin administration, dogs were divided into categories according to aspirin responsiveness: responders, non-responders, and inconsistent responders. Results Low dose aspirin increased closure times significantly (62% by Day 10, P<0.001), with an equal distribution among aspirin responsiveness categories, 8 dogs per group. Platelet COX-1 mean fluorescent intensity (MFI) increased significantly during treatment, 13% on Day 3 (range, −29.7%–136.1%) (P=0.047) and 72% on Day 10 (range, −0.37–210.36%) (P<0.001). Platelet COX-2 MFI increased significantly by 34% (range, −29.2–270.4%) on Day 3 (P = 0.003) and 74% (range, −19.7–226.2%) on Day 10 (P<0.001). Urinary 11-dTXB2 concentrations significantly (P=0.005, P<0.001) decreased at both time points. There was no difference between aspirin responsiveness and either platelet COX expression or thromboxane production. Conclusions and Clinical Importance Low dose aspirin consistently inhibits platelet function in approximately one third of healthy dogs, despite decreased thromboxane synthesis and increased platelet COX expression in most dogs. Pre-treatment COX isoform expression did not predict aspirin resistance. PMID:23278865

Ratio of mean platelet volume to platelet count is a potential surrogate marker predicting liver cirrhosis.

PubMed

Iida, Hiroya; Kaibori, Masaki; Matsui, Kosuke; Ishizaki, Morihiko; Kon, Masanori

2018-01-27

To provide a simple surrogate marker predictive of liver cirrhosis (LC). Specimens from 302 patients who underwent resection for hepatocellular carcinoma between January 2006 and December 2012 were retrospectively analyzed. Based on pathologic findings, patients were divided into groups based on whether or not they had LC. Parameters associated with hepatic functional reserve were compared in these two groups using Mann-Whitney U -test for univariate analysis. Factors differing significantly in univariate analyses were entered into multivariate logistic regression analysis. There were significant differences between the LC group ( n = 100) and non-LC group ( n = 202) in prothrombin activity, concentrations of alanine aminotransferase, aspartate aminotransferase, total bilirubin, albumin, cholinesterase, type IV collagen, hyaluronic acid, indocyanine green retention rate at 15 min, maximal removal rate of technitium-99m diethylene triamine penta-acetic acid-galactosyl human serum albumin and ratio of mean platelet volume to platelet count (MPV/PLT). Multivariate analysis showed that prothrombin activity, concentrations of alanine aminotransferase, aspartate aminotransferase, total bilirubin and hyaluronic acid, and MPV/PLT ratio were factors independently predictive of LC. The area under the curve value for MPV/PLT was 0.78, with a 0.8 cutoff value having a sensitivity of 65% and a specificity of 78%. The MPV/PLT ratio, which can be determined simply from the complete blood count, may be a simple surrogate marker predicting LC.

Quantitative, functional, morphological and ultrastructural recovery of platelets as predictor for cryopreservation.

PubMed

Balint, Bela; Vucetić, Dusan; Trajković-Lakić, Zlatija; Petakov, Marijana; Bugarski, Diana; Brajusković, Goran; Taseski, Jovan

2002-01-01

Cryopreservation of platelets is of great interest, since it could extend the shelf life of therapeutic platelet concentrates and facilitate stockpiling and inventory control in blood banking. Despite the use of many cryopreservation procedures the optimal cryopreservation procedure is not defined yet. We have compared the cryopreservation of human platelets by various protocols employing controlled-rate and non-controlled-rate freezing procedures in combination with different concentrations of DMSO (6% and 10%) or 5% DMSO + 6% HES combination. After storage for 1 to 3 months, samples were thawed and analyzed. Measurements included cell recovery, platelet viability according to hypotonic shock response (HSR), platelet aggregation with ADP, morphological and ultrastructural properties of defrozen platelets. Our findings show that the application of our original procedure for controlled-rate freezing consisting of six cooling steps (cooling rate 1 degree C/min) with compensation of released heat of fusion (cooling rate 2 degrees C/min) has significantly influenced the quality of thawed platelets. At the same time, a concentration of 6% DMSO proved to be the most effective. In summary, cryopreservation of human platelets using controlled-rate freezing procedure in combination with lower (6%) DMSO concentration resulted in less damage from freezing and higher recovered function of platelets.

Role of Tumor Suppressor P53 in Megakaryopoiesis and Platelet Function

PubMed Central

Apostolidis, Pani A.; Woulfe, Donna S.; Chavez, Massiel; Miller, William M.; Papoutsakis, Eleftherios T.

2011-01-01

The pathobiological role of p53 has been widely studied, however its role in normophysiology is relatively unexplored. We previously showed that p53 knock-down increased ploidy in megakaryocytic cultures. This study aims to examine the effect of p53 loss on in vivo megakaryopoiesis, platelet production and function, and to investigate the basis for greater ploidy in p53−/− megakaryocytic cultures. Here, we used flow cytometry to analyze ploidy, DNA synthesis and apoptosis in murine cultured and bone marrow megakaryocytes following thrombopoietin administration and to analyze fibrinogen binding to platelets in vitro. Culture of p53−/− marrow cells for 6 days with thrombopoietin gave rise to 1.7-fold more megakaryocytes, 26.1±3.6% of which reached ploidy classes ≥64N compared to 8.2±0.9% of p53+/+ megakaryocytes. This was due to 30% greater DNA synthesis in p53−/− megakaryocytes and 31% greater apoptosis in p53+/+ megakaryocytes by day 4 of culture. Although the bone marrow and spleen steady-state megakaryocytic content and ploidy were similar in p53+/+ and p53−/− mice, thrombopoietin administration resulted in increased megakaryocytic polyploidization in p53−/− mice. Although their platelet counts were normal, p53−/− mice exhibited significantly longer bleeding times and p53−/− platelets were less sensitive than p53+/+ platelets to agonist-induced fibrinogen binding and P-selectin secretion. In summary, our in vivo and ex-vivo studies indicate that p53 loss leads to increased polyploidization during megakaryopoiesis. Our findings also suggest for the first time a direct link between p53 loss and the development of fully functional platelets resulting in hemostatic deficiencies. PMID:22024107

Platelet-Derived S100A8/A9 and Cardiovascular Disease in Systemic Lupus Erythematosus.

PubMed

Lood, Christian; Tydén, Helena; Gullstrand, Birgitta; Jönsen, Andreas; Källberg, Eva; Mörgelin, Matthias; Kahn, Robin; Gunnarsson, Iva; Leanderson, Tomas; Ivars, Fredrik; Svenungsson, Elisabet; Bengtsson, Anders A

2016-08-01

Levels of S100A8/A9, a proinflammatory and prothrombotic protein complex, are increased in several diseases, and high levels predispose to cardiovascular disease (CVD). Recently, platelet S100A8/A9 synthesis was described in mice and humans in relation to CVD. The aim of this study was to investigate the role of platelet S100A8/A9 in systemic lupus erythematosus (SLE), a disease with markedly increased cardiovascular morbidity, as well as the exact platelet distribution of the S100A8/A9 proteins. The occurrence and distribution of platelet S100A8/A9 protein were detected by enzyme-linked immunosorbent assay, electron microscopy, Western blotting, and flow cytometry in healthy controls (n = 79) and in 2 individual cohorts of SLE patients (n = 148 and n = 318, respectively) and related to cardiovascular morbidity. We observed that human platelets expressed S100A8/A9 proteins, and that these were localized in close proximity to intracellular membranes and granules as well as on the cell surface upon activation with physiologic and pathophysiologic stimuli. Interestingly, S100A8/A9 was enriched at sites of membrane interactions, indicating a role of S100A8/A9 in cell-cell communication. S100A8/A9 levels were highly regulated by interferon-α, both in vivo and in vitro. Patients with SLE had increased platelet S100A8/A9 content compared with healthy individuals. Increased levels of platelet S100A8/A9 were associated with CVD, particularly myocardial infarction (odds ratio 4.8, 95% confidence interval 1.5-14.9, P = 0.032 [adjusted for age, sex, and smoking]). Platelets contain S100A8/A9 in membrane-enclosed vesicles, enabling rapid cell surface deposition upon activation. Furthermore, platelet S100A8/A9 protein levels were increased in SLE patients, particularly in those with CVD, and may be a future therapeutic target. © 2016, American College of Rheumatology.

Synthesis and antiplatelet activity of thioaryloxyacids analogues of clofibric acid.

PubMed

Ammazzalorso, Alessandra; Amoroso, Rosa; Baraldi, Mario; Bettoni, Giancarlo; Braghiroli, Daniela; De Filippis, Barbara; Giampietro, Letizia; Tricca, Maria L; Vezzalini, Francesca

2005-09-01

The thiophene-, benzothiazole- and pyridine-thioaryloxyacids analogues of clofibric acid were synthesized and their antiplatelet activity was screened. Some compounds exhibited antiaggregating properties. The platelet-related haemostasis was measured on a PFA-100 analyzer using bull blood.

Strategy for the hemocompatibility testing of microparticles.

PubMed

Braune, S; Basu, S; Kratz, K; Johansson, J Bäckemo; Reinthaler, M; Lendlein, A; Jung, F

2016-01-01

Polymer-based microparticles are applied as non-thrombogenic or thrombogenic materials in a wide variety of intra- or extra-corporeal medical devices. As demanded by the regulatory agencies, the hemocompatibility of these blood contacting biomaterials has to be evaluated in vitro to ensure that the particle systems appropriately fulfill the envisioned function without causing undesired events such as thrombosis or inflammation. Currently described in vitro assays for hemocompatibility testing of particles comprise tests with different single cell types (e.g. erythrocytes or leukocytes), varying concentrations/dilutions of the used blood cells or whole blood, which are not standardized.Here, we report about an in vitro dynamic test system for studying the hemocompatibility of polymeric microparticles utilizing fresh human whole blood from apparently healthy subjects, collected and processed under standardized conditions. Spherical poly(ether imide) microparticles with an average diameter of 140±30 μm were utilized as model systems. Reported as candidate materials for the removal of uremic toxins, these microparticles are anticipated to facilitate optimal flow conditions in a dialyzer with minimal backflow and blood cell damage. Pristine (PEI) and potassium hydroxide (PEI-KOH) functionalized microparticles exhibited similarly nanoporous surfaces (PEI: ØExternal pore = 90±60 nm; PEI-KOH ØExternal pore = 150±130 nm) but varying water wettabilities (PEI: θadv = 112±10° PEI-KOH θadv = 60±2°). The nanoporosity of the microparticle surfaces allows the exchange of toxic solutes from blood towards the interconnective pores in the particle core, while an immigration of the substantially larger blood cells is inhibited.Sterilized PEI microparticles were incorporated -air-free -in a syringe-based test system and exposed to whole blood for 60 minutes under gentle agitation. Thereafter, thrombi formation on the particles surfaces were analyzed microscopically. In the collected whole blood the non-adherent/circulating single blood cells were quantified via a differentiated complete blood cell count and the activation of platelets (P-Selectin expression, secretion and release), platelet function (PFA100 closure time) as well as thrombin formation (thrombin-antithrombin-complex) was analyzed. Free hemoglobin (HGB) levels were quantified as a measure of hemolysis.Microscopic evaluation revealed thrombi formation and particle aggregates for all tested microparticles. Reduction of circulating blood cells differed significantly between the particle types. Particularly, platelet and monocyte counts decreased up to 50% compared to the control (syringe filled with whole blood but without microparticles). In accordance, platelet activation, thrombin levels and degrees of hemolysis were clearly elevated in the particle loaded test systems and allowed a differentiation between the particle types. Increased PFA100 closure times (as activating agent a combination of collagen/ADP was used) indicated a similarly reduced ability of platelets to adhere and form stable aggregates independent from the particle type tested. This observation is most probably a consequence of the strong thrombus formation in the test system, which is associated with a reduction of the circulating blood cells.The reported in vitro dynamic whole blood test system allowed the sensitive analysis of the hemocompatibility of polymer-based microparticles and was successfully validated for porous PEI microparticles with different water wettabilities. Beyond the qualitative and quantitative analysis of cell-material interactions, the test also allowed the functional evaluation of platelets in whole blood.

Novel HIT antibody detection method using Sonoclot® coagulation analyzer.

PubMed

Wanaka, Keiko; Asada, Reiko; Miyashita, Kumiko; Kaneko, Makoto; Endo, Hirokazu; Yatomi, Yutaka

2015-01-01

Since heparin-induced thrombocytopenia (HIT), caused by the generation of antibodies against platelet factor 4 (PF4)/heparin complexes (HIT antibodies), may induce serious complications due to thrombosis, a prompt diagnosis is desirable. Functional tests with platelet activation to detect HIT antibodies are useful for diagnosis of HIT, in particular (14)C-selotonin release assay (SRA). However, they are complicated and so can be performed only in limited laboratories. We tested if a blood coagulation test using Sonoclot® analyzer can serve for the detection of HIT antibodies. A murine monoclonal antibody (HIT-MoAb) against PF4/heparin complexes was used as an alternative to human HIT antibodies. To the mixture of HIT-MoAb and heparin (0.5 U/mL, final), whole blood obtained from a healthy volunteer was added, and then the activated clotting time (ACT), clot rate (CR), and area under the curve (AUC) were measured with Sonoclot® analyzer for 30minutes. The HIT-MoAb (30 to 100μg/mL, final) concentration dependently suppressed the anticoagulation activity (prolongation of ACT and decrease of CR and AUC) of heparin. The suppression of anticoagulation effect of heparin by HIT-MoAb was demonstrated by measurements using Sonoclot® analyzer. This method may provide a new tool for screening of HIT antibodies. Copyright © 2014 Elsevier Ltd. All rights reserved.

Randomized controlled trial comparing impact on platelet reactivity of twice-daily with once-daily aspirin in people with Type 2 diabetes.

PubMed

Bethel, M A; Harrison, P; Sourij, H; Sun, Y; Tucker, L; Kennedy, I; White, S; Hill, L; Oulhaj, A; Coleman, R L; Holman, R R

2016-02-01

Reduced aspirin efficacy has been demonstrated in people with Type 2 diabetes. Because increased platelet reactivity and/or turnover are postulated mechanisms, we examined whether higher and/or more frequent aspirin dosing might reduce platelet reactivity more effectively. Participants with Type 2 diabetes (n = 24) but without known cardiovascular disease were randomized in a three-way crossover design to 2-week treatment periods with aspirin 100 mg once daily, 200 mg once daily or 100 mg twice daily. The primary outcome was platelet reactivity, assessed using the VerifyNow(™) ASA method. Relationships between platelet reactivity and aspirin dosing were examined using generalized linear mixed models with random subject effects. Platelet reactivity decreased from baseline with all doses of aspirin. Modelled platelet reactivity was more effectively reduced with aspirin 100 mg twice daily vs. 100 mg once daily, but not vs. 200 mg once daily. Aspirin 200 mg once daily did not differ from 100 mg once daily. Aspirin 100 mg twice daily was also more effective than once daily as measured by collagen/epinephrine-stimulated platelet aggregation and urinary thromboxane levels, with a similar trend measured by serum thromboxane levels. No episodes of bleeding occurred. In Type 2 diabetes, aspirin 100 mg twice daily reduced platelet reactivity more effectively than 100 mg once daily, and numerically more than 200 mg once daily. Clinical outcome trials evaluating primary cardiovascular disease prevention with aspirin in Type 2 diabetes may need to consider using a more frequent dosing schedule. © 2015 The Authors. Diabetic Medicine © 2015 Diabetes UK.

[Essential thrombocythemia and cerebral ischemic accident: discussion of two cases].

PubMed

Alecu, C; Abraham, P; Ternisien, C; Enon, B; Saumet, J L

1999-10-01

Not only the total number of platelets but their normal or abnormal function are essential points to be analyzed in case of stroke associated with thrombocytemia. When possible the treatment of arterial episodes in thrombocytemia should not be surgical. Anti-platelet agents and the rigorous control of the different risk factors are warranted to limit the activation of abnormal platelets on early endothelial lesions and thereby limit the risk of recurrent accidents. We report two typical cases illustrating these different points.

Comparison of platelet function and viscoelastic test results between healthy dogs and dogs with naturally occurring chronic kidney disease.

PubMed

Dudley, Alicia; Byron, Julie K; Burkhard, Mary Jo; Warry, Emma; Guillaumin, Julien

2017-05-01

OBJECTIVE To compare platelet function and viscoelastic test results between healthy dogs and dogs with chronic kidney disease (CKD) to assess whether dogs with CKD have platelet dysfunction and altered blood coagulation. ANIMALS 10 healthy control dogs and 11 dogs with naturally occurring CKD. PROCEDURES Blood and urine were collected once from each dog for a CBC, serum biochemical analysis, urinalysis, and determination of the urine protein-to-creatinine ratio, prothrombin time, activated partial thromboplastin time, plasma fibrinogen concentration, and antithrombin activity. Closure time was determined by use of a platelet function analyzer and a collagen-ADP platelet agonist. Thromboelastography (TEG) variables (reaction time, clotting time, α angle, maximum amplitude, and global clot strength [G value]) were determined by use of recalcified nonactivated TEG. Platelet expression of glycoprotein Ib (GPIb; receptor for von Willebrand factor), integrin αIIbβ3 (αIIbβ3; receptor for fibrinogen), and P-selectin (marker for platelet activation) was assessed by flow cytometry. RESULTS Compared with healthy control dogs, the median closure time was prolonged, the median maximum amplitude and G value were increased, and the median clotting time was decreased for dogs with CKD. Platelet expression of both αIIbβ3 and P-selectin was also significantly increased for dogs with CKD, compared with that for control dogs. Platelet expression of GPIb, αIIbβ3, and P-selectin was not correlated with closure time or any TEG variable. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that dogs with CKD frequently had evidence of platelet dysfunction and hypercoagulability that were not totally attributable to alterations in platelet surface expression of GPIb, αIIbβ3, and P-selectin.

Platelet microparticles: detection and assessment of their paradoxical functional roles in disease and regenerative medicine.

PubMed

Burnouf, Thierry; Goubran, Hadi Alphonse; Chou, Ming-Li; Devos, David; Radosevic, Mirjana

2014-07-01

There is increasing research on and clinical interest in the physiological role played by platelet microparticles (PMPs). PMPs are 0.1-1-μm fragments shed from plasma membranes of platelets that are undergoing activation, stress, or apoptosis. They have a phospholipid-based structure and express functional receptors from platelet membranes. As they are the most abundant microparticles in the blood and they express the procoagulant phosphatidylserine, PMPs likely complement, if not amplify, the functions of platelets in hemostasis, thrombosis, cancer, and inflammation, but also act as promoters of tissue regeneration. Their size and structure make them instrumental in platelet-cell communications as a delivery tool of platelet-borne bioactive molecules including growth factors, other signaling molecules and micro (mi)RNA. PMPs can therefore be a pathophysiological threat or benefit to the cellular environment when interacting with the blood vasculature. There is also increasing evidence that PMP generation is triggered during blood collection, separation into components, and storage, a phenomenon potentially leading to thrombotic and inflammatory side effects in transfused patients. Evaluating PMPs requires strict pre-analytical and analytical procedures to avoid artifactual generation and ensure accurate assessment of the number, size repartitioning, and functional properties. This review describes the physical and functional methods developed for analyzing and quantifying PMPs. It then presents the functional roles of PMPs as markers or triggers of diseases like thrombosis, atherosclerosis, and cancer, and discusses the possible detrimental immunological impact of their generation in blood components. Finally we review the potential function of PMPs in tissue regeneration and the prospects for their use in therapeutic strategies for human health. Copyright © 2014 Elsevier Ltd. All rights reserved.

In vitro and in vivo assessment of platelet function in healthy dogs during administration of a low-dose aspirin regimen.

PubMed

Haines, Jillian M; Thomason, John M; Seage, Eileen C; Wills, Robert W; Bulla, Camilo; Lunsford, Kari V; Mackin, Andrew J

2016-02-01

To assess the in vitro and in vivo platelet function of healthy dogs during administration of a low-dose aspirin regimen. 16 dogs. Dogs received aspirin (1 mg/kg, PO, q 24 h) for 7 days. Blood and urine samples were collected before (day 1; baseline) and on days 3 and 7 of the low-dose aspirin regimen. Platelet function was evaluated by use of turbidimetric and conventional impedance aggregometry, multiple-electrode impedance aggregometry, a platelet function analyzer (PFA), and determination of urine 11-dehydro-thromboxane B2 concentration. Turbidimetric aggregometry results were compared with the results obtained by the other 4 methods. Fourteen days after cessation of aspirin, platelet-rich plasma was incubated with acetylsalicylic acid and platelet function was assessed by turbidimetric aggregometry to determine whether this technique could accurately identify dogs that responded to the low-dose aspirin regimen. Of the 16 dogs, 13 had turbidimetric and conventional impedance aggregometry results that were decreased by > 25% from baseline on days 3 and 7, and 4 and 7 dogs had PFA closure times > 300 seconds on days 3 and 7, respectively. The median urine 11-dehydro-thromboxane B2 concentration-to-creatinine concentration ratio decreased by 49% between days 1 and 7. Turbidimetric aggregometry results were correlated with conventional impedance aggregometry results. There was poor agreement between the turbidimetric aggregometry and PFA results. The multiple-electrode impedance aggregometry protocol failed to reliably detect aspirin-induced platelet dysfunction. In vitro incubation of platelet-rich plasma with acetylsalicylic acid followed by turbidimetric aggregometry did not predict whether dogs responded to the low-dose aspirin regimen. Results indicated that the response to a low-dose aspirin regimen varied among healthy dogs.

On-pump Cardiac Surgery Enhances Platelet Renewal and Impairs Aspirin Pharmacodynamics: Effects of Improved Dosing Regimens.

PubMed

Cavalca, V; Rocca, B; Veglia, F; Petrucci, G; Porro, B; Myasoedova, V; De Cristofaro, R; Turnu, L; Bonomi, A; Songia, P; Cavallotti, L; Zanobini, M; Camera, M; Alamanni, F; Parolari, A; Patrono, C; Tremoli, E

2017-11-01

On-pump cardiac surgery may trigger inflammation and accelerate platelet cyclooxygenase-1 renewal, thereby modifying low-dose aspirin pharmacodynamics. Thirty-seven patients on standard aspirin 100 mg once-daily were studied before surgery and randomized within 36 hours postsurgery to 100 mg once-daily, 100 mg twice-daily, or 200 mg once-daily for 90 days. On day 7 postsurgery, immature and mature platelets, platelet mass, thrombopoietin, glycocalicin, leukocytes, C-reactive protein, and interleukin-6 significantly increased. Interleukin-6 significantly correlated with immature platelets. At day 7, patients randomized to 100 mg once-daily showed a significant increase in serum thromboxane (TX)B 2 within the 24-hour dosing interval and urinary TXA 2 metabolite (TXM) excretion. Aspirin 100 mg twice-daily lowered serum TXB 2 and prevented postsurgery TXM increase (P < 0.01), without affecting prostacyclin metabolite excretion. After cardiac surgery, shortening the dosing interval, but not doubling the once-daily dose, rescues the impaired antiplatelet effect of low-dose aspirin and prevents platelet activation associated with acute inflammation and enhanced platelet turnover. © 2017 American Society for Clinical Pharmacology and Therapeutics.

Dietary flavanols and procyanidin oligomers from cocoa (Theobroma cacao) inhibit platelet function.

PubMed

Murphy, Karen J; Chronopoulos, Andriana K; Singh, Indu; Francis, Maureen A; Moriarty, Helen; Pike, Marilyn J; Turner, Alan H; Mann, Neil J; Sinclair, Andrew J

2003-06-01

Flavonoids may be partly responsible for some health benefits, including antiinflammatory action and a decreased tendency for the blood to clot. An acute dose of flavanols and oligomeric procyanidins from cocoa powder inhibits platelet activation and function over 6 h in humans. This study sought to evaluate whether 28 d of supplementation with cocoa flavanols and related procyanidin oligomers would modulate human platelet reactivity and primary hemostasis and reduce oxidative markers in vivo. Thirty-two healthy subjects were assigned to consume active (234 mg cocoa flavanols and procyanidins/d) or placebo (< or = 6 mg cocoa flavanols and procyanidins/d) tablets in a blinded parallel-designed study. Platelet function was determined by measuring platelet aggregation, ATP release, and expression of activation-dependent platelet antigens by using flow cytometry. Plasma was analyzed for oxidation markers and antioxidant status. Plasma concentrations of epicatechin and catechin in the active group increased by 81% and 28%, respectively, during the intervention period. The active group had significantly lower P selectin expression and significantly lower ADP-induced aggregation and collagen-induced aggregation than did the placebo group. Plasma ascorbic acid concentrations were significantly higher in the active than in the placebo group (P < 0.05), whereas plasma oxidation markers and antioxidant status did not change in either group. Cocoa flavanol and procyanidin supplementation for 28 d significantly increased plasma epicatechin and catechin concentrations and significantly decreased platelet function. These data support the results of acute studies that used higher doses of cocoa flavanols and procyanidins.

Effects of polyphenol-rich extract from berries of Aronia melanocarpa on the markers of oxidative stress and blood platelet activation.

PubMed

Olas, Beata; Kedzierska, Magdalena; Wachowicz, Barbara; Stochmal, Anna; Oleszek, Wieslaw

2010-01-01

Bioactive substances found in numerous foods can be successfully and safely used to modify various cellular functions and affect the oxidative stress. Aronia melanocarpa fruits (Rosaceae) are one of the richest plant sources of phenolic substances shown to have anti-inflammatory, antitumor, antioxidative and antiplatelet activities. We investigated antioxidant properties of the extract from berries of A. melanocarpa by the estimation of the selected and other biomarkers of oxidative stress, i.e. the level of 8-epi-prostaglandin F(2) (8-EPI) (by immunoassay kit) and the amount of glutathione (by HPLC method) in control platelets and platelets treated with H(2)O(2). The expression of alpha(IIb)beta(3) (a marker of platelet activation) was measured by flow cytometer. The antioxidative and antiplatelet properties of the tested extract were also compared with the action of a well characterized antioxidative and antiplatelet commercial monomeric polyphenol-resveratrol. The extract from berries of A. melanocarpa (at the highest tested concentration -100 microg/ml) decreased the production of 8-EPI (a marker of lipid peroxidation) in control blood platelets and platelets treated with H(2)O(2) (2 mM). A combined action of the tested plant extract and H(2)O(2) evoked a significant increase of reduced form of glutathione in platelets compared with cells treated with H(2)O(2) only. Moreover, the tested plant extract (at the highest used concentration -100 microg/ml) reduced the expression of alpha(IIb)beta(3) on blood platelets. Comparative studies indicate that the tested plant extract was found to be more reactive in blood platelets than the solution of pure resveratrol.

Incidence and risk factors of bleeding-related adverse events in patients with chronic lymphocytic leukemia treated with ibrutinib.

PubMed

Lipsky, Andrew H; Farooqui, Mohammed Z H; Tian, Xin; Martyr, Sabrina; Cullinane, Ann M; Nghiem, Khanh; Sun, Clare; Valdez, Janet; Niemann, Carsten U; Herman, Sarah E M; Saba, Nakhle; Soto, Susan; Marti, Gerald; Uzel, Gulbu; Holland, Steve M; Lozier, Jay N; Wiestner, Adrian

2015-12-01

Ibrutinib is associated with bleeding-related adverse events of grade ≤ 2 in severity, and infrequently with grade ≥ 3 events. To investigate the mechanisms of bleeding and identify patients at risk, we prospectively assessed platelet function and coagulation factors in our investigator-initiated trial of single-agent ibrutinib for chronic lymphocytic leukemia. At a median follow-up of 24 months we recorded grade ≤ 2 bleeding-related adverse events in 55% of 85 patients. No grade ≥ 3 events occurred. Median time to event was 49 days. The cumulative incidence of an event plateaued by 6 months, suggesting that the risk of bleeding decreases with continued therapy. At baseline, von Willebrand factor and factor VIII levels were often high and normalized on treatment. Platelet function measured via the platelet function analyzer (PFA-100™) was impaired in 22 patients at baseline and in an additional 19 patients on ibrutinib (often transiently). Collagen and adenosine diphosphate induced platelet aggregation was tested using whole blood aggregometry. Compared to normal controls, response to both agonists was decreased in all patients with chronic lymphocytic leukemia, whether on ibrutinib or not. Compared to untreated chronic lymphocytic leukemia patients, response to collagen showed a mild further decrement on ibrutinib, while response to adenosine diphosphate improved. All parameters associated with a significantly increased risk of bleeding-related events were present at baseline, including prolonged epinephrine closure time (HR 2.74, P=0.012), lower levels of von Willebrand factor activity (HR 2.73, P=0.009) and factor VIII (HR 3.73, P=0.0004). In conclusion, both disease and treatment-related factors influence the risk of bleeding. Patients at greater risk for bleeding of grade ≤ 2 can be identified by clinical laboratory tests and counseled to avoid aspirin, non-steroidal anti-inflammatory drugs and fish oils. ClinicalTrials.gov identifier NCT01500733. Copyright© Ferrata Storti Foundation.

Incidence and risk factors of bleeding-related adverse events in patients with chronic lymphocytic leukemia treated with ibrutinib

PubMed Central

Lipsky, Andrew H.; Farooqui, Mohammed Z.H.; Tian, Xin; Martyr, Sabrina; Cullinane, Ann M.; Nghiem, Khanh; Sun, Clare; Valdez, Janet; Niemann, Carsten U.; Herman, Sarah E. M.; Saba, Nakhle; Soto, Susan; Marti, Gerald; Uzel, Gulbu; Holland, Steve M.; Lozier, Jay N.; Wiestner, Adrian

2015-01-01

Ibrutinib is associated with bleeding-related adverse events of grade ≤2 in severity, and infrequently with grade ≥3 events. To investigate the mechanisms of bleeding and identify patients at risk, we prospectively assessed platelet function and coagulation factors in our investigator-initiated trial of single-agent ibrutinib for chronic lymphocytic leukemia. At a median follow-up of 24 months we recorded grade ≤2 bleeding-related adverse events in 55% of 85 patients. No grade ≥3 events occurred. Median time to event was 49 days. The cumulative incidence of an event plateaued by 6 months, suggesting that the risk of bleeding decreases with continued therapy. At baseline, von Willebrand factor and factor VIII levels were often high and normalized on treatment. Platelet function measured via the platelet function analyzer (PFA-100™) was impaired in 22 patients at baseline and in an additional 19 patients on ibrutinib (often transiently). Collagen and adenosine diphosphate induced platelet aggregation was tested using whole blood aggregometry. Compared to normal controls, response to both agonists was decreased in all patients with chronic lymphocytic leukemia, whether on ibrutinib or not. Compared to untreated chronic lymphocytic leukemia patients, response to collagen showed a mild further decrement on ibrutinib, while response to adenosine diphosphate improved. All parameters associated with a significantly increased risk of bleeding-related events were present at baseline, including prolonged epinephrine closure time (HR 2.74, P=0.012), lower levels of von Willebrand factor activity (HR 2.73, P=0.009) and factor VIII (HR 3.73, P=0.0004). In conclusion, both disease and treatment-related factors influence the risk of bleeding. Patients at greater risk for bleeding of grade ≤2 can be identified by clinical laboratory tests and counseled to avoid aspirin, non-steroidal anti-inflammatory drugs and fish oils. ClinicalTrials.gov identifier NCT01500733 PMID:26430171

Platelet Function Tests

MedlinePlus

... Time and International Normalized Ratio (PT/INR) PSEN1 Quantitative Immunoglobulins Red Blood Cell (RBC) Antibody Identification Red ... 100 – FAQ. Florida Hospital Cancer Institute, Clinical and Research Laboratories [On-line information]. Available online at http:// ...

Use of thromboelastography to tailor dual-antiplatelet therapy in patients undergoing treatment of intracranial aneurysms with the Pipeline embolization device.

PubMed

McTaggart, Ryan A; Choudhri, Omar A; Marcellus, Mary L; Brennan, Tom; Steinberg, Gary K; Dodd, Robert L; Do, Huy M; Marks, Michael P

2015-06-01

Platelet function testing is controversial and not well studied in patients with neurovascular disease. To evaluate the performance of thromboelastography (TEG) as a platelet function test in neurovascular patients treated with the Pipeline embolization device (PED). A prospective protocol was instituted for platelet function testing in patients undergoing repair of intracranial aneurysms with the PED. All patients received dual antiplatelet therapy (DAT) and their response to both P2Y12 inhibitors and aspirin was quantified with TEG. Each patient's DAT induction strategy was tailored based on the percentage ADP-induced and percentage arachidonic acid-induced platelet inhibition reported by TEG. Data collected included clinical presentation, aneurysm characteristics, treatment details, and periprocedural events. Patients were followed up clinically and/or angiographically at 30 days, 6 months, and 1 year. Thirty-four PED procedures were performed on 31 patients. TEG results altered the DAT strategy in 35% of patients. Technical success with the Pipeline placement was 100%. Two patients had minor strokes and five had transient ischemic attacks (TIAs). There have been no hemorrhagic complications. No patient had permanent neurologic deficits. Six of eight (75%) of patients with thromboembolic/TIA events were ADP-induced hyporesponders by TEG. Our 6- and 12-month angiographic occlusion rates were 78.9% and 89.5%, respectively. The 19 major branches covered by the PED that were assessed by follow-up imaging have all remained patent. Platelet function testing with TEG altered our DAT induction strategy in a significant number of cases. No hemorrhagic or disabling thromboembolic complications were seen in this series. Future studies should compare methods of platelet function testing and, possibly, no platelet function testing in neurovascular patients undergoing flow diversion and/or stent-assisted treatment of intracranial aneurysms. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Acetaminophen and meloxicam inhibit platelet aggregation and coagulation in blood samples from humans.

PubMed

Martini, Angela K; Rodriguez, Cassandra M; Cap, Andrew P; Martini, Wenjun Z; Dubick, Michael A

2014-12-01

Acetaminophen (Ace) and meloxicam (Mel) are the two types of analgesic and antipyretic medications. This study investigated the dose responses of acetaminophen and meloxicam on platelet aggregation and coagulation function in human blood samples. Blood samples were collected from six healthy humans and processed to make platelet-adjusted (100 × 10 cells/μl) blood samples. Acetaminophen (Tylenol, Q-PAP, 100 mg/ml) was added at the doses of 0 μg/ml (control), 214 μg/ml (the standard dose, 1 ×), 4 ×, 8 ×, 10 ×, 12 ×, 16 ×, and 20 ×. Similarly, meloxicam (Metacam, 5 mg/ml) was added at doses of 0 μg/ml (control), 2.85 μg/ml (the standard dose, 1 ×), 4 ×, 8 ×, 10 ×, 12 ×, 16 ×, and 20 ×. Fifteen minutes after the addition of acetaminophen and/or meloxicam, platelet aggregation was stimulated with collagen (2 μg/ml) or arachidonic acid (0.5 mmol/l) and assessed using a Chrono-Log 700 aggregometer. Coagulation function was assessed by prothrombin time (PT), activated partial thromboplastin time (aPTT), and using Rotem thrombelastogram. A robust inhibition by acetaminophen and/or meloxicam was observed in arachidonic acid-stimulated platelet aggregation starting at 1 × dose. Collagen-stimulated platelet aggregation was inhibited by ACE starting at 1 × (78 ± 10% of control), and by meloxicam starting at 4 × (72 ± 5% of control, both P < 0.05). The inhibitions by acetaminophen and meloxicam combined were similar to those by acetaminophen or meloxicam. aPTT was prolonged by meloxicam starting at 4 ×. No changes were observed in PT or any of Rotem measurements by acetaminophen and/or meloxicam. Acetaminophen and meloxicam compromised platelet aggregation and aPTT. Further effort is warranted to characterize the effects of acetaminophen and meloxicam on bleeding in vivo.

Reversible electropermeabilisation of human and rat blood platelets: evaluation of morphological and functional integrity 'in vitro' and 'in vivo'.

PubMed

Hughes, K; Crawford, N

1989-06-06

A high-voltage discharge procedure has been developed for permeabilising the plasma membranes of both human and rat blood platelets. The cells can be resealed by incubation at 37 degrees C, show less than 4% loss of lactate dehydrogenase (LDH) implying minimal cell lysis and also have well maintained morphological and functional integrity. The prototype apparatus used at field strengths between 6 and 8 kV/cm produces membrane pores which allow free diffusion of low molecular weight substances such as adenine nucleotides, inositol phosphate and fluorescent dyes. Two properties, namely Ca2+-induced secretion of granule stored 5-hydroxytryptamine (5HT) and inositol 1,4,5-trisphosphate (IP3)-induced release of intracellularly sequestered 45Ca, which are both well expressed immediately after permeabilisation, are essentially abolished after resealing. The efficiency of permeabilisation and resealing can be simply monitored by shifts in 'apparent platelet volume' using a resistive particle counter (Coulter). Permeabilised platelets show a shift in modal volumes from a control range 4-7 fl to 10-15 fl. Resealing restores these modal volumes to the original control range. Encapsulation of the fluorochrome, Lucifer yellow (Mr 550), during permeabilisation revealed that after resealing greater than 85% of rat platelets, and close to 100% human platelets, contained the encapsulated dye. The initial rates and % aggregation responses of both human and rat platelets to collagen, thrombin and the thromboxane A2-mimetic U46619 remained essentially normal after permeabilisation and resealing further illustrating the maintenance of functional competence following treatment. Resealed rat platelets reinfused into the circulation after labelling with [111In]indium oxine gave survival curves similar to those of control platelets. Therefore, this reversible permeabilisation procedure may allow the use of autologous or heterologous platelets as carrier vehicles for the delivery of drugs and other agents 'in vivo'.

Identification and Characterization of Anti-Platelet Antibodies in Idiopathic Thrombocytopenic Purpura Patients

PubMed Central

Aghabeigi, N; Lindsey, N; Zamani, A; Shishaeyan, B

2012-01-01

Background: The autoimmune disease known as Idiopathic (immune thrombocytopenic purpura thrombocytopenic purpura (ITP) is clinically defined by a low numbers of platelets in the circulation blood. This study aimed to isolate autoantibodies made against the platelet glycoproteins using platelets from healthy volunteers, to determine their specificity and further elucidate their effects on platelet function. Methods: This study used a phage display system to recognize Fab anti-platelet antibodies. Anti-platelet After isolation, the anti-platelet Fab-expressing phage was characterized by ELISA and Western blotting. The Fab-bearing phage pool obtained from five rounds of panning was analysed in order to determine its anti-platelet reactivity. Of the phage colonies obtained, 100 colonies of different sizes were randomly selected for reaction with whole platelets, using M13 phage as a negative control. Results: Twelve colonies of them had strong reactions against the whole platelet preparation, but only four colonies showed substantial reactivity against the lysed platelet preparation (lysate). Three of the four colonies showed three bands representing proteins with different molecular weights. The fourth colony showed only a single band. The final experiment to characterise the protein isolated from the phage library was a DNA gel agarose test. Conclusion: Each colony showed a DNA band that corresponded with the molecular size marker for 5.4 kbase pairs, and this suggested the presence of heavy and light antibody chains in the phage. PMID:23113135

Comparison of the effects of 7.2% hypertonic saline and 20% mannitol on whole blood coagulation and platelet function in dogs with suspected intracranial hypertension - a pilot study.

PubMed

Yozova, Ivayla D; Howard, Judith; Henke, Diana; Dirkmann, Daniel; Adamik, Katja N

2017-06-19

Hyperosmolar therapy with either mannitol or hypertonic saline (HTS) is commonly used in the treatment of intracranial hypertension (ICH). In vitro data indicate that both mannitol and HTS affect coagulation and platelet function in dogs. The aim of this study was to compare the effects of 20% mannitol and 7.2% HTS on whole blood coagulation using rotational thromboelastometry (ROTEM®) and platelet function using a platelet function analyzer (PFA®) in dogs with suspected ICH. Thirty client-owned dogs with suspected ICH needing osmotherapy were randomized to receive either 20% mannitol (5 ml/kg IV over 15 min) or 7.2% HTS (4 ml/kg IV over 5 min). ROTEM® (EXTEM® and FIBTEM® assays) and PFA® analyses (collagen/ADP cartridges) were performed before (T 0 ), as well as 5 (T 5 ), 60 (T 60 ) and 120 (T 120 ) minutes after administration of HTS or mannitol. Data at T 5 , T 60 and T 120 were analyzed as a percentage of values at T 0 for comparison between groups, and as absolute values for comparison between time points, respectively. No significant difference was found between the groups for the percentage change of any parameter at any time point except for FIBTEM® clotting time. Within each group, no significant difference was found between time points for any parameter except for FIBTEM® clotting time in the HTS group, and EXTEM® and FIBTEM® maximum clot firmness in the mannitol group. Median ROTEM® values lay within institutional reference intervals in both groups at all time points, whereas median PFA® values were above the reference intervals at T 5 (both groups) and T 60 (HTS group). Using currently recommended doses, mannitol and HTS do not differ in their effects on whole blood coagulation and platelet function in dogs with suspected ICH. Moreover, no relevant impairment of whole blood coagulation was found following treatment with either solution, whereas a short-lived impairment of platelet function was found after both solutions.

Identification of Tspan9 as a novel platelet tetraspanin and the collagen receptor GPVI as a component of tetraspanin microdomains

PubMed Central

Protty, Majd B.; Watkins, Nicholas A.; Colombo, Dario; Thomas, Steven G.; Heath, Victoria L.; Herbert, John M. J.; Bicknell, Roy; Senis, Yotis A.; Ashman, Leonie K.; Berditchevski, Fedor; Ouwehand, Willem H.; Watson, Steve P.; Tomlinson, Michael G.

2008-01-01

Platelets are essential for wound healing and inflammatory processes, but can also play a deleterious role by causing heart attack and stroke. Normal platelet activation is dependent on tetraspanins, a superfamily of glycoproteins that function as ‘organisers’ of cell membranes by recruiting other receptors and signalling proteins into tetraspanin-enriched microdomains. However, our understanding of how tetraspanin microdomains regulate platelets is hindered by the fact that only four of the 33 mammalian tetraspanins have been identified in platelets. This is because of a lack of antibodies to most tetraspanins and difficulties in measuring mRNA, due to low levels in this anucleate cell. To identify potentially platelet-expressed tetraspanins, mRNA was measured in their nucleated progenitor cell, the megakaryocyte, using serial analysis of gene expression and DNA microarrays. Amongst 19 tetraspanins identified in megakaryocytes, Tspan9, a previously uncharacterized tetraspanin, was relatively specific to these cells. Through generating the first Tspan9 antibodies, Tspan9 expression was found to be tightly regulated in platelets. The relative levels of CD9, CD151, Tspan9 and CD63 were 100, 14, 6 and 2 respectively. Since CD9 was expressed at 49000 cell surface copies per platelet, this suggested a copy number of 2800 Tspan9 molecules. Finally, Tspan9 was shown to be a component of tetraspanin microdomains that included the collagen receptor GPVI (glycoprotein VI) and integrin α6β1, but not the von Willebrand receptor GPIbα or the integrins αIIbβ3 or α2β1. These findings suggest a role for Tspan9 in regulating platelet function in concert with other platelet tetraspanins and their associated proteins. PMID:18795891

Does whole blood coagulation analysis reflect developmental haemostasis?

PubMed

Ravn, Hanne Berg; Andreasen, Jo Bønding; Hvas, Anne-Mette

2017-04-01

: Developmental haemostasis has been well documented over the last 3 decades and age-dependent reference ranges have been reported for a number of plasmatic coagulation parameters. With the increasing use of whole blood point-of-care tests like rotational thromboelastometry (ROTEM) and platelet function tests, an evaluation of age-dependent changes is warranted for these tests as well. We obtained blood samples from 149 children, aged 1 day to 5.9 years, and analysed conventional plasmatic coagulation tests, including activated partial prothrombin time, prothrombin time, and fibrinogen (functional). Whole blood samples were analysed using ROTEM to assess overall coagulation capacity and Multiplate analyzer to evaluate platelet aggregation. Age-dependent changes were analysed for all variables. We found age-dependent differences in all conventional coagulation tests (all P values < 0.05), but there was no sign of developmental changes in whole blood coagulation assessment when applying ROTEM, apart from clotting time in the EXTEM assay (P < 0.03). Despite marked differences in mean platelet aggregation between age groups, data did not reach statistical significance. Citrate-anticoagulated blood showed significantly reduced platelet aggregation compared with blood anticoagulated with heparin or hirudin (all P values < 0.003). We confirmed previous developmental changes in conventional plasmatic coagulation test. However, these age-dependent changes were not displayed in whole blood monitoring using ROTEM or Multiplate analyzer. Type of anticoagulant had a significant influence on platelet aggregation across all age groups.

Comparison of growth factor and platelet concentration from commercial platelet-rich plasma separation systems.

PubMed

Castillo, Tiffany N; Pouliot, Michael A; Kim, Hyeon Joo; Dragoo, Jason L

2011-02-01

Clinical studies claim that platelet-rich plasma (PRP) shortens recovery times because of its high concentration of growth factors that may enhance the tissue repair process. Most of these studies obtained PRP using different separation systems, and few analyzed the content of the PRP used as treatment. This study characterized the composition of single-donor PRP produced by 3 commercially available PRP separation systems. Controlled laboratory study. Five healthy humans donated 100 mL of blood, which was processed to produce PRP using 3 PRP concentration systems (MTF Cascade, Arteriocyte Magellan, Biomet GPS III). Platelet, white blood cell (WBC), red blood cell, and fibrinogen concentrations were analyzed by automated systems in a clinical laboratory, whereas ELISA determined the concentrations of platelet-derived growth factor αβ and ββ (PDGF-αβ, PDGF-ββ), transforming growth factor β1 (TGF-β1), and vascular endothelial growth factor (VEGF). There was no significant difference in mean PRP platelet, red blood cell, active TGF-β1, or fibrinogen concentrations among PRP separation systems. There was a significant difference in platelet capture efficiency. The highest platelet capture efficiency was obtained with Cascade, which was comparable with Magellan but significantly higher than GPS III. There was a significant difference among all systems in the concentrations of WBC, PDGF-αβ, PDGF-ββ, and VEGF. The Cascade system concentrated leukocyte-poor PRP, compared with leukocyte-rich PRP from the GPS III and Magellan systems. The GPS III and Magellan concentrate leukocyte-rich PRP, which results in increased concentrations of WBCs, PDGF-αβ, PDGF-ββ, and VEGF as compared with the leukocyte-poor PRP from Cascade. Overall, there was no significant difference among systems in the platelet concentration, red blood cell, active TGF-β1, or fibrinogen levels. Products from commercially available PRP separation systems produce differing concentrations of growth factors and WBCs. Further research is necessary to determine the clinical relevance of these findings.

In vitro storage characteristics of platelet concentrates suspended in 70% SSP+(TM) additive solution versus plasma over a 14-day storage period.

PubMed

Saunders, C; Rowe, G; Wilkins, K; Holme, S; Collins, P

2011-08-01

The non-paired two-arm study compared the in vitro storage characteristics of platelets suspended as concentrates in either 100% plasma or a mixture of additive solution (SSP+™, MacoPharma, Mouveaux, France) and autologous plasma in a 70:30 ratio over a 14-day storage period. The buffy coat-derived pooled platelet concentrates were sampled on days 1, 2, 3, 6, 8, 10 and 14 and tests performed to determine platelet morphology, function, metabolism, activation and apoptosis-like activity. Swirling remained strong (score=3) in SSP+™, whilst scores of 1 and 0 were noted for plasma units by end of storage. In contrast to units in plasma, pH levels remained above seven in SSP+™ units, increasing after day 10. Percent positive expression of CD62P was similar in both groups on day 1 (median of 54% and 56% for plasma (n=13) and SSP+™ (n=12), respectively), with SSP+™ units showing a more moderate increase in activation after day 10. A progressive decrease in mitochondrial membrane potential was evident in both groups from day 1, whilst annexin V binding was relatively stable from days 1 to 3, with median values remaining below 6%. Subsequent to this, the percentage of platelets binding annexin V increased to approximately 30% by day 14. Platelets suspended in a medium of 70:30 SSP+™ to plasma ratio performed at least as well as platelets in 100% autologous plasma for up to 10 days of storage. Further, results are suggestive of an apoptosis-like process being involved in the platelet storage lesion. © 2011 The Author(s). Vox Sanguinis © 2011 International Society of Blood Transfusion.

Effects of the NO/soluble guanylate cyclase/cGMP system on the functions of human platelets.

PubMed

Makhoul, Stephanie; Walter, Elena; Pagel, Oliver; Walter, Ulrich; Sickmann, Albert; Gambaryan, Stepan; Smolenski, Albert; Zahedi, René P; Jurk, Kerstin

2018-06-01

Platelets are circulating sentinels of vascular integrity and are activated, inhibited, or modulated by multiple hormones, vasoactive substances or drugs. Endothelium- or drug-derived NO strongly inhibits platelet activation via activation of the soluble guanylate cyclase (sGC) and cGMP elevation, often in synergy with cAMP-elevation by prostacyclin. However, the molecular mechanisms and diversity of cGMP effects in platelets are poorly understood and sometimes controversial. Recently, we established the quantitative human platelet proteome, the iloprost/prostacyclin/cAMP/protein kinase A (PKA)-regulated phosphoproteome, and the interactions of the ADP- and iloprost/prostacyclin-affected phosphoproteome. We also showed that the sGC stimulator riociguat is in vitro a highly specific inhibitor, via cGMP, of various functions of human platelets. Here, we review the regulatory role of the cGMP/protein kinase G (PKG) system in human platelet function, and our current approaches to establish and analyze the phosphoproteome after selective stimulation of the sGC/cGMP pathway by NO donors and riociguat. Present data indicate an extensive and diverse NO/riociguat/cGMP phosphoproteome, which has to be compared with the cAMP phosphoproteome. In particular, sGC/cGMP-regulated phosphorylation of many membrane proteins, G-proteins and their regulators, signaling molecules, protein kinases, and proteins involved in Ca 2+ regulation, suggests that the sGC/cGMP system targets multiple signaling networks rather than a limited number of PKG substrate proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

Diagnosis of Glanzmann thrombasthenia by whole blood impedance analyzer (MEA) vs. light transmission aggregometry.

PubMed

Albanyan, A; Al-Musa, A; AlNounou, R; Al Zahrani, H; Nasr, R; AlJefri, A; Saleh, M; Malik, A; Masmali, H; Owaidah, T

2015-08-01

Glanzmann thrombasthenia (GT) is a rare inherited platelet disorder that is characterized by spontaneous or postprocedural bleeding. The diagnosis of GT depends on identifying the dysfunction of the platelets. The aim of this study was to compare a whole blood impedance Multiplate analyzer (MEA) with the standard method, light transmission aggregometry (LTA) in diagnosis of GT. Fifteen patients with GT were assessed on MEA and LTA using arachidonic acid (ASPI: 15 mm), (TRAP: 1 mm), collagen (100 μg/mL), ADP (0.2 mm), and ristocetin (Risto: 10 mg/mL). Whole blood samples were collected in sodium citrate and hirudin vacuum, blood collection tubes and tested within 4 h. Platelet-rich plasma was used for LTA using platelet agonists (ristocetin 1.5 mg/mL) (arachidonic acid 0.5 mg/mL) (ADP 2.5 mg/mL) and (collagen 1 mg/mL). The platelet count and PFA-100 results were (average and SD) 319 ± 93 × 10(9) L and 252 ± 34 s, respectively. Flow cytometry analysis showed that all samples are positive for CD42a and CD42b, whereas 9/15 samples were negative for CD61 and CD41. The other six patients had either partial or full expression of CD61/CD41. Aggregation analysis using both methods showed that all samples had no aggregation response to any of the agonists used apart from six samples which, using only the MEA, showed minimal aggregation in response to collagen (average = 14.3 ± 7 μg, which may suggest ability to detect qualitative abnormality of GPIIb/IIIa). These results suggest that the MEA is sensitive for the detection of Glanzmann thrombasthenia. Furthermore, MEA may also be able to differentiate between the subtypes of Glanzmann thrombasthenia. © 2014 John Wiley & Sons Ltd.

Polyphosphate colocalizes with factor XII on platelet-bound fibrin and augments its plasminogen activator activity

PubMed Central

Lionikiene, Ausra S.; Georgiev, Georgi; Klemmer, Anja; Brain, Chelsea; Kim, Paul Y.

2016-01-01

Activated factor XII (FXIIa) has plasminogen activator capacity but its relative contribution to fibrinolysis is considered marginal compared with urokinase and tissue plasminogen activator. Polyphosphate (polyP) is released from activated platelets and mediates FXII activation. Here, we investigate the contribution of polyP to the plasminogen activator function of αFXIIa. We show that both polyP70, of the chain length found in platelets (60-100 mer), and platelet-derived polyP significantly augment the plasminogen activation capacity of αFXIIa. PolyP70 stimulated the autoactivation of FXII and subsequent plasminogen activation, indicating that once activated, αFXIIa remains bound to polyP70. Indeed, complex formation between polyP70 and αFXIIa provides protection against autodegradation. Plasminogen activation by βFXIIa was minimal and not enhanced by polyP70, highlighting the importance of the anion binding site. PolyP70 did not modulate plasmin activity but stimulated activation of Glu and Lys forms of plasminogen by αFXIIa. Accordingly, polyP70 was found to bind to FXII, αFXIIa, and plasminogen, but not βFXIIa. Fibrin and polyP70 acted synergistically to enhance αFXIIa-mediated plasminogen activation. The plasminogen activator activity of the αFXIIa-polyP70 complex was modulated by C1 inhibitor and histidine-rich glycoprotein, but not plasminogen activator inhibitors 1 and 2. Platelet polyP and FXII were found to colocalize on the activated platelet membrane in a fibrin-dependent manner and decorated fibrin strands extending from platelet aggregates. We show that in the presence of platelet polyP and the downstream substrate fibrin, αFXIIa is a highly efficient and favorable plasminogen activator. Our data are the first to document a profibrinolytic function of platelet polyP. PMID:27694320

Meloxicam, 15 mg/day, spares platelet function in healthy volunteers.

PubMed

de Meijer, A; Vollaard, H; de Metz, M; Verbruggen, B; Thomas, C; Novakova, I

1999-10-01

To study the influence of meloxicam, a cyclooxygenase-2 (COX-2) preferential nonsteroidal anti-inflammatory drug, on serum thromboxane and platelet function in healthy volunteers with use of the maximum recommended daily dosage of 15 mg/day. This study used an open, randomized crossover design. Indomethacin (INN, indometacin) was given as a positive control for nonsteroidal anti-inflammatory drug-induced inhibition of platelet function. The following variables were recorded: thromboxane B2 serum concentrations by radioimmunoassay, platelet aggregation by whole blood aggregometry in response to collagen 1.1 microg/L and to arachidonic acid 0.35 mmol/L, and closure time with use of the PFA-100. Serum thromboxane B2 at baseline was 535+/-233 nmol/L (mean +/- SD) and was reduced for 95% by indomethacin to 26+/-19 nmol/L (P < .001) and for 66% by meloxicam to 183+/-62 nmol/L (P < .001). Maximal platelet aggregation in response to collagen at baseline was 18.7+/-1.6 ohms (ohms). It was reduced by indomethacin to 7.3+/-4.5 ohms (P < .001), but not by meloxicam (19+/-2.5 ohms). Platelet aggregation in response to arachidonic acid at baseline was 12.2+/-2.0 ohms. It was reduced by indomethacin in all subjects to 0 ohms, but not by meloxicam (11+/-2.4 ohms). Closure time at baseline was 128+/-24 seconds and was prolonged by indomethacin to 286+/-38 seconds (P < .001). Meloxicam caused a minor prolongation of the closure time (141+/-32 seconds; P < .05). Meloxicam, 15 mg/day caused a major reduction of maximum thromboxane production but no reduction in collagen- or arachidonic acid-induced platelet aggregation and only minor increase of the closure time.

A new platelet cryoprecipitate glue promoting bone formation after ectopic mesenchymal stromal cell-loaded biomaterial implantation in nude mice.

PubMed

Trouillas, Marina; Prat, Marie; Doucet, Christelle; Ernou, Isabelle; Laplace-Builhé, Corinne; Blancard, Patrick Saint; Holy, Xavier; Lataillade, Jean-Jacques

2013-01-04

This study investigated the promising effect of a new Platelet Glue obtained from Cryoprecipitation of Apheresis Platelet products (PGCAP) used in combination with Mesenchymal Stromal Cells (MSC) loaded on ceramic biomaterials to provide novel strategies enhancing bone repair. PGCAP growth factor content was analyzed by ELISA and compared to other platelet and plasma-derived products. MSC loaded on biomaterials (65% hydroxyapatite/35% beta-TCP or 100% beta-TCP) were embedded in PGCAP and grown in presence or not of osteogenic induction medium for 21 days. Biomaterials were then implanted subcutaneously in immunodeficient mice for 28 days. Effect of PGCAP on MSC was evaluated in vitro by proliferation and osteoblastic gene expression analysis and in vivo by histology and immunohistochemistry. We showed that PGCAP, compared to other platelet-derived products, allowed concentrating large amount of growth factors and cytokines which promoted MSC and osteoprogenitor proliferation. Next, we found that PGCAP improves the proliferation of MSC and osteogenic-induced MSC. Furthermore, we demonstrated that PGCAP up-regulates the mRNA expression of osteogenic markers (Collagen type I, Osteonectin, Osteopontin and Runx2). In vivo, type I collagen expressed in ectopic bone-like tissue was highly enhanced in biomaterials embedded in PGCAP in the absence of osteogenic pre-induction. Better results were obtained with 65% hydroxyapatite/35% beta-TCP biomaterials as compared to 100% beta-TCP. We have demonstrated that PGCAP is able to enhance in vitro MSC proliferation, osteoblastic differentiation and in vivo bone formation in the absence of osteogenic pre-induction. This clinically adaptable platelet glue could be of interest for improving bone repair.

A new platelet cryoprecipitate glue promoting bone formation after ectopic mesenchymal stromal cell-loaded biomaterial implantation in nude mice

PubMed Central

2013-01-01

Introduction This study investigated the promising effect of a new Platelet Glue obtained from Cryoprecipitation of Apheresis Platelet products (PGCAP) used in combination with Mesenchymal Stromal Cells (MSC) loaded on ceramic biomaterials to provide novel strategies enhancing bone repair. Methods PGCAP growth factor content was analyzed by ELISA and compared to other platelet and plasma-derived products. MSC loaded on biomaterials (65% hydroxyapatite/35% beta-TCP or 100% beta-TCP) were embedded in PGCAP and grown in presence or not of osteogenic induction medium for 21 days. Biomaterials were then implanted subcutaneously in immunodeficient mice for 28 days. Effect of PGCAP on MSC was evaluated in vitro by proliferation and osteoblastic gene expression analysis and in vivo by histology and immunohistochemistry. Results We showed that PGCAP, compared to other platelet-derived products, allowed concentrating large amount of growth factors and cytokines which promoted MSC and osteoprogenitor proliferation. Next, we found that PGCAP improves the proliferation of MSC and osteogenic-induced MSC. Furthermore, we demonstrated that PGCAP up-regulates the mRNA expression of osteogenic markers (Collagen type I, Osteonectin, Osteopontin and Runx2). In vivo, type I collagen expressed in ectopic bone-like tissue was highly enhanced in biomaterials embedded in PGCAP in the absence of osteogenic pre-induction. Better results were obtained with 65% hydroxyapatite/35% beta-TCP biomaterials as compared to 100% beta-TCP. Conclusions We have demonstrated that PGCAP is able to enhance in vitro MSC proliferation, osteoblastic differentiation and in vivo bone formation in the absence of osteogenic pre-induction. This clinically adaptable platelet glue could be of interest for improving bone repair. PMID:23290259

An anti-von Willebrand factor aptamer reduces platelet adhesion among patients receiving aspirin and clopidogrel in an ex vivo shear-induced arterial thrombosis.

PubMed

Arzamendi, Dabit; Dandachli, Firas; Théorêt, Jean-François; Ducrocq, Gregory; Chan, Mark; Mourad, Walid; Gilbert, James C; Schaub, Robert G; Tanguay, Jean-François; Merhi, Yahye

2011-01-01

The von Willebrand factor (vWF) aptamer, ARC1779 that blocks the binding of vWF A1-domain to platelet glycoprotein 1b (GPIb) at high shear, may deliver a site-specific antithrombotic effect. We investigated the efficiency of ARC1779 on platelet function in patients with coronary artery disease (CAD) on double antiplatelet therapy. Blood from patients taking aspirin and clopidogrel and from normal volunteers was treated ex vivo with ARC1779 or abciximab, either prior to perfusion (pretherapy) or 10 minutes following the initiation of perfusion (posttherapy) on damaged arteries. Under pre- but not posttherapy, platelet adhesion was significantly reduced by ARC1779 at 83 and 250 nmol/L and by abciximab (100 nmol/L) versus placebo (4.8, 3.8, and 2.9 vs 7.3 platelets × 10(6)/cm(2), P < .05). In contrast to abciximab, ARC1779 did not significantly affect platelet aggregation, P-selectin expression, and platelet-leukocyte binding. These proof-of-concept data may constitute the framework for randomized clinical investigations of this novel antiplatelet therapy among patients with CAD.

Plateletworks platelet function test compared to the thromboelastograph for prediction of postoperative outcomes.

PubMed

Ostrowsky, Jacob; Foes, Jennifer; Warchol, Mark; Tsarovsky, Gary; Blay, Jessica

2004-06-01

Approximately 3.5 million units of platelets are transfused in the United States each year to patients undergoing open-heart surgery with cardiopulmonary bypass (CPB). CPB is a known contributor to platelet loss and platelet dysfunction leading to disruption of hemostasis. Impaired hemostasis results in excess bleeding in 5-25% of all patients undergoing CPB. For this reason, it may be beneficial to measure platelet number and function in these patients. The purpose of this study was to compare the Plateletworks platelet function analyzer to the thromboelastograph (TEG) in predicting postoperatiave hemostatic outcomes as measured by blood product use and chest tube (CT) drainage. This study consisted of 35 adult patients undergoing cardiac surgery with cardiopulmonary bypass at Rush-Presbyterian-Saint Luke's Medical Center (RPSLMC). The Plateletworks and TEG tests were performed preoperatively, after protamine was given, and 24 hours postoperatively on all patients. Plateletworks demonstrated a statistically significant change in platelet function as shown by the adenosine diphosphate (ADP) reagent tube from the preoperative period to the removal of the aortic cross clamp (p = .011). The TEG did not demonstrate a significant change in the k-time and maximum amplitude (MA), but did show a significant change in the alpha-angle from the pre-operative to postoperatiave sample (p = .035). A correlation was found between Plateletworks collagen reagent tubes preoperatively and CT drainage (p = .048, r -0.324). No statistical correlation was established between TEG parameters and CT drainage at any time interval. TEG preoperative MA showed a correlation to receipt of blood products (p = .016). When comparing the Plateletworks to the TEG in this study, the Plateletworks system was a more useful predictor of blood product use and chest tube drainage.

Detection of mild inherited disorders of blood coagulation: current options and personal recommendations.

PubMed

Lippi, Giuseppe; Pasalic, Leonardo; Favaloro, Emmanuel J

2015-08-01

Although assessment of prior personal and familial bleeding history is an important aspect of the diagnosis of bleeding disorders, patients with mild inherited bleeding disorders are sometimes clinically asymptomatic until presented with a hemostatic challenge. However, bleeding may occur after incursion of trauma or surgery, so detection of these conditions reflects an important facet of clinical and laboratory practice. Mild bleeding disorders may be detected as a result of family studies or following identification of abnormal values in first-line screening tests such as activated partial thromboplastin time, prothrombin time, fibrinogen and global platelet function screen testing, such as the platelet function analyzer. Following determination of abnormal screening tests, subsequent investigation should follow a systematic approach that targets specific diagnostic tests, and including factor assays, full platelet function assays and more extensive specialized hemostasis testing. The current report provides a personal overview on inherited disorders of blood coagulation and their detection.

The pharmacological modulation of thrombin-induced cerebral thromboembolism in the rabbit.

PubMed Central

May, G. R.; Paul, W.; Crook, P.; Butler, K. D.; Page, C. P.

1992-01-01

1. Intracarotid (i.c.) administration of thrombin induced a marked accumulation of 111indium-labelled platelets and 125I-labelled fibrinogen within the cranial vasculature of anaesthetized rabbits. 2. Thrombin (100 iu kg-1, i.c.) - induced platelet accumulation was completely abolished by pretreatment with desulphatohirudin (CGP 39393; 1 mg kg-1 i.c., 1 min prior to thrombin). Administration of CGP 39393 1 or 20 min after thrombin produced a significant reduction in platelet accumulation. 3. Intravenous (i.v.) administration of the platelet activating factor (PAF) receptor antagonist BN 52021 (10 mg kg-1) 5 min prior to thrombin (100 iu kg-1, i.c.) had no effect on platelet accumulation. 4. An inhibitor of NO biosynthesis, L-NG-nitro arginine methyl ester (L-NAME; 100 mg kg-1, i.c.), had no significant effect on the cranial platelet accumulation response to thrombin (10 iu kg-1, i.c.) when administered 5 min prior to thrombin. 5. Defibrotide (32 or 64 mg kg-1 bolus i.c. followed by 32 or 64 mg kg-1 h-1, i.c., infusion for 45 min) treatment begun 20 min after thrombin (100 iu kg-1, i.c.) did not significantly modify the cranial platelet accumulation response. 6. Cranial platelet accumulation induced by thrombin (100 iu kg-1, i.c.) was significantly reversed by the fibrinolytic drugs urokinase (20 iu kg-1, i.c., infusion for 45 min), anisoylated plasminogen streptokinase activator complex (APSAC) (200 micrograms kg-1, i.v. bolus) or recombinant tissue plasminogen activator (rt-PA; 100 micrograms kg-1, i.c. bolus followed by 20 micrograms kg-1 min-1, i.c., infusion for 45 min) administered 20 min after thrombin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1504722

Pathogen inactivation treatment of plasma and platelet concentrates and their predicted functionality in massive transfusion protocols.

PubMed

Arbaeen, Ahmad F; Schubert, Peter; Serrano, Katherine; Carter, Cedric J; Culibrk, Brankica; Devine, Dana V

2017-05-01

Trauma transfusion packages for hemorrhage control consist of red blood cells, plasma, and platelets at a set ratio. Although pathogen reduction improves the transfusion safety of platelet and plasma units, there is an associated reduction in quality. This study aimed to investigate the impact of riboflavin/ultraviolet light-treated plasma or platelets in transfusion trauma packages composed of red blood cell, plasma, and platelet units in a ratio of 1:1:1 in vitro by modeling transfusion scenarios for trauma patients and assessing function by rotational thromboelastometry. Pathogen-reduced or untreated plasma and buffy coat platelet concentrate units produced in plasma were used in different combinations with red blood cells in trauma transfusion packages. After reconstitution of these packages with hemodiluted blood, the hemostatic functionality was analyzed by rotational thromboelastometry. Hemostatic profiles of pathogen-inactivated buffy coat platelet concentrate and plasma indicated decreased activity compared with their respective controls. Reconstitution of hemodiluted blood (hematocrit = 20%) with packages that contained treated or nontreated components resulted in increased alpha and maximum clot firmness and enhanced clot-formation time. Simulating transfusion scenarios based on 30% blood replacement with a transfusion trauma package resulted in a nonsignificant difference in rotational thromboelastometry parameters between packages containing treated and nontreated blood components (p ≥ 0.05). Effects of pathogen inactivation treatment were evident when the trauma package percentage was 50% or greater and contained both pathogen inactivation-treated plasma and buffy coat platelet concentrate. Rotational thromboelastometry investigations suggest that there is relatively little impact of pathogen inactivation treatment on whole blood clot formation unless large amounts of treated components are used. © 2017 AABB.

Evaluation of Aspirin and Clopidogrel resistance in patients with Acute Coronary Syndrome by using Adenosine Diposphate Test and Aspirin Test

PubMed Central

O, Ibrahim; M, Oteh; A, A Syukur; HH, Che Hassan; W, S Fadilah; Rahman, MM

2013-01-01

Objectives: To evaluate Aspirin and Clopidogrel resistance/non-responders in patients with acute coronary syndrome (ACS) by using adenosine diposphate and aspirin tests. Methodology: In the study patients with ACS loaded with 300 mg of clopidogrel and 300 mg aspirin and patients on stable daily dose of 75 mg of clopidogrel (more than 3 days) underwent PCI. Response to clopidogrel and Aspirin was assessed by Adenosine Diphosphate (ADP) Test (20 µmol/L) and Aspirin Test (Acetyl Acid) (ASP) 20 µmol/L, respectively, using the Multiplate Platelet Function Analyzer (Dynabyte Medical, Munich, Germany). Results: Sixty four patients were included in this study out of which 57 were with ACS and 7 scheduled for percutaneous coronary intervention (PCI) electively. The proportion of Aspirin good responders and adequate responders were 76.56% and 18.75%, respectively while adequate response and good response to Clopidogrel accounted for 29.7 and 48.4%, respectively Hyperlipidaemia was only co-morbidity associated with higher AUC ADP value (p: 0.046). Hypertriglyceridaemia and serum calcium were weakly correlated with higher AUC ADP serum calcium r=0.08, triglyceride r=0.12. Patients admitted for scheduled PCI and on stable dose of 75mg clopidogrel exhibited lower AUC ADP value as compared to those admitted with acute coronary syndrome given loading dose of 300mg of Clopidogrel. Post loading dose measurement of anti-platelet therapy among ACS patients using the Multiplate Platelet Function Analyzer showed comparable results with other methods. Conclusions : As determined by Multiplate Platelet Function Analyzer, Aspirin resistance/non-responders in this study in acute coronary syndrome patients accounted for 4.69% while Non-responders in Clopidogrel was 21.9%. PMID:24353516

Effect of extraction method on the concentrations of selected bioactive compounds in mandarin juice.

PubMed

Nogata, Yoichi; Ohta, Hideaki; Sumida, Takashi; Sekiya, Keizo

2003-12-03

A mandarin-type citrus fruit, ponkan (Citrus reticulata), was processed by in-line, chopper pulper, and hand-press extractions to investigate the effect of extraction method on the concentrations of bioactive compounds in processed juice. Concentrations of polymethoxylated flavones (tangeretin, nobiletin, and sinensetin) and beta-cryptoxanthin in juice, and inhibitory activities against arachidonate cyclooxygenase and lipoxygenases of the juice extract were analyzed. The juice processed by hand-press extraction contained the largest amounts of nobiletin (3.56 mg/100 mL), tangeretin (4.10 mg/100 mL), and sinensetin (0.13 mg/100 mL). Concentrations of beta-cryptoxanthin were 0.66, 0.59, 0.55, and 0.50 mg/100 mL in chopper pulper, in-line (5/64 in.), in-line (8/64 in.) and hand-press juices, respectively. Both extracts of in-line juices showed greater inhibitory activity toward platelet 12-lipoxygenase than the others. The inhibitory effect of hand-press juice extract on platelet cyclooxygenase activity was remarkable among juice extracts. All juice extracts effectively inhibited polymorphonuclear 5-lipoxygenase activity at nearly the same rate.

Aerobic exercise training lowers platelet reactivity and improves platelet sensitivity to prostacyclin in pre- and postmenopausal women.

PubMed

Lundberg Slingsby, M H; Nyberg, M; Egelund, J; Mandrup, C M; Frikke-Schmidt, R; Kirkby, N S; Hellsten, Y

2017-12-01

Essentials It is unknown how regular exercise affects platelet function after menopause. We studied the effect of 3-months of high-intensity exercise in pre- and postmenopausal women. Platelet sensitivity to the inhibitory effect of arterially infused prostacyclin was increased. Reduced basal platelet reactivity was seen in the premenopausal women only. Background The risk of atherothrombotic events increases after the menopause. Regular physical activity has been shown to reduce platelet reactivity in younger women, but it is unknown how regular exercise affects platelet function after the menopause. Objectives To examine the effects of regular aerobic exercise in late premenopausal and recent postmenopausal women by testing basal platelet reactivity and platelet sensitivity to prostacyclin and nitric oxide. Methods Twenty-five sedentary, but healthy, late premenopausal and 24 matched recently postmenopausal women, mean (95% confidence interval) 49.1 (48.2-49.9) and 53.7 (52.5-55.0) years old, participated in an intervention study: 3-month high-intensity supervised aerobic spinning-cycle training (1 h, × 3/week). Basal platelet reactivity was analyzed in platelet-rich plasma from venous blood as agonist-induced % aggregation. In a subgroup of 13 premenopausal and 14 postmenopausal women, platelet reactivity was tested ex vivo after femoral arterial infusion of prostacyclin, acetylcholine, a cyclooxygenase inhibitor, and after acute one-leg knee extensor exercise. Results Basal platelet reactivity (%aggregation) to TRAP-6 (1 μm) was higher in the postmenopausal, 59% (50-68), than the premenopausal women, 45% (35-55). Exercise training reduced basal platelet reactivity to collagen (1 μg mL -1 ) in the premenopausal women only: from 63% (55-71%) to 51% (41-62%). After the training intervention, platelet aggregation was more inhibited by the arterial prostacyclin infusion and the acute exercise in both premenopausal and postmenopausal women. Conclusions These results highlight previously unknown cardioprotective aspects of regular aerobic exercise in premenopausal and postmenopausal women, improving their regulation of platelet reactivity through an increased platelet sensitivity to prostacyclin, which may counterbalance the increased atherothrombotic risk associated with the menopause. © 2017 International Society on Thrombosis and Haemostasis.

The disulfide isomerase ERp57 is required for fibrin deposition in vivo.

PubMed

Zhou, J; Wu, Y; Wang, L; Rauova, L; Hayes, V M; Poncz, M; Essex, D W

2014-11-01

ERp57 is required for platelet function; however, whether ERp57 contributes to fibrin generation is unknown. Using an inhibitory anti-ERp57 antibody (mAb1), Pf4-Cre/ERp57(fl/fl) mice, Tie2-Cre/ERp57(fl/fl) mice, and mutants of ERp57, we analyzed the function of ERp57 in laser-induced thrombosis. Fibrin deposition was decreased in Pf4-Cre/ERp57(fl/fl) mice, consistent with a role for platelet ERp57 in fibrin generation. Fibrin deposition was further decreased with infusion of mAb1 and in Tie2-Cre/ERp57(fl/fl) mice, consistent with endothelial cells also contributing to fibrin deposition. Infusion of eptibifatide inhibited platelet and fibrin deposition, confirming a role for platelets in fibrin deposition. Infusion of recombinant ERp57 corrected the defect in fibrin deposition but not platelet accumulation, suggesting a direct effect of ERp57 on coagulation. mAb1 inhibited thrombin generation in vitro, consistent with a requirement for ERp57 in coagulation. Platelet accumulation was decreased to similar extents in Pf4-Cre/ERp57(fl/fl) mice, Tie2-Cre/ERp57(fl/fl) mice and normal mice infused with mAb1. Infusion of completely inactivated ERp57 or ERp57 with a non-functional second active site inhibited fibrin deposition and platelet accumulation, indicating that the isomerase activity of the second active site is required for these processes. ERp57 regulates thrombosis via multiple targets. © 2014 International Society on Thrombosis and Haemostasis.

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow.

PubMed

Barendrecht, Arjan D; Verhoef, Johan J F; Pignatelli, Silvia; Pasterkamp, Gerard; Heijnen, Harry F G; Maas, Coen

2017-07-10

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the formation of thrombi that occlude the vasculature and injure organs, with life-threatening consequences. This motivates scientific research on platelet function and the development of methods to track cell-biological processes as they occur under flow conditions. A variety of flow models are available for the study of platelet adhesion and aggregation, two key phenomena in platelet biology. This work describes a method to study real-time platelet degranulation under flow during activation. The method makes use of a flow chamber coupled to a syringe-pump setup that is placed under a wide-field, inverted, LED-based fluorescence microscope. The setup described here allows for the simultaneous excitation of multiple fluorophores that are delivered by fluorescently labeled antibodies or fluorescent dyes. After live-cell imaging experiments, the cover glasses can be further processed and analyzed using static microscopy (i.e., confocal microscopy or scanning electron microscopy).

Influence of Brownian motion on blood platelet flow behavior and adhesive dynamics near a planar wall.

PubMed

Mody, Nipa A; King, Michael R

2007-05-22

We used the platelet adhesive dynamics computational method to study the influence of Brownian motion of a platelet on its flow characteristics near a surface in the creeping flow regime. Two important characterizations were done in this regard: (1) quantification of the platelet's ability to contact the surface by virtue of the Brownian forces and torques acting on it, and (2) determination of the relative importance of Brownian motion in promoting surface encounters in the presence of shear flow. We determined the Peclet number for a platelet undergoing Brownian motion in shear flow, which could be expressed as a simple linear function of height of the platelet centroid, H from the surface Pe (platelet) = . (1.56H + 0.66) for H > 0.3 microm. Our results demonstrate that at timescales relevant to shear flow in blood Brownian motion plays an insignificant role in influencing platelet motion or creating further opportunities for platelet-surface contact. The platelet Peclet number at shear rates >100 s-1 is large enough (>200) to neglect platelet Brownian motion in computational modeling of flow in arteries and arterioles for most practical purposes even at very close distances from the surface. We also conducted adhesive dynamics simulations to determine the effects of platelet Brownian motion on GPIbalpha-vWF-A1 single-bond dissociation dynamics. Brownian motion was found to have little effect on bond lifetime and caused minimal bond stressing as bond rupture forces were calculated to be less than 0.005 pN. We conclude from our results that, for the case of platelet-shaped cells, Brownian motion is not expected to play an important role in influencing flow characteristics, platelet-surface contact frequency, and dissociative binding phenomena under flow at physiological shear rates (>50 s(-1)).

Hydroxychavicol, a novel betel leaf component, inhibits platelet aggregation by suppression of cyclooxygenase, thromboxane production and calcium mobilization.

PubMed

Chang, M C; Uang, B J; Tsai, C Y; Wu, H L; Lin, B R; Lee, C S; Chen, Y J; Chang, C H; Tsai, Y L; Kao, C J; Jeng, J H

2007-09-01

Platelet hyperactivity is important in the pathogenesis of cardiovascular diseases. Betel leaf (PBL) is consumed by 200-600 million betel quid chewers in the world. Hydroxychavicol (HC), a betel leaf component, was tested for its antiplatelet effect. We tested the effect of HC on platelet aggregation, thromboxane B(2) (TXB(2)) and reactive oxygen species (ROS) production, cyclooxygenase (COX) activity, ex vivo platelet aggregation and mouse bleeding time and platelet plug formation in vivo. The pharmacokinetics of HC in rats was also assessed. HC inhibited arachidonic acid (AA) and collagen-induced platelet aggregation and TXB(2) production. HC inhibited the thrombin-induced TXB(2) production, but not platelet aggregation. SQ29548, suppressed collagen- and thrombin-induced TXB(2) production, but not thrombin-induced platelet aggregation. HC also suppressed COX-1/COX-2 enzyme activity and the AA-induced ROS production and Ca(2+) mobilization. HC further inhibited the ex vivo platelet aggregation of platelet-rich plasma (>100 nmole/mouse) and prolonged platelet plug formation (>300 nmole/mouse) in mesenteric microvessels, but showed little effect on bleeding time in mouse tail. Moreover, pharmacokinetics analysis found that more than 99% of HC was metabolized within 3 min of administration in Sprague-Dawley rats in vivo. HC is a potent COX-1/COX-2 inhibitor, ROS scavenger and inhibits platelet calcium signaling, TXB(2) production and aggregation. HC could be a potential therapeutic agent for prevention and treatment of atherosclerosis and other cardiovascular diseases through its anti-inflammatory and antiplatelet effects, without effects on haemostatic functions.

Favorable effects of berry consumption on platelet function, blood pressure, and HDL cholesterol.

PubMed

Erlund, Iris; Koli, Raika; Alfthan, Georg; Marniemi, Jukka; Puukka, Pauli; Mustonen, Pirjo; Mattila, Pirjo; Jula, Antti

2008-02-01

Berries are a particularly rich source of polyphenols. They also contain other bioactive substances, such as vitamin C. Previous studies indicated that the consumption of polyphenol-rich foods (eg, cocoa, tea, and red wine) may induce beneficial changes in pathways related to cardiovascular health. Whether the consumption of berries has similar effects is unknown. We aimed to investigate the effects of berry consumption on hemostatic function, serum lipids, and blood pressure (BP). Middle-aged unmedicated subjects (n = 72) with cardiovascular risk factors consumed moderate amounts of berry or control products for 8 wk in a single-blind, randomized, placebo-controlled intervention trial. Berry consumption inhibited platelet function as measured with a platelet function analyzer (using collagen and ADP as platelet activator) [changes: 11% and -1.4% in the berry and control groups, respectively; P = 0.018, analysis of covariance (ANCOVA)]. Plasma biomarkers of platelet activation, coagulation, and fibrinolysis did not change during the intervention. Serum HDL-cholesterol concentrations increased significantly more (P = 0.006, ANCOVA) in the berry than in the control group (5.2% and 0.6%, respectively), but total cholesterol and triacylglycerol remained unchanged. Systolic BP decreased significantly (P = 0.050, ANCOVA); the decrease mostly occurred in subjects with high baseline BP (7.3 mm Hg in highest tertile; P = 0.024, ANCOVA). Polyphenol and vitamin C concentrations in plasma increased, whereas other nutritional biomarkers (ie, folate, tocopherols, sodium, and potassium) were unaffected. The consumption of moderate amounts of berries resulted in favorable changes in platelet function, HDL cholesterol, and BP. The results indicate that regular consumption of berries may play a role in the prevention of cardiovascular disease.

Influence of HbA1c levels on platelet function profiles associated with tight glycemic control in patients presenting with hyperglycemia and an acute coronary syndrome. A subanalysis of the CHIPS Study ("Control de HIperglucemia y Actividad Plaquetaria en Pacientes con Síndrome Coronario Agudo").

PubMed

Vivas, David; García-Rubira, Juan C; Bernardo, Esther; Angiolillo, Dominick J; Martín, Patricia; Calle-Pascual, Alfonso; Núñez-Gil, Iván; Macaya, Carlos; Fernández-Ortiz, Antonio

2013-02-01

Patients with hyperglycemia, an acute coronary syndrome and poor glycemic control have increased platelet reactivity and poor prognosis. However, it is unclear the influence of a tight glycemic control on platelet reactivity in these patients. This is a subanalysis of the CHIPS study. This trial randomized patients with hyperglycemia to undergo an intensive glucose control (target blood glucose 80-120 mg/dL), or conventional glucose control (target blood glucose <180 mg/dL). We analyzed platelet function at discharge on the subgroup of patients with poor glycemic control, defined with admission levels of HbA1c higher than 6.5%. The primary endpoint was maximal platelet aggregation following stimuli with 20 μM ADP. We also measured aggregation following collagen, epinephrine, and thrombin receptor-activated peptide, as well as P2Y12 reactivity index and surface expression of glycoprotein IIb/IIIa and P-selectin. A total of 67 patients presented HbA1c ≥ 6.5% (37 intensive, 30 conventional), while 42 had HbA1c < 6.5% (20 intensive, 22 conventional). There were no differences in baseline characteristics between groups. At discharge, patients with HbA1c ≥6.5% had significantly reduced MPA with intensive glucose control compared with conventional control (46.1 ± 22.3 vs. 60.4 ± 20.0%; p = 0.004). Similar findings were shown with other measures of platelet function. However, glucose control strategy did not affect platelet function parameters in patients with HbA1c < 6.5%. Intensive glucose control in patients presenting with an acute coronary syndrome and hyperglycemia results in a reduction of platelet reactivity only in the presence of elevated HbA1c levels.

Alterations on the morphology, nitric oxide synthesis and activity of platelets reproduced in rats as possible biomarkers for depression are reversed by fluoxetine.

PubMed

González-Trujano, María Eva; Alvarado-Vásquez, Noé; Mendoza-Sotelo, José; López, Guadalupe; Estrada-Camarena, Erika; Martínez-Mota, Lucia; Moreno, Julia

2012-08-01

Biochemical markers associated with the prognosis of depression in humans are being described in the literature, whereas experimental studies in animal models in search for antidepressant strategies are lacking. The aim of this study was to evaluate platelet morphology, platelet activity and nitric oxide (NO) synthesis as possible biomarkers of depressive-like behavior by using FST alone and in the presence of fluoxetine. Naïve rats were compared to those receiving vehicle or fluoxetine at 10mg/kg i.p. in acute, subchronic and chronic administration in the FST. After behavioral assessment, platelets were isolated from blood samples and analyzed by flow cytometry to determine the platelet mitochondrial membrane potential and NO synthesis. In addition, HPLC and electron microscopy were used to examine 5-HT and tryptophan levels and morphology of platelets, respectively. Rats receiving vehicle and exposed to FST showed depressive-like behavior at all the times tested; after chronic FST rats showed a similar pattern of alteration in platelet morphology and in the studied as possible biochemical markers as those previously recognized in depressive humans. Depressive-like behavior in rats exposed to FST was prevented in the presence of fluoxetine administration at all the times tested and associated with the prevention of alterations in platelet morphology, platelet activity and NO synthesis, and/or in 5-HT concentrations. The results of the present study suggest that platelet function and morphology might be relevant markers for the prognosis of depression and the search for functional treatments. Besides, the relevance of FST as model to study this psychiatric illness is reinforced. Copyright © 2012 Elsevier Inc. All rights reserved.

The effect of the perfluorocarbon emulsion Oxycyte on platelet count and function in the treatment of decompression sickness in a swine model.

PubMed

Cronin, William A; Senese, Angela L; Arnaud, Francoise G; Regis, David P; Auker, Charles R; Mahon, Richard T

2016-09-01

Decompression from elevated ambient pressure is associated with platelet activation and decreased platelet counts. Standard treatment for decompression sickness (DCS) is hyperbaric oxygen therapy. Intravenous perfluorocarbon (PFC) emulsion is a nonrecompressive therapy being examined that improves mortality in animal models of DCS. However, PFC emulsions are associated with a decreased platelet count. We used a swine model of DCS to study the effect of PFC therapy on platelet count, function, and hemostasis. Castrated male swine (n = 50) were fitted with a vascular port, recovered, randomized, and compressed to 180 feet of sea water (fsw) for 31 min followed by decompression at 30 fsw/min. Animals were observed for DCS, administered 100% oxygen, and treated with either emulsified PFC Oxycyte (DCS-PFC) or isotonic saline (DCS-NS). Controls underwent the same procedures, but were not compressed (Sham-PFC and Sham-NS). Measurements of platelet count, thromboelastometry, and coagulation were obtained 1 h before compression and 1, 24, 48, 96, 168 and 192 h after treatment. No significant changes in normalized platelet counts were observed. Prothrombin time was elevated in DCS-PFC from 48 to 192 h compared with DCS-NS, and from 96 to 192 h compared with Sham-PFC. Normalized activated partial thromboplastin time was also elevated in DCS-PFC from 168 to 192 h compared with Sham-PFC. No bleeding events were noted. DCS treated with PFC (Oxycyte) does not impact platelet numbers, whole blood clotting by thromboelastometry, or clinical bleeding. Late changes in prothrombin time and activated partial thromboplastin time associated with PFC use in both DCS therapy and controls warrant further investigation.

Associations of MDR1, TBXA2R, PLA2G7, and PEAR1 genetic polymorphisms with the platelet activity in Chinese ischemic stroke patients receiving aspirin therapy.

PubMed

Peng, Ling-Ling; Zhao, Yuan-Qi; Zhou, Zi-Yi; Jin, Jing; Zhao, Min; Chen, Xin-Meng; Chen, Ling-Yan; Cai, Ye-Feng; Li, Jia-Li; Huang, Min

2016-11-01

Aspirin resistance has an incidence of 5%-65% in patients with ischemic stroke, who receive the standard dose of aspirin, but the platelet function is inadequately inhibited, thereby leading to thrombotic events. Numerous evidence shows that thromboxane A 2 receptor (TXA 2 receptor, encoded by TBXA2R), lipoprotein-associated phospholipase A 2 (Lp-PLA 2 , encoded by PLA2G7) and platelet endothelial aggregation receptor-1 (PEAR1, encoded by PEAR1) are crucial in regulating platelet activation, and P-glycoprotein (P-gp, encoded by MDR1) influences the absorption of aspirin in the intestine. In this study we examined the correlation between MDR1, TBXA2R, PLA2G7, PEAR1 genetic polymorphisms and platelet activity in Chinese ischemic stroke patients receiving aspirin therapy. A total of 283 ischemic stroke patients receiving 100 mg aspirin for 7 d were genotyped for polymorphisms in MDR1 C3435T, TBXA2R (rs1131882), PLA2G7 (rs1051931, rs7756935), and PEAR1 (rs12566888, rs12041331). The platelet aggregation response was measured using an automatic platelet aggregation analyzer and a commercially available TXB 2 ELISA kit. Thirty-three patients (11.66%) were insensitive to aspirin treatment. MDR1 3435TT genotype carriers, whose arachidonic acid (AA) or adenosine diphosphate (ADP)-induced platelet aggregation was lower than that of CC+CT genotype carriers, were less likely to suffer from aspirin resistance (odds ratio=0.421, 95% CI: 0.233-0.759). The TBXA2R rs1131882 CC genotype, which was found more frequently in the aspirin-insensitive group (81.8% vs 62.4%) than in the sensitive group, was identified as a risk factor for aspirin resistance (odds ratio=2.712, 95% CI: 1.080-6.810) with a higher level of AA-induced platelet aggregation. Due to the combined effects of PLA2G7 rs1051931 and rs7756935, carriers of the AA-CC haplotype had a higher level of ADP-induced platelet aggregation, and were at considerably higher risk of aspirin resistance than noncarriers (odds ratio=8.233, 95% CI: 1.590-42.638). A considerable portion (11.66%) of Chinese ischemic stroke patients are insensitive to aspirin treatment, which may be correlated with the MDR1 C3435T, TBXA2R (rs1131882), and PLA2G7 (rs1051931-rs7756935) polymorphisms.

Platelet Function Tests: Preanalytical Variables, Clinical Utility, Advantages, and Disadvantages.

PubMed

Hvas, Anne-Mette; Grove, Erik Lerkevang

2017-01-01

Platelet function tests are mainly used in the diagnostic work-up of platelet disorders. During the last decade, the additional use of platelet function tests to evaluate the effect of antiplatelet therapy has also emerged in an attempt to identify patients with an increased risk of arterial thrombosis. Furthermore, platelet function tests are increasingly used to measure residual effect of antiplatelet therapy prior to surgery with the aim of reducing the risk of bleeding. To a limited extend, platelet function tests are also used to evaluate hyperaggregability as a potential marker of a prothrombotic state outside the setting of antiplatelet therapy. This multifaceted use of platelet function tests and the development of simpler point-of-care tests with narrower application have increased the use of platelet function testing and also facilitated the use of platelet function tests outside the highly specialized laboratories. The present chapter describes the preanalytical variables, which should be taken into account when planning platelet function testing. Also, the most widely used platelet function tests are introduced, and their clinical utility and their relative advantages and disadvantages are discussed.

Effects of clopidogrel and aspirin in combination versus aspirin alone on platelet activation and major receptor expression in diabetic patients: the PLavix Use for Treatment Of Diabetes (PLUTO-Diabetes) trial.

PubMed

Serebruany, Victor L; Malinin, Alex I; Pokov, Alex; Barsness, Gregory; Hanley, Dan F

2008-01-01

Clopidogrel is widely used in diabetic patients after vascular events; however, the ability of this thienopyridine to yield additional antiplatelet protection on top of aspirin has never been explored in a controlled study with comprehensive assessment of platelet activity. The objective of this study was to compare the antiplatelet profiles of clopidogrel + aspirin in combination (C + ASA) versus aspirin alone (ASA) in patients with type 2 diabetes mellitus. Seventy patients with documented diabetes already treated with antecedent aspirin were randomly assigned to receive C + ASA or ASA in the PLUTO-Diabetes trial. Platelet studies included adenosine diphosphate-, collagen-, and arachidonic acid-induced aggregometry; PFA-100 (Dade-Behring, Miami, FL) and Ultegra (Accumetrics, San Diego, CA) analyzers; and expression of 6 major receptors by flow cytometry at baseline and at day 30 after randomization. There were no differences in the baseline clinical and platelet characteristics between the C + ASA and ASA groups, or subsequent significant changes in platelet biomarkers in the ASA group, except for diminished collagen-induced aggregation (P = .02). In contrast, when compared with the ASA group, therapy with C + ASA resulted in significant inhibition of platelet activity assessed by adenosine diphosphate aggregation (P = .0001); closure time prolongation (P = .0003) and reduction of platelet activation units with Ultegra (P = .0001); and expression of platelet/endothelial cell adhesion molecule 1 (P = .002), glycoprotein IIb/IIIa antigen (P = .0002), and activity (P = .0001). Treatment with C + ASA for 1 month provides significantly greater inhibition of platelet activity than ASA alone in diabetic patients in this small randomized trial. However, despite dual antiplatelet regimen, diabetic patients exhibit high residual activity of some platelet biomarkers, including unaffected protease-activated receptor 1 receptor expression.

The use of platelet-rich plasma to treat chronic tendinopathies: A technical analysis.

PubMed

Kaux, Jean-François; Emonds-Alt, Thibault

2018-05-01

Platelet-rich plasma (PRP) is blood plasma with a high concentration of autologous platelets which constitute an immense reservoir of growth factors. The clinical use of PRP is widespread in various medical applications. Although highly popular with athletes, the use of PRP for the treatment of tendinopathies remains scientifically controversial, particularly due to the diversity of products that go by the name of "PRP." To optimize its use, it is important to look at the various stages of obtaining PRP. In this literature review, we take a closer look at eight parameters which may influence the quality of PRP: 1) anticoagulants used to preserve the best platelet function, 2) the speed of centrifugation used to extract the platelets, 3) the platelet concentrations obtained, 4) the impact of the concentration of red and while blood cells on PRP actions, 5) platelet activators encouraging platelet degranulation and, hence, the release of growth factors, and 6) the use or nonuse of local anesthetics when carrying out infiltration. In addition to these parameters, it may be interesting to analyze other variables such as 7) the use of ultrasound guidance during the injection with a view to determining the influence they have on potential recovery.

Platelet biomechanics, platelet bioenergetics, and applications to clinical practice and translational research.

PubMed

George, Mitchell J; Bynum, James; Nair, Prajeeda; Cap, Andrew P; Wade, Charles E; Cox, Charles S; Gill, Brijesh S

2018-07-01

The purpose of this review is to explore the relationship between platelet bioenergetics and biomechanics and how this relationship affects the clinical interpretation of platelet function devices. Recent experimental and technological advances highlight platelet bioenergetics and biomechanics as alternative avenues for collecting clinically relevant data. Platelet bioenergetics drive energy production for key biomechanical processes like adhesion, spreading, aggregation, and contraction. Platelet function devices like thromboelastography, thromboelastometry, and aggregometry measure these biomechanical processes. Platelet storage, stroke, sepsis, trauma, or the activity of antiplatelet drugs alters measures of platelet function. However, the specific mechanisms governing these alterations in platelet function and how they relate to platelet bioenergetics are still under investigation.

Flow cytometric assessment of activation of peripheral blood platelets in dogs with normal platelet count and asymptomatic thrombocytopenia.

PubMed

Żmigrodzka, M; Guzera, M; Winnicka, A

2016-01-01

Platelets play a crucial role in hemostasis. Their activation has not yet been evaluated in healthy dogs with a normal and low platelet count. The aim of this study was to determine the influence of activators on platelet activation in dogs with a normal platelet count and asymptomatic thrombocytopenia. 72 clinically healthy dogs were enrolled. Patients were allocated into three groups. Group 1 consisted of 30 dogs with a normal platelet count, group 2 included 22 dogs with a platelet count between 100 and 200×109/l and group 3 consisted of 20 dogs with a platelet count lower than 100×109/l. Platelet rich-plasma (PRP) was obtained from peripheral blood samples using tripotassium ethylenediaminetetraacetic acid (K3-EDTA) as anticoagulant. Next, platelets were stimulated using phorbol-12-myristate-13-acetate or thrombin, stabilized using procaine or left unstimulated. The expression of CD51 and CD41/CD61 was evaluated. Co-expression of CD41/CD61 and Annexin V served as a marker of platelet activation. The expression of CD41/CD61 and CD51 did not differ between the 3 groups. Thrombin-stimulated platelets had a significantly higher activity in dogs with a normal platelet count than in dogs with asymptomatic thrombocytopenia. Procaine inhibited platelet activity in all groups. In conclusion, activation of platelets of healthy dogs in vitro varied depending on the platelet count and platelet activator.

Fibrinogen concentrate as first-line therapy in aortic surgery reduces transfusion requirements in patients with platelet counts over or under 100×109/L

PubMed Central

Solomon, Cristina; Rahe-Meyer, Niels

2015-01-01

Background Administration of fibrinogen concentrate, targeting improved maximum clot firmness (MCF) of the thromboelastometric fibrin-based clot quality test (FIBTEM) is effective as first-line haemostatic therapy in aortic surgery. We performed a post-hoc analysis of data from a randomised, placebo-controlled trial of fibrinogen concentrate, to investigate whether fibrinogen concentrate reduced transfusion requirements for patients with platelet counts over or under 100×109/L. Material and methods Aortic surgery patients with coagulopathic bleeding after cardiopulmonary bypass were randomised to receive either fibrinogen concentrate (n=29) or placebo (n=32). Platelet count was measured upon removal of the aortic clamp, and coagulation and haematology parameters were measured peri-operatively. Transfusion of allogeneic blood components was recorded and compared between groups. Results After cardiopulmonary bypass, haemostatic and coagulation parameters worsened in all groups; plasma fibrinogen level (determined by the Clauss method) decreased by 43–58%, platelet count by 53–64%, FIBTEM maximum clot firmness (MCF) by 38–49%, FIBTEM maximum clot elasticity (MCE) by 43–54%, extrinsically activated test (EXTEM) MCF by 11–22%, EXTEM MCE by 25–41% and the platelet component of the clot by 23–39%. Treatment with fibrinogen concentrate (mean dose 7–9 g in the 4 groups) significantly reduced post-operative allogeneic blood component transfusion requirements when compared to placebo both for patients with a platelet count ≥100×109/L and for patients with a platelet count <100×109/L. Discussion FIBTEM-guided administration of fibrinogen concentrate reduced transfusion requirements when used as a first-line haemostatic therapy during aortic surgery in patients with platelet counts over or under 100×109/L. PMID:25369608

Dabigatran affects thrombin-dependent platelet aggregation after a week-long therapy.

PubMed

Sokol, Juraj; Nehaj, Frantisek; Ivankova, Jela; Mokan, Michal; Mokan, Marian; Stasko, Jan

2018-05-29

Dabigatran is a direct thrombin inhibitor. As the main adverse event is bleeding, it is relevant whether dabigatran has additional effects on platelet function. If so, it could affect the bleeding risk. We aimed to assess in vitro aggregation in patients with atrial fibrillation (AF) receiving dabigatran. We evaluated 32 AF patients treated with dabigatran (study group) and 18 non-anticoagulated non-AF blood donors (control group). We assessed light transmittance platelet aggregation (LTA) with 100 nmol/L γ-thrombin in both groups. The LTA was performed at two time-points in our dabigatran group of patients. The thrombin-induced platelet aggregation was significantly lower two hours after dabigatran was taken compared to baseline measurement (9% ± 6% vs. 29% ± 21%) in our study group. Moreover, we observed that the baseline value of platelet aggregation in patients on dabigatran treatment was significantly lower compared to healthy volunteers (29% ± 21% vs. 89 ± 8). However, one subanalysis showed that this significant reduction in platelet aggregation at baseline was only observed in patients who received dabigatran for over a week. The thrombin-induced platelet aggregation is reduced in non-valvular AF patients receiving dabigatran after a week-long therapy.

The Protective Effect of Poloxamer-188 on Platelet Functions.

PubMed

Guler, Nil; Abro, Schuharazad; Emanuele, Marty; Iqbal, Omer; Hoppensteadt, Debra; Fareed, Jawed

2017-11-01

Poloxamer-188 (MST-188) is effective in the repair/recovery of damaged cell membranes. MST-188 is a promising agent for protecting blood cell viability. The aim of the study is to test the hypothesis that MST-188 can extend the duration of platelet function. Blood samples were collected from 20 healthy volunteers. MST-188 (10 or 2 mg/mL) containing platelet-rich plasma (PRP) was prepared with 2 procedures. First, PRP prepared from MST-188 added whole blood (WB); second, MST-188 was added to PRP. These were referred to MST-188-WB preparation (WBP) and MST-188-PRP preparation (PRPP), respectively. For control, saline was used in the same manner. Agonist-induced aggregation (AIA) studies were performed at 30, 180, and 300 minutes using Platelet Aggregation Profiler (PAP-8) aggregometer (Bio/Data Corporation, Horsham, Pennsylvania) and Adenosine diphosphate (ADP), arachidonic acid, collagen, and epinephrine as agonists at final concentration of 20 µM, 500 µg/mL, 0.19 mg/mL, and 100 µM, respectively. There was a protective effect of MST-188 on ADP and collagen AIA. At 300 minutes, ADP AIA was found to be 50.2% higher than saline control in 2-mg WBP, 43% at 10-mg PRPP, and 10.4% at 2-mg PRPP. Protective effect of on collagen AIA was 65.9% in 2-mg WBP, 42.74% at 10-mg PRPP, and 11.42% at 2-mg PRPP. In comparison between 30 and 300 minutes, MST-188 showed significant protection in terms of ADP and collagen receptors and for both types of preparations (WBP and PRPP). The protective effects of MST-188 on ADP- and collagen-induced platelet aggregation may contribute to the preservation of platelet functionality upon storage in blood banks.

Disorders of Platelet Function

PubMed Central

Huebsch, Lothar B.; Harker, Laurence A.

1981-01-01

Platelets play an important role in hemostasis, and alterations in platelet function may be the cause of abnormal bleeding in a wide variety of congenital and acquired clinical disorders. Platelet dysfunction may be classified as disorders of (1) substrate connective tissue, (2) adhesion, (3) aggregation and (4) platelet-release reaction. The congenital defects of platelet function, although uncommon, have provided important insights into platelet physiology and pathophysiology and, as a group, are less common, better characterized and more readily classified than the acquired defects. The severity of bleeding resulting from platelet dysfunction varies greatly and is substantially increased when another defect of hemostasis coexists. A disorder of platelet function is suspected on the basis of the history and physical examination and is confirmed by the finding of a prolonged bleeding time in the presence of an adequate number of platelets. A specific diagnosis often requires measurements of the factor VIII and von Willebrand factor complex and other tests of platelet function. Some of these tests may be available only in specialized laboratories. Therapy for bleeding episodes resulting from platelet dysfunction is directed at (1) removing or treating the underlying cause of the platelet disorder; (2) replacing the missing plasma cofactors needed to support normal platelet function (such as by the transfusion of cryoprecipitate in patients with von Willebrand disease, and (3) transfusing functional platelets in the form of platelet concentrates in patients with disorders of intrinsic platelet dysfunction. ImagesFigure 1.Figure 2.Figure 3. PMID:7013276

The role of platelet and endothelial GARP in thrombosis and hemostasis.

PubMed

Vermeersch, Elien; Denorme, Frederik; Maes, Wim; De Meyer, Simon F; Vanhoorelbeke, Karen; Edwards, Justin; Shevach, Ethan M; Unutmaz, Derya; Fujii, Hodaka; Deckmyn, Hans; Tersteeg, Claudia

2017-01-01

Glycoprotein-A Repetitions Predominant protein (GARP or LRRC32) is present on among others human platelets and endothelial cells. Evidence for its involvement in thrombus formation was suggested by full knockout of GARP in zebrafish. To evaluate the role of GARP in platelet physiology and in thrombus formation using platelet and endothelial conditional GARP knock out mice. Platelet and endothelial specific GARP knockout mice were generated using the Cre-loxP recombination system. The function of platelets without GARP was measured by flow cytometry, spreading analysis and aggregometry using PAR4-activating peptide and collagen related peptide. Additionally, clot retraction and collagen-induced platelet adhesion and aggregation under flow were analyzed. Finally, in vivo tail bleeding time, occlusion time of the mesenteric and carotid artery after FeCl3-induced thrombosis were determined in platelet and endothelial specific GARP knock out mice. Platelet specific GARP knockout mice had normal surface GPIb, GPVI and integrin αIIb glycoprotein expression. Although GARP expression was increased upon platelet activation, platelets without GARP displayed normal agonist induced activation, spreading on fibrinogen and aggregation responses. Furthermore, absence of GARP on platelets did not influence clot retraction and had no impact on thrombus formation on collagen-coated surfaces under flow. In line with this, neither the tail bleeding time nor the occlusion time in the carotid- and mesenteric artery after FeCl3-induced thrombus formation in platelet or endothelial specific GARP knock out mice were affected. Evidence is provided that platelet and endothelial GARP are not important in hemostasis and thrombosis in mice.

Platelet Activation in Human Immunodeficiency Virus Type-1 Patients Is Not Altered with Cocaine Abuse

PubMed Central

Kiebala, Michelle; Singh, Meera V.; Piepenbrink, Michael S.; Qiu, Xing; Kobie, James J.; Maggirwar, Sanjay B.

2015-01-01

Recent work has indicated that platelets, which are anucleate blood cells, significantly contribute to inflammatory disorders. Importantly, platelets also likely contribute to various inflammatory secondary disorders that are increasingly associated with Human Immunodeficiency Virus Type-1 (HIV) infection including neurological impairments and cardiovascular complications. Indeed, HIV infection is often associated with increased levels of platelet activators. Additionally, cocaine, a drug commonly abused by HIV-infected individuals, leads to increased platelet activation in humans. Considering that orchestrated signaling mechanisms are essential for platelet activation, and that nuclear factor-kappa B (NF-κB) inhibitors can alter platelet function, the role of NF-κB signaling in platelet activation during HIV infection warrants further investigation. Here we tested the hypothesis that inhibitory kappa B kinase complex (IKK) activation would be central for platelet activation induced by HIV and cocaine. Whole blood from HIV-positive and HIV-negative individuals, with or without cocaine abuse was used to assess platelet activation via flow cytometry whereas IKK activation was analyzed by performing immunoblotting and in vitro kinase assays. We demonstrate that increased platelet activation in HIV patients, as measured by CD62P expression, is not altered with reported cocaine use. Furthermore, cocaine and HIV do not activate platelets in whole blood when treated ex vivo. Finally, HIV-induced platelet activation does not involve the NF-κB signaling intermediate, IKKβ. Platelet activation in HIV patients is not altered with cocaine abuse. These results support the notion that non-IKK targeting approaches will be better suited for the treatment of HIV-associated inflammatory disorders. PMID:26076359

The role of platelet and endothelial GARP in thrombosis and hemostasis

PubMed Central

Vermeersch, Elien; Denorme, Frederik; Maes, Wim; De Meyer, Simon F.; Vanhoorelbeke, Karen; Edwards, Justin; Shevach, Ethan M.; Unutmaz, Derya; Fujii, Hodaka; Deckmyn, Hans; Tersteeg, Claudia

2017-01-01

Background Glycoprotein-A Repetitions Predominant protein (GARP or LRRC32) is present on among others human platelets and endothelial cells. Evidence for its involvement in thrombus formation was suggested by full knockout of GARP in zebrafish. Objectives To evaluate the role of GARP in platelet physiology and in thrombus formation using platelet and endothelial conditional GARP knock out mice. Methods Platelet and endothelial specific GARP knockout mice were generated using the Cre-loxP recombination system. The function of platelets without GARP was measured by flow cytometry, spreading analysis and aggregometry using PAR4-activating peptide and collagen related peptide. Additionally, clot retraction and collagen-induced platelet adhesion and aggregation under flow were analyzed. Finally, in vivo tail bleeding time, occlusion time of the mesenteric and carotid artery after FeCl3-induced thrombosis were determined in platelet and endothelial specific GARP knock out mice. Results Platelet specific GARP knockout mice had normal surface GPIb, GPVI and integrin αIIb glycoprotein expression. Although GARP expression was increased upon platelet activation, platelets without GARP displayed normal agonist induced activation, spreading on fibrinogen and aggregation responses. Furthermore, absence of GARP on platelets did not influence clot retraction and had no impact on thrombus formation on collagen-coated surfaces under flow. In line with this, neither the tail bleeding time nor the occlusion time in the carotid- and mesenteric artery after FeCl3-induced thrombus formation in platelet or endothelial specific GARP knock out mice were affected. Conclusions Evidence is provided that platelet and endothelial GARP are not important in hemostasis and thrombosis in mice. PMID:28278197

Effect of aliskiren and valsartan combination versus aliskiren monotherapy on hemostatic biomarkers in hypertensive diabetics: Aliskiren and Valsartan Impact in Diabetics pilot trial.

PubMed

Serebruany, Victor L; Pokov, Alex N; Aradi, Daniel; Can, Mehmet; DiNicolantonio, James; Kipshidze, Nodar; Atar, Dan

2014-01-01

Valsartan is known to inhibit platelet activity in both in vitro and ex vivo clinical setting, whereas aliskiren in vitro modulates antithrombin-III in plasma. The authors tested how aliskiren and valsartan combination versus aliskiren monotherapy will affect hemostatic biomarkers in mild-to-moderate hypertensive diabetics in the frame of the Aliskiren and Valsartan Impact in Diabetics (AVID) trial. A total of 52 patients with type 2 diabetes and mild-to-moderate hypertension were equally randomized to aliskiren (150-300 mg/d) and valsartan (160 mg/d) versus aliskiren (150-300 mg/d) alone for 4 weeks. A total of 25 biomarkers were serially measured, of which 16 are related to platelet function, 6 to coagulation, and 3 to fibrinolysis. Aliskiren monotherapy has no significant impact on any of the assessed biomarkers. In contrast, valsartan on top of aliskiren provided significant inhibition of ADP-induced platelet aggregation (P=0.032), decreased shear-induced activation measured with PFA-100 analyzer (P=0.041), and diminished expression of GP IIb/IIIa activity (P=0.027) measured by PAC-1 antibody, GP Ib (CD42b, P=0.033), vitronectin receptor (CD51/61, P=0.046), P-selectin (CD62p, P=0.026), lysosome-associated membrane protein (CD107a, P=0.042), and CD40-ligand (CD154, P=0.048). In AVID trial, valsartan in combination with aliskiren mildly but significantly inhibited platelets, confirming previous observations. In contrast, aliskiren monotherapy does not enhance antithrombin activity, suggesting that previous data probably represent a laboratory artifact. Importantly, these randomized data were generated on top of low-dose daily aspirin, supporting extra benefit for combination use of angiotensin receptor blockers and renin inhibitors in high-risk diabetic population.

Hydroxychavicol, a novel betel leaf component, inhibits platelet aggregation by suppression of cyclooxygenase, thromboxane production and calcium mobilization

PubMed Central

Chang, M C; Uang, B J; Tsai, C Y; Wu, H L; Lin, B R; Lee, C S; Chen, Y J; Chang, C H; Tsai, Y L; Kao, C J; Jeng, J H

2007-01-01

Background and purpose: Platelet hyperactivity is important in the pathogenesis of cardiovascular diseases. Betel leaf (PBL) is consumed by 200-600 million betel quid chewers in the world. Hydroxychavicol (HC), a betel leaf component, was tested for its antiplatelet effect. Experimental approach: We tested the effect of HC on platelet aggregation, thromboxane B2 (TXB2) and reactive oxygen species (ROS) production, cyclooxygenase (COX) activity, ex vivo platelet aggregation and mouse bleeding time and platelet plug formation in vivo. The pharmacokinetics of HC in rats was also assessed. Key results: HC inhibited arachidonic acid (AA) and collagen-induced platelet aggregation and TXB2 production. HC inhibited the thrombin-induced TXB2 production, but not platelet aggregation. SQ29548, suppressed collagen- and thrombin-induced TXB2 production, but not thrombin-induced platelet aggregation. HC also suppressed COX-1/COX-2 enzyme activity and the AA-induced ROS production and Ca2+ mobilization. HC further inhibited the ex vivo platelet aggregation of platelet-rich plasma (>100 nmole/mouse) and prolonged platelet plug formation (>300 nmole/mouse) in mesenteric microvessels, but showed little effect on bleeding time in mouse tail. Moreover, pharmacokinetics analysis found that more than 99% of HC was metabolized within 3 min of administration in Sprague-Dawley rats in vivo. Conclusions and implications: HC is a potent COX-1/COX-2 inhibitor, ROS scavenger and inhibits platelet calcium signaling, TXB2 production and aggregation. HC could be a potential therapeutic agent for prevention and treatment of atherosclerosis and other cardiovascular diseases through its anti-inflammatory and antiplatelet effects, without effects on haemostatic functions. PMID:17641677

Combined aspirin and cilostazol treatment is associated with reduced platelet aggregation and prevention of exercise-induced platelet activation.

PubMed

Cleanthis, M; Bhattacharya, V; Smout, J; Ashour, H; Stansby, G

2009-05-01

Cilostazol has proven efficacy in increasing walking distance in claudicants, but it has not been demonstrated to be more effective than placebo in secondary cardiovascular prevention. The direct effect of exercise on platelet function remains less well defined. We have investigated the effect of combination treatment with aspirin and cilostazol on platelet activity in claudicants subjected to repeated treadmill exercise. Nineteen claudicants completed a double-blind, randomised, controlled, cross-over trial. Each subject received a 2-week course of aspirin (75mg) and placebo and aspirin and cilostazol (100mg twice daily). Following each 2-week treatment period, patients participated in a standardised treadmill test (3.2kmh(-1), 10 degrees incline) walking to maximal claudication distance. The exercise was repeated thrice in total, and blood was sampled before and after exercise. Platelet activation was measured using free platelet counting aggregation, flow cytometry for surface markers of platelet activation and soluble P-selectin assay. Compared to aspirin and placebo, combination treatment with aspirin and cilostazol was associated with reduced arachidonic-acid-induced platelet aggregation (p<0.01, Wilcoxon signed-rank test). Aspirin and placebo treatment were associated with elevated P-selectin expression, platelet-monocyte aggregation and reduced CD42b expression (p<0.05, Wilcoxon signed-rank test) post-exercise. No difference was seen in spontaneous platelet aggregation whilst soluble P-selectin was reduced post-exercise with combination treatment with aspirin and cilostazol (p<0.05, Wilcoxon signed-rank test). Combination treatment with aspirin and cilostazol results in suppression of platelet activation and reduces the effect of exercise on platelets. The benefit seen may be a result of cilostazol enhancing the inhibitory effect of aspirin on the cyclo-oxygenase pathway.

Platelet-rich plasma combined with agarose as a bioactive scaffold to enhance cartilage repair: an in vitro study.

PubMed

Yin, Zhaowei; Yang, Xiaofei; Jiang, Yiqiu; Xing, Linzi; Xu, Yang; Lu, Yiming; Ding, Peng; Ma, Junxin; Xu, Yan; Gui, Jianchao

2014-03-01

The purpose of this study was to determine whether the platelet-rich plasma-agarose gel scaffold could be a bioactive scaffold capable of growth factors release for cartilage repair. Porcine chondrocytes were seeded in agarose gel and platelet-rich plasma-agarose gel. During the 28-days culture, microstructure of hydrogels and morphologies of chondrocytes seeded in the hydrogels were observed using scanning electron microscope; viability of chondrocytes in gels was examined by live/dead assay; qualitative and quantitative analysis of glycosaminoglycan, collagen and DNA were assessed by histological, immunohistochemical staining and biochemical assay; gene expression was measured by real-time polymerase chain reaction. In vitro cartilage ring models were used to evaluate the integration of the scaffolds, and the integration strength was analyzed by mechanical push-out tests. Scanning electron microscope revealed both scaffolds had highly uniform porous structure. Live/dead scaffolds showed 100% cells alive in both groups. After 28-days culture, glycosaminoglycan, collagen, DNA content and chondrocyte-related genes expression in platelet-rich plasma-agarose gel were significantly higher than pure agarose gel. Integration strength in platelet-rich plasma-agarose gel was also higher compared to pure agarose gel. Platelet-rich plasma showed a positive effect on chondrocytes proliferation, differentiation and integration between native cartilage and engineered tissue when combined with agarose gel. Our findings suggest that platelet-rich plasma-agarose gel scaffold is a promising bioactive scaffold for future cartilage tissue engineering and future clinical works.

Immobilization and detection of platelet-derived extracellular vesicles on functionalized silicon substrate: cytometric and spectrometric approach.

PubMed

Gajos, Katarzyna; Kamińska, Agnieszka; Awsiuk, Kamil; Bajor, Adrianna; Gruszczyński, Krzysztof; Pawlak, Anna; Żądło, Andrzej; Kowalik, Artur; Budkowski, Andrzej; Stępień, Ewa

2017-02-01

Among the various biomarkers that are used to diagnose or monitor disease, extracellular vesicles (EVs) represent one of the most promising targets in the development of new therapeutic strategies and the application of new diagnostic methods. The detection of circulating platelet-derived microvesicles (PMVs) is a considerable challenge for laboratory diagnostics, especially in the preliminary phase of a disease. In this study, we present a multistep approach to immobilizing and detecting PMVs in biological samples (microvesicles generated from activated platelets and human platelet-poor plasma) on functionalized silicon substrate. We describe the application of time-of-flight secondary ion mass spectrometry (TOF-SIMS) and spectroscopic ellipsometry methods to the detection of immobilized PMVs in the context of a novel imaging flow cytometry (ISX) technique and atomic force microscopy (AFM). This novel approach allowed us to confirm the presence of the abundant microvesicle phospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) on a surface with immobilized PMVs. Phosphatidylcholine groups (C 5 H 12 N + ; C 5 H 15 PNO 4 + ) were also detected. Moreover, we were able to show that ellipsometry permitted the immobilization of PMVs on a functionalized surface to be evaluated. The sensitivity of the ISX technique depends on the size and refractive index of the analyzed microvesicles. Graphical abstract Human platelets activated with thrombin (in concentration 1IU/mL) generate population of PMVs (platelet derived microvesicles), which can be detected and enumerated with fluorescent-label method (imaging cytometry). Alternatively, PMVs can be immobilized on the modified silicon substrate which is functionalized with a specific IgM murine monoclonal antibody against human glycoprotein IIb/IIIa complex (PAC-1). Immobilized PMVs can be subjected to label-free analyses by means ellipsometry, atomic force microscopy (AFM) and time-of-flight secondary ion mass spectrometry (TOF-SIMS).

Ultrasound-guided platelet-rich plasma injection for distal biceps tendinopathy.

PubMed

Barker, Scott L; Bell, Simon N; Connell, David; Coghlan, Jennifer A

2015-04-01

Distal biceps tendinopathy is an uncommon cause of elbow pain. The optimum treatment for cases refractory to conservative treatment is unclear. Platelet-rich plasma has been used successfully for other tendinopathies around the elbow. Six patients with clinical and radiological evidence of distal biceps tendinopathy underwent ultrasound-guided platelet-rich plasma (PRP) injection. Clinical examination findings, visual analogue score (VAS) for pain and Mayo Elbow Performance scores were recorded. The Mayo Elbow Performance Score improved from 68.3 (range 65 to 85) (fair function) to 95 (range 85 to 100) (excellent function). The VAS at rest improved from a mean of 2.25 (range 2 to 5) pre-injection to 0. The VAS with movement improved from a mean of 7.25 (range 5 to 8) pre-injection to 1.3 (range 0 to 2). No complications were noted. Ultrasound-guided PRP injection appears to be a safe and effective treatment for recalcitrant cases of distal biceps tendinopathy. Further investigation with a randomized controlled trial is needed to fully assess its efficacy.

Polyphosphate colocalizes with factor XII on platelet-bound fibrin and augments its plasminogen activator activity.

PubMed

Mitchell, Joanne L; Lionikiene, Ausra S; Georgiev, Georgi; Klemmer, Anja; Brain, Chelsea; Kim, Paul Y; Mutch, Nicola J

2016-12-15

Activated factor XII (FXIIa) has plasminogen activator capacity but its relative contribution to fibrinolysis is considered marginal compared with urokinase and tissue plasminogen activator. Polyphosphate (polyP) is released from activated platelets and mediates FXII activation. Here, we investigate the contribution of polyP to the plasminogen activator function of αFXIIa. We show that both polyP 70 , of the chain length found in platelets (60-100 mer), and platelet-derived polyP significantly augment the plasminogen activation capacity of αFXIIa. PolyP 70 stimulated the autoactivation of FXII and subsequent plasminogen activation, indicating that once activated, αFXIIa remains bound to polyP 70 Indeed, complex formation between polyP 70 and αFXIIa provides protection against autodegradation. Plasminogen activation by βFXIIa was minimal and not enhanced by polyP 70 , highlighting the importance of the anion binding site. PolyP 70 did not modulate plasmin activity but stimulated activation of Glu and Lys forms of plasminogen by αFXIIa. Accordingly, polyP 70 was found to bind to FXII, αFXIIa, and plasminogen, but not βFXIIa. Fibrin and polyP 70 acted synergistically to enhance αFXIIa-mediated plasminogen activation. The plasminogen activator activity of the αFXIIa-polyP 70 complex was modulated by C1 inhibitor and histidine-rich glycoprotein, but not plasminogen activator inhibitors 1 and 2. Platelet polyP and FXII were found to colocalize on the activated platelet membrane in a fibrin-dependent manner and decorated fibrin strands extending from platelet aggregates. We show that in the presence of platelet polyP and the downstream substrate fibrin, αFXIIa is a highly efficient and favorable plasminogen activator. Our data are the first to document a profibrinolytic function of platelet polyP. © 2016 by The American Society of Hematology.

Rearranged EML4-ALK fusion transcripts sequester in circulating blood platelets and enable blood-based crizotinib response monitoring in non-small-cell lung cancer

PubMed Central

Nilsson, R. Jonas A.; Karachaliou, Niki; Berenguer, Jordi; Gimenez-Capitan, Ana; Schellen, Pepijn; Teixido, Cristina; Tannous, Jihane; Kuiper, Justine L.; Drees, Esther; Grabowska, Magda; van Keulen, Marte; Heideman, Danielle A.M.; Thunnissen, Erik; Dingemans, Anne-Marie C.; Viteri, Santiago; Tannous, Bakhos A.; Drozdowskyj, Ana; Rosell, Rafael; Smit, Egbert F.; Wurdinger, Thomas

2016-01-01

Purpose: Non-small-cell lung cancers harboring EML4-ALK rearrangements are sensitive to crizotinib. However, despite initial response, most patients will eventually relapse, and monitoring EML4-ALK rearrangements over the course of treatment may help identify these patients. However, challenges associated with serial tumor biopsies have highlighted the need for blood-based assays for the monitoring of biomarkers. Platelets can sequester RNA released by tumor cells and are thus an attractive source for the non-invasive assessment of biomarkers. Methods: EML4-ALK rearrangements were analyzed by RT-PCR in platelets and plasma isolated from blood obtained from 77 patients with non-small-cell lung cancer, 38 of whom had EML4-ALK-rearranged tumors. In a subset of 29 patients with EML4-ALK-rearranged tumors who were treated with crizotinib, EML4-ALK rearrangements in platelets were correlated with progression-free and overall survival. Results: RT-PCR demonstrated 65% sensitivity and 100% specificity for the detection of EML4-ALK rearrangements in platelets. In the subset of 29 patients treated with crizotinib, progression-free survival was 3.7 months for patients with EML4-ALK+ platelets and 16 months for those with EML4-ALK− platelets (hazard ratio, 3.5; P = 0.02). Monitoring of EML4-ALK rearrangements in the platelets of one patient over a period of 30 months revealed crizotinib resistance two months prior to radiographic disease progression. Conclusions: Platelets are a valuable source for the non-invasive detection of EML4-ALK rearrangements and may prove useful for predicting and monitoring outcome to crizotinib, thereby improving clinical decisions based on radiographic imaging alone. PMID:26544515

Rearranged EML4-ALK fusion transcripts sequester in circulating blood platelets and enable blood-based crizotinib response monitoring in non-small-cell lung cancer.

PubMed

Nilsson, R Jonas A; Karachaliou, Niki; Berenguer, Jordi; Gimenez-Capitan, Ana; Schellen, Pepijn; Teixido, Cristina; Tannous, Jihane; Kuiper, Justine L; Drees, Esther; Grabowska, Magda; van Keulen, Marte; Heideman, Danielle A M; Thunnissen, Erik; Dingemans, Anne-Marie C; Viteri, Santiago; Tannous, Bakhos A; Drozdowskyj, Ana; Rosell, Rafael; Smit, Egbert F; Wurdinger, Thomas

2016-01-05

Non-small-cell lung cancers harboring EML4-ALK rearrangements are sensitive to crizotinib. However, despite initial response, most patients will eventually relapse, and monitoring EML4-ALK rearrangements over the course of treatment may help identify these patients. However, challenges associated with serial tumor biopsies have highlighted the need for blood-based assays for the monitoring of biomarkers. Platelets can sequester RNA released by tumor cells and are thus an attractive source for the non-invasive assessment of biomarkers. EML4-ALK rearrangements were analyzed by RT-PCR in platelets and plasma isolated from blood obtained from 77 patients with non-small-cell lung cancer, 38 of whom had EML4-ALK-rearranged tumors. In a subset of 29 patients with EML4-ALK-rearranged tumors who were treated with crizotinib, EML4-ALK rearrangements in platelets were correlated with progression-free and overall survival. RT-PCR demonstrated 65% sensitivity and 100% specificity for the detection of EML4-ALK rearrangements in platelets. In the subset of 29 patients treated with crizotinib, progression-free survival was 3.7 months for patients with EML4-ALK+ platelets and 16 months for those with EML4-ALK- platelets (hazard ratio, 3.5; P = 0.02). Monitoring of EML4-ALK rearrangements in the platelets of one patient over a period of 30 months revealed crizotinib resistance two months prior to radiographic disease progression. Platelets are a valuable source for the non-invasive detection of EML4-ALK rearrangements and may prove useful for predicting and monitoring outcome to crizotinib, thereby improving clinical decisions based on radiographic imaging alone.

PDGFRα promoter polymorphisms and expression patterns influence risk of development of imatinib-induced thrombocytopenia in chronic myeloid leukemia: A study from India.

PubMed

Guru, Sameer Ahmad; Mir, Rashid; Bhat, Musadiq; Najar, Imtiyaz; Zuberi, Mariyam; Sumi, Mamta; Masroor, Mirza; Gupta, Naresh; Saxena, Alpana

2017-10-01

Platelet-derived growth factor receptor has been implicated in many malignant and non-malignant diseases. Platelet-derived growth factor receptor-α is a tyrosine kinase and a side target for imatinib, a revolutionary drug for the treatment of chronic myeloid leukemia that has dramatically improved the survival of chronic myeloid leukemia patients. Given the importance of platelet-derived growth factor receptor in platelet development and its inhibition by imatinib, it was intriguing to analyze the role of platelet-derived growth factor receptor-α in relation to imatinib treatment in the development of imatinib-induced thrombocytopenia in chronic myeloid leukemia patients. We hypothesized that two known functional polymorphisms, +68GA insertion/deletion and -909C/A, in the promoter region of the platelet-derived growth factor receptor-α gene may affect the susceptibility of chronic myeloid leukemia patients receiving imatinib treatment to the development of thrombocytopenia. A case-control study was conducted among a cohort of chronic myeloid leukemia patients admitted to the Lok Nayak Hospital, New Delhi, India. A set of 100 patients of chronic myeloid leukemia in chronic phase and 100 age- and sex-matched healthy controls were studied. After initiation of imatinib treatment, the hematological response of chronic myeloid leukemia patients was monitored regularly for 2 years, in which the development of thrombocytopenia was the primary end point. Platelet-derived growth factor receptor-α promoter polymorphisms +68GA ins/del and -909C/A were studied by allele-specific polymerase chain reaction. Platelet-derived growth factor receptor-α messenger RNA expression was evaluated by quantitative real-time polymerase chain reaction. The messenger RNA expression results were expressed as 2 -Δct ± standard deviation. The distribution of +68GA ins/del promoter polymorphism genotypes differed significantly between the thrombocytopenic and non-thrombocytopenic chronic myeloid leukemia patient groups (p < 0.0001). Moreover, +68GA del/del and ins/del genotypes in imatinib-treated chronic myeloid leukemia patients were associated with an increased risk of developing thrombocytopenia, with odds ratios 6.5 (95% confidence interval = 2.02-0.89, p = 0.001) and 6.0 (95% confidence interval = 2.26-15.91, p = 0.0002), respectively. Similarly, -909C/A promoter polymorphism genotype distribution also differed significantly between thrombocytopenic and non-thrombocytopenic chronic myeloid leukemia patient groups (p = 0.02), and a significantly increased risk of imatinib-induced thrombocytopenia was associated with -909C/A polymorphism mutant homozygous (AA) genotypes the odds ratio being 7.7 (95% confidence interval 1.50 to 39.91, p = 0.009). However, no significant risk of imatinib-induced thrombocytopenia was found to be associated with heterozygous genotype (-909C/A) with odds ratio 1.9 (95% confidence interval = 0.86-4.56, p = 1.14). Platelet-derived growth factor receptor-α messenger RNA expression was significantly higher in chronic myeloid leukemia patients compared to controls (p = 0.008). Moreover, patients with imatinib-induced thrombocytopenia had a significantly lower platelet-derived growth factor receptor-α messenger RNA expression, compared to patients without thrombocytopenia (p = 0.01). A differential expression of platelet-derived growth factor receptor-α messenger RNA was observed with respect to different +68 GA ins/del and -909C/A polymorphism genotypes. The +68GA deletion allele and -909A allele were significantly associated with lower expression of platelet-derived growth factor receptor-α messenger RNA. The platelet-derived growth factor receptor-α +68GA del/del, +68GA ins/del, and -909AA genotypes are associated with an increased risk of developing thrombocytopenia in imatinib-treated chronic myeloid leukemia patients. A significantly lower platelet-derived growth factor receptor-α messenger RNA expression accompanies the +68GA deletion allele in an allele dose-dependent manner. Platelet-derived growth factor receptor-α -909AA genotype is also associated with lower expression of platelet-derived growth factor receptor-α. The downregulation of platelet-derived growth factor receptor-α expression may play a causative role in imatinib-induced thrombocytopenia, a common side effect, in the subset of chronic myeloid leukemia patients with platelet-derived growth factor receptor-α +68 GA ins/del, +68 GA del/del, and -909C/A genotypes.

Monitoring aspirin therapy in children after interventional cardiac catheterization: laboratory measures, dose response, and clinical outcomes.

PubMed

Schmugge, Markus; Speer, Oliver; Kroiss, Sabine; Knirsch, Walter; Kretschmar, Oliver; Rand, Margaret L; Albisetti, Manuela

2015-07-01

Very few studies have investigated dose response of aspirin and agreement of different platelet function assays in children. One hundred five children were studied at baseline and after interventional cardiac catheterization during aspirin treatment and, in cases of aspirin resistance (AR), after dose increase. Results from arachidonate-induced aggregation (AA) were compared with aggregation induced by ADP, PFA-100 closure times (CTs), urinary 11-dehydro-thromboxane B2 (urinary 11-dhTxB2) levels, and Impact-R % surface coverage. Aspirin at 2-5 mg/kg/day inhibited platelet function in a large majority. While 19 % showed bruising and mild epistaxis, no thrombotic complications were recorded. AR was detected by AA in seven children (6.7 %). After dose increase, the majority showed inhibition by aspirin. Infants had higher urinary 11-dhTxB2 baseline levels; this assay showed some correlation with AA. Both assays manifested high sensitivity and specificity for aspirin while inferior results were found for the other assays. With the PFA-100, 15.2 % of patients were found to have AR, but this corresponded to AR by AA in only one of seven children. While there was poor agreement among assays, AA and urinary 11-dhTxB2 show good specificity for the monitoring of aspirin therapy in children. Aspirin at 2-5 mg/kg inhibits platelet function; AR in children is rare and can be overcome by dose increase.

Biologic variability and correlation of platelet function testing in healthy dogs.

PubMed

Blois, Shauna L; Lang, Sean T; Wood, R Darren; Monteith, Gabrielle

2015-12-01

Platelet function tests are influenced by biologic variability, including inter-individual (CVG ) and intra-individual (CVI ), as well as analytic (CVA ) variability. Variability in canine platelet function testing is unknown, but if excessive, would make it difficult to interpret serial results. Additionally, the correlation between platelet function tests is poor in people, but not well described in dogs. The aims were to: (1) identify the effect of variation in preanalytic factors (venipuncture, elapsed time until analysis) on platelet function tests; (2) calculate analytic and biologic variability of adenosine diphosphate (ADP) and arachidonic acid (AA)-induced thromboelastograph platelet mapping (TEG-PM), ADP-, AA-, and collagen-induced whole blood platelet aggregometry (WBA), and collagen/ADP and collagen/epinephrine platelet function analysis (PFA-CADP, PFA-CEPI); and (3) determine the correlation between these variables. In this prospective observational trial, platelet function was measured once every 7 days, for 4 consecutive weeks, in 9 healthy dogs. In addition, CBC, TEG-PM, WBA, and PFA were performed. Overall coefficients of variability ranged from 13.3% to 87.8% for the platelet function tests. Biologic variability was highest for AA-induced maximum amplitude generated during TEG-PM (MAAA; CVG = 95.3%, CVI = 60.8%). Use of population-based reference intervals (RI) was determined appropriate only for PFA-CADP (index of individuality = 10.7). There was poor correlation between most platelet function tests. Use of population-based RI appears inappropriate for most platelet function tests, and tests poorly correlate with one another. Future studies on biologic variability and correlation of platelet function tests should be performed in dogs with platelet dysfunction and those treated with antiplatelet therapy. © 2015 American Society for Veterinary Clinical Pathology.

A Prospective, Randomized, Open-Label, Blinded, Endpoint Study Exploring Platelet Response to Half-Dose Prasugrel and Ticagrelor in Patients with the Acute Coronary Syndrome: HOPE-TAILOR Study.

PubMed

Jin, Cai De; Kim, Moo Hyun; Bang, Junghee; Serebruany, Victor

The optimal dosing of novel oral P2Y12 receptor platelet inhibitors such as prasugrel or ticagrelor is unclear and especially challenging in East Asians. We hypothesize that half-dose prasugrel and ticagrelor may be sufficient for long-term maintenance management in Korean patients with the acute coronary syndrome (ACS) compared with conventional dosages. HOPE-TAILOR (Half Dose of Prasugrel and Ticagrelor in Platelet Response after Acute Coronary Syndromes) is a prospective, randomized, open-label, blinded, endpoint (PROBE) single-center, clinical trial. A total of 100 patients with ACS undergoing drug-eluting stent implantation will be randomly assigned to prasugrel, ticagrelor, or clopidogrel, and the patients in each treatment group will receive 1-month therapy with 100 mg q.d. aspirin plus prasugrel 10 mg q.d., ticagrelor 90 mg b.i.d., or clopidogrel 75 mg q.d., followed by half-dose prasugrel 5 mg q.d. or ticagrelor 45 mg b.i.d. for maintenance treatment but without clopidogrel dose reduction. The primary endpoint will be optimal platelet reactivity 3 months after coronary intervention, defined by VerifyNow Analyzer (PRU: 85-208) and vasodilator-stimulated phosphoprotein P2Y12 flow cytometry assay (platelet reactivity indices: 16-50%). Clinical outcomes will also be assessed, including major efficacy (composite of cardiac death, nonfatal myocardial infarction, repeat revascularization, or stroke) and safety (bleeding ≥2 according to the Bleeding Academic Research Consortium). HOPE-TAILOR is a prospective, randomized, open-label, blinded, endpoint study to explore the efficacy and safety of novel P2Y12 receptor inhibitors administered orally at half the dose in Korean patients with ACS. The results will be available late in 2017. © 2017 S. Karger AG, Basel.

Congenital platelet function defects

MedlinePlus

Platelet storage pool disorder; Glanzmann's thrombasthenia; Bernard-Soulier syndrome; Platelet function defects - congenital ... This disorder may also cause severe bleeding. Platelet storage pool disorder (also called platelet secretion disorder) occurs ...

In vitro effects of fermented papaya (Carica papaya, L.) on platelets obtained from patients with type 2 diabetes.

PubMed

Raffaelli, F; Nanetti, L; Montecchiani, G; Borroni, F; Salvolini, E; Faloia, E; Ferretti, G; Mazzanti, L; Vignini, A

2015-02-01

Oxidative stress is associated with insulin resistance pathogenesis, insulin secretion deficiency, and complication onset. Fermented papaya preparation (FPP), a dietary supplement obtained by fermentation of the papaya fruit, may be used as an antioxidant in the prevention of diabetic complications. Platelets from 30 patients with type 2 diabetes mellitus (DM 2) and 15 healthy subjects were analyzed to evaluate the in vitro effects of FPP incubation. Na(+)/K(+)-adenosine triphosphatase (ATPase) activity, membrane fluidity, total antioxidant capacity (TAC), superoxide dismutase (SOD) activity, and conjugated diene levels were determined. In vitro FPP incubation improved platelet function, by enhancing Na(+)/K(+)-ATPase activity and membrane fluidity, and ameliorated the antioxidant system functionality, through an increase in TAC and SOD activity and a parallel decrease in conjugated diene levels in patients with DM 2. Our data suggest that the incubation with FPP may have a protective effect on platelets from patients with DM 2, by preventing the progression of oxidative damage associated with diabetes and its complications. Copyright © 2014 Elsevier B.V. All rights reserved.

Type I Glanzmann thrombasthenia caused by an apparently silent beta3 mutation that results in aberrant splicing and reduced beta3 mRNA.

PubMed

Xie, Jingli; Pabón, Dina; Jayo, Asier; Butta, Nora; González-Manchón, Consuelo

2005-05-01

We report a novel genetic defect in a patient with type I Glanzmann thrombasthenia. Flow cytometry analysis revealed undetectable levels of platelet glycoproteins alphaIIb and beta3, although residual amounts of both proteins were detectable in immunoblotting analysis. Sequence analysis of reversely transcribed platelet beta3 mRNA showed a 100-base pair deletion in the 3'-boundary of exon 11, that results in a frame shift and appearance of a premature STOP codon. Analysis of the corresponding genomic DNA fragment revealed the presence of a homozygous C1815T transition in exon 11. The mutation does not change the amino acid residue but it creates an ectopic consensus splice donor site that is used preferentially, causing splicing out of part of exon 11. The parents of the proband, heterozygous for this mutation, were asymptomatic and had reduced platelet content of alphaIIbbeta3. PCR-based relative quantification of beta3 mRNA failed to detect the mutant transcript in the parents and showed a marked reduction in the patient. The results suggest that the thrombasthenic phenotype is, mainly, the result of the reduced availability of beta3-mRNA, most probably due to activation of the nonsense-mediated mRNA decay mechanism. They also show the convenience of analyzing both genomic DNA and mRNA, in order to ascertain the functional consequences of single nucleotide substitutions.

Nanodiamonds activate blood platelets and induce thromboembolism.

PubMed

Kumari, Sharda; Singh, Manoj K; Singh, Sunil K; Grácio, José J A; Dash, Debabrata

2014-03-01

Nanodiamonds (NDs) have been evaluated for a wide range of biomedical applications. Thus, thorough investigation of the biocompatibility of NDs has become a research priority. Platelets are highly sensitive and are one of the most abundant cell types found in blood. They have a central role in hemostasis and arterial thrombosis. In this study, we aim to investigate the direct and acute effects of carboxylated NDs on platelet function. In this study, pro-coagulant parameters such as platelet aggregability, intracellular Ca(2+) flux, mitochondrial transmembrane potential (ΔΨm), generation of reactive oxygen species, surface exposure of phosphatidylserine, electron microscopy, cell viability assay and in vivo thromboembolism were analyzed in great detail. Carboxylated NDs evoked significant activation of human platelets. When administered intravenously in mice, NDs were found to induce widespread pulmonary thromboembolism, indicating the remarkable thrombogenic potential of this nanomaterial. Our findings raise concerns regarding the putative biomedical applications of NDs pertaining to diagnostics and therapeutics, and their toxicity and prothrombotic properties should be critically evaluated.

Comparison of point-of-care methods for preparation of platelet concentrate (platelet-rich plasma).

PubMed

Weibrich, Gernot; Kleis, Wilfried K G; Streckbein, Philipp; Moergel, Maximilian; Hitzler, Walter E; Hafner, Gerd

2012-01-01

This study analyzed the concentrations of platelets and growth factors in platelet-rich plasma (PRP), which are likely to depend on the method used for its production. The cellular composition and growth factor content of platelet concentrates (platelet-rich plasma) produced by six different procedures were quantitatively analyzed and compared. Platelet and leukocyte counts were determined on an automatic cell counter, and analysis of growth factors was performed using enzyme-linked immunosorbent assay. The principal differences between the analyzed PRP production methods (blood bank method of intermittent flow centrifuge system/platelet apheresis and by the five point-of-care methods) and the resulting platelet concentrates were evaluated with regard to resulting platelet, leukocyte, and growth factor levels. The platelet counts in both whole blood and PRP were generally higher in women than in men; no differences were observed with regard to age. Statistical analysis of platelet-derived growth factor AB (PDGF-AB) and transforming growth factor β1 (TGF-β1) showed no differences with regard to age or gender. Platelet counts and TGF-β1 concentration correlated closely, as did platelet counts and PDGF-AB levels. There were only rare correlations between leukocyte counts and PDGF-AB levels, but comparison of leukocyte counts and PDGF-AB levels demonstrated certain parallel tendencies. TGF-β1 levels derive in substantial part from platelets and emphasize the role of leukocytes, in addition to that of platelets, as a source of growth factors in PRP. All methods of producing PRP showed high variability in platelet counts and growth factor levels. The highest growth factor levels were found in the PRP prepared using the Platelet Concentrate Collection System manufactured by Biomet 3i.

In vivo quantitation of platelet deposition on human peripheral arterial bypass grafts using indium-111-labeled platelets. Effect of dipyridamole and aspirin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pumphrey, C.W.; Chesebro, J.H.; Dewanjee, M.K.

Indium-111-labeled autologous platelets, injected 48 hours after operation, were used to evaluate the thrombogenicity of prosthetic material and the effect of platelet inhibitor therapy in vivo. Dacron double-velour (Microvel) aortofemoral artery bifurcation grafts were placed in 16 patients and unilateral polytetrafluoroethylene femoropopliteal grafts were placed in 10 patients. Half the patients in each group received platelet inhibitors before operation (dipyridamole, 100 mg 4 times a day) and after operation (dipyridamole, 75 mg, and acetylsalicylic acid, 325 mg 3 times a day); the rest of the patients served as control subjects. Five-minute scintigrams of the graft region were taken with amore » gamma camera interfaced with a computer 48, 72, and 96 hours after injection of the labeled platelets. Platelet deposition was estimated from the radioactivities of the grafts and expressed as counts per 100 pixels per microcurie injected. Dipyridamole and aspirin therapy significantly reduced the number of platelets deposited on Dacron grafts and prevented platelet accumulation over 3 days. With the small amount of platelet deposition on polytetrafluoroethylene femoropopliteal artery grafts even in control patients, platelet inhibitor therapy had no demonstrable effect on platelet deposition on these grafts. It is concluded that (1) platelet deposition on vascular grafts in vivo can be quantitated by noninvasive methods, and (2) dipyridamole and aspirin therapy reduced platelet deposition on Dacron aortofemoral artery grafts.« less

Messenger RNA profiling of human platelets by microarray hybridization.

PubMed

Bugert, Peter; Dugrillon, Alex; Günaydin, Ayse; Eichler, Hermann; Klüter, Harald

2003-10-01

Platelets are generally believed to be inactive in terms of de novo protein synthesis. On the other hand, the presence of ribosomes and mRNA molecules is well established. Many studies have used reverse transcriptase (RT) -PCR for detection of gene transcripts in platelets. As RT-PCR is a very sensitive method, any leukocyte contamination of platelet preparations can lead to false results. We performed three filtration procedures to minimize leukocyte contamination of pooled buffy-coat platelet concentrates prior to RNA isolation. Furthermore, by applying a genomic PCR approach with 50 amplification cycles we demonstrated that nucleated cells were not detectable. Microarray hybridization was used to analyze 9,850 individual human genes in RNA from purified platelets. In total we identified 1,526 (15.5%) positive genes. The data were confirmed in six individual experiments each performed on a PC pooled from four individual blood donations. Genes specific for nucleated blood cells such as CD4, CD83 and others were negative and verified the purity of PC. Overrepresentation of positive genes was found in the functional categories of glycoproteins/integrins (22.6% vs. 15.5%, p=0.029) and receptors (20.7% vs. 15.5%, p<0.001). Gene transcripts encoding RANTES, GRO-alpha, MIP-1alpha, MIP-1beta, and others were found at high levels of signal intensity and confirmed literature data. This work provides a mRNA profile of human platelets and a complete list of results can be downloaded from the website of our institute www.ma.uni-heidelberg.de/inst/iti/plt_array.xls. The knowledge about gene transcripts may have an impact on the characterization of novel proteins and their functions in platelets.

Platelet response to aspirin 50 and 100 mg in patients with coronary heart disease over a five-year period.

PubMed

Berent, Robert; Auer, Johann; Franklin, Barry; Schmid, Peter; von Duvillard, Serge P

2011-09-01

Aspirin has been shown to decrease cardiovascular (CV) events by ∼25%. Despite aspirin therapy 10% to 20% of patients with arterial vascular disease develop atherothrombotic events. A meta-analysis of antiplatelet therapy showed a progressive decrease in clinical efficacy of aspirin after 2 years. Whether this is due to a decreased sensitivity to aspirin during long-term therapy remains unclear. A prospective randomized clinical trial with serial monitoring over 5 years was conducted in 100 patients with documented coronary heart disease. We investigated whether long-term treatment with aspirin 50 and 100 mg affects platelet response similarly. Occurrence of CV events was documented. Platelet sensitivity to aspirin, prostacyclin, and adenosine diphosphate-, collagen-, and epinephrine-induced platelet aggregation were evaluated over time. In addition, β-thromboglobulin and inflammatory markers were measured. Four patients were lost to follow-up and 10 patients died. Eleven patients developed nonfatal CV events. In the 2 groups platelet response to aspirin and the referenced variables remained unchanged over 5 years. In patients who developed CV events, the last monitoring interval revealed no difference in platelet response to aspirin. However, patients with nonfatal and fatal CV events showed increased inflammatory markers versus patients without CV events independent of aspirin 50 or 100 mg intake. In conclusion, our study revealed no difference in antiplatelet response to aspirin 50 versus 100 mg or CV events over 5 years in patients with coronary heart disease. Copyright © 2011 Elsevier Inc. All rights reserved.

Association of measured platelet reactivity with changes in P2Y12 receptor inhibitor therapy and outcomes after myocardial infarction: Insights into routine clinical practice from the TReatment with ADP receptor iNhibitorS: Longitudinal Assessment of Treatment Patterns and Events after Acute Coronary Syndrome (TRANSLATE-ACS) study.

PubMed

Bagai, Akshay; Peterson, Eric D; McCoy, Lisa A; Effron, Mark B; Zettler, Marjorie E; Stone, Gregg W; Henry, Timothy D; Cohen, David J; Schulte, Phillip J; Anstrom, Kevin J; Wang, Tracy Y

2017-05-01

Little is known about the use of platelet function testing to guide choice of P2Y 12 receptor inhibitor therapy in routine clinical practice. We studied 671 myocardial infarction (MI) patients treated with percutaneous coronary intervention in the TRANSLATE-ACS Registry who had VerifyNow platelet function testing performed while on clopidogrel treatment during their index hospitalization (April 2010-October 2012). High platelet reactivity (>208 platelet reactivity units [PRU]) was present in 261 (38.9%) patients. Clopidogrel was switched in-hospital to prasugrel in 80 (30.7%) patients with high platelet reactivity and 18 (4.4%) patients with therapeutic platelet reactivity (≤208 PRU). Among high platelet reactivity patients, switch to prasugrel was associated with lower major adverse cardiovascular events (death, MI, stroke, or unplanned revascularization) at 1year (10.0% vs 22.7%, P=.02; adjusted odds ratio [OR] 0.39, 95% CI 0.18-0.85, P=.02) and no significant difference in Bleeding Academic Research Consortium type 2 or higher bleeding (23.8% vs 22.1%, P=.77; adjusted OR 0.91, 95% CI 0.48-1.7, P=.77) compared with patients continued on clopidogrel. No significant differences in major adverse cardiovascular event (22.2% vs 12.8%, P=.25; adjusted OR 1.8, 95% CI 0.47-7.3, P=.38) or bleeding (22.2% vs 19.4%, P=.77; adjusted OR 1.3, 95% CI 0.27-6.8, P=.72) were observed among therapeutic platelet reactivity patients between switching and continuation on clopidogrel. Only one-third of percutaneous coronary intervention-treated MI patients with high on-clopidogrel platelet reactivity were switched to a more potent P2Y 12 receptor inhibitor. Intensification of antiplatelet therapy was associated with lower risk of ischemic events at 1year among HPR patients. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization of platelet adhesion under flow using microscopic image sequence analysis.

PubMed

Machin, M; Santomaso, A; Cozzi, M R; Battiston, M; Mazzuccato, M; De Marco, L; Canu, P

2005-07-01

A method for quantitative analysis of platelet deposition under flow is discussed here. The model system is based upon perfusion of blood platelets over an adhesive substrate immobilized on a glass coverslip acting as the lower surface of a rectangular flow chamber. The perfusion apparatus is mounted onto an inverted microscope equipped with epifluorescent illumination and intensified CCD video camera. Characterization is based on information obtained from a specific image analysis method applied to continuous sequences of microscopical images. Platelet recognition across the sequence of images is based on a time-dependent, bidimensional, gaussian-like pdf. Once a platelet is located,the variation of its position and shape as a function of time (i.e., the platelet history) can be determined. Analyzing the history we can establish if the platelet is moving on the surface, the frequency of this movement and the distance traveled before its resumes the velocity of a non-interacting cell. Therefore, we can determine how long the adhesion would last which is correlated to the resistance of the platelet-substrate bond. This algorithm enables the dynamic quantification of trajectories, as well as residence times, arrest and release frequencies for a high numbers of platelets at the same time. Statistically significant conclusions on platelet-surface interactions can then be obtained. An image analysis tool of this kind can dramatically help the investigation and characterization of the thrombogenic properties of artificial surfaces such as those used in artificial organs and biomedical devices.

Platelet ERK5 is a Redox Switch and Triggers Maladaptive Platelet Responses and Myocardial Infarct Expansion

PubMed Central

Cameron, Scott J.; Ture, Sara K.; Mickelsen, Deanne; Chakrabarti, Enakshi; Modjeski, Kristina L.; McNitt, Scott; Seaberry, Micheal; Field, David J.; Le, Nhat-Tu; Abe, Jun-ichi; Morrell, Craig N.

2015-01-01

Background Platelets have a pathophysiologic role in the ischemic microvascular environment of acute coronary syndromes (ACS). Compared to platelet activation in normal healthy conditions, less attention is given to mechanisms of platelet activation in diseased states. Platelet function and mechanisms of activation in ischemic and reactive oxygen species (ROS) rich environments may not be the same as in normal healthy conditions. Extracellular Regulated Protein Kinase 5 (ERK5) is a Mitogen Activated Protein Kinase (MAPK) family member activated in hypoxic, ROS rich environments, and in response to receptor signaling mechanisms. Prior studies suggest a protective effect of ERK5 in endothelial and myocardial cells following ischemia. We present evidence that platelets express ERK5 and platelet ERK5 has an adverse effect on platelet activation via selective receptor-dependent and receptor-independent ROS mediated mechanisms in ischemic myocardium. Methods and Results Using isolated human platelets and a mouse model of myocardial infarction (MI), we found that platelet ERK5 is activated post-MI and platelet specific ERK5−/− mice have less platelet activation, reduced MI size, and improved post-MI heart function. Furthermore, the expression of downstream ERK5 regulated proteins is reduced in ERK5−/− platelets post-MI. Conclusions ERK5 functions as a platelet activator in ischemic conditions and platelet ERK5 maintains the expression of some platelet proteins following MI, leading to infarct expansion. This demonstrates that platelet function in normal healthy conditions is different from platelet function in chronic ischemic and inflammatory conditions. Platelet ERK5 may be a target for acute therapeutic intervention in the thrombotic and inflammatory post-MI environment. PMID:25934838

Clinical determinants of clopidogrel responsiveness in a heterogeneous cohort of Puerto Rican Hispanics.

PubMed

Hernandez-Suarez, Dagmar F; Scott, Stuart A; Tomey, Matthew I; Melin, Kyle; Lopez-Candales, Angel; Buckley, Charlotte E; Duconge, Jorge

2017-09-01

Clopidogrel is by far the most prescribed platelet adenosine diphosphate (ADP) antagonist in Puerto Rico despite the advent of newer agents (prasugrel and ticagrelor). Given the paucity of data on clopidogrel responsiveness in Hispanics, we sought to determine the association between clinical characteristics and platelet reactivity in Puerto Rican patients on clopidogrel therapy. A total of 100 Puerto Rican patients on clopidogrel therapy were enrolled and allocated into two groups: Group I, without high on-treatment platelet reactivity (HTPR); and Group II, with HTPR. Platelet function was measured ex vivo using the VerifyNow® P2Y12 assay. The cohort was comprised of Hispanic patients with coronary artery disease (57%), peripheral artery disease (32%), carotid artery stenosis (7%), cerebral artery aneurysm (2%), and stroke (2%). Mean platelet reactivity was 200 ± 61 P2Y12 reaction units (PRUs) (range: 8-324), and 35% of patients had HTPR (PRUs ⩾ 230). Multivariable logistic regression analysis determined that diabetes mellitus (DM) [odds ratio (OR) = 3.27; 95% confidence interval (CI): 1.20-8.96], use of proton-pump inhibitors (PPIs) (OR = 3.60; 95% CI: 1.09-11.82), and calcium channel blockers (CCBs) (OR = 3.10; 95% CI: 1.09-8.83) were independent predictors of HTPR ( p < 0.05) after adjusting for other clinical variables. In a sample of 100 Puerto Rican Hispanic patients on clopidogrel, 35% had HTPR. Furthermore, DM, PPIs and CCBs predicted HTPR. Clinical outcome data are needed to identify appropriate PRU thresholds for risk prediction in the Puerto Rican population.

Influence of Brownian Motion on Blood Platelet Flow Behavior and Adhesive Dynamics near a Planar Wall

PubMed Central

Mody, Nipa A.; King, Michael R.

2008-01-01

We used the Platelet Adhesive Dynamics computational method to study the influence of Brownian motion of a platelet on its flow characteristics near a surface in the creeping flow regime. Two important characterizations were done in this regard: (1) quantification of the platelet’s ability to contact the surface by virtue of the Brownian forces and torques acting on it, and (2) determination of the relative importance of Brownian motion in promoting surface encounters in the presence of shear flow. We determined the Peclet number for a platelet undergoing Brownian motion in shear flow, which could be expressed as a simple linear function of height of the platelet centroid, H from the surface Pe (platelet) = γ. · (1.56H + 0.66) for H > 0.3 μm. Our results demonstrate that at timescales relevant to shear flow in blood, Brownian motion plays an insignificant role in influencing platelet motion or creating further opportunities for platelet-surface contact. The platelet Peclet number at shear rates > 100 s-1 is large enough (> 200) to neglect platelet Brownian motion in computational modeling of flow in arteries and arterioles for most practical purposes even at very close distances from the surface. We also conducted adhesive dynamics simulations to determine the effects of platelet Brownian motion on GPIbα-vWF-A1 single-bond dissociation dynamics. Brownian motion was found to have little effect on bond lifetime and caused minimal bond stressing as bond rupture forces were calculated to be less than 0.005 pN. We conclude from our results that for the case of platelet-shaped cells, Brownian motion is not expected to play an important role in influencing flow characteristics, platelet-surface contact frequency and dissociative binding phenomena under flow at physiological shear rates (> 50 s-1). PMID:17417890

Autonomous role of Wiskott-Aldrich syndrome platelet deficiency in inducing autoimmunity and inflammation.

PubMed

Sereni, Lucia; Castiello, Maria Carmina; Marangoni, Francesco; Anselmo, Achille; di Silvestre, Dario; Motta, Sara; Draghici, Elena; Mantero, Stefano; Thrasher, Adrian J; Giliani, Silvia; Aiuti, Alessandro; Mauri, Pierluigi; Notarangelo, Luigi D; Bosticardo, Marita; Villa, Anna

2018-02-06

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency characterized by eczema, infections, and susceptibility to autoimmunity and malignancies. Thrombocytopenia is a constant finding, but its pathogenesis remains elusive. To dissect the basis of the WAS platelet defect, we used a novel conditional mouse model (CoWas) lacking Wiskott-Aldrich syndrome protein (WASp) only in the megakaryocytic lineage in the presence of a normal immunologic environment, and in parallel we analyzed samples obtained from patients with WAS. Phenotypic and functional characterization of megakaryocytes and platelets in mutant CoWas mice and patients with WAS with and without autoantibodies was performed. Platelet antigen expression was examined through a protein expression profile and cluster proteomic interaction network. Platelet immunogenicity was tested by using ELISAs and B-cell and platelet cocultures. CoWas mice showed increased megakaryocyte numbers and normal thrombopoiesis in vitro, but WASp-deficient platelets had short lifespan and high expression of activation markers. Proteomic analysis identified signatures compatible with defects in cytoskeletal reorganization and metabolism yet surprisingly increased antigen-processing capabilities. In addition, WASp-deficient platelets expressed high levels of surface and soluble CD40 ligand and were capable of inducing B-cell activation in vitro. WASp-deficient platelets were highly immunostimulatory in mice and triggered the generation of antibodies specific for WASp-deficient platelets, even in the context of a normal immune system. Patients with WAS also showed platelet hyperactivation and increased plasma soluble CD40 ligand levels correlating with the presence of autoantibodies. Overall, these findings suggest that intrinsic defects in WASp-deficient platelets decrease their lifespan and dysregulate immune responses, corroborating the role of platelets as modulators of inflammation and immunity. Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Functional factor XIII-A is exposed on the stimulated platelet surface

PubMed Central

Mitchell, Joanne L.; Lionikiene, Ausra S.; Fraser, Steven R.; Whyte, Claire S.; Booth, Nuala A.

2014-01-01

Factor XIII (FXIII) stabilizes thrombi against fibrinolysis by cross-linking α2-antiplasmin (α2AP) to fibrin. Cellular FXIII (FXIII-A) is abundant in platelets, but the extracellular functions of this pool are unclear because it is not released by classical secretion mechanisms. We examined the function of platelet FXIII-A using Chandler model thrombi formed from FXIII-depleted plasma. Platelets stabilized FXIII-depleted thrombi in a transglutaminase-dependent manner. FXIII-A activity on activated platelets was unstable and was rapidly lost over 1 hour. Inhibiting platelet activation abrogated the ability of platelets to stabilize thrombi. Incorporating a neutralizing antibody to α2AP into FXIII-depleted thrombi revealed that the stabilizing effect of platelet FXIII-A on lysis was α2AP dependent. Platelet FXIII-A activity and antigen were associated with the cytoplasm and membrane fraction of unstimulated platelets, and these fractions were functional in stabilizing FXIII-depleted thrombi against lysis. Fluorescence confocal microscopy and flow cytometry revealed exposure of FXIII-A on activated membranes, with maximal signal detected with thrombin and collagen stimulation. FXIII-A was evident in protruding caps on the surface of phosphatidylserine-positive platelets. Our data show a functional role for platelet FXIII-A through exposure on the activated platelet membrane where it exerts antifibrinolytic function by cross-linking α2AP to fibrin. PMID:25331118

Inhibition of Glycoprotein VI Clustering by Collagen as a Mechanism of Inhibiting Collagen-Induced Platelet Responses: The Example of Losartan

PubMed Central

Jiang, Peng; Loyau, Stéphane; Tchitchinadze, Maria; Ropers, Jacques; Jondeau, Guillaume; Jandrot-Perrus, Martine

2015-01-01

Exposure of platelets to collagen triggers the formation of a platelet clot. Pharmacological agents capable of inhibiting platelet activation by collagen are thus of potential therapeutic interest. Thrombus formation is initiated by the interaction of the GPIb-V-IX complex with collagen-bound vWF, while GPVI interaction with collagen triggers platelet activation that is reinforced by ADP and thromboxane A2. Losartan is an angiotensin II (Ang II) type I receptor (AT1R) antagonist proposed to have an antiplatelet activity via the inhibition of both the thromboxane A2 (TXA2) receptor (TP) and the glycoprotein VI (GPVI). Here, we characterized in vitro the effects of losartan at different doses on platelet responses: losartan inhibited platelet aggregation and secretion induced by 1 μg.mL-1 and 10 μg.mL-1 of collagen with an IC50 of ~ 6 μM. Losartan inhibited platelet responses induced by the GPVI specific collagen related peptide but not by the α2β1 specific peptide. However, losartan did not inhibit the binding of recombinant GPVI to collagen, which is not in favor of a simple competition. Indeed, the clustering of GPVI observed in flow cytometry and using the Duolink methodology, was inhibited by losartan. The impact of a therapeutic dose of losartan (100 mg/day) on platelet responses was analyzed ex vivo in a double blind study. No statistically significant differences were observed between losartan-treated (n=25) and non-treated (n=30) patients in terms of collagen and U46619-induced platelet activation. These data indicate that in treated patients, losartan does not achieve a measurable antiplatelet effect but provide the proof of concept that inhibiting collagen-induced GPVI clustering is of pharmacological interest to obtain an antithrombotic efficacy. Trial Registration ClinicalTrials.gov NCT00763893 PMID:26052700

Direct factor IXa inhibition with the RNA-aptamer pegnivacogin reduces platelet reactivity in vitro and residual platelet aggregation in patients with acute coronary syndromes.

PubMed

Staudacher, Dawid L; Putz, Vera; Heger, Lukas; Reinöhl, Jochen; Hortmann, Marcus; Zelenkofske, Steven L; Becker, Richard C; Rusconi, Christopher P; Bode, Christoph; Ahrens, Ingo

2017-04-01

Residual platelet reactivity is a predictor of poor prognosis in patients with acute coronary syndromes (ACSs) undergoing percutaneous coronary intervention. Thrombin is a major platelet activator and upon initiation of the coagulation cascade, it is subsequently produced downstream of factor IXa, which itself is known to be increased in ACS. Pegnivacogin is a novel RNA-aptamer based factor IXa inhibitor featuring a reversal agent, anivamersen. We hypothesized that pegnivacogin could reduce platelet reactivity. Whole blood samples from healthy volunteers were incubated in vitro in the presence and absence of pegnivacogin and platelet reactivity was analysed. In addition, platelet aggregometry was performed in blood samples from ACS patients in the RADAR trial featuring the intravenous administration of pegnivacogin as well as reversal by anivamersen. In vitro, pegnivacogin significantly reduced adenosine diphosphate-induced CD62P-expression (100% vs. 89.79±4.04%, p=0.027, n=9) and PAC-1 binding (100% vs. 83.02±4.08%, p=0.010, n=11). Platelet aggregation was reduced (97.71±5.30% vs. 66.53±9.92%, p=0.013, n=10) as evaluated by light transmission aggregometry. In the presence of the RNA-aptamer reversal agent anivamersen, neither CD62P-expression nor platelet aggregation was attenuated. In patients with ACS treated with aspirin and clopidogrel, residual platelet aggregation was significantly reduced 20 min after intravenous bolus of 1 mg/kg pegnivacogin (100% versus 43.21±8.23%, p=0.020). Inhibition of factor IXa by pegnivacogin decreases platelet activation and aggregation in vitro. This effect was negated by anivamersen. In ACS patients, platelet aggregation was significantly reduced after intravenous pegnivacogin. An aptamer-based anticoagulant inhibiting factor IXa therefore might be a promising antithrombotic strategy in ACS patients.

Combination antiplatelet therapy with aspirin and clopidogrel: the role of antecedent and concomitant doses of aspirin. An analysis of 711 patients.

PubMed

Serebruany, Victor L; Malinin, Alex I; Atar, Dan

2007-01-01

Numerous randomized studies have shown that the combination of clopidogrel with aspirin yields better clinical outcomes than monotherapy in patients with acute vascular events. However, the impact of the aspirin dose on the antiplatelet potency of clopidogrel is unclear. We sought to compare the antiplatelet profile of aspirin 81 mg (n = 252) versus aspirin 325 mg (n = 459) before and during conventional clopidogrel loading (300 mg), and/or clopidogrel maintenance (75 mg/daily) therapy. Secondary post hoc analysis of an existing dataset consisting of 711 patients after coronary stenting (n = 601) and ischemic stroke (n = 110) treated previously with aspirin for at least 1 month, and then with aspirin + clopidogrel for at least 7 days was performed. Platelet assessments include conventional and whole blood aggregometry, rapid cartridge-based analyzers, and expression of platelet/endothelial cell adhesion molecule-1, P-selectin, and GPIIb/IIIa activity by flow cytometry measured before and after addition of clopidogrel. There was a small but consistent yet non-significant trend towards more potent platelet inhibition with aspirin 325 mg compared to aspirin 81 mg for every platelet activation parameter before addition of clopidogrel. However, after loading and/or 1 week of chronic treatment with clopidogrel + aspirin, measured platelet parameters became very similar between the groups, and identical for collagen-induced aggregation and PFA-100 analyzer readings. Before addition of clopidogrel, aspirin 325 mg has a tendency to provide stronger platelet inhibition than aspirin 81 mg. However, when clopidogrel and aspirin are used in combination, the higher aspirin dose does not translate into superior antiplatelet action. Given that the existing body of evidence supports the comparable efficacy and, particularly, superior safety of lower versus higher doses of aspirin, aspirin 81 mg should be the dose used in combination with clopidogrel. 2007 S. Karger AG, Basel

Platelet-derived S100 family member myeloid-related protein-14 regulates thrombosis

PubMed Central

Wang, Yunmei; Fang, Chao; Gao, Huiyun; Bilodeau, Matthew L.; Zhang, Zijie; Croce, Kevin; Liu, Shijian; Morooka, Toshifumi; Sakuma, Masashi; Nakajima, Kohsuke; Yoneda, Shuichi; Shi, Can; Zidar, David; Andre, Patrick; Stephens, Gillian; Silverstein, Roy L.; Hogg, Nancy; Schmaier, Alvin H.; Simon, Daniel I.

2014-01-01

Expression of the gene encoding the S100 calcium–modulated protein family member MRP-14 (also known as S100A9) is elevated in platelets from patients presenting with acute myocardial infarction (MI) compared with those from patients with stable coronary artery disease; however, a causal role for MRP-14 in acute coronary syndromes has not been established. Here, using multiple models of vascular injury, we found that time to arterial thrombotic occlusion was markedly prolonged in Mrp14–/– mice. We observed that MRP-14 and MRP-8/MRP-14 heterodimers (S100A8/A9) are expressed in and secreted by platelets from WT mice and that thrombus formation was reduced in whole blood from Mrp14–/– mice. Infusion of WT platelets, purified MRP-14, or purified MRP-8/MRP-14 heterodimers into Mrp14–/– mice decreased the time to carotid artery occlusion after injury, indicating that platelet-derived MRP-14 directly regulates thrombosis. In contrast, infusion of purified MRP-14 into mice deficient for both MRP-14 and CD36 failed to reduce carotid occlusion times, indicating that CD36 is required for MRP-14–dependent thrombosis. Our data identify a molecular pathway of thrombosis that involves platelet MRP-14 and CD36 and suggest that targeting MRP-14 has potential for treating atherothrombotic disorders, including MI and stroke. PMID:24691441

Evaluation of a BED-SIDE platelet function assay: performance and clinical utility.

PubMed

Lau, Wei C; Walker, C Ty; Obilby, David; Wash, Mark M; Carville, David G M; Guyer, Kirk E; Bates, Eric R

2002-01-01

Platelets have a pivotal role in the initial defense against insult to the vasculature and are also recognized of critical importance in the acute care settings of percutaneous coronary intervention and cardiopulmonary bypass. In these environments both platelet count and function may be markedly compromised. Unfortunately, current assays to evaluate the parameters of platelet count and function are of limited utility for bed-side testing. Moreover, it is suggested that there may be significant inter patient variation in response to antiplatelet therapy that may be exacerbated by other agents (e.g. heparin) that are routinely administered during cardiac intervention. Here we describe a practical, rapid and user-friendly whole blood platelet function assay that has been developed for use in bed-side settings. Platelet agonists were formulated with an anticoagulant and lyophilized in blood collection tubes standardised to receive a l mL fresh whole blood sample. In the presence of an agonist, platelets are activated and interact (aggregate). Using traditional cell counting principles, non-aggregated platelets are counted whereas aggregated platelets are not. The percentage (%) of functional platelets in reference to a baseline tube may then be determined. Results are available within four minutes. Platelet aggregation in whole blood demonstrated good correlation with turbidometric aggregometry for both ADP (r=0.91) and collagen (r=0.88). Moreover, in clinical settings where antiplatelet agents were administered, this rapid, bed-side, platelet function assay demonstrated utility in monitoring patient response to these therapies. This novel bed-side assay of platelet function is extremely suitable for the clinical environment with a rapid turn-around time. In addition, it provides a full haematology profile, including platelet count, and should permit enhancement of transfusion and interventional decisions.

Glycoprotein Ibα receptor instability is associated with loss of quality in platelets produced in culture.

PubMed

Robert, Amélie; Boyer, Lucie; Pineault, Nicolas

2011-03-01

The development of culture processes for hematopoietic progenitors could lead to the development of a complementary source of platelets for therapeutic purposes. However, functional characterization of culture-derived platelets remains limited, which raises some uncertainties about the quality of platelets produced in vitro. The aim of this study was to define the proportion of functional platelets produced in cord blood CD34+ cell cultures. Toward this, the morphological and functional properties of culture-derived platelet-like particles (PLPs) were critically compared to that of blood platelets. Flow cytometry combined with transmission electron microscopy analyses revealed that PLPs formed a more heterogeneous population of platelets at a different stage of maturation than blood platelets. The majority of PLPs harbored the fibrinogen receptor αIIbβ3, but a significant proportion failed to maintain glycoprotein (GP)Ibα surface expression, a component of the vWF receptor essential for platelet functions. Importantly, GPIbα extracellular expression correlated closely with platelet function, as the GPIIb+ GPIbα+ PLP subfraction responded normally to agonist stimulation as evidenced by α-granule release, adhesion, spreading, and aggregation. In contrast, the GPIIb+ GPIbα⁻ subfraction was unresponsive in most functional assays and appeared to be metabolically inactive. The present study confirms that functional platelets can be generated in cord blood CD34+ cell cultures, though these are highly susceptible to ectodomain shedding of receptors associated with loss of function. Optimization of culture conditions to prevent these deleterious effects and to homogenize PLPs is necessary to improve the quality and yields of culture-derived platelets before they can be recognized as a suitable complementary source for therapeutic purposes.

Are the changes in the peripheral brain-derived neurotrophic factor levels due to platelet activation?

PubMed Central

Serra-Millàs, Montserrat

2016-01-01

Brain-derived neurotrophic factor (BDNF) plays an important role in central nervous system development, neurogenesis and neuronal plasticity. BDNF is also expressed in several non-neuronal tissues, and it could play an important role in other processes, such as cancer, angiogenesis, etc. Platelets are the major source of peripheral BDNF. However, platelets also contain high amounts of serotonin; they express specific surface receptors during activation, and a multitude of pro-inflammatory and immunomodulatory bioactive compounds are secreted from the granules. Until recently, there was insufficient knowledge regarding the relationship between BDNF and platelets. Recent studies showed that BDNF is present in two distinct pools in platelets, in α-granules and in the cytoplasm, and only the BDNF in the granules is secreted following stimulation, representing 30% of the total BDNF in platelets. BDNF has an important role in the pathophysiology of depression. Low levels of serum BDNF have been described in patients with major depressive disorder, and BDNF levels increased with chronic antidepressant treatment. Interestingly, there is an association between depression and platelet function. This review analyzed studies that evaluated the relationship between BDNF and platelet activation and the effect of treatments on both parameters. Only a few studies consider this possible confounding factor, and it could be very important in diseases such as depression, which show changes in both parameters. PMID:27014600

Global analysis of the rat and human platelet proteome – the molecular blueprint for illustrating multi-functional platelets and cross-species function evolution

PubMed Central

Yu, Yanbao; Leng, Taohua; Yun, Dong; Liu, Na; Yao, Jun; Dai, Ying; Yang, Pengyuan; Chen, Xian

2013-01-01

Emerging evidences indicate that blood platelets function in multiple biological processes including immune response, bone metastasis and liver regeneration in addition to their known roles in hemostasis and thrombosis. Global elucidation of platelet proteome will provide the molecular base of these platelet functions. Here, we set up a high throughput platform for maximum exploration of the rat/human platelet proteome using integrated proteomics technologies, and then applied to identify the largest number of the proteins expressed in both rat and human platelets. After stringent statistical filtration, a total of 837 unique proteins matched with at least two unique peptides were precisely identified, making it the first comprehensive protein database so far for rat platelets. Meanwhile, quantitative analyses of the thrombin-stimulated platelets offered great insights into the biological functions of platelet proteins and therefore confirmed our global profiling data. A comparative proteomic analysis between rat and human platelets was also conducted, which revealed not only a significant similarity, but also an across-species evolutionary link that the orthologous proteins representing ‘core proteome’, and the ‘evolutionary proteome’ is actually a relatively static proteome. PMID:20443191

Medication History versus Point-of-Care Platelet Activity Testing in Patients with Intracerebral Hemorrhage.

PubMed

Maas, Matthew B; Naidech, Andrew M; Kim, Minjee; Batra, Ayush; Manno, Edward M; Sorond, Farzaneh A; Prabhakaran, Shyam; Liotta, Eric M

2018-05-01

We evaluated whether reduced platelet activity detected by point-of-care (POC) testing is a better predictor of hematoma expansion and poor functional outcomes in patients with intracerebral hemorrhage (ICH) than a history of antiplatelet medication exposure. Patients presenting with spontaneous ICH were enrolled in a prospective observational cohort study that collected demographic, clinical, laboratory, and radiographic data. We measured platelet activity using the PFA-100 (Siemens AG, Germany) and VerifyNow-ASA (Accumetrics, CA) systems on admission. We performed univariate and adjusted multivariate analyses to assess the strength of association between those measures and (1) hematoma growth at 24 hours and (2) functional outcomes measured by the modified Rankin Scale (mRS) at 3 months. We identified 278 patients for analysis (mean age 65 ± 15, median ICH score 1 [interquartile range 0-2]), among whom 164 underwent initial neuroimaging within 6 hours of symptom onset. Univariate association with hematoma growth was stronger for antiplatelet medication history than POC measures, which was confirmed in multivariable models (β 3.64 [95% confidence interval [CI] 1.02-6.26], P = .007), with a larger effect size measured in the under 6-hour subgroup (β 7.20 [95% CI 3.35-11.1], P < .001). Moreover, antiplatelet medication history, but not POC measures of platelet activity, was independently associated with poor outcome at 3 months (mRS 4-6) in the under 6-hour subgroup (adjusted OR 3.6 [95% CI 1.2-11], P = .023). A history of antiplatelet medication use better identifies patients at risk for hematoma growth and poor functional outcomes than POC measures of platelet activity after spontaneous ICH. Copyright © 2018 National Stroke Association. Published by Elsevier Inc. All rights reserved.

A Nitric Oxide-Releasing Heparin Conjugate for Delivery of a Combined Antiplatelet/Anticoagulant Agent

PubMed Central

2015-01-01

Heparin is a widely used anticoagulant due to its ability to inhibit key components in the coagulation cascade such as Factor Xa and thrombin (Factor IIa). Its potential to preferentially bind to antithrombin (ATIII) results in a conformational change and activation that leads to the prevention of fibrin formation from fibrinogen and ultimately obstructs a hemostatic plug from forming. Nitric oxide (NO) exhibits potent antiplatelet activity attributed to its capacity to increase the amount of cyclic guanosine monophosphate (cGMP) within platelets, which decreases the Ca2+ concentration required for platelet activation. Currently there is no single agent that combines the functions of both antiplatelet and anticoagulant (anti-Xa and anti-IIa) activities to effectively block both the extrinsic and the intrinsic coagulation pathways. The research reported herein demonstrates the ability to combine the physiological capabilities of both heparin and NO into one functional compound via use of a spermine derivative of heparin, thus enabling formation of a novel diazeniumdiolate (NONOate). The heparin–spermine NONOate has a half-life of 85 min at 25 °C (pH 7.4). The heparin backbone of the conjugate maintains its anticoagulant activity as demonstrated via an anti-Xa assay, providing an anticoagulant conversion of 3.6 μg/mL of the heparin–spermine–NONO conjugate being equivalent to 2.5 μg/mL (0.50 IU/mL) of underivatized heparin in terms of anti-Xa activity. Using standard platelet aggregometry, it is shown that the functionality of the NO release portion of the heparin conjugate prevents (nearly 100%) platelet aggregation in the presence of adenosine diphosphate (ADP, platelet agonist). PMID:24423090

Platelets from patients with the Quebec platelet disorder contain and secrete abnormal amounts of urokinase-type plasminogen activator.

PubMed

Kahr, W H; Zheng, S; Sheth, P M; Pai, M; Cowie, A; Bouchard, M; Podor, T J; Rivard, G E; Hayward, C P

2001-07-15

The Quebec platelet disorder (QPD) is an autosomal dominant platelet disorder associated with delayed bleeding and alpha-granule protein degradation. The degradation of alpha-granule, but not plasma, fibrinogen in patients with the QPD led to the investigation of their platelets for a protease defect. Unlike normal platelets, QPD platelets contained large amounts of fibrinolytic serine proteases that had properties of plasminogen activators. Western blot analysis, zymography, and immunodepletion experiments indicated this was because QPD platelets contained large amounts of urokinase-type plasminogen activator (u-PA) within a secretory compartment. u-PA antigen was not increased in all QPD plasmas, whereas it was increased more than 100-fold in QPD platelets (P <.00009), which contained increased u-PA messenger RNA. Although QPD platelets contained 2-fold more plasminogen activator inhibitor 1 (PAI-1) (P <.0008) and 100-fold greater u-PA-PAI-1 complexes (P <.0002) than normal platelets, they contained excess u-PA activity, predominantly in the form of two chain (tcu-PA), which required additional PAI-1 for full inhibition. There was associated proteolysis of plasminogen in QPD platelets, to forms that comigrated with plasmin. When similar amounts of tcu-PA were incubated with normal platelet secretory proteins, many alpha-granule proteins were proteolyzed to forms that resembled degraded QPD platelet proteins. These data implicate u-PA in the pathogenesis of alpha-granule protein degradation in the QPD. Although patients with the QPD have normal to increased u-PA levels in their plasma, without evidence of systemic fibrinogenolysis, their increased platelet u-PA could contribute to bleeding by accelerating fibrinolysis within the hemostatic plug. QPD is the only inherited bleeding disorder in humans known to be associated with increased u-PA.

Ultrasound-guided platelet-rich plasma injection for distal biceps tendinopathy

PubMed Central

Bell, Simon N; Connell, David; Coghlan, Jennifer A

2015-01-01

Background Distal biceps tendinopathy is an uncommon cause of elbow pain. The optimum treatment for cases refractory to conservative treatment is unclear. Platelet-rich plasma has been used successfully for other tendinopathies around the elbow. Methods Six patients with clinical and radiological evidence of distal biceps tendinopathy underwent ultrasound-guided platelet-rich plasma (PRP) injection. Clinical examination findings, visual analogue score (VAS) for pain and Mayo Elbow Performance scores were recorded. Results The Mayo Elbow Performance Score improved from 68.3 (range 65 to 85) (fair function) to 95 (range 85 to 100) (excellent function). The VAS at rest improved from a mean of 2.25 (range 2 to 5) pre-injection to 0. The VAS with movement improved from a mean of 7.25 (range 5 to 8) pre-injection to 1.3 (range 0 to 2). No complications were noted. Discussion Ultrasound-guided PRP injection appears to be a safe and effective treatment for recalcitrant cases of distal biceps tendinopathy. Further investigation with a randomized controlled trial is needed to fully assess its efficacy. PMID:27582965

Aspirin Inhibits Platelet-Derived Sphingosine-1-Phosphate Induced Endothelial Cell Migration.

PubMed

Polzin, Amin; Knoop, Betül; Böhm, Andreas; Dannenberg, Lisa; Zurek, Mark; Zeus, Tobias; Kelm, Malte; Levkau, Bodo; Rauch, Bernhard H

2018-01-01

Aspirin plays a crucial role in the prevention of cardiovascular diseases. We previously described that aspirin has effects beyond inhibition of platelet aggregation, as it inhibited thrombin-mediated release of sphingosine-1-phosphate (S1P) from human platelets. S1P is a bioactive lipid with important functions on inflammation and apoptosis. In endothelial cells (EC), S1P is a key regulator of cell migration. In this study, we aimed to analyze the effects of aspirin on platelet-induced EC migration. Human umbilical EC migration was measured by Boyden chamber assay. EC migration was induced by platelet supernatants of thrombin receptor-activating peptide-1 (AP1) stimulated platelets. To investigate the S1P receptor subtype that promotes EC migration, specific inhibitors of S1P receptor subtypes were applied. S1P induced EC migration in a concentration-dependent manner. EC migration induced by AP1-stimulated platelet supernatants was reduced by aspirin. S1P1 receptor inhibition almost completely abolished EC migration induced by activated platelets. The inhibition of S1P2 or S1P3 receptor had no effect. Aspirin inhibits EC migration induced by activated platelets that is in part due to S1P and mediated by the endothelial S1P1 receptor. The clinical significance of this novel mechanism of aspirin action has to be investigated in future studies. © 2017 S. Karger AG, Basel.

Survival protein anoctamin-6 controls multiple platelet responses including phospholipid scrambling, swelling, and protein cleavage.

PubMed

Mattheij, Nadine J A; Braun, Attila; van Kruchten, Roger; Castoldi, Elisabetta; Pircher, Joachim; Baaten, Constance C F M J; Wülling, Manuela; Kuijpers, Marijke J E; Köhler, Ralf; Poole, Alastair W; Schreiber, Rainer; Vortkamp, Andrea; Collins, Peter W; Nieswandt, Bernhard; Kunzelmann, Karl; Cosemans, Judith M E M; Heemskerk, Johan W M

2016-02-01

Scott syndrome is a rare bleeding disorder, characterized by altered Ca(2+)-dependent platelet signaling with defective phosphatidylserine (PS) exposure and microparticle formation, and is linked to mutations in the ANO6 gene, encoding anoctamin (Ano)6. We investigated how the complex platelet phenotype of this syndrome is linked to defective expression of Anos or other ion channels. Mice were generated with heterozygous of homozygous deficiency in Ano6, Ano1, or Ca(2+)-dependent KCa3.1 Gardos channel. Platelets from these mice were extensively analyzed on molecular functions and compared with platelets from a patient with Scott syndrome. Deficiency in Ano1 or Gardos channel did not reduce platelet responses compared with control mice (P > 0.1). In 2 mouse strains, deficiency in Ano6 resulted in reduced viability with increased bleeding time to 28.6 min (control 6.4 min, P < 0.05). Platelets from the surviving Ano6-deficient mice resembled platelets from patients with Scott syndrome in: 1) normal collagen-induced aggregate formation (P > 0.05) with reduced PS exposure (-65 to 90%); 2) lowered Ca(2+)-dependent swelling (-80%) and membrane blebbing (-90%); 3) reduced calpain-dependent protein cleavage (-60%); and 4) moderately affected apoptosis-dependent PS exposure. In conclusion, mouse deficiency of Ano6 but not of other channels affects viability and phenocopies the complex changes in platelets from hemostatically impaired patients with Scott syndrome. © FASEB.

Effects of hormones on platelet aggregation.

PubMed

Farré, Antonio López; Modrego, Javier; Zamorano-León, José J

2014-04-01

Platelets and their activation/inhibition mechanisms play a central role in haemostasis. It is well known agonists and antagonists of platelet activation; however, during the last years novel evidences of hormone effects on platelet activation have been reported. Platelet functionality may be modulated by the interaction between different hormones and their platelet receptors, contributing to sex differences in platelet function and even in platelet-mediated vascular damage. It has suggested aspects that apparently are well established should be reviewed. Hormones effects on platelet activity are included among them. This article tries to review knowledge about the involvement of hormones in platelet biology and activity.

LH750 hematology analyzers to identify malaria and dengue and distinguish them from other febrile illnesses.

PubMed

Sharma, P; Bhargava, M; Sukhachev, D; Datta, S; Wattal, C

2014-02-01

Tropical febrile illnesses such as malaria and dengue are challenging to differentiate clinically. Automated cellular indices from hematology analyzers may afford a preliminary rapid distinction. Blood count and VCS parameters from 114 malaria patients, 105 dengue patients, and 105 febrile controls without dengue or malaria were analyzed. Statistical discriminant functions were generated, and their diagnostic performances were assessed by ROC curve analysis. Three statistical functions were generated: (i) malaria-vs.-controls factor incorporating platelet count and standard deviations of lymphocyte volume and conductivity that identified malaria with 90.4% sensitivity, 88.6% specificity; (ii) dengue-vs.-controls factor incorporating platelet count, lymphocyte percentage and standard deviation of lymphocyte conductivity that identified dengue with 81.0% sensitivity and 77.1% specificity; and (iii) febrile-controls-vs.-malaria/dengue factor incorporating mean corpuscular hemoglobin concentration, neutrophil percentage, mean lymphocyte and monocyte volumes, and standard deviation of monocyte volume that distinguished malaria and dengue from other febrile illnesses with 85.1% sensitivity and 91.4% specificity. Leukocyte abnormalities quantitated by automated analyzers successfully identified malaria and dengue and distinguished them from other fevers. These economic discriminant functions can be rapidly calculated by analyzer software programs to generate electronic flags to trigger-specific testing. They could potentially transform diagnostic approaches to tropical febrile illnesses in cost-constrained settings. © 2013 John Wiley & Sons Ltd.

Platelet adhesion in breast cancer: development and application of a novel assay.

PubMed

Caine, Graham J; Nadar, Sunil K; Lip, Gregory Y H; Stonelake, Paul S; Blann, Andrew D

2004-09-01

The increased risk of thromboembolism in cancer may be related to a prothrombotic or hypercoagulable state, with abnormalities of haemostasis and platelet activation. To further investigate the role of platelets in this disease, we developed and applied a new assay to detect and quantify platelet adhesion to the well-defined subendothelial substrate, fibrinogen. Platelet-rich plasma was obtained from 31 females with breast cancer (13 metastatic, 18 benign), and 30 healthy female controls, re-suspended to 2 x 10(8) cells/ml and 100 microl and incubated for 1 h in microtitre plates pre-coated with fibrinogen (5 mg/ml). The supernatant was carefully aspirated, lysed with Triton X-100 and stored at -70 degrees C as supernatant-platelet lysate. The microtitre wells were carefully washed with saline, bound platelets lysed with Triton, and the lysate stored at -70 degrees C as bound-platelet lysate. P-selectin was determined in supernatant-platelet lysate and bound-platelet lysate for each patient by enzyme-linked immunosorbent assay. Interpreting differences in P-selectin in different lysates as reflective of adhesion, patients with cancer had increased platelet adhesion (absolute and percentage, both P < 0.001) compared with healthy controls. There was also more adhesion (P < 0.001) in metastatic disease compared with non-metastatic disease. Patients with breast carcinomas, and, in particular, those with metastatic disease, have a higher degree of platelet adhesion, which may by quantified by a novel method based on cell lysis. This increase in platelet adhesiveness may be related to an increased risk of thromboembolism in these patients.

VERifyNow in DIabetes high-on-treatment platelet reactivity: a pharmacodynamic study on switching from clopidogrel to prasugrel.

PubMed

Cubero Gómez, José M; Acosta Martínez, Juan; Mendias Benítez, Crsitina; Díaz De La Llera, Luis S; Fernández-Quero, Mónica; Guisado Rasco, Agustí; Villa Gil-Ortega, Manuel; Sánchez González, Ángel

2015-12-01

Diabetic patients with an acute coronary syndrome undergoing percutaneous coronary intervention frequently exhibit high platelet reactivity while on clopidogrel. We hypothesized that in diabetic patients undergoing percutaneous coronary intervention, who exhibit high-platelet-reactivity after standard treatment with clopidogrel, a 60-mg prasugrel loading dose is superior to standard treatment with clopidogrel for optimal P2Y12 inhibition within the first 24-36 h post-angioplasty. VERDI was a prospective, randomized, single-centre, single-blind, parallel-design study (NCT01684813). Consecutive diabetic patients with an non-ST-segment elevation acute coronary syndrome undergoing percutaneous coronary intervention and loaded with clopidogrel were considered for platelet reactivity assessment immediately before angioplasty with the VerifyNow assay measured in P2Y12 reaction units (PRU). Fifty of 63 screened patients (79.4%) had high platelet reactivity (PRU ≥ 208) and were randomized to receive a 60-mg prasugrel loading dose (n = 25) versus clopidogrel standard dose (n = 25). Platelet function was assessed again 24 hours post-angioplasty. Prasugrel achieved greater platelet inhibition than clopidogrel 24 hours post-angioplasty (median [interquartile range], 38 [9-72] vs 285 [240-337], respectively; P < 0.001). The non-high-platelet-reactivity rate (PRU < 208) at 24 h post-angioplasty (primary end point) was higher with prasugrel; 25 patients (100%) in the prasugrel group achieved optimal antiaggregation vs 4 patients (16%) in the clopidogrel group (P < 0.001). No significant acute bleeding was documented in either group. Among type 2 diabetic patients suffering an acute coronary syndrome with high-platelet-reactivity undergoing percutaneous coronary intervention, switching from clopidogrel to prasugrel was superior to standard treatment with clopidogrel for the achievement of optimal antiaggregation within the first 24 hours post-angioplasty.

Microfluidics and Coagulation Biology

PubMed Central

Colace, Thomas V.; Tormoen, Garth W.

2014-01-01

The study of blood ex vivo can occur in closed or open systems, with or without flow. Microfluidic devices facilitate measurements of platelet function, coagulation biology, cellular biorheology, adhesion dynamics, pharmacology, and clinical diagnostics. An experimental session can accommodate 100s to 1000s of unique clotting events. Using microfluidics, thrombotic events can be studied on defined surfaces of biopolymers, matrix proteins, and tissue factor under constant flow rate or constant pressure drop conditions. Distinct shear rates can be created on a device with a single perfusion pump. Microfluidic devices facilitated the determination of intraluminal thrombus permeability and the discovery that platelet contractility can be activated by a sudden decrease in flow. Microfluidics are ideal for multicolor imaging of platelets, fibrin, and phosphatidylserine and provide a human blood analog to the mouse injury models. Overall, microfluidic advances offer many opportunities for research, drug testing under relevant hemodynamic conditions, and clinical diagnostics. PMID:23642241

Platelets and hemophilia: A review of the literature.

PubMed

Riedl, Julia; Ay, Cihan; Pabinger, Ingrid

2017-07-01

Hemophilia A and B are inherited bleeding disorders due to deficiencies of the clotting factors VIII and IX, respectively. The severity of the disease correlates with remaining factor levels, although individual differences in bleeding tendency are seen despite similar factor levels. While thrombin generation is severely impaired in persons with hemophilia, primary hemostasis, i.e. platelet function, has been generally considered to be normal. However, some studies reported prolonged bleeding times in hemophilia, suggesting that also primary hemostasis is affected. In several other studies different aspects of platelet function in hemophilia have been investigated in more detail and various alterations were discovered, such as increased platelet P-selectin expression, a lower number of procoagulant, so-called 'coated' platelets, lower aggregation upon co-incubation with tissue factor, or reduced platelet contractile forces during clot formation in comparison to healthy individuals. An influence of platelet function on clinical phenotype was suggested, which might contribute in part to variations in bleeding tendency in hemophilic patients with similar factor levels. However, the available evidence is currently limited and no clear correlations between platelet function parameters and clinical phenotypes have been demonstrated. The impact of alterations of platelet function in hemophilia remains to be better defined. Another interesting role of platelets in hemophilia has been reported recently by establishing a novel gene-therapeutic strategy using platelets as a delivery system for FVIII, showing promising results in animal models. This review gives an overview on the currently published literature on platelet function and the potential roles of platelets in hemophilia. Copyright © 2017 Elsevier Ltd. All rights reserved.

Intraplatelet reactive oxygen species (ROS) correlate with the shedding of adhesive receptors, microvesiculation and platelet adhesion to collagen during storage: Does endogenous ROS generation downregulate platelet adhesive function?

PubMed

Ghasemzadeh, Mehran; Hosseini, Ehteramolsadat; Roudsari, Zahra Oushyani; Zadkhak, Parvin

2018-03-01

Platelets storage lesion is mainly orchestrated by platelet activating signals during storage. Reactive oxygen species (ROS) are being considered as important signaling molecules modulating platelet function while their production has also been shown to be augmented by platelet activation. This study investigated to what extent endogenous ROS generation during platelet storage could be correlated with platelet receptor shedding, microvesiculation and adhesive function. 10 PRP-platelet concentrates were subjected to flow cytometry analysis to examine the generation of intraplatelet ROS on days 1, 5 and 7 after storage. In 5 day-stored platelets considering 40% of ROS generation as a cutoff point, samples were divided into two groups of those with higher or lower levels of ROS. The expression of adhesion receptors (GPVI, GPIbα), the amount of microparticles and phosphatidylserine exposure in each group were then examined by flow cytometry. Platelet receptor shedding and adhesion to collagen matrix were respectively measured by western blotting and microscopic assays. Our data showed lowered expression of GPIbα (p < 0.05) and GPVI in samples with ROS > 40% than those with ROS ≤ 40%, whereas receptors shedding and microvesiculation were (p < 0.05) elevated in platelets with higher levels of ROS. Functionally, we observed significantly (p < 0.05) lower levels of platelet adhesion to collagen matrix in samples with ROS generation more than 40%. Taken together, we showed correlations between intraplatelet ROS generation and either platelet receptors or microparticle shedding as well as platelet adhesive capacity to collagen. These findings suggest that augmented ROS generation during storage might be relevant to down-regulation of platelet adhesive function. Copyright © 2018 Elsevier Ltd. All rights reserved.

Platelet-Derived Short-Chain Polyphosphates Enhance the Inactivation of Tissue Factor Pathway Inhibitor by Activated Coagulation Factor XI.

PubMed

Puy, Cristina; Tucker, Erik I; Ivanov, Ivan S; Gailani, David; Smith, Stephanie A; Morrissey, James H; Gruber, András; McCarty, Owen J T

2016-01-01

Factor (F) XI supports both normal human hemostasis and pathological thrombosis. Activated FXI (FXIa) promotes thrombin generation by enzymatic activation of FXI, FIX, FX, and FV, and inactivation of alpha tissue factor pathway inhibitor (TFPIα), in vitro. Some of these reactions are now known to be enhanced by short-chain polyphosphates (SCP) derived from activated platelets. These SCPs act as a cofactor for the activation of FXI and FV by thrombin and FXIa, respectively. Since SCPs have been shown to inhibit the anticoagulant function of TFPIα, we herein investigated whether SCPs could serve as cofactors for the proteolytic inactivation of TFPIα by FXIa, further promoting the efficiency of the extrinsic pathway of coagulation to generate thrombin. Purified soluble SCP was prepared by size-fractionation of sodium polyphosphate. TFPIα proteolysis was analyzed by western blot. TFPIα activity was measured as inhibition of FX activation and activity in coagulation and chromogenic assays. SCPs significantly accelerated the rate of inactivation of TFPIα by FXIa in both purified systems and in recalcified plasma. Moreover, platelet-derived SCP accelerated the rate of inactivation of platelet-derived TFPIα by FXIa. TFPIα activity was not affected by SCP in recalcified FXI-depleted plasma. Our data suggest that SCP is a cofactor for TFPIα inactivation by FXIa, thus, expanding the range of hemostatic FXIa substrates that may be affected by the cofactor functions of platelet-derived SCP.

Effects of an antiandrogenic oral contraceptive pill compared with metformin on blood coagulation tests and endothelial function in women with the polycystic ovary syndrome: influence of obesity and smoking.

PubMed

Luque-Ramírez, Manuel; Mendieta-Azcona, Covandonga; del Rey Sánchez, José M; Matíes, Milagro; Escobar-Morreale, Héctor F

2009-03-01

To study the blood clotting tests and endothelial function of polycystic ovary syndrome (PCOS) patients and non-hyperandrogenic women, and their changes during PCOS treatment, as a function of the presence of obesity and smoking. Case-control study followed by a randomized clinical trial. Blood clotting and endothelial function were analyzed in 40 PCOS patients and 20 non-hyperandrogenic women. Thirty-four PCOS women were randomized to an oral contraceptive containing 35 microg ethinyl-estradiol plus 2 mg cyproterone acetate (Diane(35)Diario) or metformin (850 mg twice daily), monitoring the changes on these parameters during 24 weeks of treatment. The influence of obesity and smoking was also analyzed. Blood clotting and endothelial function tests were similar among PCOS patients and controls with the exception of a higher platelet count in the former. Obesity increased circulating fibrinogen levels, prothrombin activity and platelet counts, and reduced prothrombin and activated partial thromboplastin times. Smoking increased fibrinogen levels, platelet counts, and prothrombin activity, and reduced prothrombin time, in relation to the larger waist circumference of smokers. Irrespective of the treatment received, PCOS patients showed a decrease in prothrombin time and an increase in prothrombin activity, with a parallel increase in homocysteine levels in metformin users. The activated partial thromboplastin time decreased markedly in the patients treated with Diane(35)Diario. Finally, flow-mediated dilation improved in non-smokers irrespective of the drug received, but worsened in smokers. Oral contraceptives and metformin may exert deleterious effects on blood clotting tests of PCOS women, yet the effects of metformin appear to be milder. Because smoking potentiates some of these effects and deteriorates endothelial function, smoking cessation should be promoted in PCOS patients.

Early storage lesions in apheresis platelets are induced by the activation of the integrin αIIbβ₃ and focal adhesion signaling pathways.

PubMed

Thiele, Thomas; Iuga, Cristina; Janetzky, Susann; Schwertz, Hansjorg; Gesell Salazar, Manuela; Fürll, Birgit; Völker, Uwe; Greinacher, Andreas; Steil, Leif

2012-12-05

Production and storage of platelet concentrates (PC) induce protein changes in platelets leading to impaired platelet function. This study aimed to identify signaling pathways involved in the development of early platelet storage lesions in apheresis-PCs stored in plasma or additive solution (PAS). Apheresis-PCs from four donors were stored in plasma or in PAS at 22°C (n=4 each). Platelets were analyzed at day 0 (production day) and after 1, 6 and 9 days of storage. Platelet response to agonists (TRAP, collagen, ADP) and to hypotonic shock decreased, CD62P expression increased in both storage media over time. Using DIGE 1550 protein spots were monitored and compared to baseline values at day 0. Platelets in plasma displayed changes in 352 spots (166/day 1, 263/day 6 and 201/day 9); in PAS 325 spots changed (202/day 1, 221/day 6, 200/day 9). LC-ESI-MS/MS analysis of 405 platelet proteins revealed 32 proteins changed during storage in plasma (9/day 1, 15/day 6 and 26/day 9) and 28 in PAS (5/day 1, 20/day 6, 26/day 9). Ingenuity pathway analysis found integrin-αII(b)β(3) and focal adhesion signaling pathways involved in early alterations, being confirmed by Western blotting. Corresponding mRNAs in platelets were identified by next generation sequencing for 84 changed proteins. Integrin-αII(b)β(3) and focal adhesion signaling cause irreversible early storage lesions in apheresis platelets. This article is part of a Special Issue entitled: Integrated omics. Copyright © 2012 Elsevier B.V. All rights reserved.

Relationship between ADAMTS13 activity, von Willebrand factor antigen levels and platelet function in the early and late phases after TIA or ischaemic stroke.

PubMed

McCabe, Dominick J H; Murphy, Stephen J X; Starke, Richard; Harrison, Paul; Brown, Martin M; Sidhu, Paul S; Mackie, Ian J; Scully, Marie; Machin, Samuel J

2015-01-15

Reduced ADAMTS13 activity is seen in thrombotic thrombocytopenic purpura (TTP), and may lead to accumulation of prothrombotic ultra-large von Willebrand factor (ULVWF) multimers in vivo. ADAMTS13 activity and its relationship with VWF antigen (VWF:Ag) levels and platelet function in 'non-TTP related' TIA or ischaemic stroke has not been comprehensively studied. In this prospective pilot observational analytical case-control study, ADAMTS13 activity and VWF:Ag levels were quantified in platelet poor plasma in 53 patients in the early phase (≤ 4 weeks) and 34 of these patients in the late phase (≥ 3 months) after TIA or ischaemic stroke on aspirin. Data were compared with those from 22 controls not on aspirin. The impact of ADAMTS13 on platelet function in whole blood was quantified by measuring Collagen-ADP (C-ADP) and Collagen-Epinephrine closure times on a platelet function analyser (PFA-100(®)). Median ADAMTS13 activity was significantly reduced in the early phase (71.96% vs. 95.5%, P <0.01) but not in the late phase after TIA or stroke compared with controls (86.3% vs. 95.5%, P=0.19). There was a significant inverse relationship between ADAMTS13 activity and VWF:Ag levels in the early phase (r=-0.31; P=0.024), but not in the late phase after TIA or stroke (P=0.74). There was a positive correlation between ADAMTS13 activity and C-ADP closure times in early phase patients only, likely mediated via VWF:Ag levels. ADAMTS13 activity is reduced and VWF:Ag expression is increased within 4 weeks of TIA or ischaemic stroke onset, and can promote enhanced platelet adhesion and aggregation in response to stimulation with collagen and ADP via VWF-mediated pathways. These data improve our understanding of the dynamic haemostatic and thrombotic profiles of ischaemic cerebrovascular disease (CVD) patients, and are important in view of the potential future role that ADAMTS13 may have to play as an anti-thrombotic agent in CVD. Copyright © 2014 Elsevier B.V. All rights reserved.

Clinical determinants of clopidogrel responsiveness in a heterogeneous cohort of Puerto Rican Hispanics

PubMed Central

Hernandez-Suarez, Dagmar F.; Scott, Stuart A.; Tomey, Matthew I.; Melin, Kyle; Lopez-Candales, Angel; Buckley, Charlotte E.; Duconge, Jorge

2017-01-01

Background: Clopidogrel is by far the most prescribed platelet adenosine diphosphate (ADP) antagonist in Puerto Rico despite the advent of newer agents (prasugrel and ticagrelor). Given the paucity of data on clopidogrel responsiveness in Hispanics, we sought to determine the association between clinical characteristics and platelet reactivity in Puerto Rican patients on clopidogrel therapy. Study population: A total of 100 Puerto Rican patients on clopidogrel therapy were enrolled and allocated into two groups: Group I, without high on-treatment platelet reactivity (HTPR); and Group II, with HTPR. Platelet function was measured ex vivo using the VerifyNow® P2Y12 assay. Results: The cohort was comprised of Hispanic patients with coronary artery disease (57%), peripheral artery disease (32%), carotid artery stenosis (7%), cerebral artery aneurysm (2%), and stroke (2%). Mean platelet reactivity was 200 ± 61 P2Y12 reaction units (PRUs) (range: 8–324), and 35% of patients had HTPR (PRUs ⩾ 230). Multivariable logistic regression analysis determined that diabetes mellitus (DM) [odds ratio (OR) = 3.27; 95% confidence interval (CI): 1.20–8.96], use of proton-pump inhibitors (PPIs) (OR = 3.60; 95% CI: 1.09–11.82), and calcium channel blockers (CCBs) (OR = 3.10; 95% CI: 1.09–8.83) were independent predictors of HTPR (p < 0.05) after adjusting for other clinical variables. Conclusions: In a sample of 100 Puerto Rican Hispanic patients on clopidogrel, 35% had HTPR. Furthermore, DM, PPIs and CCBs predicted HTPR. Clinical outcome data are needed to identify appropriate PRU thresholds for risk prediction in the Puerto Rican population. PMID:28675986

[Adjusting Platelet Counts for Platelet Aggregation Tests].

PubMed

Ling, Li-Qin; Yang, Xin-Chun; Chen, Hao; Liu, Chao-Nan; Chen, Si; Jiang, Hong; Jin, Ya-Xiong; Zhou, Jing

2018-03-01

To explore a better method to adjust platelet counts for light transmission aggregometry (LTA). Blood samples from 36 healthy participants aged from 18 to 50 yr. were collected.Platelet-rich plasma (PRP) was diluted using platelet-poor plasma (PPP) and physiological saline (PS),respectively,in a ratio of 1.5,2,2.5 and 3 times. Platelet aggregation was induced by adenosine diphosphate (ADP),arachidonic acid (ARA),collagen (COL), epinephrine (EPI),or ristocetin (RIS). The maximal aggregation rates (MAs) of different approaches were compared. We also compared the MAs induced by RIS between PRP-obtained-PPP and whole blood-obtained-PPP (2 100× g, 5 min). Compared with the original PRP,the MAs induced by ADP,ARA,and EPI decreased in PPP-adjusted PRP (significant at 2-3 times dilution ratio, P <0.05),but not in PS-adjusted PRP ( P >0.05). The MA induced by RIS decreased in PS-adjusted PRP (significant at all dilution ratios, P <0.05),but not in PPP-adjusted PRP ( P >0.05). No changes in the MA induced by COL were found in PS-adjusted PRP and PPP-adjusted PRP ( P >0.05). Whole blood-obtained-PPP (2 100× g, 5 min) had the same MA induced by ristocetin compared with PRP-obtained-PPP ( P >0.05). PS is recommended for adjusting platelets counts for platelet aggregation induced by ADP,ARA,COL and EPI. Whole blood-obtained-PPP (2 100 × g, 5 min) is recommended for RIS-induced aggregation as a matter of convenience. Copyright© by Editorial Board of Journal of Sichuan University (Medical Science Edition).

Comparison of the in vitro effects of saline, hypertonic hydroxyethyl starch, hypertonic saline, and two forms of hydroxyethyl starch on whole blood coagulation and platelet function in dogs.

PubMed

Wurlod, Virginie A; Howard, Judith; Francey, Thierry; Schweighauser, Ariane; Adamik, Katja N

2015-01-01

To compare the in vitro effects of hypertonic solutions and colloids to saline on coagulation in dogs. In vitro experimental study. Veterinary teaching hospital. Twenty-one adult dogs. Blood samples were diluted with saline, 7.2% hypertonic saline solution with 6% hydroxyethylstarch with an average molecular weight of 200 kDa and a molar substitution of 0.4 (HH), 7.2% hypertonic saline (HTS), hydroxyethyl starch (HES) 130/0.4 or hydroxyethyl starch 600/0.75 at ratios of 1:22 and 1:9, and with saline and HES at a ratio of 1:3. Whole blood coagulation was analyzed using rotational thromboelastometry (extrinsic thromboelastometry-cloting time (ExTEM-CT), maximal clot firmness (MCF) and clot formation time (CFT) and fibrinogen function TEM-CT (FibTEM-CT) and MCF) and platelet function was analyzed using a platelet function analyzer (closure time, CTPFA ). All parameters measured were impaired by saline dilution. The CTPFA was prolonged by 7.2% hypertonic saline solution with 6% hydroxyethylstarch with an average molecular weight of 200 kDa and a molar substitution of 0.4 (HH) and HTS but not by HES solutions. At clinical dilutions equivalent to those generally administered for shock (saline 1:3, HES 1:9, and hypertonic solutions 1:22), CTPFA was more prolonged by HH and HTS than other solutions but more by saline than HES. No difference was found between the HES solutions or the hypertonic solutions. ExTEM-CFT and MCF were impaired by HH and HTS but only mildly by HES solutions. At clinically relevant dilutions, no difference was found in ExTEM-CFT between HTS and saline or in ExTEM-MCF between HH and saline. No consistent difference was found between the 2 HES solutions but HH impaired ExTEM-CFT and MCF more than HTS. At high dilutions, FibTEM-CT and -MCF and ExTEM-CT were impaired by HES. Hypertonic solutions affect platelet function and whole blood coagulation to a greater extent than saline and HES. At clinically relevant dilutions, only CTPFA was markedly more affected by hypertonic solutions than by saline. At high dilutions, HES significantly affects coagulation but to no greater extent than saline at clinically relevant dilutions. © Veterinary Emergency and Critical Care Society 2015.

Platelet Dynamics during Natural and Pharmacologically Induced Torpor and Forced Hypothermia

PubMed Central

de Vrij, Edwin L.; Vogelaar, Pieter C.; Goris, Maaike; Houwertjes, Martin C.; Herwig, Annika; Dugbartey, George J.; Boerema, Ate S.; Strijkstra, Arjen M.; Bouma, Hjalmar R.; Henning, Robert H.

2014-01-01

Hibernation is an energy-conserving behavior in winter characterized by two phases: torpor and arousal. During torpor, markedly reduced metabolic activity results in inactivity and decreased body temperature. Arousal periods intersperse the torpor bouts and feature increased metabolism and euthermic body temperature. Alterations in physiological parameters, such as suppression of hemostasis, are thought to allow hibernators to survive periods of torpor and arousal without organ injury. While the state of torpor is potentially procoagulant, due to low blood flow, increased viscosity, immobility, hypoxia, and low body temperature, organ injury due to thromboembolism is absent. To investigate platelet dynamics during hibernation, we measured platelet count and function during and after natural torpor, pharmacologically induced torpor and forced hypothermia. Splenectomies were performed to unravel potential storage sites of platelets during torpor. Here we show that decreasing body temperature drives thrombocytopenia during torpor in hamster with maintained functionality of circulating platelets. Interestingly, hamster platelets during torpor do not express P-selectin, but expression is induced by treatment with ADP. Platelet count rapidly restores during arousal and rewarming. Platelet dynamics in hibernation are not affected by splenectomy before or during torpor. Reversible thrombocytopenia was also induced by forced hypothermia in both hibernating (hamster) and non-hibernating (rat and mouse) species without changing platelet function. Pharmacological torpor induced by injection of 5′-AMP in mice did not induce thrombocytopenia, possibly because 5′-AMP inhibits platelet function. The rapidness of changes in the numbers of circulating platelets, as well as marginal changes in immature platelet fractions upon arousal, strongly suggest that storage-and-release underlies the reversible thrombocytopenia during natural torpor. Possibly, margination of platelets, dependent on intrinsic platelet functionality, governs clearance of circulating platelets during torpor. PMID:24722364

How platelets safeguard vascular integrity

PubMed Central

Ho-Tin-Noé, Benoit; Demers, Mélanie; Wagner, Denisa D

2011-01-01

Summary The haemostatic role of platelets was established in the 1880s by Bizzozero who observed their ability to adhere and aggregate at sites of vascular injury. It was only some 80 years later that the function of platelets in maintaining the structural integrity of intact blood vessels was reported by Danielli. Danielli noted that platelets help preserve the barrier function of endothelium during organ perfusion. Subsequent studies have demonstrated further that platelets are continuously needed to support intact mature blood vessels. More recently, platelets were shown to safeguard developing vessels, lymphatics, as well as the microvasculature at sites of leukocyte infiltration, including inflamed organs and tumours. Interestingly, from a mechanistic point of view, the supporting role of platelets in these various vessels does not necessarily involve the well-understood process of platelet plug formation but, rather, may rely on secretion of the various platelet granules and their many active components. The present review focuses on these nonconventional aspects of platelet biology and function by presenting situations in which platelets intervene to maintain vascular integrity and discusses possible mechanisms of their actions. We propose that modulating these newly described platelet functions may help treat haemorrhage as well as treat cancer by increasing the efficacy of drug delivery to tumours. PMID:21781242

RhoG protein regulates platelet granule secretion and thrombus formation in mice.

PubMed

Goggs, Robert; Harper, Matthew T; Pope, Robert J; Savage, Joshua S; Williams, Christopher M; Mundell, Stuart J; Heesom, Kate J; Bass, Mark; Mellor, Harry; Poole, Alastair W

2013-11-22

Rho GTPases such as Rac, RhoA, and Cdc42 are vital for normal platelet function, but the role of RhoG in platelets has not been studied. In other cells, RhoG orchestrates processes integral to platelet function, including actin cytoskeletal rearrangement and membrane trafficking. We therefore hypothesized that RhoG would play a critical role in platelets. Here, we show that RhoG is expressed in human and mouse platelets and is activated by both collagen-related peptide (CRP) and thrombin stimulation. We used RhoG(-/-) mice to study the function of RhoG in platelets. Integrin activation and aggregation were reduced in RhoG(-/-) platelets stimulated by CRP, but responses to thrombin were normal. The central defect in RhoG(-/-) platelets was reduced secretion from α-granules, dense granules, and lysosomes following CRP stimulation. The integrin activation and aggregation defects could be rescued by ADP co-stimulation, indicating that they are a consequence of diminished dense granule secretion. Defective dense granule secretion in RhoG(-/-) platelets limited recruitment of additional platelets to growing thrombi in flowing blood in vitro and translated into reduced thrombus formation in vivo. Interestingly, tail bleeding times were normal in RhoG(-/-) mice, suggesting that the functions of RhoG in platelets are particularly relevant to thrombotic disorders.

Novel and unexpected clearance mechanisms for cold platelets

PubMed Central

Rumjantseva, Viktoria; Hoffmeister, Karin M.

2015-01-01

Storage at room temperature is limited to 5 days because of the risk of bacterial growth and loss of platelet functionality. Platelet refrigeration remains impossible, because once chilled, platelets are rapidly removed from circulation. Chilling platelets (<4 h) clusters glycoprotein (GP) Ibα receptors, and β2 integrins on hepatic macrophages recognize clustered βGlcNAc residues leading to rapid clearance of acutely chilled platelets. Prolonged refrigeration increases the exposure of galactose residues such that, unexpectedly, hepatocytes remove platelets using their asialoglycoprotein receptors. Here we review current knowledge of the mechanisms of platelet removal, the existing knowledge of refrigerated platelet function, and methods to preserve platelet concentrates long-term for transfusion. PMID:19932055

Thrombin-stimulated platelet aggregation is inhibited by kallikrein in a time- and concentration-dependent manner.

PubMed

Veloso, D

2003-01-01

Many in vitro studies have shown that activation of prekallikrein (PK) to kallikrein (KAL) in normal plasma triggers rapid activation of the coagulation cascade. In agreement, the coagulation activation is impaired in PK-deficient plasma. Paradoxically, PK-deficient patients show a tendency to thrombosis. To investigate the discrepancy between the in vitro and in vivo findings, we analyzed the effect of KAL on the rate of platelet aggregation. For this research, physiologic concentrations of washed human platelets were incubated for 5 and/or 10 min with approximately 2.2 to 88 nM human plasma KAL (< 1/100 to approximately 1/3 of PK concentrations in plasma) prior to the addition of high concentrations of alpha-thrombin (54 nM) or fibrinogen plus ADP. KAL concentrations were arbitrarily selected on the assumption that concentrations of free KAL (the enzymatically active species) were minute in normal plasma and higher when KAL production was enhanced, and/or inhibitors were depleted. Full platelet aggregation was that seen in the absence of KAL or PK. Inhibition of platelet aggregation stimulated by thrombin was markedly increased with increased KAL concentrations and incubation times. The degree of inhibition by KAL was smaller when ADP was the agonist. The data suggest that KAL may play a role in the modulation of platelet aggregation in vivo under normal conditions as well as when prolonged, high concentrations of KAL occur in blood. The data may also help to explain the intriguing observation that PK-deficient patients show a tendency to thrombotic episodes and myocardial infarction whereas in vitro assays predict bleeding.

The Small GTPase Rif Is Dispensable for Platelet Filopodia Generation in Mice

PubMed Central

Goggs, Robert; Savage, Joshua S.; Mellor, Harry; Poole, Alastair W.

2013-01-01

Background Formation of filopodia and other shape change events are vital for platelet hemostatic function. The mechanisms regulating filopodia formation by platelets are incompletely understood however. In particular the small GTPase responsible for initiating filopodia formation by platelets remains elusive. The canonical pathway involving Cdc42 is not essential for filopodia formation in mouse platelets. The small GTPase Rif (RhoF) provides an alternative route to filopodia generation in other cell types and is expressed in both human and mouse platelets. Hypothesis/Objective We hypothesized that Rif might be responsible for generating filopodia by platelets and generated a novel knockout mouse model to investigate the functional role of Rif in platelets. Methodology/Principal Findings Constitutive RhoF−/− mice are viable and have normal platelet, leukocyte and erythrocyte counts and indices. RhoF−/− platelets form filopodia and spread normally on various agonist surfaces in static conditions and under arterial shear. In addition, RhoF−/− platelets have normal actin dynamics, are able to activate and aggregate normally and secrete from alpha and dense granules in response to collagen related peptide and thrombin stimulation. Conclusions The small GTPase Rif does not appear to be critical for platelet function in mice. Functional overlap between Rif and other small GTPases may be responsible for the non-essential role of Rif in platelets. PMID:23359340

The antiplatelet effect of higher loading and maintenance dose regimens of clopidogrel: the PRINC (Plavix Response in Coronary Intervention) trial.

PubMed

Gladding, Patrick; Webster, Mark; Zeng, Irene; Farrell, Helen; Stewart, Jim; Ruygrok, Peter; Ormiston, John; El-Jack, Seif; Armstrong, Guy; Kay, Patrick; Scott, Douglas; Gunes, Arzu; Dahl, Marja-Liisa

2008-12-01

This study evaluated the antiplatelet effect of a higher loading and maintenance dose regimen of clopidogrel and a possible drug interaction with verapamil. Clopidogrel loading doses above 600 mg have not resulted in more rapid or complete platelet inhibition. Higher maintenance dosages may be more effective than 75 mg/day. A double-blind, randomized, placebo-controlled trial was undertaken in 60 patients undergoing percutaneous coronary intervention. All patients received clopidogrel 600 mg at the start of the procedure. Using a 2 x 2 design, patients were allocated to clopidogrel 600 mg given 2 h later or matching placebo, and to verapamil 5 mg intra-arterial or placebo. Platelet function was measured using the VerifyNow P2Y12 analyzer (Accumetrics Ltd., San Diego, California) at 2, 4, and 7 h. Patients were further randomized to receive a clopidogrel 75 or 150 mg once daily, with platelet function assessed after 1 week. Two hours after the second dose of clopidogrel or placebo, platelet inhibition was 42 +/- 27% with clopidogrel, compared with 24 +/- 22% with placebo (p = 0.0006). By 5 h after the second dose, platelet inhibition was 49 +/- 30% with clopidogrel, compared with 29 +/- 22% with placebo (p = 0.01). No drug interaction was seen with verapamil. A clopidogrel maintenance dosage of 150 mg daily for 1 week resulted in greater platelet inhibition than 75 mg daily (50 +/- 28% vs. 29 +/- 19%, p = 0.01). In an unselected population undergoing percutaneous coronary intervention a clopidogrel 1,200-mg loading dose, given as two 600-mg doses 2 h apart, results in more rapid and complete platelet inhibition than a single 600-mg dose. A maintenance dosage of 150 mg daily produces greater platelet inhibition than 75 mg daily. (The PRINC trial; ACTRN12606000129583).

Olive Oil-Based Lipid Emulsions Do Not Influence Platelet Receptor Expression in Comparison to Medium and Long Chain Triglycerides In vitro.

PubMed

Stoetzer, Carsten; Nickel, Katja; Weißig, Annette; Großheim, Marieke; Scheinichen, Dirk; Doll, Thorben; Jüttner, Björn

2016-11-01

Lipid emulsions influence platelet aggregation and receptor expression. However, the effect on platelet function is not fully explained. Therefore, the aim of this study was to examine the influence of the lipids Lipofundin ® , Lipidem ® and ClinOleic ® on surface expressions of P-selectin, GPIb and GPIIb/IIIa on platelets in vitro. Whole blood was incubated in two different concentrations (0.06 and 0.6 mg/ml) of LCT/MCT, n-3/LCT/MCT and LCT-MUFA for 30 min, followed by activation with TRAP-6 or ADP for flow-cytometric assay. Rates of P-selectin, GPIb and GPIIb/IIIa expression were analyzed. There was a significant increase in GPIIb/IIIa- and P-selectin-expression after incubation with LCT/MCT and n-3/LCT/MCT at the concentration of 0.6 mg/ml, without and after stimulation with TRAP-6 and ADP. GPIb was significantly decreased. Accordingly, LCT-MUFA had no effect on receptor expression of platelets in vitro. We demonstrated that LCT-MUFA did not activate receptor expression of platelets whereas LCT/MCT significantly increased platelet aggregation in vitro. This finding should be noted for parenteral nutrition of intensive care patients and, in the future, might provide further insight into the pathogenic pathways of acute thromboembolic events. However, prospectively designed clinical studies are needed to support our results.

Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue.

PubMed

Trugilho, Monique Ramos de Oliveira; Hottz, Eugenio Damaceno; Brunoro, Giselle Villa Flor; Teixeira-Ferreira, André; Carvalho, Paulo Costa; Salazar, Gustavo Adolfo; Zimmerman, Guy A; Bozza, Fernando A; Bozza, Patrícia T; Perales, Jonas

2017-05-01

Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV) infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P) translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet proteome in dengue, and sheds light on new mechanisms of platelet activation and platelet-mediated immune and inflammatory responses.

Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue

PubMed Central

Teixeira-Ferreira, André; Carvalho, Paulo Costa; Salazar, Gustavo Adolfo; Zimmerman, Guy A.; Perales, Jonas

2017-01-01

Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV) infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P) translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet proteome in dengue, and sheds light on new mechanisms of platelet activation and platelet-mediated immune and inflammatory responses. PMID:28542641

Protein kinase Cι/λ is dispensable for platelet function in thrombosis and hemostasis in mice.

PubMed

Beck, Sarah; Leitges, Michael; Stegner, David

2017-10-01

Platelet activation at sites of vascular injury is crucial for hemostasis, but it may also cause myocardial infarction or ischemic stroke. Upon platelet activation, cytoskeletal reorganization is essential for platelet secretion and thrombus formation. Members of the protein kinase C family, which includes 12 isoforms, are involved in most platelet responses required for thrombus formation. The atypical protein kinase Cι/λ (PKCι/λ) has been implicated as an important mediator of cell polarity, carcinogenesis and immune cell responses. PKCι/λ is known to be associated with the small GTPase Cdc42, an important mediator of multiple platelet functions; however, its exact function in platelets is not known. To study the role of PKCι/λ, we generated platelet- and megakaryocyte-specific PKCι/λ knockout mice (Prkci fl/fl, Pf4-Cre ) and used them to investigate the function of PKCι/λ in platelet activation and aggregation in vitro and in vivo. Surprisingly, lack of PKCι/λ had no detectable effect on platelet spreading and function in vitro and in vivo under all tested conditions. These results indicate that PKCι/λ is dispensable for Cdc42-triggered processes and for thrombosis and hemostasis in mice. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of functional VEGF receptors on human platelets.

PubMed

Selheim, Frode; Holmsen, Holm; Vassbotn, Flemming S

2002-02-13

Platelets secrete platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) upon stimulation. We have demonstrated that platelets have functionally active PDGF alpha-receptors, a transmembrane tyrosine kinase involved in negative feedback regulation. Here we demonstrate the presence of the related VEGF receptors fms-like tyrosine kinase-1 and kinase-insert domain region on human platelets. VEGF itself did not cause platelet aggregation. However, addition of exogenous VEGF to SFRLLN or thrombin-stimulated platelets potentiated platelet aggregation. Moreover, thrombin-induced phosphoinositide 3-kinase and mitogen-activated protein kinase activity were enhanced in the presence of VEGF.

Fibrinogen adsorption, platelet adhesion and activation on mixed hydroxyl-/methyl-terminated self-assembled monolayers.

PubMed

Rodrigues, Sofia N; Gonçalves, Inês C; Martins, M C L; Barbosa, Mário A; Ratner, Buddy D

2006-11-01

The effect of surface wettability on fibrinogen adsorption, platelet adhesion and platelet activation was investigated using self-assembled monolayers (SAMs) containing different ratios of longer chain methyl- and shorter chain hydroxyl-terminated alkanethiols (C15CH3 vs. C11OH) on gold. Protein adsorption studies were performed using radiolabeled human fibrinogen (HFG). Platelet adhesion and activation studies with and without pre-adsorbed fibrinogen, albumin and plasma were assessed using scanning electron microscopy (SEM) and a glutaraldehyde-induced fluorescence technique (GIFT). Results demonstrated a linear decrease of HFG adsorption with the increase of OH groups on the monolayer (increase of the hydrophilicity). Platelet adhesion and activation also decrease with increase of hydrophilicity of surface. Concerning SAMs pre-immersed in proteins, fibrinogen adsorption was related with high platelet adhesion and activation. The passivant effect of albumin on platelet adhesion and activation was only demonstrated on SAMs contained C11OH. When all the blood proteins are present (plasma) platelet adhesion was almost absent on SAMs with 65% and 100% C11OH. This could be explained by the higher albumin affinity of the SAMs with 65% C11OH and the lower total protein adsorption associated with SAMs with 100% C11OH.

Inhibitory activity of aspirin on von Willebrand factor-induced platelet aggregation.

PubMed

Homoncik, M; Jilma, B; Eichelberger, B; Panzer, S

2000-09-01

The effect of aspirin (ASA) on vWF induced platelet - platelet interaction is unknown. We therefore tested the response of platelets to von Willebrand factor (vWF) coated beads induced platelet aggregation before and after i.v. and oral ASA. 1000 mg ASA was infused to 10 healthy individuals and after a wash-out period 7 volunteers received 100 mg ASA orally over a period of 11 days. Prior to ASA and in regular intervals thereafter we tested the reactivity to vWF-coated beads to assess platelet adhesion/aggregation and the fade-out time of ASA effects on platelets. Considerable interindividual variability in response to vWF-coated beads was observed, both before ASA and after treatment with ASA. The maximal response to vWF-coated beads (Tmax), the time lag, and the slope of the curve were significantly affected by i.v. ASA, whereas 100 mg of ASA had only inconstant effect on Tmax and slope. The absolute reduction of Tmax after ASA depended on the pre-ASA level, while the percentage of the reduction was similar in all individuals. Thus, platelet aggregation induced by vWF-coated beads is impaired by ASA. Furthermore, our data indicate a large interindividual variability of the response to ASA shortly after treatment induction, which becomes more constant after prolonged treatment.

Association of Cytochrome P450 2C19 Genotype With the Antiplatelet Effect and Clinical Efficacy of Clopidogrel Therapy

PubMed Central

Shuldiner, Alan R.; O'Connell, Jeffrey R.; Bliden, Kevin P.; Gandhi, Amish; Ryan, Kathleen; Horenstein, Richard B.; Damcott, Coleen M.; Pakyz, Ruth; Tantry, Udaya S.; Gibson, Quince; Pollin, Toni I.; Post, Wendy; Parsa, Afshin; Mitchell, Braxton D.; Faraday, Nauder; Herzog, William; Gurbel, Paul A.

2013-01-01

Context Clopidogrel therapy improves cardiovascular outcomes in patients with acute coronary syndromes and following percutaneous coronary intervention by inhibiting adenosine diphosphate (ADP)–dependent platelet activation. However, nonresponsiveness is widely recognized and is related to recurrent ischemic events. Objective To identify gene variants that influence clopidogrel response. Design, Setting, and Participants In the Pharmacogenomics of Antiplatelet Intervention (PAPI) Study (2006-2008), we administered clopidogrel for 7 days to 429 healthy Amish persons and measured response by ex vivo platelet aggregometry. A genome-wide association study was performed followed by genotyping the loss-of-function cytochrome P450 (CYP) 2C19*2 variant (rs4244285). Findings in the PAPI Study were extended by examining the relation of CYP2C19*2 genotype to platelet function and cardiovascular outcomes in an independent sample of 227 patients undergoing percutaneous coronary intervention. Main Outcome Measure ADP-stimulated platelet aggregation in response to clopidogrel treatment and cardiovascular events. Results Platelet response to clopidogrel was highly heritable (h2=0.73; P<.001). Thirteen single-nucleotide polymorphisms on chromosome 10q24 within the CYP2C18-CYP2C19-CYP2C9-CYP2C8 cluster were associated with diminished clopidogrel response, with a high degree of statistical significance (P=1.5 × 10−13 for rs12777823, additive model). The rs12777823 polymorphism was in strong linkage disequilibrium with the CYP2C19*2 variant, and was associated with diminished clopidogrel response, accounting for 12% of the variation in platelet aggregation to ADP (P=4.3 × 10−11). The relation between CYP2C19*2 genotype and platelet aggregation was replicated in clopidogrel-treated patients undergoing coronary intervention (P=.02). Furthermore, patients with the CYP2C19*2 variant were more likely (20.9% vs 10.0%) to have a cardiovascular ischemic event or death during 1 year of follow-up (hazard ratio, 2.42; 95% confidence interval, 1.18-4.99; P=.02). Conclusion CYP2C19*2 genotype was associated with diminished platelet response to clopidogrel treatment and poorer cardiovascular outcomes. PMID:19706858

Effects of pathogen reduction systems on platelet microRNAs, mRNAs, activation, and function

PubMed Central

Osman, Abdimajid; Hitzler, Walter E.; Meyer, Claudius U.; Landry, Patricia; Corduan, Aurélie; Laffont, Benoit; Boilard, Eric; Hellstern, Peter; Vamvakas, Eleftherios C.

2015-01-01

Pathogen reduction (PR) systems for platelets, based on chemically induced cross-linking and inactivation of nucleic acids, potentially prevent transfusion transmission of infectious agents, but can increase clinically significant bleeding in some clinical studies. Here, we documented the effects of PR systems on microRNA and mRNA levels of platelets stored in the blood bank, and assessed their impact on platelet activation and function. Unlike platelets subjected to gamma irradiation or stored in additive solution, platelets treated with Intercept (amotosalen + ultraviolet-A [UVA] light) exhibited significantly reduced levels of 6 of the 11 microRNAs, and 2 of the 3 anti-apoptotic mRNAs (Bcl-xl and Clusterin) that we monitored, compared with platelets stored in plasma. Mirasol (riboflavin + UVB light) treatment of platelets did not produce these effects. PR neither affected platelet microRNA synthesis or function nor induced cross-linking of microRNA-sized endogenous platelet RNA species. However, the reduction in the platelet microRNA levels induced by Intercept correlated with the platelet activation (p < 0.05) and an impaired platelet aggregation response to ADP (p < 0.05). These results suggest that Intercept treatment may induce platelet activation, resulting in the release of microRNAs and mRNAs from platelets. The clinical implications of this reduction in platelet nucleic acids secondary to Intercept remain to be established. PMID:24749844

Impact of a pneumatic tube system transport on hemostasis parameters measurement: the experiment of Cochin universitary hospital (AP-HP, Paris, France).

PubMed

Calmette, Leyla; Ibrahim, Firas; Gouin, Isabelle; Horellou, Marie-Hélène; Mazoyer, Élisabeth; Fontenay, Michaela; Flaujac, Claire

2017-02-01

Samples transported by pneumatic tube system are submitted to forces of acceleration and deceleration which can affect laboratory parameters. At Cochin hospital, majority of samples of hemostasis, except for platelets tests, are transported by pneumatic tube system. The objective of this study was to evaluate the impact of a pneumatic tube system (PTS) transport compared to hand-delivered transport on samples and to qualify Cochin hospital PTS according to requirements of standard ISO 15189. A bibliographical study was made and showed that pneumatic tube system particularly influences platelets tests. Four citrate tubes were collected in 5 healthy volunteers in the maternity: 2 tubes were transported by PTS and 2 others were hand-delivered to the laboratory. Five coagulation tests were analyzed: prothrombine time (PT), activated partial thromboplastin time (aPTT), factor (F) V, FVIII and platelet closure time with PFA-100TM collagen/epinephrine. For each volunteer, the results obtained by PTS and by hand-delivered transport were compared with formula usually used for biological analysis retake: 2.8 x standard deviation of reproductibility variation coefficient (SH GTA 01, COFRAC). This study did not show an impact of PTS on PT, aPTT, FV and FVIII. For PFA-100TM collagen/epinephrine, we noted an impact on 2/5 volunteers. These results, in agreement with the literature, led to the conclusion that Cochin hospital PTS is in compliance to transport samples for usual coagulation tests except platelet tests. This study allowed to issue French recommendations for PTS transport of hemostasis tubes qualification available on "Groupe français d'hémostase et thrombose" Web site.

Evaluation of Plasma Platelet Microparticles in Thrombotic Thrombocytopenic Purpura.

PubMed

Tahmasbi, Leila; Karimi, Mehran; Kafiabadi, Sedigheh Amini; Nikougoftar, Mahin; Haghpanah, Sezaneh; Ranjbaran, Reza; Moghadam, Mohamad

2017-01-01

Platelet microparticles (PMPs) have a procoagulant activity about 50-100 times greater than active platelets due to high expression of negatively charged phospholipids on their surfaces. In this study, we evaluated microparticle immunophenotyping and also plasma PMPs level in patients with Thrombotic Thrombocytopenic Purpura (TTP) in Southern Iran. We had two study groups: 15 TTP patients and 15 healthy control group and PMPs from platelet concentrate (PC) at the 5 th day of storage. Microparticles were prepared in two steps, by low and high centrifugation followed by size confirmation via 'Dynamic Light Scattering (DLS)' Zetasizer. Immunophenotyping of PMPs was done via flow cytometry, using a FACS Calibur flow cytometer (BD, USA). PMPs counts were obtained using Partec-cyflow and Polysciences Microbeads (1 micron in diameter). Results were analyzed using FlowJo 7.6 (Treestar, USA) and Partec FlowMax software. Our results showed that the majority of microparticles in TTP patients and normal individuals were PMPs and also demonstrated that the plasma PMPs level in TTP patients was higher than the normal control group ( P -value<0.001). It seems that elevated PMPs level in TTP patients could be related to thrombotic events. Nevertheless, more studies are needed to confirm these results. © 2017 by the Association of Clinical Scientists, Inc.

Platelets generated from human embryonic stem cells are functional in vitro and in the microcirculation of living mice

PubMed Central

Lu, Shi-Jiang; Li, Feng; Yin, Hong; Feng, Qiang; Kimbrel, Erin A; Hahm, Eunsil; Thon, Jonathan N; Wang, Wei; Italiano, Joseph E; Cho, Jaehyung; Lanza, Robert

2011-01-01

Platelets play an essential role in hemostasis and atherothrombosis. Owing to their short storage time, there is constant demand for this life-saving blood component. In this study, we report that it is feasible to generate functional megakaryocytes and platelets from human embryonic stem cells (hESCs) on a large scale. Differential-interference contrast and electron microscopy analyses showed that ultrastructural and morphological features of hESC-derived platelets were indistinguishable from those of normal blood platelets. In functional assays, hESC-derived platelets responded to thrombin stimulation, formed microaggregates, and facilitated clot formation/retraction in vitro. Live cell microscopy demonstrated that hESC-platelets formed lamellipodia and filopodia in response to thrombin activation, and tethered to each other as observed in normal blood. Using real-time intravital imaging with high-speed video microscopy, we have also shown that hESC-derived platelets contribute to developing thrombi at sites of laser-induced vascular injury in mice, providing the first evidence for in vivo functionality of hESC-derived platelets. These results represent an important step toward generating an unlimited supply of platelets for transfusion. Since platelets contain no genetic material, they are ideal candidates for early clinical translation involving human pluripotent stem cells. PMID:21221130

N-octanoyl-dopamine is a potent inhibitor of platelet function.

PubMed

Ait-Hsiko, Lamia; Kraaij, Tineke; Wedel, Johannes; Theisinger, Bastian; Theisinger, Sonja; Yard, Benito; Bugert, Peter; Schedel, Angelika

2013-01-01

Dopamine (DA) is a co-agonist for platelet activation; yet, donor DA treatment is associated with improved transplantation outcome in renal and heart recipients. Recently, N-octanoyl-dopamine (NOD) was developed which displays superior effects compared to DA in terms of graft protecting properties. Whereas DA is a known platelet co-agonist, the effect of NOD on platelet function is unknown. This is a hypothesis generating study with the aim to assess the effects and molecular mechanisms of NOD and NOD-like compounds on platelet function. The influence of DA, NOD, and NOD-like compounds on platelet responses to classical agonists (adenosine 5'-diphosphate (ADP), U46619) was investigated in six healthy donors by applying whole blood aggregometry (Multiplate®) and flow cytometry for Pac-1, CD62P, and CD63 expression. Changes in platelet cAMP concentrations were assessed by ELISA. While DA showed synergy in platelet activation by ADP and U46619, NOD caused significant inhibition of platelet function both in whole blood aggregometry and flow cytometry. The inhibitory effect of NOD was not mediated via cAMP levels. The nonredox-active NOD-analog N-octanoyl-tyramine had no effects on platelet function. Acetylated NOD conferred to NOD by intracellular esterases showed similar inhibitory effects as NOD. In contrast to DA, NOD is a potent inhibitor of platelet function most likely through intracellular redox-active processes. This adds to the overall protective effect of NOD on pre-transplantation injury and makes NOD an attractive candidate compound for donor or organ conditioning prior to transplantation.

JAM-A protects from thrombosis by suppressing integrin αIIbβ3-dependent outside-in signaling in platelets

PubMed Central

Naik, Meghna U.; Stalker, Timothy J.; Brass, Lawrence F.

2012-01-01

Mounting evidence suggests that agonist-initiated signaling in platelets is closely regulated to avoid excessive responses to injury. A variety of physiologic agonists induce a cascade of signaling events termed as inside-out signaling that culminate in exposure of high-affinity binding sites on integrin αIIbβ3. Once platelet activation has occurred, integrin αIIbβ3 stabilizes thrombus formation by providing agonist-independent “outside-in” signals mediated in part by contractile signaling. Junctional adhesion molecule A (JAM-A), a member of the cortical thymocyte marker of the Xenopus (CTX) family, was initially identified as a receptor for a platelet stimulatory mAb. Here we show that JAM-A in resting platelets functions as an endogenous inhibitor of platelet function. Genetic ablation of Jam-A in mice enhances thrombotic function of platelets in vivo. The absence of Jam-A results in increase in platelet aggregation ex vivo. This gain of function is not because of enhanced inside-out signaling because granular secretion, Thromboxane A2 (TxA2) generation, as well as fibrinogen receptor activation, are normal in the absence of Jam-A. Interestingly, integrin outside-in signaling such as platelet spreading and clot retraction is augmented in Jam-A–deficient platelets. We conclude that JAM-A normally limits platelet accumulation by inhibiting integrin outside-in signaling thus preventing premature platelet activation. PMID:22271446

Inhibition of cyclooxygenase-independent platelet aggregation by sodium salicylate.

PubMed

Violi, F; Alessandri, C; Praticò, D; Guzzo, A; Ghiselli, A; Balsano, F

1989-06-15

The effect of acetylsalicylic acid (ASA) on platelet aggregation (PA) and thromboxane A2 (TxA2) formation was investigated in vitro and ex vivo after 1 g or 300 mg ASA administration to healthy subjects. 50-100 microM ASA inhibited PA by single aggregating agent such as platelet aggregating factor (PAF) or epinephrine and reduced to less than or equal to 5% of control platelet TxB2 formation, but did not influence PA by epinephrine plus PAF. The latter was inhibited by increasing ASA concentration. In samples incubated with 100 microM ASA and stimulated with epinephrine plus PAF, PA could be inhibited by the addition of 100-300 microM sodium salicylate. After 300 mg-1 g ASA administration to healthy subjects, the inhibition of PA by epinephrine plus PAF was more marked by highest doses of ASA. This study suggests that aspirin inhibits PA with a cyclooxygenase-independent mechanism; this effect is mediated, at least in vitro, by salicylic acid.

Rare platelet GPCR variants: what can we learn?

PubMed

Nisar, S P; Jones, M L; Cunningham, M R; Mumford, A D; Mundell, S J

2015-07-01

Platelet-expressed GPCRs are critical regulators of platelet function. Pharmacological blockade of these receptors forms a powerful therapeutic tool in the treatment and prevention of arterial thrombosis associated with coronary atherosclerosis and ischaemic stroke. However, anti-thrombotic drug therapy is associated with high inter-patient variability in therapeutic response and adverse bleeding side effects. In order to optimize the use of existing anti-platelet drugs and to develop new therapies, more detailed knowledge is required relating to the molecular mechanisms that regulate GPCR and therefore platelet function. One approach has been to identify rare, function-disrupting mutations within key platelet proteins in patients with bleeding disorders. In this review, we describe how an integrated functional genomics strategy has contributed important structure-function information about platelet GPCRs with specific emphasis upon purinergic and thromboxane A2 receptors. We also discuss the potential implications these findings have for pharmacotherapy and for understanding the molecular basis of mild bleeding disorders. © 2014 The British Pharmacological Society.

Mechanism of platelet functional changes and effects of anti-platelet agents on in vivo hemostasis under different gravity conditions.

PubMed

Li, Suping; Shi, Quanwei; Liu, Guanglei; Zhang, Weilin; Wang, Zhicheng; Wang, Yuedan; Dai, Kesheng

2010-05-01

Serious thrombotic and hemorrhagic problems or even fatalities evoked by either microgravity or hypergravity occur commonly in the world. We recently reported that platelet functions are inhibited in microgravity environments and activated under high-G conditions, which reveals the pathogenesis for gravity change-related hemorrhagic and thrombotic diseases. However, the mechanisms of platelet functional variations under different gravity conditions remain unclear. In this study we show that the amount of filamin A coimmunoprecipitated with GPIbalpha was enhanced in platelets exposed to modeled microgravity and, in contrast, was reduced in 8 G-exposed platelets. Hypergravity induced actin filament formation and redistribution, whereas actin filaments were reduced in platelets treated with modeled microgravity. Furthermore, intracellular Ca2+ levels were elevated by hypergravity. Pretreatment of platelets with the cell-permeable Ca2+ chelator BAPTA-AM had no effect on cytoskeleton reorganization induced by hypergravity but significantly reduced platelet aggregation induced by ristocetin/hypergravity. Two anti-platelet agents, aspirin and tirofiban, effectively reversed the shortened tail bleeding time and reduced the death rate of mice exposed to hypergravity. Furthermore, the increased P-selectin surface expression was obviously reduced in platelets from mice treated with aspirin/hypergravity compared with those from mice treated with hypergravity alone. These data suggest that the actin cytoskeleton reorganization and intracellular Ca2+ level play key roles in the regulation of platelet functions in different gravitational environments. The results with anti-platelet agents not only further confirm the activation of platelets in vivo but also suggest a therapeutic potential for hypergravity-induced thrombotic diseases.

Pathophysiological consequences of receptor mistraffic: Tales from the platelet P2Y12 receptor.

PubMed

Cunningham, Margaret R; Aungraheeta, Riyaad; Mundell, Stuart J

2017-07-05

Genetic variations in G protein-coupled receptor (GPCR) genes can disrupt receptor function in a wide variety of human genetic diseases, including platelet bleeding disorders. Platelets are critical for haemostasis with inappropriate platelet activation leading to the development of arterial thrombosis, which can result in heart attack and stroke whilst decreased platelet activity is associated with an increased risk of bleeding. GPCRs expressed on the surface of platelets play key roles in regulating platelet activity and therefore function. Receptors include purinergic receptors (P2Y 1 and P2Y 12 ), proteinase-activated receptor (PAR1 and PAR4) and thromboxane receptors (TPα), among others. Pharmacological blockade of these receptors forms a powerful therapeutic tool in the treatment and prevention of arterial thrombosis. With the advance of genomic technologies, there has been a substantial increase in the identification of naturally occurring rare and common GPCR variants. These variants include single-nucleotide polymorphisms (SNPs) and insertion or deletions that have the potential to alter GPCR expression or function. A number of defects in platelet GPCRs that disrupt receptor function have now been characterized in patients with mild bleeding disorders. This review will focus on rare, function-disrupting variants of platelet GPCRs with particular emphasis upon mutations in the P2Y 12 receptor gene that affect receptor traffic to modulate platelet function. Further this review will outline how the identification and characterization of function-disrupting GPCR mutations provides an essential link in translating our detailed understanding of receptor traffic and function in cell line studies into relevant human biological systems. Copyright © 2017. Published by Elsevier B.V.

Platelet count recovery after intravenous immunoglobulin predicts a favorable outcome in children with immune thrombocytopenia

PubMed Central

Ji, Mi Hong; Kim, Sung Jin; Ahn, Hyo Seop

2016-01-01

Background Childhood immune thrombocytopenic purpura (ITP) is a common acquired bleeding disorder. Even though most children recover, either spontaneously or with therapy, 10-20% of newly diagnosed ITP cases have a chronic course beyond 12 months. This study evaluated whether clinical and laboratory findings can predict the response to intravenous immunoglobulin (IVIG) and progression to persistent or chronic ITP in children. Methods During the period between March 2003 and June 2015, we retrospectively analyzed 72 children, newly diagnosed with ITP, who received IVIG treatment. Peripheral blood counts were obtained at diagnosis and at 1, 3, 6, and 12 months after IVIG treatment. Results After 6 months of IVIG treatment, 14 of 72 patients (19.4%) had persistent ITP, and after 12 months, 7 of 40 patients (17.5%) had chronic ITP. Age at diagnosis, gender, history of viral infection, or vaccination before disease onset were not statistically correlated with platelet recovery at 6 and 12 months. However, a platelet count recovery of ≥100×103/µL at 1 and 3 months was significantly correlated with platelet recovery at 6 (P<0.001 and P<0.001, respectively) and 12 (P=0.007 and P=0.004, respectively) months. Conclusion This study demonstrated that early platelet count recovery, at 1 and 3 months after IVIG treatment, predicts a short disease duration and a favorable outcome in children with newly diagnosed ITP. Further investigation in a larger group of patients is warranted to validate these findings. PMID:27382553

Contact activation of blood coagulation on a defined kaolin/collagen surface in a microfluidic assay.

PubMed

Zhu, Shu; Diamond, Scott L

2014-12-01

Generation of active Factor XII (FXIIa) triggers blood clotting on artificial surfaces and may also enhance intravascular thrombosis. We developed a patterned kaolin (0 to 0.3 pg/μm(2))/type 1 collagen fibril surface for controlled microfluidic clotting assays. Perfusion of whole blood (treated only with a low level of 4 μg/mL of the XIIa inhibitor, corn trypsin inhibitor) drove platelet deposition followed by fibrin formation. At venous wall shear rate (100 s(-1)), kaolin accelerated onset of fibrin formation by ~100 sec when compared to collagen alone (250 sec vs. 350 sec), with little effect on platelet deposition. Even with kaolin present, arterial wall shear rate (1000 s(-1)) delayed and suppressed fibrin formation compared to venous wall shear rate. A comparison of surfaces for extrinsic activation (tissue factor TF/collagen) versus contact activation (kaolin/collagen) that each generated equal platelet deposition at 100 s(-1) revealed: (1) TF surfaces promoted much faster fibrin onset (at 100 sec) and more endpoint fibrin at 600 sec at either 100 s(-1) or 1000 s(-1), and (2) kaolin and TF surfaces had a similar sensitivity for reduced fibrin deposition at 1000 s(-1) (compared to fibrin formed at 100 s(-1)) despite differing coagulation triggers. Anti-platelet drugs inhibiting P2Y1, P2Y12, cyclooxygenase-1 or activating IP-receptor or guanylate cyclase reduced platelet and fibrin deposition on kaolin/collagen. Since FXIIa or FXIa inhibition may offer safe antithrombotic therapy, especially for biomaterial thrombosis, these defined collagen/kaolin surfaces may prove useful in drug screening tests or in clinical diagnostic assays of blood under flow conditions. Copyright © 2014 Elsevier Ltd. All rights reserved.

Prealbumin, platelet factor 4 and S100A12 combination at baseline predicts good response to TNF alpha inhibitors in Rheumatoid Arthritis.

PubMed

Nguyen, Minh Vu Chuong; Baillet, Athan; Romand, Xavier; Trocmé, Candice; Courtier, Anaïs; Marotte, Hubert; Thomas, Thierry; Soubrier, Martin; Miossec, Pierre; Tébib, Jacques; Grange, Laurent; Toussaint, Bertrand; Lequerré, Thierry; Vittecoq, Olivier; Gaudin, Philippe

2018-06-06

Tumour necrosis factor-alpha inhibitors (TNFi) are effective treatments for Rheumatoid Arthritis (RA). Responses to treatment are barely predictable. As these treatments are costly and may induce a number of side effects, we aimed at identifying a panel of protein biomarkers that could be used to predict clinical response to TNFi for RA patients. Baseline blood levels of C-reactive protein, platelet factor 4, apolipoprotein A1, prealbumin, α1-antitrypsin, haptoglobin, S100A8/A9 and S100A12 proteins in bDMARD naive patients at the time of TNFi treatment initiation were assessed in a multicentric prospective French cohort. Patients fulfilling good EULAR response at 6 months were considered as responders. Logistic regression was used to determine best biomarker set that could predict good clinical response to TNFi. A combination of biomarkers (prealbumin, platelet factor 4 and S100A12) was identified and could predict response to TNFi in RA with sensitivity of 78%, specificity of 77%, positive predictive values (PPV) of 72%, negative predictive values (NPV) of 82%, positive likelihood ratio (LR+) of 3.35 and negative likelihood ratio (LR-) of 0.28. Lower levels of prealbumin and S100A12 and higher level of platelet factor 4 than the determined cutoff at baseline in RA patients are good predictors for response to TNFi treatment globally as well as to Infliximab, Etanercept and Adalimumab individually. A multivariate model combining 3 biomarkers (prealbumin, platelet factor 4 and S100A12) accurately predicted response of RA patients to TNFi and has potential in a daily practice personalized treatment. Copyright © 2018. Published by Elsevier Masson SAS.

Thrombocytopenia and Platelet Dysfunction in Acute Tropical Infectious Diseases.

PubMed

Hapsari Putri, Indri; Tunjungputri, Rahajeng N; De Groot, Philip G; van der Ven, Andre J; de Mast, Quirijn

2018-06-18

Thrombocytopenia is a well-known manifestation of acute tropical infectious diseases. The role of platelets in infections has received much attention recently because of their emerging activities in modulation of inflammatory responses, host defense, and vascular integrity. However, while many studies have addressed thrombocytopenia in tropical infections, abnormalities in platelet function have been largely overlooked. This is an important research gap, as platelet dysfunction may contribute to the bleeding tendency that characterizes some tropical infections. The development of novel platelet function assays that can be used in thrombocytopenic conditions (e.g., flow cytometry assays) has contributed to important new insights in recent years. In this review, the importance of platelets in tropical infections is discussed with special emphasis on the underlying mechanisms and consequences of thrombocytopenia and platelet dysfunction in these infections. Special attention is paid to malaria, a disease characterized by microvascular obstruction in which bleeding is rare, and to infections in which bleeding is common, such as dengue, other viral hemorrhagic fevers, and the bacterial infection leptospirosis. Given the importance of platelet function abnormalities in these infections, the development of affordable assays for monitoring of platelet function in low-resource countries, as well as pharmacologic interventions to prevent or reverse platelet function abnormalities, might improve clinical care and the prognosis of these infections. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

Role of platelet transfusion in children with bleeding in dengue fever.

PubMed

Pothapregada, Sriram; Kamalakannan, Banupriya; Thulasingam, Mahalakshmy

2015-12-01

The indications for platelet transfusion in dengue fever are clearly defined in World Health Organization (WHO) guidelines (2011) for dengue fever, but physicians face practical difficulty in its implementation in an epidemic setting. On one hand there is an intense social pressure created by the panic-struck parents to transfuse platelets in presence of bleeding and on the other hand there is a need for its judicious use as the requirement is more than its availability. The study was aimed to assess the clinico-hematological parameters, and the requirement and need for platelet transfusion in children with dengue fever. All children (0-12 yr of age) diagnosed and confirmed with dengue fever at a tertiary care hospital in Puducherry between 1 August 2012 and 31 January 2015 were reviewed retrospectively from hospital case records as per the revised WHO guidelines for dengue fever. The diagnosis was confirmed by NS1 antigen- based ELISA test or dengue serology for IgM and IgG antibodies and the data were analyzed using SPSS 16.0 statistical software. Out of 261 cases of dengue fever, hemorrhagic manifestations were observed in 52 children (19.9%), which mainly included petechiae (38.5%), gum bleeding (34.6%) and melena (26.9%). Thrombocytopenia was seen in 211 (80.8%) cases. Bleeding manifestations were present in 20(39.2%), 8(15.7%), 13(25.5%) and 11(21.6%) cases with platelet count <50,000/mm3, 50,000-100,000/mm3, 1-1.50,000/mm3, and >1.50,000/mm3 respectively. Bleeding manifestations did not always correlate with platelet count in non-severe dengue infection in comparison to severe dengue infection. The most common mode of presentation of severe dengue infection was shock with 102(39.1%) cases and among them only 22 children (21.6%) had bleeding. About 17 children (6.5%) with severe dengue infection required platelet transfusion and out of them, 12 children (70.6%) had a platelet count <20,000/ mm3 whereas five children (29.4%) had platelet count in the range of 20,000-50,000/mm3. Platelet transfusion was required in children with severe dengue infection in the form of significant spontaneous bleed, shock and severe thrombocytopenia. Bleeding should not be considered only indicator to transfuse platelets as it occurred in children even with normal platelet counts. The community and treating physicians should be educated regarding the judicious transfusion of platelets. Unnecessary and empirical use of platelets should be completely avoided especially during an epidemic when there is scarcity in its availability.

Platelet activation, adhesion, inflammation, and aggregation potential are altered in the presence of electronic cigarette extracts of variable nicotine concentrations.

PubMed

Hom, Sarah; Chen, Li; Wang, Tony; Ghebrehiwet, Berhane; Yin, Wei; Rubenstein, David A

2016-11-01

Tobacco smoke extracts prepared from both mainstream and sidestream smoking have been associated with heightened platelet activation, aggregation, adhesion, and inflammation. Conversely, it has been shown that pure nicotine inhibits similar platelet functions. In this work, we 1) evaluated the effects of e-cigarette extracts on platelet activities and 2) elucidated the differences between the nicotine-dependent and non-nicotine dependent (e.g. fine particulate matter or toxic compounds) effects of tobacco and e-cigarette products on platelet activities. To accomplish these goals, platelets from healthy volunteers (n = 50) were exposed to tobacco smoke extracts, e-cigarette vapor extracts, and pure nicotine and changes in platelet activation, adhesion, aggregation, and inflammation were evaluated, using optical aggregation, flow cytometry, and ELISA methods. Interestingly, the exposure of platelets to e-vapor extracts induced a significant up-regulation in the expression of the pro-inflammatory gC1qR and cC1qR and induced a marked increase in the deposition of C3b as compared with traditional tobacco smoke extracts. Similarly, platelet activation, as measured by a prothrombinase based assay, and platelet aggregation were also significantly enhanced after exposure to e-vapor extracts. Finally, platelet adhesion potential toward fibrinogen, von Willebrand factor, and other platelets was also enhanced after exposure to e-cigarette vapor extracts. In the presence of pure nicotine, platelet functions were observed to be inhibited, which further suggests that other constituents of tobacco smoke and electronic vapor can antagonize platelet functions, however, the presence of nicotine in extracts somewhat perpetuated the platelet functional changes in a dose-dependent manner.

The influence of aspirin dose and glycemic control on platelet inhibition in patients with type 2 diabetes mellitus.

PubMed

Lemkes, B A; Bähler, L; Kamphuisen, P W; Stroobants, A K; Van Den Dool, E J; Hoekstra, J B; Nieuwland, R; Gerdes, V E; Holleman, F

2012-04-01

Low-dose aspirin seems to offer no benefit in the primary prevention of cardiovascular disease in type 2 diabetes mellitus (DM2). The anti-platelet effect may be diminished by poor glycemic control or inadequate dosing of aspirin. To study the effects of both glycemic control and increasing aspirin dose on platelet response to aspirin in DM2 patients and matched controls. Platelet effects of increasing doses of aspirin (30, 100 and 300 mg daily) were prospectively assessed in 94 DM2 patients and 25 matched controls by measuring thromboxane levels in urine (11-dhTxB2) and platelet aggregation using VerifyNow(®) and light transmission aggregometry (LTA). DM2 patients were stratified for glycemic control (hemoglobin-A1c [HbA1c] ≤ 53, 53-69, ≥ 69 mmol mol(-1)). At baseline, median 11-dhTxB2 excretion was higher in the poorly controlled patients (77 ng mmol(-1)), and the moderately controlled (84 ng mmol(-1)) compared with the well-controlled patients (64 ng mmol(-1)) and controls (53 ng mmol(-1)), P < 0.01. Next, 30 mg of aspirin reduced 11-dhTxB2 excretion to 31, 29 and 24 ng mmol(-1) in the poorly, moderately and well-controlled patients, respectively, and to 19 ng mmol(-1) in controls, P < 0.001. VerifyNow(®) and LTA were also incompletely suppressed in DM2 patients using 30 mg of aspirin, but 100 mg resulted in similar platelet suppression in all groups, with no additional effect of 300 mg. DM2 patients with inadequate glycemic control (HbA1c > 53 mmol mol(-1)) have higher baseline platelet activity and incomplete suppression of platelet activity with 30 mg of aspirin. However, 100 mg of aspirin leads to optimal inhibition irrespective of glycemic control, and 300 mg does not further improve platelet suppression. © 2012 International Society on Thrombosis and Haemostasis.

Effects of escalating doses of tirofiban on platelet aggregation and major receptor expression in diabetic patients: hitting the TARGET in the TENACITY trial?

PubMed

Serebruany, Victor; Malinin, Alex; Pokov, Alex; Arora, Umesh; Atar, Dan; Angiolillo, Dominick

2007-01-01

Ongoing search for the optimal dosing regimens, and valid concerns that some GPIIb/IIIa inhibitors may cause rebound platelet activation are limiting the use of these agents in patients with acute vascular events. We assessed the in vitro effects of preincubation with escalating (12.5-200 ng/mL) concentrations of tirofiban on platelet biomarkers in 20 diabetic patients. Platelet activity was assessed by ADP-, and collagen-induced conventional plasma aggregometry, and by whole blood flow cytometry measuring expression of PECAM-1, GPIb, GP IIb/IIIa antigen and activity, vitronectin, P-selectin, LAMP-1, GP 37, LAMP-3, activated and intact PAR-1 thrombin receptors, GPIV, and platelet-monocyte formation. All patients were treated with aspirin (at least 81 mg daily for 1 month); other antiplatelet agents were not allowed. Significant decrease of ADP-induced platelet aggregation was observed starting at the low 12.5 ng/mL concentration (p=0.0001), with total inhibition occurring at 50 ng/mL of tirofiban dose. Inhibition of collagen-induced platelet aggregability requires 25 ng/ml of tirofiban (p=0.002), and was complete at 100 ng/mL. Dose-dependent blockade of GP IIb/IIIa activity was observed with tirofiban concentrations over 50 ng/mL (p=0.003). Other receptors were unaffected even with the high doses of tirofiban (100-200 ng/mL). Tirofiban completely inhibits ADP- and, with the higher dose, collagen-induced platelet aggregation. Higher loading dose of tirofiban used in the ongoing TENACITY trial (100 ng/mL) may be superior with regard to clinical outcomes to the regimens used in PRISM-PLUS (25 ng/mL), or TARGET (50 ng/mL). Selective inhibition of GPIIb/IIIa activity, and lack of alternative platelet activation beyond the GP IIb/IIIa blockade may represent the therapeutic advantage of tirofiban over other agents.

Update: Acute coronary syndromes (V). Personalized antiplatelet therapy.

PubMed

Gurbel, Paul A; Rafeedheen, Rahil; Tantry, Udaya S

2014-06-01

It is well established that high on-treatment platelet reactivity to adenosine diphosphate during clopidogrel therapy is an independent risk factor for ischemic event occurrences in a postpercutaneous coronary intervention patients. However, the precise role of platelet function testing remains debated. Platelet function testing to ensure optimal platelet inhibition has been recommended by some authorities to improve outcomes in patients treated with clopidogrel. Recent prospective, randomized trials of personalized antiplatelet therapy have failed to demonstrate a benefit of platelet function testing in improving outcomes. In this review article, we discuss the mechanisms responsible for clopidogrel nonreponsiveness, recent trials of platelet function testing, and other new developments in the field of personalized antiplatelet therapy. Copyright © 2014 Sociedad Española de Cardiología. Published by Elsevier Espana. All rights reserved.

Monitoring ASA and P2Y12-specific platelet inhibition--comparison of conventional (single) and multiple electrode aggregometry.

PubMed

Krüger, Jan-Christopher; Meves, Saskia H; Kara, Kaffer; Mügge, Andreas; Neubauer, Horst

2014-10-01

Several platelet function test systems exist for the evaluation of the platelet inhibitory effect in patients on P2Y12 inhibitors and/or acetylsalicylic acid (ASA, aspirin) therapy. Studies comparing different available assays found only a poor correlation. The objective of the present study was to evaluate the correlation and agreement between single electrode (SEA) and multiple electrode (MEA) aggregometry. In whole blood arachidonic acid (AA) and adenosine diphosphate (ADP)-induced platelet aggregation was measured simultaneously using SEA (Chrono-Log) and MEA (Multiplate). We analyzed a total of 226 measurements taken from 58 patients on single ASA therapy or dual antiplatelet therapy with ASA and a thienopyridine. A cut-off value for clopidogrel/prasugrel high on-treatment platelet reactivity (HPR) of > 47 units (U) was chosen for MEA testing using hirudin and > 5 Ohm for SEA with citrate anticoagulated blood samples. The respective cut-off values for ASA HPR were > 30 U for the MEA assay and > 1 Ohm for SEA testing. There was a good correlation of the prevalence of thienopyridine-HPR in both whole blood assays (Spearman rank correlation coefficient r = 0.698) and a good inter-rate accordance (Cohen's Kappa statistic κ = 0.648). For AA-induced aggregation, the correlation of the results obtained was significant (r = 0.536; p < 0.001) and detecting ASA-HPR revealed a moderate (κ = 0.482) correlation between both impedance aggregometry assays. Platelet function testing using SEA and MEA provided both good accordance and correlation and therefore study results obtained by these two assays similarly enabled the detection of HPR of thienopyridine (and ASA) therapy.

Enhanced generation of megakaryocytes from umbilical cord blood-derived CD34(+) cells expanded in the presence of two nutraceuticals, docosahexanoic acid and arachidonic acid, as supplements to the cytokine-containing medium.

PubMed

Siddiqui, Nikhat Firdaus A; Shabrani, Namrata C; Kale, Vaijayanti P; Limaye, Lalita S

2011-01-01

Ex vivo generation of megakaryocytes (MK) from hematopoietic stem cells (HSC) is important for both basic research, to understand the mechanism of platelet biogenesis, and clinical infusions, for rapid platelet recovery in thrombocytopenic patients. We investigated the role of two nutraceuticals, docosahexanoic acid (DHA) and arachidonic acid (AA), in the in vitro generation of MK. Umbilical cord blood (UCB)-derived CD34+cells were cultured with stem cell factor (SCF) and thrombopoietin (TPO) in the presence (test) or absence (control) of the two additives. On day 10, MK and platelets generated were quantitated by morphologic, phenotypic and functional assays. The cell yield of MK and platelet numbers were significantly higher in test compared with control cells. Phenotypic analyzes and gene expression profiles confirmed these findings. Functional properties, such as colony-forming unit (CFU)-MK formation, chemotaxis and platelet activation, were found to be enhanced in cells cultured with nutraceuticals. The engraftment potential of ex vivo-expanded cells was studied in NOD/SCID mice. Mice that received MK cultured in the presence of DHA/AA engrafted better. There was a reduction in apoptosis and total reactive oxygen species (ROS) levels in the CD41(+) compartment of the test compared with control sets. The data suggest that these compounds probably exert their beneficial effect by modulating apoptotic and redox pathways. Use of nutraceuticals like DHA and AA may prove to be a useful strategy for efficient generation of MK and platelets from cord blood cells, for future use in clinics and basic research.

Effects of in vitro supplementation with Syzygium cumini (L.) on platelets from subjects affected by diabetes mellitus.

PubMed

Raffaelli, Francesca; Borroni, Francesca; Alidori, Alessandro; Tirabassi, Giacomo; Faloia, Emanuela; Rabini, Rosa Anna; Giulietti, Alessia; Mazzanti, Laura; Nanetti, Laura; Vignini, Arianna

2015-01-01

The aim of this study was to assess the in vitro effects of Syzygium cumini (L.) (Sc) incubation on platelets from patients with diabetes, in order to test its efficacy as a potential adjuvant therapy. This study was performed on 77 patients with diabetes [29 in good (DMgc) and 48 in poor glycemic control (DMpc)] and 85 controls. In patients, platelets were analyzed at recruitment and after in vitro Sc incubation (final concentration of 200 µg/ml for 3 hours at 37 °C), whereas in controls only basal evaluation was performed. Lipoperoxide and nitric oxide (NO) levels, superoxide dismutase (SOD) and Na(+)/K(+) ATPase activities, total antioxidant capacity (TAC), and membrane fluidity tested by anisotropy of fluorescent probes 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 1-6-phenyl-1,3,5-hexatriene (DPH) were determined. Collagen-induced platelet aggregation was also evaluated. In vitro Sc activity counteracts oxidative damage, by improving platelet function through augmented membrane fluidity and Na(+)/K(+) ATPase activity; it also enhances antioxidant system functionality by increasing NO levels, SOD activity, and TAC and by decreasing lipoperoxide levels both in whole samples and in DMgc and DMpc. In addition, a slight tendency towards collagen-induced platelet aggregation decrease after Sc was observed. However, all these parameters, even after improvement, did not reach the levels of control subjects. Our results suggest that Sc may have a preventive and protective effect in oxidative damage progression associated with diabetes mellitus and its complications. If our data will be confirmed, Sc supplementation might become a further tool in the management of this disease, especially in view of its easy availability, safety, low cost, and absence of side effects.

First comparative analysis concerning the plasma platelet contamination during MNC collection.

PubMed

Pfeiffer, Hella; Achenbach, Susanne; Strobel, Julian; Zimmermann, Robert; Eckstein, Reinhold; Strasser, Erwin F

2017-08-01

Monocytes can be cultured into dendritic cells with addition of autologous plasma, which is highly prone to platelet contamination due to the apheresis process. Since platelets affect the maturation process of monocytes into dendritic cells and might even lead to a diminished harvest of dendritic cells, it is very important to reduce the platelet contamination. A new collection device (Spectra Optia) was analyzed, compared to two established devices (COM.TEC, Cobe Spectra) and evaluated regarding the potential generation of source plasma. Concurrent plasma collected during leukapheresis was analyzed for residual cell contamination in a prospective study with the new Spectra Optia apheresis device (n=24) and was compared with COM.TEC and Cobe Spectra data (retrospective analysis, n=72). Donor pre-donation counts of platelets were analyzed for their predictive value of contaminating PLTs in plasma harvests. The newest apheresis device showed the lowest residual platelet count of the collected concurrent plasma (median 3.50×10 9 /l) independent of pre-donation counts. The other two devices and sets had a higher platelet contamination. The contamination of the plasma with leukocytes was very low (only 2.0% were higher than 0.5×10 9 /l). This study showed a significant reduction of platelet contamination of the concurrent plasma collected with the new Spectra Optia device. This plasma product with low residual platelets and leukocytes might also be used as plasma for fractionation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Platelet Aggregometry Testing: Molecular Mechanisms, Techniques and Clinical Implications

PubMed Central

Koltai, Katalin; Kesmarky, Gabor; Feher, Gergely; Tibold, Antal

2017-01-01

Platelets play a fundamental role in normal hemostasis, while their inherited or acquired dysfunctions are involved in a variety of bleeding disorders or thrombotic events. Several laboratory methodologies or point-of-care testing methods are currently available for clinical and experimental settings. These methods describe different aspects of platelet function based on platelet aggregation, platelet adhesion, the viscoelastic properties during clot formation, the evaluation of thromboxane metabolism or certain flow cytometry techniques. Platelet aggregometry is applied in different clinical settings as monitoring response to antiplatelet therapies, the assessment of perioperative bleeding risk, the diagnosis of inherited bleeding disorders or in transfusion medicine. The rationale for platelet function-driven antiplatelet therapy was based on the result of several studies on patients undergoing percutaneous coronary intervention (PCI), where an association between high platelet reactivity despite P2Y12 inhibition and ischemic events as stent thrombosis or cardiovascular death was found. However, recent large scale randomized, controlled trials have consistently failed to demonstrate a benefit of personalised antiplatelet therapy based on platelet function testing. PMID:28820484

[Effects of lysine clonixinate on platelet function. Comparison with other non-steroidal anti-inflammatory agents].

PubMed

Kramer, E H; Sassetti, B; Kaminker, A J; De Los Santos, A R; Martí, M L; Di Girolamo, G

2001-01-01

One of the mechanisms of action of non steroid antiinflammatory drugs (NSAIDs) consists of inhibition of prostaglandin synthesis. This explains many of the pharmacological effects and adverse events observed in medical practice. Administration of NSAIDs to patients with hemostatic disorders or perioperative conditions entails the risk of bleeding due to inhibition of platelet function. This study deals with platelet changes induced by lysine clonixinate vs diclofenac, ibuprofen and aspirin in classical tests such as platelet count, platelet factor 3 (PF3) activity and platelet aggregation with various inductors and more recent procedures such as P-selectin measurement by flow cytometry. Unlike control drugs, lysine clonixinate did not induce changes in platelet count or function when administered to healthy volunteers at the commonly used therapeutic doses.

Thioredoxin Inhibitors Attenuate Platelet Function and Thrombus Formation

PubMed Central

Metcalfe, Clive; Ramasubramoni, Anjana; Pula, Giordano; Harper, Matthew T.; Mundell, Stuart J.; Coxon, Carmen H.

2016-01-01

Thioredoxin (Trx) is an oxidoreductase with important physiological function. Imbalances in the NADPH/thioredoxin reductase/thioredoxin system are associated with a number of pathologies, particularly cancer, and a number of clinical trials for thioredoxin and thioredoxin reductase inhibitors have been carried out or are underway. Due to the emerging role and importance of oxidoreductases for haemostasis and the current interest in developing inhibitors for clinical use, we thought it pertinent to assess whether inhibition of the NADPH/thioredoxin reductase/thioredoxin system affects platelet function and thrombosis. We used small molecule inhibitors of Trx (PMX 464 and PX-12) to determine whether Trx activity influences platelet function, as well as an unbiased proteomics approach to identify potential Trx substrates on the surface of platelets that might contribute to platelet reactivity and function. Using LC-MS/MS we found that PMX 464 and PX-12 affected the oxidation state of thiols in a number of cell surface proteins. Key surface receptors for platelet adhesion and activation were affected, including the collagen receptor GPVI and the von Willebrand factor receptor, GPIb. To experimentally validate these findings we assessed platelet function in the presence of PMX 464, PX-12, and rutin (a selective inhibitor of the related protein disulphide isomerase). In agreement with the proteomics data, small molecule inhibitors of thioredoxin selectively inhibited GPVI-mediated platelet activation, and attenuated ristocetin-induced GPIb-vWF-mediated platelet agglutination, thus validating the findings of the proteomics study. These data reveal a novel role for thioredoxin in regulating platelet reactivity via proteins required for early platelet responses at sites of vessel injury (GPVI and GPIb). This work also highlights a potential opportunity for repurposing of PMX 464 and PX-12 as antiplatelet agents. PMID:27716777

Thioredoxin Inhibitors Attenuate Platelet Function and Thrombus Formation.

PubMed

Metcalfe, Clive; Ramasubramoni, Anjana; Pula, Giordano; Harper, Matthew T; Mundell, Stuart J; Coxon, Carmen H

2016-01-01

Thioredoxin (Trx) is an oxidoreductase with important physiological function. Imbalances in the NADPH/thioredoxin reductase/thioredoxin system are associated with a number of pathologies, particularly cancer, and a number of clinical trials for thioredoxin and thioredoxin reductase inhibitors have been carried out or are underway. Due to the emerging role and importance of oxidoreductases for haemostasis and the current interest in developing inhibitors for clinical use, we thought it pertinent to assess whether inhibition of the NADPH/thioredoxin reductase/thioredoxin system affects platelet function and thrombosis. We used small molecule inhibitors of Trx (PMX 464 and PX-12) to determine whether Trx activity influences platelet function, as well as an unbiased proteomics approach to identify potential Trx substrates on the surface of platelets that might contribute to platelet reactivity and function. Using LC-MS/MS we found that PMX 464 and PX-12 affected the oxidation state of thiols in a number of cell surface proteins. Key surface receptors for platelet adhesion and activation were affected, including the collagen receptor GPVI and the von Willebrand factor receptor, GPIb. To experimentally validate these findings we assessed platelet function in the presence of PMX 464, PX-12, and rutin (a selective inhibitor of the related protein disulphide isomerase). In agreement with the proteomics data, small molecule inhibitors of thioredoxin selectively inhibited GPVI-mediated platelet activation, and attenuated ristocetin-induced GPIb-vWF-mediated platelet agglutination, thus validating the findings of the proteomics study. These data reveal a novel role for thioredoxin in regulating platelet reactivity via proteins required for early platelet responses at sites of vessel injury (GPVI and GPIb). This work also highlights a potential opportunity for repurposing of PMX 464 and PX-12 as antiplatelet agents.

Prediction of Thrombus Growth: Effect of Stenosis and Reynolds Number.

PubMed

Hosseinzadegan, Hamid; Tafti, Danesh K

2017-06-01

Shear stresses play a major role in platelet-substrate interactions and thrombus formation and growth in blood flow, where under both pathological and physiological conditions platelet adhesion and accumulation occur. In this study, a shear-dependent continuum model for platelet activation, adhesion and aggregation is presented. The model was first verified under three different shear conditions and at two heparin levels. Three-dimensional simulations were then carried out to evaluate the performance of the model for severely damaged (stripped) aortas with mild and severe stenosis degrees in laminar flow regime. For these cases, linear shear-dependent functions were developed for platelet-surface and platelet-platelet adhesion rates. It was confirmed that the platelet adhesion rate is not only a function of Reynolds number (or wall shear rate) but also the stenosis severity of the vessel. General correlations for adhesion rates of platelets as functions of stenosis and Reynolds number were obtained based on these cases. Finally using the new platelet adhesion rates, the model was applied to different experimental systems and shown to agree well with measured platelet deposition.

Quantitative phase imaging of platelet: assessment of cell morphology and function

NASA Astrophysics Data System (ADS)

Vasilenko, Irina; Vlasova, Elizaveta; Metelin, Vladislav; Agadzhanjan, B.; Lyfenko, R.

2017-02-01

It is well known that platelets play a central role in hemostasis and thrombosis, they also mediate tumor cell growth, dissemination and angiogenesis. The purpose of the present experiment was to evaluate living platelet size, function and morphology simultaneously in unactivated and activated states using Phase-Interference Microscope "Cytoscan" (Moscow, Russia). We enrolled 30 healthy volunteers, who had no past history of aeteriosclerosis-related disorders, such as coronary heart disease, cerebrovascular disease, hypertention, diabetes or hyperlipidemia and 30 patients with oropharynx cancer. We observed the optic-geometrical parameters of each isolated living cell and the distribution of platelets by sizes have been analysed to detect the dynamics of cell population heterogeneity. Simultaneously we identified 4 platelet forms that have different morphological features and different parameters of size distribution. We noticed that morphological platelet types correlate with morphometric platelet parameters. The data of polymorphisms of platelet reactivity in tumor progression can be used to improve patient outcomes in the cancer prevention and treatment. Moreover morphometric and functional platelet parameters can serve criteria of the efficiency of the radio- and chemotherapy carried out. In conclusion the computer phase-interference microscope provides rapid and effective analysis of living platelet morphology and function at the same time. The use of the computer phase-interference microscope could be an easy and fast method to check the state of platelets in patients with changed platelet activation and to follow a possible pharmacological therapy to reduce this phenomenon.

DMSO inhibits human platelet activation through cyclooxygenase-1 inhibition. A novel agent for drug eluting stents?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Asmis, Lars; Tanner, Felix C.; Center for Integrative Human Physiology, University of Zuerich, Zuerich

2010-01-22

Background: DMSO is routinely infused together with hematopoietic cells in patients undergoing myeloablative therapy and was recently found to inhibit smooth muscle cells proliferation and arterial thrombus formation in the mouse by preventing tissue factor (TF), a key activator of the coagulation cascade. This study was designed to investigate whether DMSO prevents platelet activation and thus, whether it may represent an interesting agent to be used on drug eluting stents. Methods and results: Human venous blood from healthy volunteers was collected in citrated tubes and platelet activation was studied by cone and platelet analyzer (CPA) and rapid-platelet-function-assay (RPFA). CPA analysismore » showed that DMSO-treated platelets exhibit a lower adherence in response to shear stress (-15.54 {+-} 0.9427%, n = 5, P < 0.0001 versus control). Additionally, aggregometry studies revealed that DMSO-treated, arachidonate-stimulated platelets had an increased lag phase (18.0% {+-} 4.031, n = 9, P = 0.0004 versus control) as well as a decreased maximal aggregation (-6.388 {+-} 2.212%, n = 6, P = 0.0162 versus control). Inhibitory action of DMSO could be rescued by exogenous thromboxane A2 and was mediated, at least in part, by COX-1 inhibition. Conclusions: Clinically relevant concentrations of DMSO impair platelet activation by a thromboxane A2-dependent, COX-1-mediated effect. This finding may be crucial for the previously reported anti-thrombotic property displayed by DMSO. Our findings support a role for DMSO as a novel drug to prevent not only proliferation, but also thrombotic complications of drug eluting stents.« less

Complement Activation on Platelets Correlates with a Decrease in Circulating Immature Platelets in Patients with Immune Thrombocytopenic Purpura

PubMed Central

Peerschke, Ellinor I.B.; Andemariam, Biree; Yin, Wei; Bussel, James B.

2010-01-01

The role of the complement system in immune thrombocytopenic purpura (ITP) is not well defined. We examined plasma from 79 patients with ITP, 50 healthy volunteers, and 25 patients with non-immune mediated thrombocytopenia, to investigate their complement activation/fixation capacity (CAC) on immobilized heterologous platelets. Enhanced CAC was found in 46 plasma samples (59%) from patients with ITP, but no samples from patients with non-immune mediated thrombocytopenia. Plasma from healthy volunteers was used for comparison. In patients with ITP, an enhanced plasma CAC was associated with a decreased circulating absolute immature platelet fraction (A-IPF) (<15 × 109/L) (p = 0.027) and thrombocytopenia (platelet count less than 100K/μl) (p= 0.024). The positive predictive value of an enhanced CAC for a low A-IPF was 93%, with a specificity of 77%. The specificity and positive predictive values increased to 100% when plasma CAC was defined strictly by enhanced C1q and/or C4d deposition on test platelets. Although no statistically significant correlation emerged between CAC and response to different pharmacologic therapies, an enhanced response to splenectomy was noted (p <0.063). Thus, complement fixation may contribute to the thrombocytopenia of ITP by enhancing clearance of opsonized platelets from the circulation, and/or directly damaging platelets and megakaryocytes. PMID:19925495

An Inherited Platelet Function Defect in Basset Hounds

PubMed Central

Johnstone, I. B.; Lotz, F.

1979-01-01

An inherited platelet function defect occurring in a family of basset hounds has been described. The trait is transmitted as an autosomal characteristic and appears to be expressed clinically only in the homozygous state. The characteristics of this platelet defect include: 1) marked bleeding tendencies and prolonged skin bleeding times in either male or female dogs. 2) normal blood coagulation mechanism. 3) adequate numbers of circulating platelets which appear morphologically normal by light microscopy. 4) normal whole blood clot retraction. 5) deficient in vivo platelet consumption and in vitro platelet retention in glass bead columns. 6) defective ADP-induced platelet aggregation in homozygotes, apparently normal ADP response in heterozygotes, and defective collagen-induced platelet aggregation in both. PMID:509382

The origin and function of platelet glycosyltransferases

PubMed Central

Rumjantseva, Viktoria; Sørensen, Anne Louise Tølbøll; Patel-Hett, Sunita; Josefsson, Emma C.; Bennett, Eric P.; Italiano, Joseph E.; Clausen, Henrik; Hartwig, John H.; Hoffmeister, Karin M.

2012-01-01

Platelets are megakaryocyte subfragments that participate in hemostatic and host defense reactions and deliver pro- and antiangiogenic factors throughout the vascular system. Although they are anucleated cells that lack a complex secretory apparatus with distinct Golgi/endoplasmic reticulum compartments, past studies have shown that platelets have glycosyltransferase activities. In the present study, we show that members of 3 distinct glycosyltransferase families are found within and on the surface of platelets. Immunocytology and flow cytometry results indicated that megakaryocytes package these Golgi-derived glycosyltransferases into vesicles that are sent via proplatelets to nascent platelets, where they accumulate. These glycosyltransferases are active, and intact platelets glycosylate large exogenous substrates. Furthermore, we show that activation of platelets results in the release of soluble glycosyltransferase activities and that platelets contain sufficient levels of sugar nucleotides for detection of glycosylation of exogenously added substrates. Therefore, the results of the present study show that blood platelets are a rich source of both glycosyltransferases and donor sugar substrates that can be released to function in the extracellular space. This platelet-glycosylation machinery offers a pathway to a simple glycoengineering strategy improving storage of platelets and may serve hitherto unknown biologic functions. PMID:22613794

38 CFR 4.117 - Schedule of ratings-hemic and lymphatic systems.

Code of Federal Regulations, 2014 CFR

2014-07-01

... platelets or red cells at least once every six weeks, or; infections recurring at least once every six weeks 100 Requiring transfusion of platelets or red cells at least once every three months, or; infections recurring at least once every three months 60 Requiring transfusion of platelets or red cells at least once...

38 CFR 4.117 - Schedule of ratings-hemic and lymphatic systems.

Code of Federal Regulations, 2012 CFR

2012-07-01

... platelets or red cells at least once every six weeks, or; infections recurring at least once every six weeks 100 Requiring transfusion of platelets or red cells at least once every three months, or; infections recurring at least once every three months 60 Requiring transfusion of platelets or red cells at least once...

38 CFR 4.117 - Schedule of ratings-hemic and lymphatic systems.

Code of Federal Regulations, 2013 CFR

2013-07-01

... platelets or red cells at least once every six weeks, or; infections recurring at least once every six weeks 100 Requiring transfusion of platelets or red cells at least once every three months, or; infections recurring at least once every three months 60 Requiring transfusion of platelets or red cells at least once...

38 CFR 4.117 - Schedule of ratings-hemic and lymphatic systems.

Code of Federal Regulations, 2011 CFR

2011-07-01

... platelets or red cells at least once every six weeks, or; infections recurring at least once every six weeks 100 Requiring transfusion of platelets or red cells at least once every three months, or; infections recurring at least once every three months 60 Requiring transfusion of platelets or red cells at least once...

The combined effect of platelet storage media and intercept pathogen reduction technology on platelet activation/activability and cellular apoptosis/necrosis: Lisbon-RBS experience.

PubMed

Carvalho, Helena; Alguero, Carmen; Santos, Matilde; de Sousa, Gracinda; Trindade, Helder; Seghatchian, Jerard

2006-04-01

Platelets are known to undergo shape change, activation, a release reaction and apoptosis/necrosis during processing and storage, all of which are collectively known as the platelet storage lesion. Any additional processing may have some deleterious impact on platelet activability and functional integrity, which need to be investigated. This preliminary investigation was undertaken to establish the combined effects of standard platelet storage media and the intercept pathogen reduction technology on platelet activation and activability during 7 day storage, using buffy-coat derived platelets in standard storage media containing 35% plasma (N=24). P-selectin (CD62p) expression, a classical marker of platelet activation, and phosphatidylserine (PS) exposure on the platelet surface membrane, a hallmark of cellular necrosis/apoptosis, were both measured by flow cytometry. The results reveal significant increases in activation, from an average of 22.7% on day 1 before treatment to 31.6% on day 2 after treatment and 58.7% at the end of storage. Concomitantly, the basal expression of PS was slightly increased from 1.9% to 2.8% at day 2 after treatment and 7.3% at the end of storage. However, the functional reserve of platelets during storage, which reflects their capability to undergo activation and the release reaction when platelets were challenged with either calcium ionophore or thrombin, was relatively well maintained. These preliminary data confirm the earlier data on the use of intercept, and for the first time, based on the assessment of platelet functional integrity, suggest that platelet functional reserve is relatively well maintained, with little change in the formation of apoptotic cells.

A randomized controlled trial of aspirin and exertional heat stress activation of platelets in firefighters during exertion in thermal protective clothing.

PubMed

Hostler, David; Suyama, Joe; Guyette, Francis X; Moore, Charity G; Pryor, Riana R; Khorana, Priya; McEntire, Serina J; Comer, Diane; Reis, Steven E

2014-01-01

Platelet aggregation is enhanced in firefighters following short bouts of work in thermal protective clothing (TPC). We sought to determine if aspirin therapy before and/or following exertion in TPC prevents platelet activation. In a double-blind, placebo-controlled study, 102 firefighters were randomized to receive daily therapy (81 mg aspirin or placebo) for 14 days before and a single dose (325 mg aspirin or placebo) following exercise in TPC resulting in four potential assignments: aspirin before and after exercise (AA), placebo before and after exercise (PP), aspirin before and placebo after exercise (AP), and placebo before and aspirin after exercise (PA). Platelet closure time (PCT) was measured with a platelet function analyzer before the 2-week treatment, after the 2 week treatment period, immediately after exercise, and 30, 60, and 90 minutes later. Baseline PCT did not differ between groups. PCT changed over time in all four groups (p < 0.001) rising to a median of >300 seconds [IQR 99, 300] in AA and >300 [92, 300] in AP prior to exercise. Following exercise, median PCT decreased to in all groups. Median PCT returned to >300 seconds 30 minutes later in AA and AP and rose to 300 seconds in PA 60 minutes after exercise. Daily aspirin therapy blunts platelet activation during exertional heat stress and single-dose aspirin therapy following exertional heat stress reduces platelet activation within 60 minutes.

[Influence of raising oxygen content on function of platelet concentrate during preservation].

PubMed

Zhan, Tong; Xiao, Jian-Yu; Tao, Jing; Miao, Xi-Feng; Liu, Yan-Cun; Tang, Rong-Cai

2006-08-01

To explore the influence of raising oxygen (dissolved oxygen) content on function of platelet concentrate, the platelet concentrate was prepared by a CS-3000 plus blood cell separator. Experiments were divided into 2 groups: test group and control group. After raising oxygen content in platelet plasma under sterile operation, the platelet samples of two groups were preserved in oscillator with horizontal oscillation at 22 +/- 2 degrees C. The platelet count, platelet aggregation rate, lactic acid content and CD62p expression level of platelet were detected on 0, 1, 2, 3, 4, 5 days of platelet preservation. The results showed that the platelet count and platelet aggregation rate decreased with prolongation of preserved time, while the lactic acid content and CD62p expression level of platelet increased gradually. Compared with control group, there were significant differences in aggregation rate of platelet preserved for 2-3 days, and in CD62p expression level of platelet preserved for 1-3 days, while significant difference was found in lactic acid content of platelet preserved for 1-3 days. It is concluded that raising content of oxygen in platelet plasma can provide more oxygen to compensate oxygen supply deficiency for platelet metabolism and improve the efficiency of platelet oxygenic metabolism and the quality of platelet during preservation.

Tangeretin regulates platelet function through inhibition of phosphoinositide 3-kinase and cyclic nucleotide signaling.

PubMed

Vaiyapuri, Sakthivel; Ali, Marfoua S; Moraes, Leonardo A; Sage, Tanya; Lewis, Kirsty R; Jones, Chris I; Gibbins, Jonathan M

2013-12-01

Dietary flavonoids have long been appreciated in reducing cardiovascular disease risk factors, but their mechanisms of action are complex in nature. In this study, the effects of tangeretin, a dietary flavonoid, were explored on platelet function, signaling, and hemostasis. Tangeretin inhibited agonist-induced human platelet activation in a concentration-dependent manner. It inhibited agonist-induced integrin αIIbβ3 inside-out and outside-in signaling, intracellular calcium mobilization, and granule secretion. Tangeretin also inhibited human platelet adhesion and subsequent thrombus formation on collagen-coated surfaces under arterial flow conditions in vitro and reduced hemostasis in mice. Further characterization to explore the mechanism by which tangeretin inhibits platelet function revealed distinctive effects of platelet signaling. Tangeretin was found to inhibit phosphoinositide 3-kinase-mediated signaling and increase cGMP levels in platelets, although phosphodiesterase activity was unaffected. Consistent with increased cGMP levels, tangeretin increased the phosphorylation of vasodilator-stimulated phosphoprotein at S239. This study provides support for the ability and mechanisms of action of dietary flavonoids to modulate platelet signaling and function, which may affect the risk of thrombotic disease.

Early impact of prescription Omega-3 fatty acids on platelet biomarkers in patients with coronary artery disease and hypertriglyceridemia.

PubMed

Serebruany, Victor L; Miller, Michael; Pokov, Alex N; Lynch, Donald; Jensen, Jesper K; Hallén, Jonas; Atar, Dan

2011-01-01

Prescription omega-3-acid ethyl esters (PO-3A) have been tested for outcome benefits in patients with coronary artery disease (CAD), arrhythmias and heart failure. Some evidence suggests that PO-3A may exert their benefit via inhibiting platelets. We tested the hypothesis that PO-3A may inhibit platelet activity in patients with documented stable CAD, beyond the antiplatelet properties of aspirin and statins. Thirty patients with documented CAD and triglycerides over 250 mg/dl treated with aspirin (70-160 mg/daily) and statins (simvastatin equivalence dose: 5-40 mg/daily) were randomized 1:1:1 to Omacor™ 1 g/day (DHA/EPA ratio 1.25:1.0), Omacor 2 g/day, or a placebo for 2 weeks. Platelet tests including aggregometry and flow cytometry and cartridge analyzer readings were performed at baseline and at 1 and 2 weeks following PO-3A therapy. ADP-induced platelet aggregation (p = 0.037), GP IIb/IIIa antigen (p = 0.031) and activity (p = 0.024), and P-selectin (p = 0.041) were significantly reduced after PO-3A, while platelet/endothelial cell adhesion molecule (p = 0.09), vitronectin receptor (p = 0.16), formation of platelet-monocyte microparticles (p = 0.19) and the VerifyNow IIb/IIIa test (p = 0.27) only exhibited nonsignificant trends suggestive of reduced platelet activity. Finally, collagen- and arachidonic acid-induced aggregation, closure time with the PFA-100 device and expression of thrombospondin (CD36), GP Ib (CD42b), LAMP-3 (CD63), LAMP-1 (CD107a), CD40-ligand (CD154), GP37 (CD165), and PAR-1 receptor intact (SPAN 12) and cleaved (WEDE-15) epitopes were not affected by 2 weeks of PO-3A. Independently of the dose and already at 1 week, short-term therapy with PO-3A provided a modest reduction of platelet activity biomarkers, despite concomitant aspirin and statin therapy, when compared to a placebo. The effect of PO-3A is unique, differs from other known antiplatelet agents and suggests potential pleiotropism. These preliminary randomized data call for confirmation in prospective studies. Copyright © 2011 S. Karger AG, Basel.

Acquired platelet function defect

MedlinePlus

... Some cases cannot be prevented. Alternative Names Acquired qualitative platelet disorders; Acquired disorders of platelet function Images ... Todd Gersten, MD, Hematology/Oncology, Florida Cancer Specialists & Research Institute, Wellington, FL. Review provided by VeriMed Healthcare ...

A critical role for the regulation of Syk from agglutination to aggregation in human platelets.

PubMed

Shih, Chun-Ho; Chiang, Tin-Bin; Wang, Wen-Jeng

2014-01-10

Agglucetin, a tetrameric glycoprotein (GP) Ibα agonist from Formosan Agkistrodon acutus venom, has been characterized as an agglutination inducer in human washed platelets (WPs). In platelet-rich plasma (PRP), agglucetin dramatically elicits a biphasic response of agglutination and subsequent aggregation. For clarifying the intracellular signaling events from agglutination to aggregation in human platelets, we examined the essential signaling molecules involved through the detection of protein tyrosine phosphorylation (PTP). In WPs, an anti-GPIbα monoclonal antibody (mAb) AP1, but not a Src kinase inhibitor PP1, completely inhibited agglucetin-induced agglutination. However, PP1 but not AP1 had a potent suppression on platelet aggregation by a GPVI activator convulxin. The PTP analyses showed agglucetin alone can cause a weak pattern involving sequential phosphorylation of Lyn/Fyn, Syk, SLP-76 and phospholipase Cγ2 (PLCγ2). Furthermore, a Syk-selective kinase inhibitor, piceatannol, significantly suppressed the aggregating response in agglucetin-activated PRP. Analyzed by flow cytometry, the binding capacity of fluorophore-conjugated PAC-1, a mAb recognizing activated integrin αIIbβ3, was shown to increase in agglucetin-stimulated platelets. Again, piceatannol but not PP1 had a concentration-dependent suppression on agglucetin-induced αIIbβ3 exposure. Moreover, the formation of signalosome, including Syk, SLP-76, VAV, adhesion and degranulation promoting adapter protein (ADAP) and PLCγ2, are required for platelet aggregation in agglucetin/fibrinogen-activated platelets. In addition, GPIbα-ligation via agglucetin can substantially promote the interactions between αIIbβ3 and fibrinogen. Therefore, the signal pathway of Lyn/Fyn/Syk/SLP-76/ADAP/VAV/PLCγ2/PKC is sufficient to trigger platelet aggregation in agglucetin/fibrinogen-pretreated platelets. Importantly, Syk may function as a major regulator for the response from GPIbα-initiated agglutination to integrin αIIbβ3-dependent aggregation in human platelets. Copyright © 2013 Elsevier Inc. All rights reserved.

P-selectin ligation induces platelet activation and enhances microaggregate and thrombus formation.

PubMed

Théorêt, Jean-François; Yacoub, Daniel; Hachem, Ahmed; Gillis, Marc-Antoine; Merhi, Yahye

2011-09-01

Platelet P-selectin is a thrombo-inflammatory molecule involved in platelet activation and aggregation. This may occur via the adhesive function of P-selectin and its potential capacity to trigger intracellular signaling. However, its impact on platelet function remains elusive. This study was therefore designed to investigate the relationship between the signaling potential of platelet P-selectin and its function in platelet physiology. Human and mouse platelets were freshly isolated from whole blood. Platelet activation was assessed using flow cytometry and western blot analysis, while platelet physiological responses were evaluated through aggregation, microaggregate formation and in a thrombosis model in wild-type and P-selectin-deficient (CD62P(-/-)) mice. Interaction of P-selectin with its high-affinity ligand, a recombinant soluble form of P-Selectin Glycoprotein Ligand-1 (rPSGL-1), enhances platelet activation, adhesion and microaggregate formation. This augmented platelet microaggregates requires an intact cytoskeleton, but occurs independently of platelet α(IIb)β(3). Thrombus formation and microaggregate were both enhanced by rPSGL-1 in wild-type, but not in CD62P(-/-) mice. In addition, CD62P(-/-) mice exhibited thrombosis abnormalities without an α(IIb)β(3) activation defect. This study demonstrates that the role of platelet P-selectin is not solely adhesive; its binding to PSGL-1 induces platelet activation that enhances platelet aggregation and thrombus formation. Therefore, targeting platelet P-selectin or its ligand PSGL-1 could provide a potential therapeutic approach in the management of thrombotic disorders. Copyright © 2011 Elsevier Ltd. All rights reserved.

PPARbeta/delta agonists modulate platelet function via a mechanism involving PPAR receptors and specific association/repression of PKCalpha--brief report.

PubMed

Ali, Ferhana Y; Hall, Matthew G; Desvergne, Béatrice; Warner, Timothy D; Mitchell, Jane A

2009-11-01

Peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) is a nuclear receptor found in platelets. PPARbeta/delta agonists acutely inhibit platelet function within a few minutes of addition. As platelets are anucleated, the effects of PPARbeta/delta agonists on platelets must be nongenomic. Currently, the particular role of PPARbeta/delta receptors and their intracellular signaling pathways in platelets are not known. We have used mice lacking PPARbeta/delta (PPARbeta/delta(-/-)) to show the effects of the PPARbeta/delta agonist GW501516 on platelet adhesion and cAMP levels are mediated specifically by PPARbeta/delta, however GW501516 had no PPARbeta/delta-specific effect on platelet aggregation. Studies in human platelets showed that PKCalpha, which can mediate platelet activation, was bound and repressed by PPARbeta/delta after platelets were treated with GW501516. These data provide evidence of a novel mechanism by which PPAR receptors influence platelet activity and thereby thrombotic risk.

Identification of a specific intronic PEAR1 gene variant associated with greater platelet aggregability and protein expression

PubMed Central

Yanek, Lisa R.; Yang, Xiao Ping; Mathias, Rasika; Herrera-Galeano, J. Enrique; Suktitipat, Bhoom; Qayyum, Rehan; Johnson, Andrew D.; Chen, Ming-Huei; Tofler, Geoffrey H.; Ruczinski, Ingo; Friedman, Alan D.; Gylfason, Arnaldur; Thorsteinsdottir, Unnur; Bray, Paul F.; O'Donnell, Christopher J.; Becker, Diane M.; Becker, Lewis C.

2011-01-01

Genetic variation is thought to contribute to variability in platelet function; however, the specific variants and mechanisms that contribute to altered platelet function are poorly defined. With the use of a combination of fine mapping and sequencing of the platelet endothelial aggregation receptor 1 (PEAR1) gene we identified a common variant (rs12041331) in intron 1 that accounts for ≤ 15% of total phenotypic variation in platelet function. Association findings were robust in 1241 persons of European ancestry (P = 2.22 × 10−8) and were replicated down to the variant and nucleotide level in 835 persons of African ancestry (P = 2.31 × 10−27) and in an independent sample of 2755 persons of European descent (P = 1.64 × 10−5). Sequencing confirmed that variation at rs12041331 accounted most strongly (P = 2.07 × 10−6) for the relation between the PEAR1 gene and platelet function phenotype. A dose-response relation between the number of G alleles at rs12041331 and expression of PEAR1 protein in human platelets was confirmed by Western blotting and ELISA. Similarly, the G allele was associated with greater protein expression in a luciferase reporter assay. These experiments identify the precise genetic variant in PEAR1 associated with altered platelet function and provide a plausible biologic mechanism to explain the association between variation in the PEAR1 gene and platelet function phenotype. PMID:21791418

Evaluation of the antioxidant properties of N-acetylcysteine in human platelets: prerequisite for bioconversion to glutathione for antioxidant and antiplatelet activity.

PubMed

Gibson, Kyle R; Neilson, Ilene L; Barrett, Fiona; Winterburn, Tim J; Sharma, Sushma; MacRury, Sandra M; Megson, Ian L

2009-10-01

N-Acetylcysteine (NAC) is a frequently used "antioxidant" in vitro, but the concentrations applied rarely correlate with those encountered with oral dosing in vivo. Here, we investigated the in vitro antioxidant and antiplatelet properties of NAC at concentrations (10-100 microM) that are achievable in plasma with tolerable oral dosing. The impact of NAC pretreatment (2 hours) on aggregation of platelets from healthy volunteers in response to thrombin and adenosine diphosphate and on platelet-derived nitric oxide (NO) was examined. NAC was found to be a weak reducing agent and a poor antioxidant compared with glutathione (reduced form) (GSH). However, platelets treated with NAC showed enhanced antioxidant activity and depression of reactive oxygen species generation associated with increases in intraplatelet GSH levels. An approximately 2-fold increase in NO synthase-derived nitrite was observed with 10 microM NAC treatment, but the effect was not concentration dependent. Finally, NAC significantly reduced both thrombin-induced and adenosine diphosphate-induced platelet aggregation. NAC should be considered a weak antioxidant that requires prior conversion to GSH to convey antioxidant and antithrombotic benefit at therapeutically relevant concentrations. Our results suggest that NAC might be an effective antiplatelet agent in conditions where increased oxidative stress contributes to heightened risk of thrombosis but only if the intraplatelet machinery to convert it to GSH is functional.

The effect of variation in donor platelet function on transfusion outcome: a semirandomized controlled trial.

PubMed

Kelly, Anne M; Garner, Stephen F; Foukaneli, Theodora; Godec, Thomas R; Herbert, Nina; Kahan, Brennan C; Deary, Alison; Bakrania, Lekha; Llewelyn, Charlotte; Ouwehand, Willem H; Williamson, Lorna M; Cardigan, Rebecca A

2017-07-13

The effect of variation in platelet function in platelet donors on patient outcome following platelet transfusion is unknown. This trial assessed the hypothesis that platelets collected from donors with highly responsive platelets to agonists in vitro assessed by flow cytometry (high-responder donors) are cleared more quickly from the circulation than those from low-responder donors, resulting in lower platelet count increments following transfusion. This parallel group, semirandomized double-blinded trial was conducted in a single center in the United Kingdom. Eligible patients were those 16 or older with thrombocytopenia secondary to bone marrow failure, requiring prophylactic platelet transfusion. Patients were randomly assigned to receive a platelet donation from a high- or low-responder donor when both were available, or when only 1 type of platelet was available, patients received that. Participants, investigators, and those assessing outcomes were masked to group assignment. The primary end point was the platelet count increment 10 to 90 minutes following transfusion. Analysis was by intention to treat. Fifty-one patients were assigned to receive platelets from low-responder donors, and 49 from high-responder donors (47 of which were randomized and 53 nonrandomized). There was no significant difference in platelet count increment 10 to 90 minutes following transfusion in patients receiving platelets from high-responder (mean, 21.0 × 10 9 /L; 95% confidence interval [CI], 4.9-37.2) or low-responder (mean, 23.3 × 10 9 /L; 95% CI, 7.8-38.9) donors (mean difference, 2.3; 95% CI, -1.1 to 5.7; P = .18). These results support the current policy of not selecting platelet donors on the basis of platelet function for prophylactic platelet transfusion. © 2017 by The American Society of Hematology.

Detection of HIT antibody dependent platelet aggregation using novel surface imprinting approach.

PubMed

Hussain, Munawar; Northoff, Hinnak; Gehring, Frank K

2016-01-15

We present a fast, robust and straightforward spin force assisted surface imprinting approach for activated platelets and demonstrate that Heparin induced thrombocytopenia (HIT) platelet aggregation can be measured by this approach. A critical and challenging step in functional assays for HIT is platelet separation from the healthy donor's platelet-rich plasma (PRP). Our approach using surface imprinted polymer (MIP) for measurements on a quartz crystal microbalance with dissipation (QCM-D) enables monitoring of platelet aggregation directly in PRP thus eliminating the challenge of platelet separation. This is the first report of platelet imprinting. We also provide proof of principle that QCM-D technology can be applied for functional measurements of HIT antibodies. Copyright © 2015 Elsevier B.V. All rights reserved.

An evaluation of the ELT-8 hematology analyzer.

PubMed

Raik, E; McPherson, J; Barton, L; Hewitt, B S; Powell, E G; Gordon, S

1982-04-01

The TMELT-8 Hematology Analyzer is a fully automated cell counter which utilizes laser light scattering and hydrodynamic focusing to provide an 8 parameter whole blood count. The instrument consists of a sample handler with ticket printer, and a data handler with visual display unit, It accepts 100 microliter samples of venous or capillary blood and prints the values for WCC, RCC, Hb, Hct, MCV, MCH, MCHC and platelet count on to a standard result card. All operational and quality control functions, including graphic display of relative cell size distribution, can be obtained from the visual display unit and can also be printed as a permanent record if required. In a limited evaluation of the ELT-8, precision, linearity, accuracy, lack of sample carry-over and user acceptance were excellent. Reproducible values were obtained for all parameters after overnight storage of samples. Reagent usage and running costs were lower than for the Coulter S and the Coulter S Plus. The ease of processing capillary samples was considered to be a major advantage. The histograms served to alert the operator to a number of abnormalities, some of which were clinically significant.

Hydrogen peroxide (H/sub 2/O/sub 2/) stimulates the active transport of 5-hydroxytryptamine (5-HT) into platelets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bosin, T.R.

1986-03-01

Platelets function in a variety of physiological and pathological processes which may be altered by oxidant injury. One such process is the active transport 5-HT, which is an important mechanism in the control of circulating 5-HT levels. Exposure of mouse platelets (10/sup 8//ml) to H/sub 2/O/sub 2/ caused a time-dependent and dose-dependent increase in 5-HT (10/sup -7/M) uptake. The uptake 4 and 10 min following H/sub 2/O/sub 2/ (50 ..mu..M) was 228% and 145% of control values, respectively. Fluoxetine (10/sup -6/M) blocked all 5-HT uptake and catalase (1500 U/ml) blocked the H/sub 2/O/sub 2/-stimulated uptake. Enzymatically produced H/sub 2/O/sub 2/more » (glucose/glucose oxidase) and xanthine (X)/xanthine oxidase (XO) generated oxygen radicals produced quantitatively and qualitatively similar results. The stimulatory response of platelets to X/XO generated oxidants was unaffected by superoxide dismutase (250 U/ml) but, was inhibited using heat-denatured XO, allopurinol (0.5 mM) and catalase; fluoxetine inhibited all 5-HT uptake. Platelets exposed to X/XO in the presence of chelated (EDTA, 100 ..mu..M) or unchelated FeSO/sub 4/, FeNH/sub 4/(SO/sub 4/)/sub 2/ or CuCl (50 ..mu..M) did not have altered 5-HT uptake. These data indicate that brief exposure of platelets to physiological levels of H/sub 2/O/sub 2/ results in marked, reversible stimulation of active 5-HT uptake which may represent a homeostatic defense mechanism when H/sub 2/O/sub 2/ is elevated in the platelet microenvironment.« less

Quantitative Protein Sulfenic Acid Analysis Identifies Platelet Releasate-Induced Activation of Integrin β2 on Monocytes via NADPH Oxidase.

PubMed

Li, Ru; Klockenbusch, Cordula; Lin, Liwen; Jiang, Honghui; Lin, Shujun; Kast, Juergen

2016-12-02

Physiological stimuli such as thrombin, or pathological stimuli such as lysophosphatidic acid (LPA), activate platelets. The activated platelets bind to monocytes through P-selectin-PSGL-1 interactions but also release the contents of their granules, commonly called "platelet releasate". It is known that monocytes in contact with platelet releasate produce reactive oxygen species (ROS). Reversible cysteine oxidation by ROS is considered to be a potential regulator of protein function. In a previous study, we used THP-1 monocytic cells exposed to LPA- or thrombin-induced platelet releasate and a modified biotin switch assay to unravel the biological processes that are influenced by reversible cysteine oxidation. To gain a better understanding of the redox regulation of monocytes in atherosclerosis, we have now altered the modified biotin switch to selectively quantify protein sulfenic acid, a subpopulation of reversible cysteine oxidation. Using arsenite as reducing agent in the modified biotin switch assay, we were able to quantify 1161 proteins, in which more than 100 sulfenic acid sites were identified. Bioinformatics analysis of the quantified sulfenic acid sites highlighted the relevant, previously missed biological process of monocyte transendothelial migration, which included integrin β 2 . Flow cytometry validated the activation of LFA-1 (α L β 2 ) and Mac-1 (α M β 2 ), two subfamilies of integrin β 2 complexes, on human primary monocytes following platelet releasate treatment. The activation of LFA-1 was mediated by ROS from NADPH oxidase (NOX) activation. Production of ROS and activation of LFA-1 in human primary monocytes were independent of P-selectin-PSGL-1 interaction. Our results proved the modified biotin switch assay to be a powerful tool with the ability to reveal new regulatory mechanisms and identify new therapeutic targets.

Effect of Immobilized Antithrombin III on the Thromboresistance of Polycarbonate Urethane.

PubMed

Lukas, Karin; Stadtherr, Karin; Gessner, Andre; Wehner, Daniel; Schmid, Thomas; Wendel, Hans Peter; Schmid, Christof; Lehle, Karla

2017-03-24

The surface of foils and vascular grafts made from a thermoplastic polycarbonate urethanes (PCU) (Chronoflex AR) were chemically modified using gas plasma treatment, binding of hydrogels-(1) polyethylene glycol bisdiamine and carboxymethyl dextran (PEG-DEX) and (2) polyethyleneimine (PEI)-and immobilization of human antithrombin III (AT). Their biological impact was tested in vitro under static and dynamic conditions. Static test methods showed a significantly reduced adhesion of endothelial cells, platelets, and bacteria, compared to untreated PCU. Modified PCU grafts were circulated in a Chandler-Loop model for 90 min at 37 °C with human blood. Before and after circulation, parameters of the hemostatic system (coagulation, platelets, complement, and leukocyte activation) were analyzed. PEI-AT significantly inhibited the activation of both coagulation and platelets and prevented the activation of leukocytes and complement. In conclusion, both modifications significantly reduce coagulation activation, but only PEI-AT creates anti-bacterial and anti-thrombogenic functionality.

Coated platelets function in platelet-dependent fibrin formation via integrin αIIbβ3 and transglutaminase factor XIII

PubMed Central

Mattheij, Nadine J.A.; Swieringa, Frauke; Mastenbroek, Tom G.; Berny-Lang, Michelle A.; May, Frauke; Baaten, Constance C.F.M.J.; van der Meijden, Paola E.J.; Henskens, Yvonne M.C.; Beckers, Erik A.M.; Suylen, Dennis P.L.; Nolte, Marc W.; Hackeng, Tilman M.; McCarty, Owen J.T.; Heemskerk, Johan W.M.; Cosemans, Judith M.E.M.

2016-01-01

Coated platelets, formed by collagen and thrombin activation, have been characterized in different ways: i) by the formation of a protein coat of α-granular proteins; ii) by exposure of procoagulant phosphatidylserine; or iii) by high fibrinogen binding. Yet, their functional role has remained unclear. Here we used a novel transglutaminase probe, Rhod-A14, to identify a subpopulation of platelets with a cross-linked protein coat, and compared this with other platelet subpopulations using a panel of functional assays. Platelet stimulation with convulxin/thrombin resulted in initial integrin αIIbβ3 activation, the appearance of a platelet population with high fibrinogen binding, (independently of active integrins, but dependent on the presence of thrombin) followed by phosphatidylserine exposure and binding of coagulation factors Va and Xa. A subpopulation of phosphatidylserine-exposing platelets bound Rhod-A14 both in suspension and in thrombi generated on a collagen surface. In suspension, high fibrinogen and Rhod-A14 binding were antagonized by combined inhibition of transglutaminase activity and integrin αIIbβ3. Markedly, in thrombi from mice deficient in transglutaminase factor XIII, platelet-driven fibrin formation and Rhod-A14 binding were abolished by blockage of integrin αIIbβ3. Vice versa, star-like fibrin formation from platelets of a patient with deficiency in αIIbβ3 (Glanzmann thrombasthenia) was abolished upon blockage of transglutaminase activity. We conclude that coated platelets, with initial αIIbβ3 activation and high fibrinogen binding, form a subpopulation of phosphatidylserine-exposing platelets, and function in platelet-dependent star-like fibrin fiber formation via transglutaminase factor XIII and integrin αIIbβ3. PMID:26721892

Calcium is a key constituent for maintaining the in vitro properties of platelets suspended in the bicarbonate-containing additive solution M-sol with low plasma levels.

PubMed

Wagner, Stephen J; Skripchenko, Andrey; Myrup, Andrew; Thompson-Montgomery, Dedeene; Awatefe, Helen; Moroff, Gary

2010-05-01

Commercially available additive solutions (ASs) require 30% to 35% plasma for optimal storage of platelets (PLTs). PLTs suspended in M-sol, a bicarbonate-based experimental platelet additive solution (PAS), maintain in vitro PLT properties during storage with low levels of plasma (< or =5%). Four different formulations of M-sol were prepared at the optimal pH (6.1): M-sol, M-sol without calcium, M-sol without citric acid, and M-sol without calcium and citric acid. Apheresis PLT units (100% plasma) were equally divided into five 50-mL aliquots in PL732 containers, centrifuged, and resuspended to prepare units suspended in the four different PASs (95%) with 5% plasma and 1 unit in 100% plasma. Units (n = 10) were stored under standard conditions and assayed for in vitro properties on Days 1, 5, and 7. The data were analyzed by analysis of variance for repeated measures (n = 10, p < 0.001). On Day 5 of storage, PLTs suspended in the M-sol formulation containing calcium but lacking citric acid had similar pH, extent of shape change (ESC) values, and percentage of CD62-positive PLTs and greater hypotonic shock response (HSR) and percentage of discoid PLTs compared to those of PLTs suspended in 100% plasma. In contrast, PLTs suspended in the M-sol formulation lacking calcium had lesser ESC values, greater percentage of CD62-positive PLTs, and similar HSR values and percentage of discoid PLTs compared to those of PLTs suspended in 100% plasma on Day 5 (p < 0.001). Calcium plays an important role in maintaining CD62-negative PLTs and relatively high ESC in 5% plasma. The removal of citric acid from M-sol may improve PLT storage properties with low plasma levels.

Leukemia-associated Rho guanine nucleotide exchange factor (LARG) plays an agonist specific role in platelet function through RhoA activation

PubMed Central

Zou, Siying; Teixeira, Alexandra M.; Yin, Mingzhu; Xiang, Yaozu; Xavier-Ferruccio, Juliana; Zhang, Ping-xia; Hwa, John; Min, Wang; Krause, Diane S.

2018-01-01

Summary Leukemia-Associated RhoGEF (LARG) is highly expressed in platelets, which are essential for maintaining normal hemostasis. We studied the function of LARG in murine and human megakaryocytes and platelets with Larg knockout, shRNA-mediated knockdown and small molecule-mediated inhibition. We found that LARG is important for human, but not murine, megakaryocyte maturation. Larg KO mice exhibit macrothrombocytopenia, internal bleeding in the ovaries and prolonged bleeding times. KO platelets have impaired aggregation, α-granule release and integrin α2bβ3 activation in response to thrombin and thromboxane, but not to ADP. The same agonist-specific reductions in platelet aggregation occur in human platelets treated with a LARG inhibitor. Larg KO platelets have reduced RhoA activation and myosin light chain phosphorylation, suggesting that Larg plays an agonist-specific role in platelet signal transduction. Using 2 different in vivo assays, Larg KO mice are protected from in vivo thrombus formation. Together, these results establish that LARG regulates human megakaryocyte maturation, and is critical for platelet function in both humans and mice. PMID:27345948

Leukaemia-associated Rho guanine nucleotide exchange factor (LARG) plays an agonist specific role in platelet function through RhoA activation.

PubMed

Zou, Siying; Teixeira, Alexandra M; Yin, Mingzhu; Xiang, Yaozu; Xavier-Ferrucio, Juliana; Zhang, Ping-Xia; Hwa, John; Min, Wang; Krause, Diane S

2016-08-30

Leukemia-Associated RhoGEF (LARG) is highly expressed in platelets, which are essential for maintaining normal haemostasis. We studied the function of LARG in murine and human megakaryocytes and platelets with Larg knockout (KO), shRNA-mediated knockdown and small molecule-mediated inhibition. We found that LARG is important for human, but not murine, megakaryocyte maturation. Larg KO mice exhibit macrothrombocytopenia, internal bleeding in the ovaries and prolonged bleeding times. KO platelets have impaired aggregation, α-granule release and integrin α2bβ3 activation in response to thrombin and thromboxane, but not to ADP. The same agonist-specific reductions in platelet aggregation occur in human platelets treated with a LARG inhibitor. Larg KO platelets have reduced RhoA activation and myosin light chain phosphorylation, suggesting that Larg plays an agonist-specific role in platelet signal transduction. Using two different in vivo assays, Larg KO mice are protected from in vivo thrombus formation. Together, these results establish that LARG regulates human megakaryocyte maturation, and is critical for platelet function in both humans and mice.

Neutrophil-derived S100 calcium-binding proteins A8/A9 promote reticulated thrombocytosis and atherogenesis in diabetes

PubMed Central

Kraakman, Michael J.; Lee, Man K.S.; Al-Sharea, Annas; Dragoljevic, Dragana; Barrett, Tessa J.; Montenont, Emilie; Basu, Debapriya; Heywood, Sarah; Kammoun, Helene L.; Flynn, Michelle; Whillas, Alexandra; Hanssen, Nordin M.J.; Febbraio, Mark A.; Westein, Erik; Chin-Dusting, Jaye; Cooper, Mark E.; Berger, Jeffrey S.; Goldberg, Ira J.; Nagareddy, Prabhakara R.; Murphy, Andrew J.

2017-01-01

Platelets play a critical role in atherogenesis and thrombosis-mediated myocardial ischemia, processes that are accelerated in diabetes. Whether hyperglycemia promotes platelet production and whether enhanced platelet production contributes to enhanced atherothrombosis remains unknown. Here we found that in response to hyperglycemia, neutrophil-derived S100 calcium-binding proteins A8/A9 (S100A8/A9) interact with the receptor for advanced glycation end products (RAGE) on hepatic Kupffer cells, resulting in increased production of IL-6, a pleiotropic cytokine that is implicated in inflammatory thrombocytosis. IL-6 acts on hepatocytes to enhance the production of thrombopoietin, which in turn interacts with its cognate receptor c-MPL on megakaryocytes and bone marrow progenitor cells to promote their expansion and proliferation, resulting in reticulated thrombocytosis. Lowering blood glucose using a sodium-glucose cotransporter 2 inhibitor (dapagliflozin), depleting neutrophils or Kupffer cells, or inhibiting S100A8/A9 binding to RAGE (using paquinimod), all reduced diabetes-induced thrombocytosis. Inhibiting S100A8/A9 also decreased atherogenesis in diabetic mice. Finally, we found that patients with type 2 diabetes have reticulated thrombocytosis that correlates with glycated hemoglobin as well as increased plasma S100A8/A9 levels. These studies provide insights into the mechanisms that regulate platelet production and may aid in the development of strategies to improve on current antiplatelet therapies and to reduce cardiovascular disease risk in diabetes. PMID:28504650

Autologous Platelet-Rich Plasma Preparations

PubMed Central

Schippinger, Gert; Prüller, Florian; Divjak, Manuela; Mahla, Elisabeth; Fankhauser, Florian; Rackemann, Steve; Raggam, Reinhard Bernd

2015-01-01

Background Autologous platelet-rich plasma (PRP) has been widely used for the treatment of sports injuries. It has been associated with improved healing and regeneration of soft tissues in elite athletes. Athletes are commonly receiving nonsteroidal anti-inflammatory drugs (NSAIDs). As yet, the effect of these drugs on platelet function in PRP formulations has not been taken into consideration. Hypothesis The function of platelets in PRP produced under the influence of NSAIDs is inhibited and may lessen a possible healing effect on the site of injury. Study Design Controlled laboratory study. Methods PRP was collected from patients receiving NSAIDs after elective orthopaedic surgery, and platelet function was evaluated using light transmission aggregometry (LTA). Results were compared with those obtained from healthy volunteers without a history of NSAID intake during the previous 2 weeks. Two different systems for blood collection and PRP production (Arthrex ACP double-syringe system and standard 4.5-mL sodium citrate blood collection tubes) were used and compared regarding the quality of PRP that was produced. Results For both groups, the baseline platelet counts of whole blood and the platelet counts of PRP formulations were found to be in the normal range. Both collection systems for PRP produced comparable results without significant differences between the groups. Platelet function testing with LTA revealed significantly impaired platelet aggregation in both PRP preparations, obtained from patients taking NSAIDs, irrespective of the type of NSAID (P < .001). All subjects from the control group showed normal platelet aggregation patterns when tested with LTA. Conclusion Autologous PRP produced from subjects after NSAID medication shows significantly impaired platelet function and may result in lower quality regarding the content of bioactive compounds. Clinical Relevance If required, the administration of NSAIDs should be performed after blood collection for preparation of autologous PRP; otherwise, the therapeutic effect may be limited. PMID:26665098

A dual role for integrin-linked kinase in platelets: regulating integrin function and α-granule secretion

PubMed Central

Sage, Tanya; Stevens, Joanne M.; Jordan, Peter A.; Jones, Sarah; Barrett, Natasha E.; St-Arnaud, Rene; Frampton, Jonathan; Dedhar, Shoukat; Gibbins, Jonathan M.

2008-01-01

Integrin-linked kinase (ILK) has been implicated in the regulation of a range of fundamental biological processes such as cell survival, growth, differentiation, and adhesion. In platelets ILK associates with β1- and β3-containing integrins, which are of paramount importance for the function of platelets. Upon stimulation of platelets this association with the integrins is increased and ILK kinase activity is up-regulated, suggesting that ILK may be important for the coordination of platelet responses. In this study a conditional knockout mouse model was developed to examine the role of ILK in platelets. The ILK-deficient mice showed an increased bleeding time and volume, and despite normal ultrastructure the function of ILK-deficient platelets was decreased significantly. This included reduced aggregation, fibrinogen binding, and thrombus formation under arterial flow conditions. Furthermore, although early collagen stimulated signaling such as PLCγ2 phosphorylation and calcium mobilization were unaffected in ILK-deficient platelets, a selective defect in α-granule, but not dense-granule, secretion was observed. These results indicate that as well as involvement in the control of integrin affinity, ILK is required for α-granule secretion and therefore may play a central role in the regulation of platelet function. PMID:18772455

Quantitative Characterization of Shear-Induced Platelet Receptor Shedding: Glycoprotein Ibα, Glycoprotein VI, and Glycoprotein IIb/IIIa.

PubMed

Chen, Zengsheng; Koenig, Steven C; Slaughter, Mark S; Griffith, Bartley P; Wu, Zhongjun J

2017-11-07

The structural integrity of platelet receptors is essential for platelets to play the normal hemostatic function. The high non-physiologic shear stress (NPSS) commonly exists in blood-contacting medical devices and has been shown to cause platelet receptor shedding. The loss of platelet receptors may impair the normal hemostatic function of platelets. The aim of this study was to quantify NPSS-induced shedding of three key receptors on the platelet surface. Human blood was subjected to the matrix of well-defined shear stresses and exposure times, generated by using a custom-designed blood-shearing device. The expression of three key platelet receptors, glycoprotein (GP) Ibα, GPVI, and GPIIb/IIIa, in sheared blood was quantified using flow cytometry. The quantitative relationship between the loss of each of the three receptors on the platelet surface and shear condition (shear stress level and exposure time) was explored. It was found that these relationships followed well the power law functional form. The coefficients of the power law models for the shear-induced shedding of these platelet receptors were derived with coefficients of determination (R) of 0.77, 0.73, and 0.78, respectively. The power law models with these coefficients may be potentially used to predict the shear-induced platelet receptor shedding of human blood.

[The effect of electromagnetic waves of very high frequency of molecular spectra of radiation and absorption of nitric oxide on the functional activity of platelets].

PubMed

Kirichuk, V F; Maĭborodin, A V; Volin, M V; Krenitskiĭ, A P; Tupikin, V D

2001-01-01

A study was made of the effect of electromagnetic EMI MMD-fluctuation on the frequencies of molecular spectra of radiation, and nitric oxide absorption under in vitro conditions on the functional activity of platelets in patients with unstable angina pectoris, with the help of a specially created generator. At amplitude-modulated and continuous modes of EMI MMD-irradiation of platelet-rich plasma for 5, 15 and 30 min the platelet functional activity decreases, which was shown up in reduction of their activation and fall of aggregative ability. The degree, to which platelet functional activity was inhibited, depended on the mode of irradiation and on duration of EMI MMD effect. The most obvious changes in platelet activation and in their readiness to aggregative response were observed at a continuous mode of irradiation within a 15 min interval.

In Vitro and Ex Vivo Approaches to Evaluate Next-Generation Tobacco and Non-Tobacco Products on Human Blood Platelets

PubMed Central

Spinelli, Sherry L.; Lannan, Katie L.; Loelius, Shannon G.

2017-01-01

Abstract Human blood platelets are major hemostatic regulators in the circulation and important in the mediation of chronic inflammation and immunomodulation. They are key elements that promote cardiovascular pathogenesis that leads to atherosclerosis, thrombosis, myocardial infarction, and stroke. New information on tobacco use and platelet dysregulation shows that these highly understudied vascular cells are dysregulated by tobacco smoke. Thus, platelet function studies should be an important consideration for the evaluation of existing and next-generation tobacco and non-tobacco products. Novel in vitro approaches are being sought to investigate these products and their influence on platelet function. Platelets are ideally suited for product assessment, as robust and novel in vitro translational methods are available to assess platelet function. Furthermore, the use of human biological systems has the advantage that risk predictions will better reflect the human condition. PMID:28337466

Surfactants reduce platelet-bubble and platelet-platelet binding induced by in vitro air embolism.

PubMed

Eckmann, David M; Armstead, Stephen C; Mardini, Feras

2005-12-01

The effect of gas bubbles on platelet behavior is poorly characterized. The authors assessed platelet-bubble and platelet-platelet binding in platelet-rich plasma in the presence and absence of bubbles and three surface-active compounds. Platelet-rich plasma was prepared from blood drawn from 16 volunteers. Experimental groups were surfactant alone, sparging (microbubble embolization) alone, sparging with surfactant, and neither sparging nor surfactant. The surfactants were Pluronic F-127 (Molecular Probes, Eugene, OR), Perftoran (OJSC SPC Perftoran, Moscow, Russia), and Dow Corning Antifoam 1510US (Dow Corning, Midland, MI). Videomicroscopy images of specimens drawn through rectangular glass microcapillaries on an inverted microscope and Coulter counter measurements were used to assess platelet-bubble and platelet-platelet binding, respectively, in calcium-free and recalcified samples. Histamine-induced and adenosine diphosphate-induced platelet-platelet binding were measured in unsparged samples. Differences between groups were considered significant for P < 0.05 using analysis of variance and the Bonferroni correction. Sixty to 100 platelets adhered to bubbles in sparged, surfactant-free samples. With sparging and surfactant, few platelets adhered to bubbles. Numbers of platelet singlets and multimers not adherent to bubbles were different (P < 0.05) compared both with unsparged samples and sparged samples without surfactant. No significant platelet-platelet binding occurred in uncalcified, sparged samples, although 20-30 platelets adhered to bubbles. Without sparging, histamine and adenosine diphosphate provoked platelet-platelet binding with and without surfactants present. Sparging causes platelets to bind to air bubbles and each other. Surfactants added before sparging attenuate platelet-bubble and platelet-platelet binding. Surfactants may have a clinical role in attenuating gas embolism-induced platelet-bubble and platelet-platelet binding.

Intrinsic platelet reactivity before start with clopidogrel as predictor for on-clopidogrel platelet function and long-term clinical outcome.

PubMed

Hochholzer, Willibald; Valina, Christian M; Bömicke, Timo; Amann, Michael; Stratz, Christian; Nührenberg, Thomas; Trenk, Dietmar; Neumann, Franz-Josef

2015-07-01

High on-clopidogrel platelet reactivity is associated with worse clinical outcome. Previous data suggest that intrinsic platelet reactivity before initiation of clopidogrel contributes significantly to on-clopidogrel platelet reactivity. It is unknown whether intrinsic reactivity can sufficiently predict on-clopidogrel reactivity and therefore identify patients with insufficient response to clopidogrel before initiation of treatment and at risk for worse clinical outcome. This analysis included 765 consecutive patients undergoing elective coronary stent implantation. Platelet reactivity was assessed by light transmission aggregometry (5 µM ADP) before administration of clopidogrel 600mg and after intake of first maintenance dose of clopidogrel on day 1 following coronary stenting. Patients were followed for up to seven years. The combined primary endpoint was death of any cause or non-fatal myocardial infarction. Intrinsic and on-clopidogrel platelet reactivity were significant correlated (r=0.31; p < 0.001). Among all tested clinical and genetic factors including the cytochrome P450 2C19*2 polymorphism, intrinsic platelet reactivity was the strongest predictor for on-clopidogrel platelet reactivity. However, intrinsic platelet reactivity could only explain 8 % of variability of on-clopidogrel platelet function. Only on-treatment platelet reactivity was predictive for long-term clinical outcome (HR 1.47, 95 % CI 1.05-2.05; p = 0.02) whereas intrinsic platelet reactivity was not (HR 1.03, 95 % CI 0.74-1.43; p = 0.86). In conclusion, intrinsic platelet reactivity before initiation of clopidogrel is the strongest predictor of early on-clopidogrel platelet reactivity but can only explain a minor proportion of its variability and is not significantly associated with clinical outcome. Thus, baseline testing cannot substitute on-clopidogrel platelet function testing.

Dynamics of platelet glycoprotein IIb-IIIa receptor expression and fibrinogen binding. I. Quantal activation of platelet subpopulations varies with adenosine diphosphate concentration.

PubMed Central

Frojmovic, M. M.; Mooney, R. F.; Wong, T.

1994-01-01

We have previously reported that maximal platelet activation with adenosine diphosphate (100 microM ADP) causes rapid expression of all GPIIb-IIIa receptors for fibrinogen (FgR) (< 1-3 s), measured with FITC-labeled PAC1 by flow cytometry. We have extended these studies to examine the effects of ADP concentration on the graded expression and Fg occupancy of GPIIb-IIIa receptors. Human citrated platelet-rich plasma, diluted 10-fold with Walsh-albumin-Mg+2 (2 mM), was treated with ADP (0.1-100 microM). The rates of GPIIb-IIIa receptor expression or Fg binding were measured in unstirred samples by flow cytometry, using FITC-labeled monoclonal antibodies (mAb) PAC1 and 9F9, respectively, from on-rates, using increasing times between mAb and ADP additions. Fibrinogen receptors were all expressed rapidly at low (1 microM) or high (100 microM) ADP (few seconds), whereas Fg occupancy was 50% of maximal by about 2 min. The maximal extent of GPIIb-IIIa receptor expression and Fg occupancy was determined from maximal binding (Flmax) at 30 min incubation with PAC1 or 9F9. On-rates and maximal extents of binding for either PAC1 or 9F9 probes showed identical [ADP]-response profiles ("KD" approximately 1.4 +/- 0.1 microM). However, Flmax studies showed bimodal histograms consisting of "resting" (Po) and maximally "activated" (P*) platelets for both PAC1 and 9F9 binding, with the fraction of "activated" platelets increasing with ADP concentration. The data best fit a model where platelet subpopulations are "quantally" transformed from Po to P*, expressing all GPIIb-IIIa receptors, rapidly filled by Fg, but "triggered" at critical ADP concentrations. Larger, but not the largest, platelets appear to be the most sensitive subpopulation. The implications for clinical studies are discussed, and the relationship to dynamics of aggregation are described in a companion paper. PMID:7858143

Human platelet lysate as a promising growth-stimulating additive for culturing of stem cells and other cell types.

PubMed

Shanskii, Ya D; Sergeeva, N S; Sviridova, I K; Kirakozov, M S; Kirsanova, V A; Akhmedova, S A; Antokhin, A I; Chissov, V I

2013-11-01

We compared the composition and biological activity of fetal calf serum and platelet lysate from donor platelet concentrate. In platelet lysate, the concentrations of alkaline phosphatase, lactate dehydrogenase, creatinine, and mineral metabolism parameters were lower, while parameters of lipid and protein metabolism were higher than in fetal calf serum. The concentrations of growth factors (platelet-derived (AA, AB, BB), vascular endothelial, insulin-like, and transforming growth factor β) in platelet lysate 1.7-148.7-fold surpassed the corresponding parameters in fetal calf serum. After replacement of fetal calf serum with platelet lysate in the culture medium (0, 25, 50, 75, and 100%), the count of multipotent mesenchymal stromal cells on day 7 (in comparison with day 1) increased by 154.8, 206.6, 228.2, 367.7, and 396.5%, respectively. Thus, platelet lysate can be an adequate non-xenogenic alternative for fetal calf serum.

Platelet antiheparin activity. The isolation and characterisation of platelet factor 4 released from thrombin-aggregated washed human platelets and its dissociation into subunits and the isolation of membrane-bound antiheparin activity.

PubMed

Moore, S; Pepper, D S; Cash, J D

1975-02-27

Platelet factor 4 was isolated by gel filtration from the soluble release products of thrombin-aggregated washed human platelets as a proteoglycan-platelet factor 4 complex of molecular weight 358 000, Stokes radius (r-s) of 14.0 nm, sedimentation coefficient (s) of 7.1 S and frictional ratio (f/f-o) of 3.04. The complex was dissociated at high ionic strength (I equals 0.75) and the proteoglycan separated from platelet factor 4 by gel filtration. Platelet factor 4 had a molecular weight of 27 100, r-s of 2.52 nm, s of 2.4 S and f/f-o of 1.26, was insoluble under physiological conditions but readily soluble at pH 3. Under these conditions platelet factor 4 dissociated into four subunits with a molecular weight of 6900, r-s of 1.92 nm, s of 0.8 S, and f/f-o of 1.52. Qualitative N-terminal amino acid analysis showed the presence of glutamic acid or glutamine as the major end group. Platelet factor 4 was compared with protamine sulphate, which has similar biological properties, by electrophoresis at pH 2.2, in which both migrated as single bands but with differing mobility, and by amino acid analysis which showed a more normal distribution of residues than occurred in protamine sulphate. Of the basic amino acids platelet factor 4 (molecular weight 27 100) contained 5.97% arginine, 3.18% histidine, and 12.31% lysine compared to protamine sulphate with 64.2% arginine, 0.6% lysine and no histidine. A partial specific volume (v) of 0.747 was calculated for platelet factor 4 from its amino acid analysis. A membrane fraction with antiheparin activity, an isopycnic density of 1.090-1.110 and r-s of 15-35 nm, was also isolated by sucrose density gradient centrifugation from the ultrasonicated insoluble platelet residue remaining after thrombin-induced aggregation of washed human platelets. Trypsin treatment of the membrane fraction neither solubilised nor destroyed the activity.

The influence of platelets, plasma and red blood cells on functional haemostatic assays.

PubMed

Bochsen, Louise; Johansson, Pär I; Kristensen, Annemarie T; Daugaard, Gedske; Ostrowski, Sisse R

2011-04-01

Functional whole blood haemostatic assays are used increasingly to guide transfusion therapy and monitor medical treatment and are also applied for in-vitro evaluations of the haemostatic potential of stored platelets. We investigated how the cellular and plasmatic elements, both isolated and combined, influenced the two methodologically different assays, thrombelastography (TEG) and impedance aggregometry (Multiplate). Platelet-rich plasma (200 × 10/l) or pure plasma (0 platelets), with and without added red blood cells (RBCs), hematocrit 0, 0.15 or 0.29, were produced in vitro from platelet concentrates, fresh frozen plasma and stored RBC. Pure platelets were investigated by removing plasma components from platelet concentrates by diafiltration against the platelet storage solution Intersol. Plasma was readded by diafiltration against plasma in Intersol. Haemostatic function was evaluated by TEG and Multiplate. In the TEG, increasing amounts of RBC reduced clot strength and clot kinetics (α-angle), most markedly in plasma/RBC without platelets. In contrast, RBC in a platelet concentrate matrix enhanced Multiplate aggregation in response to weak agonists (ADP and arachidonic acid). Furthermore, removing plasma from platelet concentrates eliminated the TEG response and diminished the Multiplate aggregation response, but readding plasma to the pure platelet concentrates restored the response. Each of the elements in whole blood, plasma, platelets and RBC, affected the Multiplate and TEG results differently. The results emphasize that the concentrations of all cellular and plasmatic components in whole blood should be taken into account when interpreting results obtained by TEG and multiplate.

Clinical course and prognostic factors of childhood immune thrombocytopenia: single center experience of 10 years

PubMed Central

Jung, Jae Yeob; O, A Rum; Kim, Je Keong

2016-01-01

Purpose This study aimed to evaluate the clinical course of childhood immune thrombocytopenia (ITP) and to assess the risk factors for developing chronic ITP. Methods The records of 64 children diagnosed with ITP from November 2005 and December 2014 at single center were retrospectively analyzed. Results The median age at diagnosis and the median platelet count were 1 year (range, 1 month to 15 years) and 9×109/L (range, 0–84×109/L), respectively. No patient experienced severe bleeding. Nineteen children (29.7%) spontaneously recovered their platelet count to ≥100×109/L at a median of 10 days. In total 45 patients (70.3%) received intravenous immunoglobulin (IVIG) as first-line therapy, and showed platelet recovery at 1 week. The final diagnosis of 55 (85.9%) and 9 patients (14.1%) was acute and chronic ITP, respectively. Older age, absence of prior infection and insidious onset of symptoms were significantly associated with the development of chronic ITP. Among the patients who received IVIG, those with platelet count <45×109/L at 1 month after IVIG showed a significantly higher incidence of chronic ITP compared to those with platelet count ≥45×109/L (88.8% vs. 44.4%, P<0.01). Conclusion In most patients, ITP runs a benign course and approximately 86% of them recover within 1 year of their initial diagnosis. The potential impact of the risk factors of chronic ITP on clinical practice needs to be explored and further studies are warranted to determine whether IVIG influences the course of ITP. PMID:27610182

Platelet Kinetics in Idiopathic Thrombocytopenic Purpura Patients Treated with Thrombopoietin Receptor Agonists

PubMed Central

Meyer, Oliver; Herzig, Eric; Salama, Abdulgabar

2012-01-01

Aim Thrombopoietin receptor agonists (Tpo RA) increase platelet counts in the majority of chronic autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura; ITP) patients. It is unknown whether this treatment may also improve platelet survival (PS) in these patients. Methods In order to determine platelet survival (PS), autologous platelets were labeled with 111In oxine and retransfused in six patients under treatment with Tpo RA (romiplostim n = 3; eltrombopag n = 3). Results Stable platelet counts of greater than 100 × 103/μl were observed in all 6 patients. Platelet survival was decreased in all cases (mean 2.10 days; range 0.13–3.73 days). No correlation was found between platelet count and PS. Similarly, there was no significant relationship between platelet turnover and platelet count. However, a high platelet turnover, exceeding 25 or three times the norm was observed in 2 patients who presented the lowest PS (0.13 or 0.83 days). Two patients had a moderately shortened PS (1.91 or 2.42 days), and, correspondingly, a moderately increased platelet turnover rate (63,072 or 72,872 platelets/μl/day). Conclusion These results indicate that Tpo RA may not only overcompensate platelet destruction in ITP, but may interfere with other mechanisms, which, in some cases, results in a reduced platelet destruction rate. PMID:22896760

Variability in platelet dense granule adenosine triphosphate release findings amongst patients tested multiple times as part of an assessment for a bleeding disorder.

PubMed

Badin, M S; Graf, L; Iyer, J K; Moffat, K A; Seecharan, J L; Hayward, C P M

2016-12-01

Lumi-aggregometry quantification of platelet dense granule adenosine triphosphate (ATP) release is commonly used for diagnosing platelet function disorders. As the test findings show considerable variability for healthy controls, we postulated that patient findings might also be variable and investigated patients who were assessed for dense granule ATP release defects more than once. Analyses were performed on prospectively collected data for first and second tests for subjects tested for dense granule ATP release defects more than once by the Hamilton Regional Laboratory Program (HRLMP) between January 2007 and June 2013 (cohort I). Similar analyses were performed for subjects who were recruited to a platelet disorder study (cohort II) and were assessed for ATP release defects more than once before October 2015. A total of 150 unique subjects had multiple ATP release tests. Results with individual agonists were variable for many subjects. While normal findings with all tested agonists were often confirmed by the second test (cohort I: 83%; cohort II: 100%), impaired release with multiple agonists was confirmed in only some subjects (cohort I: 34%; cohort II: 54%). Inconsistent findings were common (cohort I: 36%; cohort II: 39%). ISTH bleeding scores showed no relationship to the test findings. The finding of impaired ATP release with 2 or more agonists on both tests was not associated with an increased likelihood of a definite bleeding disorder. The variability in platelet dense granule ATP release findings amongst patients assessed for diagnostic purposes suggests that the test has limited value for diagnosing platelet disorders. © 2016 John Wiley & Sons Ltd.

Molecular cloning and characterization of rhesus monkey platelet glycoprotein Ibα, a major ligand-binding subunit of GPIb-IX-V complex.

PubMed

Qiao, Jianlin; Shen, Yang; Shi, Meimei; Lu, Yanrong; Cheng, Jingqiu; Chen, Younan

2014-05-01

Through binding to von Willebrand factor (VWF), platelet glycoprotein (GP) Ibα, the major ligand-binding subunit of the GPIb-IX-V complex, initiates platelet adhesion and aggregation in response to exposed VWF or elevated fluid-shear stress. There is little data regarding non-human primate platelet GPIbα. This study cloned and characterized rhesus monkey (Macaca Mullatta) platelet GPIbα. DNAMAN software was used for sequence analysis and alignment. N/O-glycosylation sites and 3-D structure modelling were predicted by online OGPET v1.0, NetOGlyc 1.0 Server and SWISS-MODEL, respectively. Platelet function was evaluated by ADP- or ristocetin-induced platelet aggregation. Rhesus monkey GPIbα contains 2,268 nucleotides with an open reading frame encoding 755 amino acids. Rhesus monkey GPIbα nucleotide and protein sequences share 93.27% and 89.20% homology respectively, with human. Sequences encoding the leucine-rich repeats of rhesus monkey GPIbα share strong similarity with human, whereas PEST sequences and N/O-glycosylated residues vary. The GPIbα-binding residues for thrombin, filamin A and 14-3-3ζ are highly conserved between rhesus monkey and human. Platelet function analysis revealed monkey and human platelets respond similarly to ADP, but rhesus monkey platelets failed to respond to low doses of ristocetin where human platelets achieved 76% aggregation. However, monkey platelets aggregated in response to higher ristocetin doses. Monkey GPIbα shares strong homology with human GPIbα, however there are some differences in rhesus monkey platelet activation through GPIbα engagement, which need to be considered when using rhesus monkey platelet to investigate platelet GPIbα function. Copyright © 2014 Elsevier Ltd. All rights reserved.

Differential proteomics study of platelets in asymptomatic constitutional macrothrombocytopenia: altered levels of cytoskeletal proteins.

PubMed

Karmakar, Shilpita; Saha, Sutapa; Banerjee, Debasis; Chakrabarti, Abhijit

2015-01-01

Harris platelet syndrome (HPS), also known as asymptomatic constitutional macrothrombocytopenia (ACMT), is an autosomal dominant platelet disorder characterized by mild-to-severe thrombocytopenia and giant platelets with normal platelet aggregation and absence of bleeding symptoms. We have attempted a comparative proteomics study for profiling of platelet proteins in healthy vs. pathological states to discover characteristic protein expression changes in macrothrombocytes and decipher the factors responsible for the functionally active yet morphologically distinct platelets. We have used 2-D gel-based protein separation techniques coupled with MALDI-ToF/ToF-based mass spectrometric identification and characterization of the proteins to investigate the differential proteome profiling of platelet proteins isolated from the peripheral blood samples of patients and normal volunteers. Our study revealed altered levels of actin-binding proteins such as myosin light chain, coactosin-like protein, actin-related protein 2/3 complex, and transgelin2 that hint toward the cytoskeletal changes necessary to maintain the structural and functional integrity of macrothrombocytes. We have also observed over expressed levels of peroxiredoxin2 that signifies the prevailing oxidative stress in these cells. Additionally, altered levels of protein disulfide isomerase and transthyretin provide insights into the measures adapted by the macrothrombocytes to maintain their normal functional activity. This first proteomics study of platelets from ACMT may provide an understanding of the structural stability and normal functioning of these platelets in spite of their large size. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Thrombocytopenia in the first 24 hours after birth and incidence of patent ductus arteriosus.

PubMed

Sallmon, Hannes; Weber, Sven C; Hüning, Britta; Stein, Anja; Horn, Peter A; Metze, Boris C; Dame, Christof; Bührer, Christoph; Felderhoff-Müser, Ursula; Hansmann, Georg; Koehne, Petra

2012-09-01

Experimental studies suggest that platelet-triggered ductal sealing is critically involved in definite ductus arteriosus closure. Whether thrombocytopenia contributes to persistently patent ductus arteriosus (PDA) in humans is controversial. This was a retrospective study of 1350 very low birth weight (VLBW; <1500 g) infants, including 592 extremely low birth weight (ELBW; <1000 g) infants. All infants who had a platelet count in the first 24 hours after birth and an echocardiogram performed on day of life 4 to 5 were included. The incidence of thrombocytopenia was analyzed in infants with and without PDA, and in those who did or did not undergo PDA intervention. The impact of thrombocytopenia, gestational age, birth weight, gender, and sepsis on PDA was determined by receiver operating characteristic curve, odds ratio, and regression analyses. Platelet numbers within the first 24 hours after birth did not differ between VLBW/ELBW infants with and without spontaneous ductal closure. Platelet numbers were not associated with subsequent PDA treatment. Low platelet counts were not related to failure of pharma-cologic PDA treatment and the need for subsequent surgical ligation. Lower gestational age or birth weight, male gender, and sepsis were linked to the presence of PDA in VLBW infants on day of life 4 to 5. Thrombocytopenia in the first 24 hours after birth was not associated with PDA in this largest VLBW/ELBW infant cohort studied to date. Impaired platelet function, due to immaturity and critical illness, rather than platelet number, might play a role in ductus arteriosus patency.

Palladin is involved in platelet activation and arterial thrombosis.

PubMed

Chen, Xuejiao; Fan, Xuemei; Tan, Juan; Shi, Panlai; Wang, Xiyi; Wang, Jinjin; Kuang, Ying; Fei, Jian; Liu, Junling; Dang, Suying; Wang, Zhugang

2017-01-01

The dynamics of actin cytoskeleton have been shown to play a critical role during platelet activation. Palladin is an actin-associated protein, serving as a cytoskeleton scaffold to bundle actin fibers and actin cross linker. The functional role of palladin on platelet activation has not been investigated. Here, we characterized heterozygous palladin knockout (palladin +/- ) mice to elucidate the platelet-related functions of palladin. The results showed that palladin was expressed in platelets and moderate palladin deficiency accelerated hemostasis and arterial thrombosis. The aggregation of palladin +/- platelets was increased in response to low levels of thrombin, U46619, and collagen. We also observed enhanced spreading of palladin +/- platelets on immobilized fibrinogen (Fg) and increased rate of clot retraction in platelet-rich plasma (PRP) containing palladin +/- platelets. Furthermore, the activation of the small GTPase Rac1 and Cdc42, which is associated with cytoskeletal dynamics and platelet activation signalings, was increased in the spreading and aggregating palladin +/- platelets compared to that in wild type platelets. Taken together, these findings indicated that palladin is involved in platelet activation and arterial thrombosis, implying a potent role of palladin in pathophysiology of thrombotic diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mechanism Underlying Linezolid-induced Thrombocytopenia in a Chronic Kidney Failure Mouse Model

PubMed Central

Nishijo, Nao; Tsuji, Yasuhiro; Matsunaga, Kazuhisa; Kutsukake, Masahiko; Okazaki, Fumiyasu; Fukumori, Shiro; Kasai, Hidefumi; Hiraki, Yoichi; Sakamaki, Ippei; Yamamoto, Yoshihiro; Karube, Yoshiharu; To, Hideto

2017-01-01

Objective: To investigate the relationship between renal function and linezolid (LZD)-induced thrombocytopenia and elucidate the underlying mechanism using a chronic renal disease (CRD) mouse model. Materials and Methods: CRD was induced in 5-week-old male Institute of Cancer Research (ICR) mice by 5/6 nephrectomy. After this procedure, LZD (25 and 100 mg/kg) was administered intraperitoneally once every day for 28 days. Platelet counts, white blood cell (WBC) counts, and hematocrit (HCT) levels were measured every 7 days. 2-14C-thymidine (0.185 MBq) was administrated intravenously to LZD-administered mice to evaluate the thymidine uptake ability of bone marrow. Results: Platelet counts were significantly lower in the LZD-administered CRD group than in the LZD-nonadministered groups at 14, 21, and 28 days (P < 0.05); however, these changes were not observed in LZD-administered mice with normal renal function, regardless of the duration of LZD administration. No significant changes were observed in WBC counts or HCT levels in any LZD-administered CRD mouse. Moreover, radioactive levels in bone marrow were not significantly different in each group. Conclusions: These results indicate that LZD-induced decreases in platelet counts were enhanced by renal impairment in vivo, suggesting that LZD-induced thrombocytopenia is not caused by nonimmune-mediated bone marrow suppression. PMID:28405130

Clopidogrel (Plavix) and cardiac surgical patients: implications for platelet function monitoring and postoperative bleeding.

PubMed

Tanaka, Kenichi A; Szlam, Fania; Kelly, Andrew B; Vega, J David; Levy, Jerrold H

2004-08-01

The use of clopidogrel (Plavix), an inhibitor of adenosine diphosphate (ADP)-induced platelet aggregation, has been proven to reduce ischemic events in cardiovascular patients, but little information is available for optimal monitoring of platelet function in patients receiving the drug preoperatively. In the first part of the study we compared different testing modalities (thrombelastography (TEG), platelet aggregometry, and whole blood aggregation) to assess platelet ADP receptor inhibition. Because clopidogrel is a pro-drug, we used an in vitro model of ADP inhibition with 5'-p-fluorosulfonylbenzoyladenosine (FSBA). FSBA at final concentration of 80 microM completely inhibited platelet aggregation but had no effect on TEG maximum amplitude (MA). In the second part of the study, antiplatelet effects of clopidogrel were clinically assessed and correlated to postoperative bleeding in 18 coronary bypass surgery patients. Preoperative TEG results were normal or hypercoagulable in clopidogrel-treated patients, although platelet aggregation responses to ADP were inhibited. Clopidogrel-treated patients who underwent cardiopulmonary bypass had a high incidence (84.6%) of platelet transfusion therapy due to increased chest tube drainage. In conclusion, we have demonstrated that normal preoperative TEG-MA does not preclude clopidogrel-induced ADP receptor blockade; however, TEG can be a reliable monitor for CPB-induced platelet dysfunction related to GPIIb/IIIa. For monitoring clopidogrel, it is necessary to perform more specific platelet function tests (aggregometry or platelet count ratio) using ADP as an activator.

Platelet Kainate Receptor Signaling Promotes Thrombosis by Stimulating Cyclooxygenase Activation

PubMed Central

Sun, Henry; Swaim, AnneMarie; Herrera, Jesus Enrique; Becker, Diane; Becker, Lewis; Srivastava, Kalyan; Thompson, Laura E.; Shero, Michelle R.; Perez-Tamayo, Alita; Suktitpat, Bhoom; Mathias, Rasika; Contractor, Anis; Faraday, Nauder; Morrell, Craig N.

2009-01-01

Rationale Glutamate is a major signaling molecule that binds to glutamate receptors including the ionotropic glutamate receptors; kainate (KA) receptor (KAR), the N-methyl-D-aspartate (NMDA) receptor (NMDAR), and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR). Each is well characterized in the central nervous system (CNS), but glutamate has important signaling roles in peripheral tissues as well, including a role in regulating platelet function. Objective Our previous work has demonstrated that glutamate is released by platelets in high concentrations within a developing thrombus and increases platelet activation and thrombosis. We now show that platelets express a functional KAR that drives increased agonist induced platelet activation. Methods and Results KAR induced increase in platelet activation is in part the result of activation of platelet cyclooxygenase (COX) in a Mitogen Activated Protein Kinase (MAPK) dependent manner. Platelets derived from KA receptor subunit knockout mice (GluR6−/−) are resistant to KA effects and have a prolonged time to thrombosis in vivo. Importantly, we have also identified polymorphisms in KA receptor subunits that are associated with phenotypic changes in platelet function in a large group of Caucasians and African Americans. Conclusion Our data demonstrate that glutamate regulation of platelet activation is in part COX dependent, and suggest that the KA receptor is a novel anti-thrombotic target. PMID:19679838

Gap Junctions and Connexin Hemichannels Underpin Haemostasis and Thrombosis

PubMed Central

Vaiyapuri, Sakthivel; Jones, Chris I.; Sasikumar, Parvathy; Moraes, Leonardo A.; Munger, Stephanie J.; Wright, Joy R.; Ali, Marfoua S.; Sage, Tanya; Kaiser, William J.; Tucker, Katherine L.; Stain, Christopher J.; Bye, Alexander P.; Jones, Sarah; Oviedo-Orta, Ernesto; Simon, Alexander M.; Mahaut-Smith, Martyn P.; Gibbins, Jonathan M.

2012-01-01

Background Connexins are a widespread family of membrane proteins that assemble into hexameric hemichannels, also known as connexons. Connexons regulate membrane permeability in individual cells or couple between adjacent cells to form gap junctions and thereby provide a pathway for regulated intercellular communication. We have now examined the role of connexins in platelets, blood cells that circulate in isolation, but upon tissue injury adhere to each other and the vessel wall to prevent blood loss and facilitate wound repair. Methods and Results We report the presence of connexins in platelets, notably connexin37, and that the formation of gap junctions within platelet thrombi is required for the control of clot retraction. Inhibition of connexin function modulated a range of platelet functional responses prior to platelet-platelet contact, and reduced laser induced thrombosis in vivo in mice. Deletion of the Cx37 gene (Gja4) in transgenic mice reduced platelet aggregation, fibrinogen binding, granule secretion and clot retraction indicating an important role for Cx37 hemichannels and gap junctions in platelet thrombus function. Conclusions Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of haemostasis and thrombosis and represent potential therapeutic targets. PMID:22528526

The role of point-of-care assessment of platelet function in predicting postoperative bleeding and transfusion requirements after coronary artery bypass grafting.

PubMed

Mishra, Pankaj Kumar; Thekkudan, Joyce; Sahajanandan, Raj; Gravenor, Mike; Lakshmanan, Suresh; Fayaz, Khazi Mohammed; Luckraz, Heyman

2015-01-01

OBJECTIVE platelet function assessment after cardiac surgery can predict postoperative blood loss, guide transfusion requirements and discriminate the need for surgical re-exploration. We conducted this study to assess the predictive value of point-of-care testing platelet function using the Multiplate® device. Patients undergoing isolated coronary artery bypass grafting were prospectively recruited ( n = 84). Group A ( n = 42) patients were on anti-platelet therapy until surgery; patients in Group B ( n = 42) stopped anti-platelet treatment at least 5 days preoperatively. Multiplate® and thromboelastography (TEG) tests were performed in the perioperative period. Primary end-point was excessive bleeding (>2.5 ml/kg/h) within first 3 h postoperative. Secondary end-points included transfusion requirements, re-exploration rates, intensive care unit and in-hospital stays. Patients in Group A had excessive bleeding (59% vs. 33%, P = 0.02), higher re-exploration rates (14% vs. 0%, P < 0.01) and higher rate of blood (41% vs. 14%, P < 0.01) and platelet (14% vs. 2%, P = 0.05) transfusions. On multivariate analysis, preoperative platelet function testing was the most significant predictor of excessive bleeding (odds ratio [OR]: 2.3, P = 0.08), need for blood (OR: 5.5, P < 0.01) and platelet transfusion (OR: 15.1, P < 0.01). Postoperative "ASPI test" best predicted the need for transfusion (sensitivity - 0.86) and excessive blood loss (sensitivity - 0.81). TEG results did not correlate well with any of these outcome measures. Peri-operative platelet functional assessment with Multiplate® was the strongest predictor for bleeding and transfusion requirements in patients on anti-platelet therapy until the time of surgery.

R1: Platelets and Megakaryocytes contain functional NF-κB

PubMed Central

Spinelli, Sherry L.; Casey, Ann E.; Pollock, Stephen J.; Gertz, Jacqueline M.; McMillan, David H.; Narasipura, Srinivasa D.; Mody, Nipa A.; King, Michael R.; Maggirwar, Sanjay B.; Francis, Charles W.; Taubman, Mark B.; Blumberg, Neil; Phipps, Richard P.

2010-01-01

The Nuclear Factor (NF)-κB transcription factor family is well-known for their role in eliciting inflammation and promoting cell survival. We discovered that human megakaryocytes and platelets express the majority of NF-κB family members including the regulatory Inhibitor (I)-κB and Inhibitor Kappa Kinase (IKK) molecules. Objective Investigate the presence and role of NF-κB proteins in megakaryocytes and platelets. Methods and Results Anucleate platelets exposed to NF-κB inhibitors demonstrated impaired fundamental functions involved in repairing vascular injury and thrombus formation. Specifically, NF-κB inhibition diminished lamellapodia formation, decreased clot retraction times and reduced thrombus stability. Moreover, inhibition of I-κB-α phosphorylation (BAY-11-7082) reverts fully spread platelets back to a spheroid morphology. Addition of recombinant IKK-β or I-κB-α protein to BAY inhibitor-treated platelets partially restore platelet spreading in I-κB-α inhibited platelets, and addition of active IKK-β increased endogenous I-κB-α phosphorylation levels. Conclusions These novel findings support a crucial and non-classical role for the NF-κB family in modulating platelet function and reveal that platelets are sensitive to NF-κB inhibitors. As NF-κB inhibitors are being developed as anti-inflammatory and anti-cancer agents, they may have unintended effects on platelets. Based on these data, NF-κB is also identified as a new target to dampen unwanted platelet activation. PMID:20042710

Extracellular cyclophilin A activates platelets via EMMPRIN (CD147) and PI3K/Akt signaling, which promotes platelet adhesion and thrombus formation in vitro and in vivo.

PubMed

Seizer, Peter; Ungern-Sternberg, Saskia N I V; Schönberger, Tanja; Borst, Oliver; Münzer, Patrick; Schmidt, Eva-Maria; Mack, Andreas F; Heinzmann, David; Chatterjee, Madhumita; Langer, Harald; Malešević, Miroslav; Lang, Florian; Gawaz, Meinrad; Fischer, Gunter; May, Andreas E

2015-03-01

Cyclophilin A (CyPA) is secreted under inflammatory conditions by various cell types. Whereas the important role of intracellular CyPA for platelet function has been reported, the effect of extracellular CyPA on platelet function has not been investigated yet. Inhibition of extracellular CyPA through a novel specific inhibitor MM284 reduced thrombus after ferric chloride-induced injury in vivo. In vitro extracellular CyPA enhanced thrombus formation even in CyPA(-/-) platelets. Treatment of isolated platelets with recombinant CyPA resulted in platelet degranulation in a time- and dose-dependent manner. Inhibition of the platelet surface receptor extracellular matrix metalloproteinase inducer (cluster of differentiation 147) by an anticluster of differentiation 147 monoclonal antibody significantly reduced CyPA-dependent platelet degranulation. Pretreatment of platelets with CyPA enhanced their recruitment to mouse carotid arteries after arterial injury, which could be inhibited by an anticluster of differentiation 147 monoclonal antibody (intravital microscopy). The role of extracellular CyPA in adhesion could be confirmed by infusing CyPA(-/-) platelets in CyPA(+/+) mice and by infusing CyPA(+/+) platelets in CyPA(-/-) mice. Stimulation of platelets with CyPA induced phosphorylation of Akt, which could in turn be inhibited in the presence of phosphoinositid-3-kinase inhibitors. Akt-1(-/-) platelets revealed a markedly decreased degranulation on CyPA stimulation. Finally, ADP-induced platelet aggregation was attenuated by MM284, as well as by inhibiting paracrine-secreted CyPA without directly affecting Ca(2+)-signaling. Extracellular CyPA activates platelets via cluster of differentiation 147-mediated phosphoinositid-3-kinase/Akt-signaling, leading to enhanced adhesion and thrombus formation independently of intracellular CyPA. Targeting extracellular CyPA via a specific inhibitor may be a promising strategy for platelet inhibition without affecting critical functions of intracellular CyPA. © 2014 American Heart Association, Inc.

Effect of photodynamic therapy on mouse platelets

NASA Astrophysics Data System (ADS)

Zhou, Chuannong; Chi, Shunji; Deng, Jinsheng; Zhang, Hua; Liang, Junlin; Ha, Xian-wen

1993-03-01

Normal mice received hematoporphyrin derivative (10 mg/kg iv) immediately, 24 or 48 hrs prior to red light irradiation. The blood was collected and the platelet-rich plasma was irradiated by red light (100 J/cm2). The platelets were fixed immediately, 8 or 16 hrs after irradiation, and processed for EM examination. In comparison with those of control mice, the platelets of all experimental mice showed structural changes: 16 hrs after irradiation all platelets were necrotized; 8 hrs after irradiation almost one fourth of the platelets were necrotized and the remaining were considerably damaged; immediately after irradiation a small number of platelets became necrotic and most other platelets were swollen and deformed, often with many cytoplasmic projections and considerable dilatation of the canalicular membrane system. Our findings provided a clear evidence that platelets are highly sensitive to PDT action and can be directly and rapidly injured by PDT even in the absence of vascular endothelial cells. Our results give firm support to the hypothesis that both endothelial cells and platelets may play an important role in the initiation of early vascular damage and microcirculatory alterations induced by PDT in vivo.

In vitro evaluation of COM.TEC apheresis platelet concentrates using a preparation set and pathogen inactivation over a storage period of five days.

PubMed

Moog, R; Fröhlich, A; Mayaudon, V; Lin, L

2004-01-01

The aim of the present study was to evaluate in vitro data on platelets collected by apheresis, processed on a preparation set followed by photochemical treatment (PCT). Fifteen single-donor platelet concentrates (PCs) were collected by apheresis (COM.TEC blood cell separator, Fresenius, Bad Homburg, Germany). The platelets were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in approximately 37% plasma and 63% platelet additive solution InterSol. PCT was done by exposing the platelets to amotosalen HCl followed by illumination with ultraviolet light. Blood cell counts and in vitro PLT function were measured up to 5 days. An average of 3.44 +/- 0.28 x 10(11) platelets were collected in a product volume of 351 +/- 21 mL. Plasma removal resulted in a mean platelet loss of 7.8%. After PCT, a progressive decrease in platelet function was observed. LDH level rose through storage (171 +/- 81 U/L) to levels approximating LDH levels observed post-collection (180 +/- 103 U/L). There was a gradual decrease of the platelets to respond to hypotonic shock response from 90 +/- 9 % post-plasma reduction to 48 +/- 16% at day 5. All PLT units met the European requirements for leukoreduction and the pH limit of 6.8 up to day 5 post-collection. The new preparation set was capable of producing platelet units meeting the requirements for PCT. Despite differences observed in in vitro platelet function parameters, PLTs at storage day 5 fit the German and European guidelines.

Progress in bio-manufacture of platelets for transfusion.

PubMed

Heazlewood, Shen Y; Nilsson, Susan K; Cartledge, Kellie; Be, Cheang Ly; Vinson, Andrew; Gel, Murat; Haylock, David N

2017-11-01

Blood transfusion services face an ever-increasing demand for donor platelets to meet clinical needs. Whilst strategies for increasing platelet storage life and improving the efficiency of donor platelet collection are important, in the longer term, platelets generated by bio-manufacturing processes will be required to meet demands. Production of sufficient numbers of in vitro-derived platelets for transfusion represents a significant bioengineering challenge. In this review, we highlight recent progress in this area of research and outline the main technical and biological obstacles that need to be met before this becomes feasible and economic. A critical consideration is assurance of the functional properties of these cells as compared to their fresh, donor collected, counterparts. We contend that platelet-like particles and in vitro-derived platelets that phenotypically resemble fresh platelets must deliver the same functions as these cells upon transfusion. We also note recent progress with immortalized megakaryocyte progenitor cell lines, molecular strategies for reducing expression of HLA Class I to generate universal donor platelets and the move to early clinical studies with in vitro-derived platelets.

Role of the recombinant protein of the platelet receptor for type I collagen in the release of nitric oxide during platelet aggregation.

PubMed

Chiang, T M; Wang, Y B; Kang, E S

2000-12-01

Nitric oxide plays an important role in platelet function and platelets possess the endothelial isoform of nitric oxide synthase. Several reports have indicated that nitric oxide is released upon exposure of platelets to collagen. We have reported that a non-integrin platelet protein of 65 kDa is a receptor for type I collagen. By direct measurement of NO release from washed human platelets suspended in Tyrode buffer with a ISO-NO Mark II, World Precision Instruments, Sarasota, FL, USA, p30 sensor, type I collagen, but not ADP and epinephrine, induces the release of NO in a time-dependent manner. The production of NO is inhibited either by preincubation of type I collagen with the platelet type I collagen receptor recombinant protein or by preincubation of platelets with the antibody to the receptor protein, the anti-65 antibody. However, preincubation of platelets with anti-P-selectin and anti-glycoprotein IIb/IIIa did not affect the release of NO by platelets. These results suggest that the 65 kDa platelet receptor for type I collagen is specifically linked to the generation of NO, and that the 65 kDa platelet receptor for type I collagen plays an important new role in platelet function.

Thromboelastography platelet mapping in healthy dogs using 1 analyzer versus 2 analyzers.

PubMed

Blois, Shauna L; Banerjee, Amrita; Wood, R Darren; Park, Fiona M

2013-07-01

The objective of this study was to describe the results of thromboelastography platelet mapping (TEG-PM) carried out using 2 techniques in 20 healthy dogs. Maximum amplitudes (MA) generated by thrombin (MAthrombin), fibrin (MAfibrin), adenosine diphosphate (ADP) receptor activity (MAADP), and thromboxane A2 (TxA2) receptor activity (stimulated by arachidonic acid, MAAA) were recorded. Thromboelastography platelet mapping was carried out according to the manufacturer's guidelines (2-analyzer technique) and using a variation of this method employing only 1 analyzer (1-analyzer technique) on 2 separate blood samples obtained from each dog. Mean [± standard deviation (SD)] MA values for the 1-analyzer/2-analyzer techniques were: MAthrombin = 51.9 mm (± 7.1)/52.5 mm (± 8.0); MAfibrin = 20.7 mm (± 21.8)/23.0 mm (± 26.1); MAADP = 44.5 mm (± 15.6)/45.6 mm (± 17.0); and MAAA = 45.7 mm (± 11.6)/45.0 mm (± 15.4). Mean (± SD) percentage aggregation due to ADP receptor activity was 70.4% (± 32.8)/67.6% (± 33.7). Mean percentage aggregation due to TxA2 receptor activity was 77.3% (± 31.6)/78.1% (± 50.2). Results of TEG-PM were not significantly different for the 1-analyzer and 2-analyzer methods. High correlation was found between the 2 methods for MAfibrin [concordance correlation coefficient (r) = 0.930]; moderate correlation was found for MAthrombin (r = 0.70) and MAADP (r = 0.57); correlation between the 2 methods for MAAA was lower (r = 0.32). Thromboelastography platelet mapping (TEG-PM) should be further investigated to determine if it is a suitable method for measuring platelet dysfunction in dogs with thrombopathy.

Thromboelastography platelet mapping in healthy dogs using 1 analyzer versus 2 analyzers

PubMed Central

Blois, Shauna L.; Banerjee, Amrita; Wood, R. Darren; Park, Fiona M.

2013-01-01

The objective of this study was to describe the results of thromboelastography platelet mapping (TEG-PM) carried out using 2 techniques in 20 healthy dogs. Maximum amplitudes (MA) generated by thrombin (MAthrombin), fibrin (MAfibrin), adenosine diphosphate (ADP) receptor activity (MAADP), and thromboxane A2 (TxA2) receptor activity (stimulated by arachidonic acid, MAAA) were recorded. Thromboelastography platelet mapping was carried out according to the manufacturer’s guidelines (2-analyzer technique) and using a variation of this method employing only 1 analyzer (1-analyzer technique) on 2 separate blood samples obtained from each dog. Mean [± standard deviation (SD)] MA values for the 1-analyzer/2-analyzer techniques were: MAthrombin = 51.9 mm (± 7.1)/52.5 mm (± 8.0); MAfibrin = 20.7 mm (± 21.8)/23.0 mm (± 26.1); MAADP = 44.5 mm (± 15.6)/45.6 mm (± 17.0); and MAAA = 45.7 mm (± 11.6)/45.0 mm (± 15.4). Mean (± SD) percentage aggregation due to ADP receptor activity was 70.4% (± 32.8)/67.6% (± 33.7). Mean percentage aggregation due to TxA2 receptor activity was 77.3% (± 31.6)/78.1% (± 50.2). Results of TEG-PM were not significantly different for the 1-analyzer and 2-analyzer methods. High correlation was found between the 2 methods for MAfibrin [concordance correlation coefficient (r) = 0.930]; moderate correlation was found for MAthrombin (r = 0.70) and MAADP (r = 0.57); correlation between the 2 methods for MAAA was lower (r = 0.32). Thromboelastography platelet mapping (TEG-PM) should be further investigated to determine if it is a suitable method for measuring platelet dysfunction in dogs with thrombopathy. PMID:24101802

IGF-1 facilitates thrombopoiesis primarily through Akt activation.

PubMed

Chen, Shilei; Hu, Mengjia; Shen, Mingqiang; Wang, Song; Wang, Cheng; Chen, Fang; Tang, Yong; Wang, Xinmiao; Zeng, Hao; Chen, Mo; Gao, Jining; Wang, Fengchao; Su, Yongping; Xu, Yang; Wang, Junping

2018-05-25

It is known that insulin-like growth factor-1 (IGF-1) also functions as a hematopoietic factor, while its direct effect on thrombopoiesis remains unclear. In this study, we show that IGF-1 is able to promote CD34+ cell differentiation toward megakaryocytes (MKs), as well as the facilitation of proplatelet formation (PPF) and platelet production from cultured MKs. The in vivo study demonstrates that IGF-1 administration accelerates platelet recovery in mice after 6.0Gy of irradiation and in mice that received bone marrow transplantation (BMT) following 10.0Gy of lethal irradiation. Subsequent investigations reveal that ERK1/2 and Akt activation mediate the effect of IGF-1 on thrombopoiesis. Notably, Akt activation induced by IGF-1 is more apparent than that of ERK1/2, compared with that of thrombopoietin (TPO) treatment. Moreover, the effect of IGF-1 on thrombopoiesis is independent of TPO signaling, because IGF-1 treatment can also lead to a significant increase of platelet counts in homozygous TPO receptor mutant mice. Further analysis indicates that the activation of Akt triggered by IGF-1 requires the assistance of steroid receptor coactivator-3 (SRC-3). Therefore, our data reveal a distinct role of IGF-1 in regulating thrombopoiesis, providing new insights into TPO-independent regulation of platelet generation. Copyright © 2018 American Society of Hematology.

Utility of a Fluorescence Microscopy Imaging System for Analyzing the DNA Ploidy of Pathological Megakaryocytes Including 5q- Syndrome.

PubMed

Nakahara, Takako; Suemori, Shinichiro; Tsujioka, Takayuki; Kataoka, Mikio; Kataoka, Hiromi; Shibakura, Misako; Tohyama, Kaoru

2018-06-01

To investigate megakaryocyte (MK) DNA ploidy in various hematological diseases, fluorescence microscopy imaging system (FMI) can be used to analyze DNA ploidy with cell morphology at the single-cell level by using specialized image-processing software. Here we compared DNA ploidy obtained by FMI measured with that obtained flow cytometry (FCM). With FMI, we could evaluate the DNA ploidy in long-term preserved bone marrow smear samples after staining. We next analyzed the MK DNA ploidy in 42 bone marrow smear samples including 26 myeloid neoplasm cases, and we compared the DNA ploidy and platelet counts in the patients' peripheral blood; the production of platelets was significantly high compared to DNA ploidy in the myeloproliferative neoplasms group. The FMI method revealed that the patients with 5q- syndrome exhibited relatively low DNA ploidy despite high platelet counts, and this result suggested that increased DNA ploidy is not indispensable to abundant platelet production. The FMI method for DNA ploidy will be a useful tool to clarify the relationship between DNA ploidy and platelet production by MKs.

Defining Platelet Function During Polytrauma

DTIC Science & Technology

2013-02-01

calibrated automated thrombography, 3. Platelet-induced clot contraction and using viscoelastic measures such as TEG with Platelet Mapping™ and, 4. Flow...using calibrated automated thrombography (CAT). 3. Platelet-induced clot contraction and using viscoelastic measures such as TEG with Platelet Mapping...formation (such as Hemodyne’s platelet contractile force measurement and thromboelastrography). The degree to which certain injury patterns as well as

Antithrombotic Potential of Tormentil Extract in Animal Models

PubMed Central

Marcinczyk, Natalia; Jarmoc, Dominika; Leszczynska, Agnieszka; Zakrzeska, Agnieszka; Kramkowski, Karol; Strawa, Jakub; Gromotowicz-Poplawska, Anna; Chabielska, Ewa; Tomczyk, Michal

2017-01-01

Potentilla species that have been investigated so far display pharmacological activity mainly due to the presence of polyphenols. Recently, it was shown that polyphenol-rich extract from rhizome of Potentilla erecta (tormentil extract) affects the metabolism of arachidonic acid and exerts both anti-inflammatory and anti-oxidant activities, suggesting a possible effect on thrombosis. Accordingly, the aim of the study was to evaluate the effect of tormentil extract on haemostasis in a rat model of thrombosis. Lyophilized water-methanol extract from P. erecta rhizome was administrated per os for 14 days in doses of 100, 200, and 400 mg/kg in a volume of 2 mL/kg in a 5% water solution of gummi arabici (VEH). In the in vivo experiment an electrically induced carotid artery thrombosis model with blood flow monitoring was used in Wistar rats. Collected blood samples were analyzed ex vivo functionally and biochemically for changes in haemostasis. Tormentil extract (400 mg/kg) significantly decreased thrombus weight and prolonged the time to carotid artery occlusion and bleeding time without changes in the blood pressure. In the ex vivo experiment tormentil extract (400 mg/kg) reduced thromboxane production and decreased t-PA activity, while total t-PA concentration, as well as total PAI-1 concentration and PAI-1 activity remained unchanged. Furthermore, tormentil extract (400 mg/kg) decreased bradykinin concentration and shortened the time to reach maximal optical density during fibrin generation. Prothrombin time, activated partial thromboplastin time, QUICK index, fibrinogen level, and collagen-induced aggregation remained unchanged. To investigate the involvement of platelets in the antithrombotic effect of tormentil, the extract was administrated per os for 2 days to mice and irreversible platelets activation after ferric chloride induced thrombosis was evaluated under intravital conditions using confocal microscopy system. In this model tormentil extract (400 mg/kg) significantly reduced platelet activation at the same extent as acetylsalicylic acid. Taken together, we have shown for the first time that tormentil extract inhibits arterial thrombosis in platelet- and endothelial-dependent mechanisms without hemodynamic changes. Further studies on the detailed mechanism of action of tormentil extract toward fibrinolysis and the kinin system should be carried out. PMID:28860991

Exogenous modification of platelet membranes with the omega-3 fatty acids EPA and DHA reduces platelet procoagulant activity and thrombus formation.

PubMed

Larson, Mark K; Tormoen, Garth W; Weaver, Lucinda J; Luepke, Kristen J; Patel, Ishan A; Hjelmen, Carl E; Ensz, Nicole M; McComas, Leah S; McCarty, Owen J T

2013-02-01

Several studies have implicated the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in inhibition of normal platelet function, suggesting a role for platelets in EPA- and DHA-mediated cardioprotection. However, it is unclear whether the cardioprotective mechanisms arise from alterations to platelet-platelet, platelet-matrix, or platelet-coagulation factor interactions. Our previous results led us to hypothesize that EPA and DHA alter the ability of platelets to catalyze the generation of thrombin. We tested this hypothesis by exogenously modifying platelet membranes with EPA and DHA, which resulted in compositional changes analogous to increased dietary EPA and DHA intake. Platelets treated with EPA and DHA showed reductions in the rate of thrombin generation and exposure of platelet phosphatidylserine. In addition, treatment of platelets with EPA and DHA decreased thrombus formation and altered the processing of thrombin precursor proteins. Furthermore, treatment of whole blood with EPA and DHA resulted in increased occlusion time and a sharply reduced accumulation of fibrin under flow conditions. These results demonstrate that EPA and DHA inhibit, but do not eliminate, the ability of platelets to catalyze thrombin generation in vitro. The ability of EPA and DHA to reduce the procoagulant function of platelets provides a possible mechanism behind the cardioprotective phenotype in individuals consuming high levels of EPA and DHA.

Platelet functional and transcriptional changes induced by intralipid infusion.

PubMed

Beaulieu, Lea M; Vitseva, Olga; Tanriverdi, Kahraman; Kucukural, Alper; Mick, Eric; Hamburg, Naomi; Vita, Joseph; Freedman, Jane E

2016-06-02

Multiple studies have shown the effects of long-term exposure to high-fat or western diets on the vascular system. There is limited knowledge on the acute effects of high circulating fat levels, specifically on platelets, which have a role in many processes, including thrombosis and inflammation. This study investigated the effects of acute, high-fat exposure on platelet function and transcript profile. Twenty healthy participants were given an intravenous infusion of 20% Intralipid emulsion and heparin over 6 hours. Blood samples were taken prior to and the day after infusion to measure platelet function and transcript expression levels. Platelet aggregation was not significantly affected by Intralipid infusion, but, when mitochondria function was inhibited by carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or oligomycin, platelet aggregation was higher in the post-infusion state compared to baseline. Through RNA sequencing, and verified by RT-qPCR, 902 miRNAs and 617 mRNAs were affected by Intralipid infusion. MicroRNAs increased include miR-4259 and miR-346, while miR-517b and miR-517c are both decreased. Pathway analysis identified two clusters significantly enriched, including cell motility. In conclusion, acute exposure to high fat affects mitochondrial-dependent platelet function, as well as the transcript profile.

Selective Inhibition of ADAM17 Efficiently Mediates Glycoprotein Ibα Retention During Ex Vivo Generation of Human Induced Pluripotent Stem Cell‐Derived Platelets

PubMed Central

Hirata, Shinji; Murata, Takahiko; Suzuki, Daisuke; Nakamura, Sou; Jono‐Ohnishi, Ryoko; Hirose, Hidenori; Sawaguchi, Akira; Nishimura, Satoshi; Sugimoto, Naoshi

2016-01-01

Abstract Donor‐independent platelet concentrates for transfusion can be produced in vitro from induced pluripotent stem cells (iPSCs). However, culture at 37°C induces ectodomain shedding on platelets of glycoprotein Ibα (GPIbα), the von Willebrand factor receptor critical for adhesive function and platelet lifetime in vivo, through temperature‐dependent activation of a disintegrin and metalloproteinase 17 (ADAM17). The shedding can be suppressed by using inhibitors of panmetalloproteinases and possibly of the upstream regulator p38 mitogen‐activated protein kinase (p38 MAPK), but residues of these inhibitors in the final platelet products may be accompanied by harmful risks that prevent clinical application. Here, we optimized the culture conditions for generating human iPSC‐derived GPIbα+ platelets, focusing on culture temperature and additives, by comparing a new and safe selective ADAM17 inhibitor, KP‐457, with previous inhibitors. Because cultivation at 24°C (at which conventional platelet concentrates are stored) markedly diminished the yield of platelets with high expression of platelet receptors, 37°C was requisite for normal platelet production from iPSCs. KP‐457 blocked GPIbα shedding from iPSC platelets at a lower half‐maximal inhibitory concentration than panmetalloproteinase inhibitor GM‐6001, whereas p38 MAPK inhibitors did not. iPSC platelets generated in the presence of KP‐457 exhibited improved GPIbα‐dependent aggregation not inferior to human fresh platelets. A thrombus formation model using immunodeficient mice after platelet transfusion revealed that iPSC platelets generated with KP‐457 exerted better hemostatic function in vivo. Our findings suggest that KP‐457, unlike GM‐6001 or p38 MAPK inhibitors, effectively enhances the production of functional human iPSC‐derived platelets at 37°C, which is an important step toward their clinical application. Stem Cells Translational Medicine 2017;6:720–730 PMID:28297575

Anti-platelet activity of a three-finger toxin (3FTx) from Indian monocled cobra (Naja kaouthia) venom.

PubMed

Chanda, Chandrasekhar; Sarkar, Angshuman; Sistla, Srinivas; Chakrabarty, Dibakar

2013-11-22

A low molecular weight anti-platelet peptide (6.9 kDa) has been purified from Naja kaouthia venom and was named KT-6.9. MALDI-TOF/TOF mass spectrometry analysis revealed the homology of KT-6.9 peptide sequence with many three finger toxin family members. KT-6.9 inhibited human platelet aggregation process in a dose dependent manner. It has inhibited ADP, thrombin and arachidonic acid induced platelet aggregation process in dose dependent manner, but did not inhibit collagen and ristocetin induced platelet aggregation. Strong inhibition (70%) of the ADP induced platelet aggregation by KT-6.9 suggests competition with ADP for its receptors on platelet surface. Anti-platelet activity of KT-6.9 was found to be 25 times stronger than that of anti-platelet drug clopidogrel. Binding of KT-6.9 to platelet surface was confirmed by surface plasma resonance analysis using BIAcore X100. Binding was also observed by a modified sandwich ELISA method using anti-KT-6.9 antibodies. KT-6.9 is probably the first 3 FTx from Indian monocled cobra venom reported as a platelet aggregation inhibitor. Copyright © 2013 Elsevier Inc. All rights reserved.

Gut microbial metabolite TMAO enhances platelet hyperreactivity and thrombosis risk

PubMed Central

Zhu, Weifei; Gregory, Jill C.; Org, Elin; Buffa, Jennifer A.; Gupta, Nilaksh; Wang, Zeneng; Li, Lin; Fu, Xiaoming; Wu, Yuping; Mehrabian, Margarete; Sartor, R. Balfour; McIntyre, Thomas M.; Silverstein, Roy L.; Tang, W.H. Wilson; DiDonato, Joseph A.; Brown, J. Mark; Lusis, Aldons J.; Hazen, Stanley L.

2016-01-01

SUMMARY Normal platelet function is critical to blood hemostasis and maintenance of a closed circulatory system. Heightened platelet reactivity, however, is associated with cardiometabolic diseases and enhanced potential for thrombotic events. We now show gut microbes, through generation of trimethylamine N-oxide (TMAO), directly contribute to platelet hyperreactivity and enhanced thrombosis potential. Plasma TMAO levels in subjects (N>4000) independently predicted incident (3 yr) thrombosis (heart attack, stroke) risk. Direct exposure of platelets to TMAO enhanced submaximal stimulus-dependent platelet activation from multiple agonists through augmented Ca2+ release from intracellular stores. Animal model studies employing dietary choline or TMAO, germ-free mice, and microbial transplantation, collectively confirm a role for gut microbiota and TMAO in modulating platelet hyperresponsiveness and thrombosis potential, and identify microbial taxa associated with plasma TMAO and thrombosis potential. Collectively, the present results reveal a previously unrecognized mechanistic link between specific dietary nutrients, gut microbes, platelet function, and thrombosis risk. PMID:26972052

Intracellular origin and ultrastructure of platelet-derived microparticles.

PubMed

Ponomareva, A A; Nevzorova, T A; Mordakhanova, E R; Andrianova, I A; Rauova, L; Litvinov, R I; Weisel, J W

2017-08-01

Essentials Platelet microparticles play a major role in pathologies, including hemostasis and thrombosis. Platelet microparticles have been analyzed and classified based on their ultrastructure. The structure and intracellular origin of microparticles depend on the cell-activating stimulus. Thrombin-treated platelets fall apart and form microparticles that contain cellular organelles. Background Platelet-derived microparticles comprise the major population of circulating blood microparticles that play an important role in hemostasis and thrombosis. Despite numerous studies on the (patho)physiological roles of platelet-derived microparticles, mechanisms of their formation and structural details remain largely unknown. Objectives Here we studied the formation, ultrastructure and composition of platelet-derived microparticles from isolated human platelets, either quiescent or stimulated with one of the following activators: arachidonic acid, ADP, collagen, thrombin or calcium ionophore A23187. Methods Using flow cytometry, transmission and scanning electron microscopy, we analyzed the intracellular origin, structural diversity and size distributions of the subcellular particles released from platelets. Results The structure, dimensions and intracellular origin of microparticles depend on the cell-activating stimulus. The main structural groups include a vesicle surrounded by one thin membrane or multivesicular structures. Thrombin, unlike other stimuli, induced formation of microparticles not only from the platelet plasma membrane and cytoplasm but also from intracellular structures. A fraction of these vesicular particles having an intracellular origin contained organelles, such as mitochondria, glycogen granules and vacuoles. The size of platelet-derived microparticles depended on the nature of the cell-activating stimulus. Conclusion The results obtained provide a structural basis for the qualitative differences of various platelet activators, for specific physiological and pathological effects of microparticles, and for development of advanced assays. © 2017 International Society on Thrombosis and Haemostasis.

Does Preoperative Platelet Function Predict Bleeding in Patients Undergoing Off Pump Coronary Artery Bypass Surgery?

PubMed

Berger, Peter B; Kirchner, H Lester; Wagner, Eric S; Ismail-Sayed, Ibrahim; Yahya, Salma; Benoit, Charles; Blankenship, James C; Carter, Russell; Casale, Alfred S; Green, Sandy M; Scott, Thomas D; Skelding, Kimberly A; Woods, Edward; Henry, Yvette M

2015-06-01

We sought to examine the relationship between preoperative platelet function and perioperative bleeding in patients undergoing CABG. There are many ways to measure platelet aggregability. Little is known about their correlations with one another, or with bleeding. We prospectively studied 50 patients undergoing a first isolated off-pump CABG. Thirty-four were exposed to a thienopyridine prior to surgery; 16 were not. Preoperative platelet function was measured by VerifyNow®, TEG®, AggreGuide™, Plateletworks®, vasodilator-stimulated phosphoprotein (VASP) phosphorylation, and light transmission aggregometry. Bleeding was assessed 2 ways: drop from pre- to nadir postoperative hematocrit, and chest tube drainage. Correlation coefficients were calculated using Spearman's rank-order correlation. Mean age was 62 years. Patient characteristics and surgical details were similar between the thienopyridine-exposed and non-exposed patients. The correlation coefficients between the 4 point-of-care platelet function measurements and hematocrit change ranged from -0.2274 to 0.2882. Only Plateletworks® correlated with drop in hematocrit (r = 0.2882, P = 0.0470). The correlation coefficients between each of the 4 point-of-care platelet function tests and the chest tube drainage were also poor, ranging from -0.3073 to 0.2272. Both AggreGuide™ (r = -0.3073, P = 0.0317) and VASP (r = -0.3187, P = 0.0272) were weakly but significantly correlated with chest tube drainage. The correlation among the 4 point-of-care platelet function measurements was poor, with coefficients ranging from -0.2504 to 0.1968. We observed little correlation among 4 platelet function tests, and between those assays and perioperative bleeding defined 2 different ways. Whether any of these assays should be used to guide decision making in individual patients is unclear. © 2015, Wiley Periodicals, Inc.

A point-of-care assessment of the effects of desmopressin on impaired platelet function using multiple electrode whole-blood aggregometry in patients after cardiac surgery.

PubMed

Weber, Christian F; Dietrich, Wulf; Spannagl, Michael; Hofstetter, Christian; Jámbor, Csilla

2010-03-01

Blood loss after cardiac surgery can be caused by acquired platelet dysfunction after cardiopulmonary bypass. Monitoring of platelet function is clinically important for the identification of patients experiencing such platelet dysfunction. 1-Deamino-8-D-arginine vasopressin (desmopressin acetate, DDAVP) has been shown to augment platelet function and to reduce blood loss in patients with platelet dysfunction. In this study, we examined the feasibility of whole blood multiple electrode aggregometry (MEA) for the detection of cardiopulmonary bypass-induced platelet dysfunction and investigated its ability to monitor DDAVP treatment. Fifty-eight consecutive patients with blood loss exceeding 150 mL/h in the first 2 consecutive hours after cardiac surgery were screened for suspected isolated platelet dysfunction. Twenty-two patients had suspected isolated platelet dysfunction and were enrolled in the study. Platelet dysfunction was assumed if conventional coagulation analyses (platelet count, activated partial thromboplastin time, international normalized ratio, and fibrinogen) did not show abnormal values as defined for transfusion of allogenic blood products, and no surgical cause of bleeding was suspected. Eleven patients received 0.3 microg/kg DDAVP, and 11 patients received no therapy in a nonrandomized manner. MEA was performed after stimulation with thrombin receptor-activating peptide (TRAPtest, 32 microM), adenosine diphosphate (ADPtest, 6.4 microM), and arachidonic acid (ASPItest, 0.5 mM) before and 2 hours after intervention. Conventional laboratory variables were recorded. The Mann-Whitney test was used to detect differences between the groups, and the Wilcoxon test was used to detect differences before and after intervention. All enrolled patients showed platelet dysfunction that manifested as impaired platelet aggregation in MEA before intervention. After the intervention, platelet function improved in the DDAVP group (49 U [30/72 U], median [25th/75th percentile] postintervention vs 15 U [8/21 U] preintervention for the ASPItest [P < 0.001]; 35 U [24/54 U] vs 14 U [7/28 U] for the ADPtest [P = 0.002]; and 85 U [66/115 U] vs 64 U [26/88 U] for the TRAPtest [P = 0.007]). In contrast, MEA remained unchanged in the control group (22 U [10/50 U] postintervention vs 33 U [14/57 U] preintervention for the ASPItest [P = 0.175]; 17 U [12/20 U] vs 14 U [10/28 U] for the ADPtest [P = 0.147]; and 65 U [41/89 U] vs 57 U [30/91 U] for the TRAPtest [P = 0.123]). Impaired platelet function after cardiac surgery can be assessed at the bedside using MEA. The effect of DDAVP on impaired platelet function can also be detected as significant improvement in platelet aggregation to all activators. This device might be helpful for the identification of patients who may benefit from DDAVP therapy.

Cationic PAMAM dendrimers disrupt key platelet functions

PubMed Central

Jones, Clinton F.; Campbell, Robert A.; Franks, Zechariah; Gibson, Christopher C.; Thiagarajan, Giridhar; Vieira-de-Abreu, Adriana; Sukavaneshvar, Sivaprasad; Mohammad, S. Fazal; Li, Dean Y.; Ghandehari, Hamidreza; Weyrich, Andrew S.; Brooks, Benjamin D.; Grainger, David W.

2012-01-01

Poly(amidoamine) (PAMAM) dendrimers have been proposed for a variety of biomedical applications and are increasingly studied as model nanomaterials for such use. The dendritic structure features both modular synthetic control of molecular size and shape and presentation of multiple equivalent terminal groups. These properties make PAMAM dendrimers highly functionalizable, versatile single-molecule nanoparticles with a high degree of consistency and low polydispersity. Recent nanotoxicological studies showed that intravenous administration of amine-terminated PAMAM dendrimers to mice was lethal, causing a disseminated intravascular coagulation-like condition. To elucidate the mechanisms underlying this coagulopathy, in vitro assessments of platelet functions in contact with PAMAM dendrimers were undertaken. This study demonstrates that cationic G7 PAMAM dendrimers activate platelets and dramatically alter their morphology. These changes to platelet morphology and activation state substantially altered platelet function, including increased aggregation and adherence to surfaces. Surprisingly, dendrimer exposure also attenuated platelet-dependent thrombin generation, indicating that not all platelet functions remained intact. These findings provide additional insight into PAMAM dendrimer effects on blood components and underscore the necessity for further research on the effects and mechanisms of PAMAM-specific and general nanoparticle toxicity in blood. PMID:22497592

Transcriptomic analysis of the ion channelome of human platelets and megakaryocytic cell lines.

PubMed

Wright, Joy R; Amisten, Stefan; Goodall, Alison H; Mahaut-Smith, Martyn P

2016-08-01

Ion channels have crucial roles in all cell types and represent important therapeutic targets. Approximately 20 ion channels have been reported in human platelets; however, no systematic study has been undertaken to define the platelet channelome. These membrane proteins need only be expressed at low copy number to influence function and may not be detected using proteomic or transcriptomic microarray approaches. In our recent work, quantitative real-time PCR (qPCR) provided key evidence that Kv1.3 is responsible for the voltage-dependent K+ conductance of platelets and megakaryocytes. The present study has expanded this approach to assess relative expression of 402 ion channels and channel regulatory genes in human platelets and three megakaryoblastic/erythroleukaemic cell lines. mRNA levels in platelets are low compared to other blood cells, therefore an improved method of isolating platelets was developed. This used a cocktail of inhibitors to prevent formation of leukocyte-platelet aggregates, and a combination of positive and negative immunomagnetic cell separation, followed by rapid extraction of mRNA. Expression of 34 channel-related transcripts was quantified in platelets, including 24 with unknown roles in platelet function, but that were detected at levels comparable to ion channels with established roles in haemostasis or thrombosis. Trace expression of a further 50 ion channel genes was also detected. More extensive channelomes were detected in MEG-01, CHRF-288-11 and HEL cells (195, 185 and 197 transcripts, respectively), but lacked several channels observed in the platelet. These "channelome" datasets provide an important resource for further studies of ion channel function in the platelet and megakaryocyte.

Platelet Arachidonic Acid Deficiency May Contribute to Abnormal Platelet Function During Parenteral Fish Oil Monotherapy in a Piglet Model.

PubMed

Turner, Justine M; Field, Catherine J; Goruk, Sue; Wizzard, Pamela; Dicken, Bryan J; Bruce, Aisha; Wales, Paul W

2016-05-01

Fish oil monotherapy has been an advance for treating intestinal failure-associated liver disease (IFALD). However, such patients are at risk of bleeding complications from liver disease and because fish oil can inhibit thrombosis. We have previously reported abnormal platelet function in neonatal piglets given fish oil monotherapy during parenteral nutrition (PN). The purpose of this study was to determine if abnormal fatty acid composition of the platelets could explain the prior observed antiplatelet effect. Neonatal piglets were assigned to 2 treatments: PN with fish oil monotherapy (FO; n = 4) or PN with soy oil (SO; n = 5). On day 14, plasma was collected and platelets isolated by centrifuging. The fatty acid content in plasma and platelet plug were measured using gas liquid chromatography and compared with controls (CON; n = 5). The arachidonic acid (AA) content in the FO group was on average half that of the SO group, in both the platelets (FO, 3.5% vs SO, 7.6%; P = .021; CON, 4.5%-11%) and the plasma (FO, 3.8% vs SO, 9.2%; P = .002; CON, 6.1%-9.5%). No bleeding complications were observed for any piglets during PN treatment. Using platelet mapping, we have previously shown that neonatal piglets given fish oil monotherapy have abnormal platelet function in the AA pathway. This report demonstrates that such an abnormality can be explained by platelet AA deficiency. Platelet mapping and platelet fatty acid analysis should be undertaken in human infants treated with fish oil monotherapy during PN. © 2015 American Society for Parenteral and Enteral Nutrition.

Preanalytical requirements for flow cytometric evaluation of platelet activation: choice of anticoagulant.

PubMed

Mody, M; Lazarus, A H; Semple, J W; Freedman, J

1999-06-01

Accurate assessment of in vivo or in vitro platelet activation requires optimal preanalytical conditions to prevent artefactual in vitro activation of the platelets. The choice of anticoagulant is one of the critical preanalytical conditions as anticoagulants exert different effects on the activation of platelets ex vivo. We tested the effectiveness of Diatube-H (also known as CTAD; sodium citrate, theophylline, adenosine and dipyridamole) and citrate vacutainer tubes in preventing artefactual activation of platelets and preserving functional reserve. Platelet surface expression of the CD62P (reflecting alpha granule release), CD63 (reflecting lysosomal release) and modulation of normal platelet membrane glycoproteins CD41a and CD42b, were measured in whole blood and in isolated platelets immediately after collection and at 6, 24 and 48 h after venipuncture. Samples taken into Diatube-H showed less spontaneous platelet activation than did those taken into citrate. To measure in vitro platelet functional reserve, thrombin was added as agonist to blood stored for varying periods up to 48 h. Although Diatube-H suppressed in vitro platelet activation for up to 4 h, in samples kept for 6-24 h before thrombin addition, the inhibitory effect was lost and platelets responded fully to agonist activation. Hence, Diatube-H preserved platelets and allowed for measurement of in vivo platelet activation as well as thrombin-induced in vitro platelet activation after 6-24 h, in both whole blood and isolated platelets.

Negative feedback regulation of human platelets via autocrine activation of the platelet-derived growth factor alpha-receptor.

PubMed

Vassbotn, F S; Havnen, O K; Heldin, C H; Holmsen, H

1994-05-13

Human platelets contain platelet-derived growth factor (PDGF) in their alpha-granules which is released during platelet exocytosis. We show by immunoprecipitation and 125I-PDGF binding experiments that human platelets have functionally active PDGF alpha-receptors, but not beta-receptors. The PDGF alpha-receptor (PDGFR-alpha) was identified as a 170-kDa glycosylated protein-tyrosine kinase as found in other cell types. Stimulation of platelets with 0.1 unit/ml thrombin resulted in a significant increase (2-5-fold) of the tyrosine phosphorylation of the PDGFR-alpha, as determined by immunoprecipitation with phosphotyrosine antiserum as well as with PDGFR-alpha antiserum. The observed thrombin-induced autophosphorylation of the PDGFR-alpha was inhibited by the addition of a neutralizing monoclonal PDGF antibody. Thus, our results suggest that the platelet PDGFR-alpha is stimulated in an autocrine manner by PDGF secreted during platelet activation. Preincubation of platelets with PDGF inhibited thrombin-induced platelet aggregation and secretion of ATP + ADP and beta-hexosaminidase. Thrombin-induced platelet aggregation was also reversed when PDGF was added 30 s after thrombin stimulation. Inhibition of the autocrine PDGF pathway during platelet activation by the PDGF antibody led to a potentiation of thrombin-induced beta-hexosaminidase secretion. Thus, the PDGFR-alpha takes part in a negative feedback regulation during platelet activation. Our demonstration of PDGF alpha-receptors on human platelets and its inhibitory function during platelet activation identifies a new possible role of PDGF in the regulation of thrombosis.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Akatsu, T.; Bourdelle, K.K.; Richtarch, C.

Extended defects formed after hydrogen implantation into Si and Ge (100) substrates and subsequent thermal anneals were investigated by transmission electron microscopy. The majority of the extended defects formed in both materials were platelet-like structures lying on {l_brace}100{r_brace} and {l_brace}111{r_brace} planes. We found {l_brace}100{r_brace} platelets not only parallel but also perpendicular to the surface. In Ge wafers, high density of {l_brace}311{r_brace} defects and nanobubbles with the average size of 2 nm were observed. The difference between two materials can be attributed to the weaker strength of Ge-H bond.

Glycoprotein IIB/IIIA inhibitor to reduce postpercutaneous coronary intervention myonecrosis and improve coronary flow in diabetics: the 'OPTIMIZE-IT' pilot randomized study.

PubMed

Talarico, Giovanni Paolo; Brancati, Marta; Burzotta, Francesco; Porto, Italo; Trani, Carlo; De Vita, Maria; Todaro, Daniel; Giammarinaro, Maura; Leone, Antonio Maria; Niccoli, Giampaolo; Andreotti, Felicita; Mazzari, Mario Attilio; Schiavoni, Giovanni; Crea, Filippo

2009-03-01

Subgroup analyses of trials enrolling acute coronary syndrome patients suggest that inhibition of glycoprotein IIb/IIIa can improve the outcome of diabetic patients undergoing percutaneous coronary interventions (PCIs), possibly by improving microvascular perfusion. However, the efficacy of small-molecule IIb/IIIa receptor inhibitors to improve microvascular perfusion in stable diabetic patients undergoing elective PCI has not been specifically investigated. We randomized consecutive stable diabetic patients, undergoing elective PCI, to tirofiban or placebo groups along with double antiplatelet therapy. High-dose bolus (25 microg/kg per 3 min) of tirofiban was administered immediately before PCI followed by 8 h continuous infusion (0.15 microg/kg per min). Postprocedural myonecrosis was assessed prospectively by measurement of cardiac troponin T (cTnT) at 6 and 24 h after PCI. The primary end-points were post-PCI coronary flow estimated by corrected thrombolysis in myocardial infarction frame count and post-PCI myocardial infarction. Platelet aggregation was measured by platelet function analyser-100 values. Forty-six patients entered the study (22 randomized to placebo and 24 randomized to tirofiban). The study drug was associated with a significant increase of platelet function analyser-100 values that peaked immediately after PCI and was maintained at 6 h (pre-PCI: 131 +/- 65 s; post-PCI: 222 +/- 49 s; after 6 h: 219 +/- 55 s).Post-PCI corrected thrombolysis in myocardial infarction frame count was similar in tirofiban and in placebo groups (10.2 +/- 3.6 vs. 12.0 +/- 7.6, P = 0.30, respectively). The prevalence of raised cTnT levels was similar in the two groups (25 vs. 30%, P = 0.56, respectively). At multivariate analysis, direct stenting (associated with reduced myonecrosis) and postdilatation (associated with increased myonecrosis) predicted cTnT elevation. A high-dose bolus of tirofiban in stable diabetic patients undergoing elective PCI, along with double antiplatelet therapy, was associated with a significant further inhibition of platelet aggregation which, however, did not translate in a lower incidence of post-PCI distal embolization.

Plasma Free Hemoglobin Generation Using the EOS PMP™ Oxygenator and the CentriMag® Blood Pump.

PubMed

Hodge, Ashley B; Deitemyer, Matthew A; Duffy, Victoria L; Tumin, Dmitry; Garbin, Dorothy A; Nicol, Kathleen K; Hayes, Don; Cismowski, Mary J; Yates, Andrew R

2018-06-01

Hemolysis is a known consequence of extracorporeal membrane oxygenation (ECMO) resulting from shear force within the different components of the extracorporeal circuit. The primary aim of this study was to evaluate the EOS PMP ™ oxygenator for generation of plasma free hemoglobin (PfHg) over 24 hours at nominal operating range flow rates. The EOS ECMO ™ (LivaNova, Inc.; formerly Sorin, Arvada, CO) is equipped with a plasma tight polymethylpentene (PMP) hollow fiber oxygenator. We hypothesized that PfHg generation would be elevated in circuits with higher flow rates, because of the significant pressure drop across the oxygenator according to manufacturer provided flow charts. Generated PfHg concentrations were compared with PfHg concentrations from blood not exposed to an ECMO circuit. The secondary aim was to evaluate circuit flow-rate-induced changes in platelet count and platelet function over 24 hours. Circuits contained a CentriMag ® (St. Jude Medical, St. Paul, MN) blood pump and an EOS ECMO PMP ™ oxygenator. Circuits in triplicate were run continuously for 24 hours at three flow rates [1, 3, and 5 liters per minute {LPM}]. PfHg was analyzed at baseline, 6, 12, 18, and 24 hours. Platelet count and function were measured at baseline and 24 hours. Concentrations of PfHg at baseline for circuits operating at 1, 3, and 5 LPM were 24.4 ± 4.0, 38.4 ± 28.6, and 26.7 ± 6.9 mg/dL, respectively. PfHg concentrations after 24 hours were statistically compared for the three flow rates using analysis of variance; PfHg concentrations at 1 LPM (181.4 ± 29.1 mg/dL), 3 LPM (145.9 ± 8.7 mg/dL), and 5 LPM (100.1 ± 111.3 mg/dL) circuits. The F -test was not statistically significant ( p = .632), indicating that PfHg generation at 24 hours was similar among the three flow rates. Excessive hemolysis using PfHg levels in the EOS PMP ™ membrane oxygenator was not observed.

Attenuation of Thrombosis by Crude Rice (Oryza sativa) Bran Policosanol Extract: Ex Vivo Platelet Aggregation and Serum Levels of Arachidonic Acid Metabolites

PubMed Central

Ismail, Maznah; Tohit, Eusni Rahayu Mohd; Abdullah, Rasedee; Zhang, Yi-Da

2016-01-01

Background. Vascular occlusion or thrombosis was often attributed to uncontrolled platelet activation. Influence of sugarcane policosanol extract on platelet was reported but little was known of rice bran policosanol, particularly its mechanisms of actions on platelet activities. Objective. Antiplatelet mechanisms of rice bran policosanol extract (RBE) were studied using hyperlipidemic Sprague Dawley rats. Ex vivo platelet aggregation, platelet count (PC), bleeding time (BT), and coagulation time were assayed. Serum eicosanoids and other aggregation-related metabolites levels were quantified. Design. Rats were divided into 6 groups for comparisons (vehicle control Tween 20/H2O, high dose policosanol 500 mg/kg, middle dose policosanol 250 mg/kg, low dose policosanol 100 mg/kg, and positive control aspirin 30 mg/kg). Results. Low dose 100 mg/kg of RBE inhibited aggregation by 42.32 ± 4.31% and this was comparable with the effect of 30 mg/kg aspirin, 43.91 ± 5.27%. Results showed that there were no significant differences in PC, BT, and coagulation time among various groups after RBE treatment. Serum thromboxane A2 was attenuated while prostacyclin level increased upon RBE treatment. Conclusions. RBE reduced ex vivo ADP-induced platelet aggregation without giving adverse effects. No changes in full blood count suggested that rice bran policosanol did not disturb biological blood cell production and destruction yet it reduced aggregation through different mechanisms. PMID:27800004

Acute impact of conventional and eccentric cycling on platelet and vascular function in patients with chronic heart failure.

PubMed

Haynes, Andrew; Linden, Matthew D; Chasland, Lauren C; Nosaka, Kazunori; Maiorana, Andrew; Dawson, Ellen A; Dembo, Lawrence H; Naylor, Louise H; Green, Daniel J

2017-06-01

Evidence-based guidelines recommend exercise therapy for patients with chronic heart failure (CHF). Such patients have increased atherothrombotic risk. Exercise can transiently increase platelet activation and reactivity and decrease vascular function in healthy participants, although data in CHF are scant. Eccentric (ECC) cycling is a novel exercise modality that may be particularly suited to patients with CHF, but the acute impacts of ECC cycling on platelet and vascular function are currently unknown. Our null hypothesis was that ECC and concentric (CON) cycling, performed at matched external workloads, would not induce changes in platelet or vascular function in patients with CHF. Eleven patients with heart failure with reduced ejection fraction (HFrEF) took part in discrete bouts of ECC and CON cycling. Before and immediately after exercise, vascular function was assessed by measuring diameter and flow-mediated dilation (FMD) of the brachial artery. Platelet function was measured by the flow cytometric determination of glycoprotein IIb/IIIa activation and granule exocytosis in the presence and absence of platelet agonists. ECC cycling increased baseline artery diameter (pre: 4.0 ± 0.8 mm vs. post: 4.2 ± 0.7 mm; P = 0.04) and decreased FMD%. When changes in baseline artery diameter were accounted for, the decrease in FMD post-ECC cycling was no longer significant. No changes were apparent after CON. Neither ECC nor CON cycling resulted in changes to any platelet-function measures (all P > 0.05). These results suggest that both ECC and CON cycling, at a moderate intensity and short duration, can be performed by patients with HFrEF without detrimental impacts on vascular or platelet function. NEW & NOTEWORTHY This is the first evidence to indicate that eccentric (ECC) cycling can be performed relatively safely by patients with chronic heart failure (CHF), as it did not result in impaired vascular or platelet function compared with conventional cycling. This is important, as acute exercise can transiently increase atherothrombotic risk, and ECC cycling is a novel exercise modality that may be particularly suited to patients with CHF. Copyright © 2017 the American Physiological Society.

Method of producing nano-scaled inorganic platelets

DOEpatents

Zhamu, Aruna; Jang, Bor Z.

2012-11-13

The present invention provides a method of exfoliating a layered material (e.g., transition metal dichalcogenide) to produce nano-scaled platelets having a thickness smaller than 100 nm, typically smaller than 10 nm. The method comprises (a) dispersing particles of a non-graphite laminar compound in a liquid medium containing therein a surfactant or dispersing agent to obtain a stable suspension or slurry; and (b) exposing the suspension or slurry to ultrasonic waves at an energy level for a sufficient length of time to produce separated nano-scaled platelets. The nano-scaled platelets are candidate reinforcement fillers for polymer nanocomposites.

Ultraviolet-Based Pathogen Inactivation Systems: Untangling the Molecular Targets Activated in Platelets

PubMed Central

Schubert, Peter; Johnson, Lacey; Marks, Denese C.; Devine, Dana V.

2018-01-01

Transfusions of platelets are an important cornerstone of medicine; however, recipients may be subject to risk of adverse events associated with the potential transmission of pathogens, especially bacteria. Pathogen inactivation (PI) technologies based on ultraviolet illumination have been developed in the last decades to mitigate this risk. This review discusses studies of platelet concentrates treated with the current generation of PI technologies to assess their impact on quality, PI capacity, safety, and clinical efficacy. Improved safety seems to come with the cost of reduced platelet functionality, and hence transfusion efficacy. In order to understand these negative impacts in more detail, several molecular analyses have identified signaling pathways linked to platelet function that are altered by PI. Because some of these biochemical alterations are similar to those seen arising in the context of routine platelet storage lesion development occurring during blood bank storage, we lack a complete picture of the contribution of PI treatment to impaired platelet functionality. A model generated using data from currently available publications places the signaling protein kinase p38 as a central player regulating a variety of mechanisms triggered in platelets by PI systems. PMID:29868586

Phenotypic approaches to gene mapping in platelet function disorders - identification of new variant of P2Y12, TxA2 and GPVI receptors.

PubMed

Watson, S; Daly, M; Dawood, B; Gissen, P; Makris, M; Mundell, S; Wilde, J; Mumford, A

2010-01-01

Platelet number or function disorders cause a range of bleeding symptoms from mild to severe. Patients with platelet dysfunction but normal platelet number are the most prevalent and typically have mild bleeding symptoms. The study of this group of patients is particularly difficult because of the lack of a gold-standard test of platelet function and the variable penetrance of the bleeding phenotype among affected individuals. The purpose of this short review is to discuss the way in which this group of patients can be investigated through platelet phenotyping in combination with targeted gene sequencing. This approach has been used recently to identify patients with mutations in key platelet activation receptors, namely those for ADP, collagen and thromboxane A2 (TxA2). One interesting finding from this work is that for some patients, mild bleeding is associated with heterozygous mutations in platelet proteins that are co-inherited with other genetic disorders of haemostasis such as type 1 von Willebrand's disease. Thus, the phenotype of mild bleeding may be multifactorial in some patients and may be considered to be a complex trait.

Glucose ameliorates the metabolic profile and mitochondrial function of platelet concentrates during storage in autologous plasma

PubMed Central

Amorini, Angela M.; Tuttobene, Michele; Tomasello, Flora M.; Biazzo, Filomena; Gullotta, Stefano; De Pinto, Vito; Lazzarino, Giuseppe; Tavazzi, Barbara

2013-01-01

Background It is essential that the quality of platelet metabolism and function remains high during storage in order to ensure the clinical effectiveness of a platelet transfusion. New storage conditions and additives are constantly evaluated in order to achieve this. Using glucose as a substrate is controversial because of its potential connection with increased lactate production and decreased pH, both parameters triggering the platelet lesion during storage. Materials and methods In this study, we analysed the morphological status and metabolic profile of platelets stored for various periods in autologous plasma enriched with increasing glucose concentrations (13.75, 27.5 and 55 mM). After 0, 2, 4, 6 and 8 days, high energy phosphates (ATP, GTP, ADP, AMP), oxypurines (hypoxanthine, xanthine, uric acid), lactate, pH, mitochondrial function, cell lysis and morphology, were evaluated. Results The data showed a significant dose-dependent improvement of the different parameters in platelets stored with increasing glucose, compared to what detected in controls. Interestingly, this phenomenon was more marked at the highest level of glucose tested and in the period of time generally used for platelet transfusion (0–6 days). Conclusion These results indicate that the addition of glucose during platelet storage ameliorates, in a dose-dependent manner, the biochemical parameters related to energy metabolism and mitochondrial function. Since there was no correspondence between glucose addition, lactate increase and pH decrease in our experiments, it is conceivable that platelet derangement during storage is not directly caused by glucose through an increase of anaerobic glycolysis, but rather to a loss of mitochondrial functions caused by reduced substrate availability. PMID:22682337

Formation of liquid crystalline phases in aqueous suspensions of platelet-like tripalmitin nanoparticles

NASA Astrophysics Data System (ADS)

Schmiele, Martin; Gehrer, Simone; Westermann, Martin; Steiniger, Frank; Unruh, Tobias

2014-06-01

Suspensions of platelet-like shaped tripalmitin nanocrystals stabilized by the pure lecithin DLPC and the lecithin blend S100, respectively, have been studied by small-angle x-ray scattering (SAXS) and optical observation of their birefringence at different tripalmitin (PPP) concentrations φPPP. It could be demonstrated that the platelets of these potential drug delivery systems start to form a liquid crystalline phase already at pharmaceutically relevant concentrations φPPP of less than 10 wt. %. The details of this liquid crystalline phase are described here for the first time. As in a previous study [A. Illing et al., Pharm. Res. 21, 592 (2004)] some platelets are found to self-assemble into lamellar stacks above a critical tripalmitin concentration \\varphi _{PPP}^{st} of 4 wt. %. In this study another critical concentration \\varphi _{PPP}^{lc}≈ 7 wt. % for DLPC and \\varphi _{PPP}^{lc}≈ 9 wt. % for S100 stabilized dispersions, respectively, has been observed. \\varphi _{PPP}^{lc} describes the transition from a phase of randomly oriented stacked lamellae and remaining non-assembled individual platelets to a phase in which the stacks and non-assembled platelets exhibit an overall preferred orientation. A careful analysis of the experimental data indicates that for concentrations above \\varphi _{PPP}^{lc} the stacked lamellae start to coalesce to rather small liquid crystalline domains of nematically ordered stacks. These liquid crystalline domains can be individually very differently oriented but possess an overall preferred orientation over macroscopic length scales which becomes successively more expressed when further increasing φPPP. The lower critical concentration for the formation of liquid crystalline domains of the DLPC-stabilized suspension compared to \\varphi _{PPP}^{lc} of the S100-stabilized suspension can be explained by a larger aspect ratio of the corresponding tripalmitin platelets. A geometrical model based on the excluded volumes of individual platelets and stacked lamellae has been developed and successfully applied to reproduce the critical volume fractions for both, the onset of stack formation and the appearance of the liquid crystalline phase.

Toward correlating structure and mechanics of platelets.

PubMed

Sorrentino, Simona; Studt, Jan-Dirk; Horev, Melanie Bokstad; Medalia, Ohad; Sapra, K Tanuj

2016-09-02

The primary physiological function of blood platelets is to seal vascular lesions after injury and form hemostatic thrombi in order to prevent blood loss. This task relies on the formation of strong cellular-extracellular matrix interactions in the subendothelial lesions. The cytoskeleton of a platelet is key to all of its functions: its ability to spread, adhere and contract. Despite the medical significance of platelets, there is still no high-resolution structural information of their cytoskeleton. Here, we discuss and present 3-dimensional (3D) structural analysis of intact platelets by using cryo-electron tomography (cryo-ET) and atomic force microscopy (AFM). Cryo-ET provides in situ structural analysis and AFM gives stiffness maps of the platelets. In the future, combining high-resolution structural and mechanical techniques will bring new understanding of how structural changes modulate platelet stiffness during activation and adhesion.

Global Proteome Analysis Identifies Active Immunoproteasome Subunits in Human Platelets*

PubMed Central

Klockenbusch, Cordula; Walsh, Geraldine M.; Brown, Lyda M.; Hoffman, Michael D.; Ignatchenko, Vladimir; Kislinger, Thomas; Kast, Juergen

2014-01-01

The discovery of new functions for platelets, particularly in inflammation and immunity, has expanded the role of these anucleate cell fragments beyond their primary hemostatic function. Here, four in-depth human platelet proteomic data sets were generated to explore potential new functions for platelets based on their protein content and this led to the identification of 2559 high confidence proteins. During a more detailed analysis, consistently high expression of the proteasome was discovered, and the composition and function of this complex, whose role in platelets has not been thoroughly investigated, was examined. Data set mining resulted in identification of nearly all members of the 26S proteasome in one or more data sets, except the β5 subunit. However, β5i, a component of the immunoproteasome, was identified. Biochemical analyses confirmed the presence of all catalytically active subunits of the standard 20S proteasome and immunoproteasome in human platelets, including β5, which was predominantly found in its precursor form. It was demonstrated that these components were assembled into the proteasome complex and that standard proteasome as well as immunoproteasome subunits were constitutively active in platelets. These findings suggest potential new roles for platelets in the immune system. For example, the immunoproteasome may be involved in major histocompatibility complex I (MHC I) peptide generation, as the MHC I machinery was also identified in our data sets. PMID:25146974

Platelet hyperaggregability in obesity: is there a role for nitric oxide impairment and oxidative stress?

PubMed

Leite, Natália Rodrigues Pereira; Siqueira de Medeiros, Mariana; Mury, Wanda Vianna; Matsuura, Cristiane; Perszel, Monique Bandeira Moss; Noronha Filho, Gerson; Brunini, Tatiana Mc; Mendes-Ribeiro, Antônio Claúdio

2016-08-01

Epidemiological evidence has shown that platelet activation markers are consistently elevated in obesity, contributing to its prothrombotic state. In order to improve the understanding of the regulation of platelet function in obesity, the aim of this study was to investigate the l-arginine-nitric oxide (NO) pathway in obese adults without other cardiovascular risk factor. Seventeen obese (body mass index [BMI] 35.9±1.0 kg/m(2) ) and eighteen age-matched normal weight subjects (BMI 22.0±0.6 kg/m(2) ) were included in this study. l-arginine influx was measured with incubation of l-[(3) H]-arginine. NO synthase (NOS) and arginase activities were determined by the citrulline assay and the conversion of l-[(14) C]-arginine to [(14) C]-urea, respectively. Cyclic guanosine monophosphate (cGMP) content was evaluated by enzyme-linked immunosorbent assay. In addition, the study analyzed: platelet aggregation; intraplatelet antioxidant enzymes, via superoxide dismutase (SOD) and catalase activities; and systemic levels of l-arginine, fibrinogen, and C-reactive protein (CRP). Obese patients presented a significant decrease of platelet l-arginine influx, NOS activity, and cGMP levels, along with platelet hyperaggregability. On the presence of NO donor, platelet aggregation was similar between the groups. The fibrinogen and CRP systemic levels were significantly higher and SOD activity was reduced in obesity. No significant differences were observed in plasma levels of l-arginine and intraplatelet arginase and catalase activities between groups. The diminished NO bioavailability associated with inflammatory status and impaired enzymatic antioxidant defence may contribute to future cardiovascular complications in obesity. © 2016 John Wiley & Sons Australia, Ltd.

Whole blood coagulation and platelet activation in the athlete: a comparison of marathon, triathlon and long distance cycling.

PubMed

Hanke, Alexander A; Staib, A; Görlinger, K; Perrey, M; Dirkmann, D; Kienbaum, P

2010-02-26

Serious thrombembolic events occur in otherwise healthy marathon athletes during competition. We tested the hypothesis that during heavy endurance sports coagulation and platelets are activated depending on the type of endurance sport with respect to its running fraction. 68 healthy athletes participating in marathon (MAR, running 42 km, n = 24), triathlon (TRI, swimming 2.5 km + cycling 90 km + running 21 km, n = 22), and long distance cycling (CYC, 151 km, n = 22) were included in the study. Blood samples were taken before and immediately after completion of competition to perform rotational thrombelastometry. We assessed coagulation time (CT), maximum clot firmness (MCF) after intrinsically activation and fibrin polymerization (FIBTEM). Furthermore, platelet aggregation was tested after activation with ADP and thrombin activating peptide 6 (TRAP) by using multiple platelet function analyzer. Complete data sets were obtained in 58 athletes (MAR: n = 20, TRI: n = 19, CYC: n = 19). CT significantly decreased in all groups (MAR -9.9%, TRI -8.3%, CYC -7.4%) without differences between groups. In parallel, MCF (MAR +7.4%, TRI +6.1%, CYC +8.3%) and fibrin polymerization (MAR +14.7%, TRI +6.1%, CYC +8.3%) were significantly increased in all groups. However, platelets were only activated during MAR and TRI as indicated by increased AUC during TRAP-activation (MAR +15.8%) and increased AUC during ADP-activation in MAR (+50.3%) and TRI (+57.5%). While coagulation is activated during physical activity irrespective of type we observed significant platelet activation only during marathon and to a lesser extent during triathlon. We speculate that prolonged running may increase platelet activity, possibly, due to mechanical alteration. Thus, particularly prolonged running may increase the risk of thrombembolic incidents in running athletes.

Whole blood coagulation and platelet activation in the athlete: A comparison of marathon, triathlon and long distance cycling

PubMed Central

2010-01-01

Introduction Serious thrombembolic events occur in otherwise healthy marathon athletes during competition. We tested the hypothesis that during heavy endurance sports coagulation and platelets are activated depending on the type of endurance sport with respect to its running fraction. Materials and Methods 68 healthy athletes participating in marathon (MAR, running 42 km, n = 24), triathlon (TRI, swimming 2.5 km + cycling 90 km + running 21 km, n = 22), and long distance cycling (CYC, 151 km, n = 22) were included in the study. Blood samples were taken before and immediately after completion of competition to perform rotational thrombelastometry. We assessed coagulation time (CT), maximum clot firmness (MCF) after intrinsically activation and fibrin polymerization (FIBTEM). Furthermore, platelet aggregation was tested after activation with ADP and thrombin activating peptide 6 (TRAP) by using multiple platelet function analyzer. Results Complete data sets were obtained in 58 athletes (MAR: n = 20, TRI: n = 19, CYC: n = 19). CT significantly decreased in all groups (MAR -9.9%, TRI -8.3%, CYC -7.4%) without differences between groups. In parallel, MCF (MAR +7.4%, TRI +6.1%, CYC +8.3%) and fibrin polymerization (MAR +14.7%, TRI +6.1%, CYC +8.3%) were significantly increased in all groups. However, platelets were only activated during MAR and TRI as indicated by increased AUC during TRAP-activation (MAR +15.8%) and increased AUC during ADP-activation in MAR (+50.3%) and TRI (+57.5%). Discussion While coagulation is activated during physical activity irrespective of type we observed significant platelet activation only during marathon and to a lesser extent during triathlon. We speculate that prolonged running may increase platelet activity, possibly, due to mechanical alteration. Thus, particularly prolonged running may increase the risk of thrombembolic incidents in running athletes. PMID:20452885

Effects of desmopressin on platelet function under conditions of hypothermia and acidosis: an in vitro study using multiple electrode aggregometry*.

PubMed

Hanke, A A; Dellweg, C; Kienbaum, P; Weber, C F; Görlinger, K; Rahe-Meyer, N

2010-07-01

Hypothermia and acidosis lead to an impairment of coagulation. It has been demonstrated that desmopressin improves platelet function under hypothermia. We tested platelet function ex vivo during hypothermia and acidosis. Blood samples were taken from 12 healthy subjects and assigned as follows: normal pH, pH 7.2, and pH 7.0, each with and without incubation with desmopressin. Platelet aggregation was assessed by multiple electrode aggregometry. Baseline was normal pH and 36 degrees C. The other samples were incubated for 30 min and measured at 32 degrees C. Acidosis significantly impaired aggregation. Desmopressin significantly increased aggregability during hypothermia and acidosis regardless of pH, but did not return it to normal values at low pH. During acidosis and hypothermia, acidosis should be corrected first; desmopressin can then be administered to improve platelet function as a bridge until normothermia can be achieved.

Rupture Forces among Human Blood Platelets at different Degrees of Activation

PubMed Central

Nguyen, Thi-Huong; Palankar, Raghavendra; Bui, Van-Chien; Medvedev, Nikolay; Greinacher, Andreas; Delcea, Mihaela

2016-01-01

Little is known about mechanics underlying the interaction among platelets during activation and aggregation. Although the strength of a blood thrombus has likely major biological importance, no previous study has measured directly the adhesion forces of single platelet-platelet interaction at different activation states. Here, we filled this void first, by minimizing surface mediated platelet-activation and second, by generating a strong adhesion force between a single platelet and an AFM cantilever, preventing early platelet detachment. We applied our setup to measure rupture forces between two platelets using different platelet activation states, and blockade of platelet receptors. The rupture force was found to increase proportionally to the degree of platelet activation, but reduced with blockade of specific platelet receptors. Quantification of single platelet-platelet interaction provides major perspectives for testing and improving biocompatibility of new materials; quantifying the effect of drugs on platelet function; and assessing the mechanical characteristics of acquired/inherited platelet defects. PMID:27146004

Cyclophilin B binding to platelets supports calcium-dependent adhesion to collagen.

PubMed

Allain, F; Durieux, S; Denys, A; Carpentier, M; Spik, G

1999-08-01

We have recently reported that cyclophilin B (CyPB), a secreted cyclosporine-binding protein, could bind to T lymphocytes through interactions with two types of binding sites. The first ones, referred to as type I, involve interactions with the conserved domain of CyPB and promote the endocytosis of surface-bound ligand, while the second type of binding sites, termed type II, are represented by glycosaminoglycans (GAG). Here, we further investigated the interactions of CyPB with blood cell populations. In addition to lymphocytes, CyPB was found to interact mainly with platelets. The binding is specific, with a dissociation constant (kd) of 9 +/- 3 nmol/L and the number of sites estimated at 960 +/- 60 per cell. Platelet glycosaminoglycans are not required for the interactions, but the binding is dramatically reduced by active cyclosporine derivatives. We then analyzed the biologic effects of CyPB and found a significant increase in platelet adhesion to collagen. Concurrently, CyPB initiates a transmembranous influx of Ca(2+) and induces the phosphorylation of the P-20 light chains of myosin. Taken together, the present results demonstrate for the first time that extracellular CyPB specifically interacts with platelets through a functional receptor related to the lymphocyte type I binding sites and might act by regulating the activity of a receptor-operated membrane Ca(2+) channel.

Fate in humans of the plasticizer, DI (2-ethylhexyl) phthalate, arising from transfusion of platelets stored in vinyl plastic bags. [plasticizer migration into human blood from vinyl plastic bags during transfusion

NASA Technical Reports Server (NTRS)

Rubin, R. J.; Schiffer, C. A.

1975-01-01

Platelet concentrates were shown to contain 18-38 mg/100 ml of a phthalate plasticizer (DEHP) which arose by migration from the vinyl plastic packs in which the plateletes were prepared and stored. Transfusion of these platelets into 6 adult patients with leukemia resulted in peak blood plasma levels of DEHP ranging from 0.34 - 0.83 mg/100 ml. The blood levels fell mono-exponentially with a mean rate of 2.83 percent per minute and a half-life of 28.0 minutes. Urine was assayed by a method that would measure unchanged DEHP as well as all phthalic acid-containing metabolities. In two patients, at most 60 and 90% of the infused dose, respectively, was excreted in the urine collected for 24 hours post-transfusion. These estimates, however, could be high due to the simultaneous excretion of DEHP remaining from previous transfusions or arising from uncontrolled environmental exposures.

Platelets: No longer bystanders in liver disease

PubMed Central

Adams, David H.; Watson, Steve P.; Lalor, Patricia F.

2016-01-01

Growing lines of evidence recognize that platelets play a central role in liver homeostasis and pathobiology. Platelets have important roles at every stage during the continuum of liver injury and healing. These cells contribute to the initiation of liver inflammation by promoting leukocyte recruitment through sinusoidal endothelium. They can activate effector cells, thus amplifying liver damage, and by modifying the hepatic cellular and cytokine milieu drive both hepatoprotective and hepatotoxic processes. Conclusion: In this review we summarize how platelets drive such pleiotropic actions and attempt to reconcile the paradox of platelets being both deleterious and beneficial to liver function; with increasingly novel methods of manipulating platelet function at our disposal, we highlight avenues for future therapeutic intervention in liver disease. (Hepatology 2016;64:1774‐1784) PMID:26934463

Endothelial progenitor cells bind and inhibit platelet function and thrombus formation.

PubMed

Abou-Saleh, Haissam; Yacoub, Daniel; Théorêt, Jean-François; Gillis, Marc-Antoine; Neagoe, Paul-Eduard; Labarthe, Benoit; Théroux, Pierre; Sirois, Martin G; Tabrizian, Maryam; Thorin, Eric; Merhi, Yahye

2009-12-01

Interactions of endothelial progenitor cells (EPCs) with vascular and blood cells contribute to vascular homeostasis. Although platelets promote the homing of EPCs to sites of vascular injury and their differentiation into endothelial cells, the functional consequences of such interactions on platelets remain unknown. Herein, we addressed the interactions between EPCs and platelets and their impact on platelet function and thrombus formation. Cultured on fibronectin in conditioned media, human peripheral blood mononuclear cells differentiated, within 10 days of culture, into EPCs, which uptake acetylated low-density lipoprotein, bind ulex-lectin, lack monocyte/leukocyte markers (CD14, P-selectin glycoprotein ligand-1, L-selectin), express progenitor/endothelial markers (CD34, vascular endothelial growth factor receptor-2, von Willebrand factor, and vascular endothelial cadherin), and proliferate in culture. These EPCs bound activated platelets via CD62P and inhibited its translocation, glycoprotein IIb/IIIa activation, aggregation, and adhesion to collagen, mainly via prostacyclin secretion. Indeed, this was associated with upregulation of cyclooxygenase-2 and inducible nitric oxide synthase. However, the effects on platelets in vitro were reversed by cyclooxygenase and cyclooxygenase-2 inhibition but not by nitric oxide or inducible nitric oxide synthase inhibition. Moreover, in a ferric chloride-induced murine arterial thrombosis model, injection of EPCs led to their incorporation into sites of injury and impaired thrombus formation, leading to an incomplete occlusion with 50% residual flow. Peripheral blood mononuclear cell-derived EPCs bind platelets via CD62P and inhibit platelet activation, aggregation, adhesion to collagen, and thrombus formation, predominantly via upregulation of cyclooxygenase-2 and secretion of prostacyclin. These findings add new insights into the biology of EPCs and define their potential roles in regulating platelet function and thrombosis.

Endothelial Progenitor Cells Bind and Inhibit Platelet Function and Thrombus Formation

PubMed Central

Abou-Saleh, Haissam; Yacoub, Daniel; Théorêt, Jean-François; Gillis, Marc-Antoine; Neagoe, Paul-Eduard; Labarthe, Benoit; Théroux, Pierre; Sirois, Martin G.; Tabrizian, Maryam; Thorin, Eric; Merhi, Yahye

2013-01-01

Background Interactions of endothelial progenitor cells (EPCs) with vascular and blood cells contribute to vascular homeostasis. Although platelets promote the homing of EPCs to sites of vascular injury and their differentiation into endothelial cells, the functional consequences of such interactions on platelets remain unknown. Herein, we addressed the interactions between EPCs and platelets and their impact on platelet function and thrombus formation. Methods and Results Cultured on fibronectin in conditioned media, human peripheral blood mononuclear cells differentiated, within 10 days of culture, into EPCs, which uptake acetylated low-density lipoprotein, bind ulex-lectin, lack monocyte/leukocyte markers (CD14, P-selectin glycoprotein ligand-1, L-selectin), express progenitor/endothelial markers (CD34, vascular endothelial growth factor receptor-2, von Willebrand factor, and vascular endothelial cadherin), and proliferate in culture. These EPCs bound activated platelets via CD62P and inhibited its translocation, glycoprotein IIb/IIIa activation, aggregation, and adhesion to collagen, mainly via prostacyclin secretion. Indeed, this was associated with upregulation of cyclooxygenase-2 and inducible nitric oxide synthase. However, the effects on platelets in vitro were reversed by cyclooxygenase and cyclooxygenase-2 inhibition but not by nitric oxide or inducible nitric oxide synthase inhibition. Moreover, in a ferric chloride–induced murine arterial thrombosis model, injection of EPCs led to their incorporation into sites of injury and impaired thrombus formation, leading to an incomplete occlusion with 50% residual flow. Conclusions Peripheral blood mononuclear cell– derived EPCs bind platelets via CD62P and inhibit platelet activation, aggregation, adhesion to collagen, and thrombus formation, predominantly via upregulation of cyclooxygenase-2 and secretion of prostacyclin. These findings add new insights into the biology of EPCs and define their potential roles in regulating platelet function and thrombosis. PMID:19917882

Dual Regulation of Glycogen Synthase Kinase 3 (GSK3)α/β by Protein Kinase C (PKC)α and Akt Promotes Thrombin-mediated Integrin αIIbβ3 Activation and Granule Secretion in Platelets*

PubMed Central

Moore, Samantha F.; van den Bosch, Marion T. J.; Hunter, Roger W.; Sakamoto, Kei; Poole, Alastair W.; Hers, Ingeborg

2013-01-01

Glycogen synthase kinase-3 is a Ser/Thr kinase, tonically active in resting cells but inhibited by phosphorylation of an N-terminal Ser residue (Ser21 in GSK3α and Ser9 in GSK3β) in response to varied external stimuli. Recent work suggests that GSK3 functions as a negative regulator of platelet function, but how GSK3 is regulated in platelets has not been examined in detail. Here, we show that early thrombin-mediated GSK3 phosphorylation (0–30 s) was blocked by PKC inhibitors and largely absent in platelets from PKCα knock-out mice. In contrast, late (2–5 min) GSK3 phosphorylation was dependent on the PI3K/Akt pathway. Similarly, early thrombin-mediated inhibition of GSK3 activity was blocked in PKCα knock-out platelets, whereas the Akt inhibitor MK2206 reduced late thrombin-mediated GSK3 inhibition and largely prevented GSK3 inhibition in PKCα knock-out platelets. More importantly, GSK3 phosphorylation contributes to platelet function as knock-in mice where GSK3α Ser21 and GSK3β Ser9 were mutated to Ala showed a significant reduction in PAR4-mediated platelet aggregation, fibrinogen binding, and P-selectin expression, whereas the GSK3 inhibitor CHIR99021 enhanced these responses. Together, these results demonstrate that PKCα and Akt modulate platelet function by phosphorylating and inhibiting GSK3α/β, thereby relieving the negative effect of GSK3α/β on thrombin-mediated platelet activation. PMID:23239877

Acidosis downregulates platelet haemostatic functions and promotes neutrophil proinflammatory responses mediated by platelets.

PubMed

Etulain, Julia; Negrotto, Soledad; Carestia, Agostina; Pozner, Roberto Gabriel; Romaniuk, María Albertina; D'Atri, Lina Paola; Klement, Giannoula Lakka; Schattner, Mirta

2012-01-01

Acidosis is one of the hallmarks of tissue injury such as trauma, infection, inflammation, and tumour growth. Although platelets participate in the pathophysiology of all these processes, the impact of acidosis on platelet biology has not been studied outside of the quality control of laboratory aggregation assays or platelet transfusion optimization. Herein, we evaluate the effect of physiologically relevant changes in extracellular acidosis on the biological function of platelets, placing particular emphasis on haemostatic and secretory functions. Platelet haemostatic responses such as adhesion, spreading, activation of αIIbβ3 integrin, ATP release, aggregation, thromboxane B2 generation, clot retraction and procoagulant activity including phosphatidilserine exposure and microparticle formation, showed a statistically significant inhibition of thrombin-induced changes at pH of 7.0 and 6.5 compared to the physiological pH (7.4). The release of alpha granule content was differentially regulated by acidosis. At low pH, thrombin or collagen-induced secretion of vascular endothelial growth factor and endostatin were dramatically reduced. The release of von Willebrand factor and stromal derived factor-1α followed a similar, albeit less dramatic pattern. In contrast, the induction of CD40L was not changed by low pH, and P-selectin exposure was significantly increased. While the generation of mixed platelet-leukocyte aggregates and the increased chemotaxis of neutrophils mediated by platelets were further augmented under acidic conditions in a P-selectin dependent manner, the increased neutrophil survival was independent of P-selectin expression. In conclusion, our results indicate that extracellular acidosis downregulates most of the haemostatic platelet functions, and promotes those involved in amplifying the neutrophil-mediated inflammatory response.

Beta-lactam antibiotic-induced platelet dysfunction: Evidence for irreversible inhibition of platelet activation in vitro and in vivo after prolonged exposure to penicillin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burroughs, S.F.; Johnson, G.J.

beta-Lactam antibiotics cause platelet dysfunction with bleeding complications. Previous in vitro studies documented reversible inhibition of agonist-receptor interaction. This mechanism is inadequate to explain the effect of beta-lactam antibiotics in vivo. Platelet function does not return to normal immediately after drug treatment, implying irreversible inhibition of platelet function. We report here evidence of irreversible platelet functional and biochemical abnormalities after in vitro and in vivo exposure to beta-lactam antibiotics. Irreversible binding of (14C)-penicillin (Pen) occurred in vitro. After 24 hours' in vitro incubation with 10 to 20 mmol/L Pen, or ex vivo after antibiotic treatment, irreversible functional impairment occurred; butmore » no irreversible inhibition of alpha 2 adrenergic receptors, measured with (3H)-yohimbine, or high-affinity thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors, measured with agonist (3H)-U46619 and antagonist (3H)-SQ29548, occurred. However, low-affinity platelet TXA2/PGH2 receptors were decreased 40% after Pen exposure in vitro or in vivo, indicating irreversible membrane alteration. Two postreceptor biochemical events were irreversibly inhibited in platelets incubated with Pen for 24 hours in vitro or ex vivo after antibiotic treatment. Thromboxane synthesis was inhibited 28.3% to 81.7%. Agonist-induced rises in cytosolic calcium ((Ca2+)i) were inhibited 40.1% to 67.5% in vitro and 26.6% to 52.2% ex vivo. Therefore, Pen binds to platelets after prolonged exposure, resulting in irreversible dysfunction attributable to inhibition of TXA2 synthesis and impairment of the rise in (Ca2+)i. The loss of low-affinity TXA2/PGH2 receptors suggests that the primary site of action of these drugs is on the platelet membrane.« less

Method of producing exfoliated graphite, flexible graphite, and nano-scaled graphene platelets

DOEpatents

Zhamu, Aruna; Shi, Jinjun; Guo, Jiusheng; Jang, Bor Z.

2010-11-02

The present invention provides a method of exfoliating a layered material (e.g., graphite and graphite oxide) to produce nano-scaled platelets having a thickness smaller than 100 nm, typically smaller than 10 nm. The method comprises (a) dispersing particles of graphite, graphite oxide, or a non-graphite laminar compound in a liquid medium containing therein a surfactant or dispersing agent to obtain a stable suspension or slurry; and (b) exposing the suspension or slurry to ultrasonic waves at an energy level for a sufficient length of time to produce separated nano-scaled platelets. The nano-scaled platelets are candidate reinforcement fillers for polymer nanocomposites. Nano-scaled graphene platelets are much lower-cost alternatives to carbon nano-tubes or carbon nano-fibers.

21 CFR 864.7040 - Adenosine triphosphate release assay.

Code of Federal Regulations, 2014 CFR

2014-04-01

... device that measures the release of adenosine triphosphate (ATP) from platelets following aggregation. This measurement is made on platelet-rich plasma using a photometer and a luminescent firefly extract. Simultaneous measurements of platelet aggregation and ATP release are used to evaluate platelet function...

21 CFR 864.7040 - Adenosine triphosphate release assay.

Code of Federal Regulations, 2011 CFR

2011-04-01

... device that measures the release of adenosine triphosphate (ATP) from platelets following aggregation. This measurement is made on platelet-rich plasma using a photometer and a luminescent firefly extract. Simultaneous measurements of platelet aggregation and ATP release are used to evaluate platelet function...

21 CFR 864.7040 - Adenosine triphosphate release assay.

Code of Federal Regulations, 2013 CFR

2013-04-01

... device that measures the release of adenosine triphosphate (ATP) from platelets following aggregation. This measurement is made on platelet-rich plasma using a photometer and a luminescent firefly extract. Simultaneous measurements of platelet aggregation and ATP release are used to evaluate platelet function...

21 CFR 864.7040 - Adenosine triphosphate release assay.

Code of Federal Regulations, 2010 CFR

2010-04-01

... device that measures the release of adenosine triphosphate (ATP) from platelets following aggregation. This measurement is made on platelet-rich plasma using a photometer and a luminescent firefly extract. Simultaneous measurements of platelet aggregation and ATP release are used to evaluate platelet function...

21 CFR 864.7040 - Adenosine triphosphate release assay.

Code of Federal Regulations, 2012 CFR

2012-04-01

... device that measures the release of adenosine triphosphate (ATP) from platelets following aggregation. This measurement is made on platelet-rich plasma using a photometer and a luminescent firefly extract. Simultaneous measurements of platelet aggregation and ATP release are used to evaluate platelet function...

Characterization of the platelet transcriptome by RNA sequencing in patients with acute myocardial infarction

PubMed Central

Eicher, John D.; Wakabayashi, Yoshiyuki; Vitseva, Olga; Esa, Nada; Yang, Yanqin; Zhu, Jun; Freedman, Jane E.; McManus, David D.; Johnson, Andrew D.

2016-01-01

Transcripts in platelets are largely produced in precursor megakaryocytes but remain physiologically-active as platelets translate RNAs and regulate protein/RNA levels. Recent studies using transcriptome sequencing (RNA-seq) characterized the platelet transcriptome in limited numbers of non-diseased individuals. Here, we expand upon these RNA-seq studies by completing RNA-seq in platelets from 32 patients with acute myocardial infarction (MI). Our goals were to characterize the platelet transcriptome using a population of patients with acute MI and relate gene expression to platelet aggregation measures and ST-segment elevation MI (STEMI) (n=16) versus non-STEMI (NSTEMI) (n=16) subtypes. Similar to other studies, we detected 9,565 expressed transcripts, including several known platelet-enriched markers (e.g., PPBP, OST4). Our RNA-seq data strongly correlated with independently ascertained platelet expression data and showed enrichment for platelet-related pathways (e.g., wound response, hemostasis, and platelet activation), as well as actin-related and post-transcriptional processes. Several transcripts displayed suggestively higher (FBXL4, ECHDC3, KCNE1, TAOK2, AURKB, ERG, and FKBP5) and lower (MIAT, PVRL3and PZP) expression in STEMI platelets compared to NSTEMI. We also identified transcripts correlated with platelet aggregation to TRAP (ATP6V1G2, SLC2A3), collagen (CEACAM1, ITGA2), and ADP (PDGFB, PDGFC, ST3GAL6). Our study adds to current platelet gene expression resources by providing transcriptome-wide analyses in platelets isolated from patients with acute MI. In concert with prior studies, we identify various genes for further study in regards to platelet function and acute MI. Future platelet RNA-seq studies examining more diverse sets of healthy and diseased samples will add to our understanding of platelet thrombotic and non-thrombotic functions. PMID:26367242

Axial postbuckling analysis of multilayer functionally graded composite nanoplates reinforced with GPLs based on nonlocal strain gradient theory

NASA Astrophysics Data System (ADS)

Sahmani, S.; Aghdam, M. M.

2017-11-01

In this paper, a new size-dependent inhomogeneous plate model is constructed to analyze the nonlinear buckling and postbuckling characteristics of multilayer functionally graded composite nanoplates reinforced with graphene platelet (GPL) nanofillers under axial compressive load. To this purpose, the nonlocal strain gradient theory of elasticity is implemented into a refined hyperbolic shear deformation plate theory. The mechanical properties of multilayer graphene platelet-reinforced composite (GPLRC) nanoplates are evaluated based upon the Halpin-Tsai micromechanical scheme. The weight fraction of randomly dispersed GPLs remain constant in each individual layer, which results in U-GPLRC nanoplate, or changes layerwise in accordance with three different functionally graded patterns, which make X-GPLRC, O-GPLRC and A-GPLRC nanoplates. Via a two-stepped perturbation technique, explicit analytical expressions for nonlocal strain gradient stability paths are established for layerwise functionally graded GPLRC nanoplates. It is demonstrated that both the nonlocal and strain gradient size dependencies are more significant for multilayer GPLRC nanoplates filling by GPL nanofillers with higher length-to-thickness and width-to-thickness ratios.

EELS Analysis of Nylon 6 Nanofibers Reinforced with Nitroxide-Functionalized Graphene Oxide.

PubMed

Leyva-Porras, César; Ornelas-Gutiérrez, C; Miki-Yoshida, M; Avila-Vega, Yazmín I; Macossay, Javier; Bonilla-Cruz, José

2014-01-01

A detailed analysis by transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS) of nitroxide-functionalized graphene oxide layers (GOFT) dispersed in Nylon 6 nanofibers is reported herein. The functionalization and exfoliation process of graphite oxide to GOFT was confirmed by TEM using electron diffraction patterns (EDP), wherein 1 to 4 graphene layers of GOFT were observed. The distribution and alignment of GOFT layers within a sample of Nylon 6 nanofiber reveals that GOFT platelets are mainly within the fiber, but some were partially protruding from it. Furthermore, Nylon 6 nanofibers exhibit an average diameter of 225 nm with several microns in length. GOFT platelets embedded into the fiber, the pristine fiber, and amorphous carbon were analyzed by EELS where each spectra [corresponding to the carbon edge (C-K)] exhibited changes in the fine structure, allowing a clear distinction between: i) GOFT single-layers, ii) Nylon-6 nanofibers, and iii) the carbon substrate. EELS analysis is presented here for the first time as a powerful tool to identify functionalized graphene single-layers (< 4 layers of GOFT) into a Nylon 6 nanofiber composite.

EELS Analysis of Nylon 6 Nanofibers Reinforced with Nitroxide-Functionalized Graphene Oxide

PubMed Central

Leyva-Porras, César; Ornelas-Gutiérrez, C.; Miki-Yoshida, M.; Avila-Vega, Yazmín I.; Macossay, Javier; Bonilla-Cruz, José

2014-01-01

A detailed analysis by transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS) of nitroxide-functionalized graphene oxide layers (GOFT) dispersed in Nylon 6 nanofibers is reported herein. The functionalization and exfoliation process of graphite oxide to GOFT was confirmed by TEM using electron diffraction patterns (EDP), wherein 1 to 4 graphene layers of GOFT were observed. The distribution and alignment of GOFT layers within a sample of Nylon 6 nanofiber reveals that GOFT platelets are mainly within the fiber, but some were partially protruding from it. Furthermore, Nylon 6 nanofibers exhibit an average diameter of 225 nm with several microns in length. GOFT platelets embedded into the fiber, the pristine fiber, and amorphous carbon were analyzed by EELS where each spectra [corresponding to the carbon edge (C-K)] exhibited changes in the fine structure, allowing a clear distinction between: i) GOFT single-layers, ii) Nylon-6 nanofibers, and iii) the carbon substrate. EELS analysis is presented here for the first time as a powerful tool to identify functionalized graphene single-layers (< 4 layers of GOFT) into a Nylon 6 nanofiber composite. PMID:24634536

Prevention of ischemia-induced myocardial platelet deposition by exogenous prostacyclin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aherne, T.; Price, D.C.; Yee, E.S.

1986-07-01

The antithrombotic effects of prostacyclin infusion on myocardial platelet deposition were studied in a canine model during and after global ischemia. Eleven isolated heart preparations were subjected to 1 hour of cardioplegic arrest under moderate hypothermia (27 to 28/sup 0/C), including a control group (n = 7) and a prostacyclin-treated group (n = 4). The hearts of four other dogs were continuously perfused for 180 minutes. Platelet deposition was measured at 15 minute intervals throughout the 3 hour study. Serial full-thickness myocardial biopsy specimens were analyzed for activity of /sup 111/In-labeled platelets with /sup 99m/Tc-labeled erythrocyte correction for tissue bloodmore » content. The pattern of platelet distribution was determined by scintiscans of each heart, taken with a gamma camera at the end of the 60 minute reperfusion period. Substantial myocardial platelet deposition was found in the control hearts after ischemia but not in the prostacyclin-treated group (p less than 0.05). Furthermore, prostacyclin infusion had a significant disaggregatory effect on intracoronary platelet deposits when the precardioplegic and postcardioplegic biopsy specimens were analyzed (p less than 0.05). Three hours of continuous perfusion did not increase tissue /sup 111/In-labeled platelet activity. Ex vivo images showed platelet deposition to be a diffuse patchy process with significantly more /sup 111/In activity in the endocardium than in the epicardium after global ischemia (p less than 0.05). These data show the potent antithrombotic properties of prostacyclin in preventing and disaggregating ischemia-induced intracoronary platelet deposition during and after cardioplegic arrest.« less

Ezetimibe inhibits platelet activation and uPAR expression on endothelial cells.

PubMed

Becher, Tobias; Schulze, Torsten J; Schmitt, Melanie; Trinkmann, Frederik; El-Battrawy, Ibrahim; Akin, Ibrahim; Kälsch, Thorsten; Borggrefe, Martin; Stach, Ksenija

2017-01-15

Lipid lowering therapy constitutes the basis of cardiovascular disease therapy. The purpose of this study was to investigate effects of ezetimibe, a selective inhibitor of intestinal cholesterol absorption, on platelets and endothelial cells in an in vitro endothelial cell model. After a 24h incubation period with ezetimibe (concentrations 1, 50, 100 and 1000ng/ml), human umbilical vein endothelial cells (HUVEC) were stimulated for 1h with lipopolysaccharide (LPS) and were then incubated in direct contact with activated platelets. Following this, the expression of CD40L and CD62P on platelets, and the expression of ICAM-1, VCAM-1, uPAR, and MT1-MMP on endothelial cells were measured by flow cytometry. Supernatants were analysed by enzyme linked immunosorbent assay for soluble MCP-1, IL-6 and MMP-1. The increased expression of uPAR on endothelial cells by proinflammatory stimulation with LPS and by direct endothelial contact with activated platelets was significantly reduced through pre-incubation with 100ng/ml and 1000ng/ml ezetimibe (p<0.05). Platelets directly incubated with ezetimibe but without endothelial cell contact showed significantly reduced CD62P and CD40L surface expression (p<0.05). Ezetimibe had no significant effects on HUVEC expression of MT1-MMP, ICAM-1 and VCAM-1 and on CD40L expression on platelets in direct contact with endothelial cells. Levels of soluble IL-6 in HUVEC supernatants were significantly lower after pre-incubation with ezetimibe. In this in vitro analysis, ezetimibe directly attenuates platelet activation and has significant endothelial cell mediated effects on selected markers of atherosclerosis. Copyright © 2016. Published by Elsevier Ireland Ltd.

Platelet glycoprotein VI binds to polymerized fibrin and promotes thrombin generation.

PubMed

Mammadova-Bach, Elmina; Ollivier, Véronique; Loyau, Stéphane; Schaff, Mathieu; Dumont, Bénédicte; Favier, Rémi; Freyburger, Geneviève; Latger-Cannard, Véronique; Nieswandt, Bernhard; Gachet, Christian; Mangin, Pierre H; Jandrot-Perrus, Martine

2015-07-30

Fibrin, the coagulation end product, consolidates the platelet plug at sites of vascular injury and supports the recruitment of circulating platelets. In addition to integrin αIIbβ3, another as-yet-unidentified receptor is thought to mediate platelet interaction with fibrin. Platelet glycoprotein VI (GPVI) interacts with collagen and several other adhesive macromolecules. We evaluated the hypothesis that GPVI could be a functional platelet receptor for fibrin. Calibrated thrombin assays using platelet-rich plasma (PRP) showed that tissue factor-triggered thrombin generation was impaired in GPVI-deficient patients and reduced by the anti-GPVI Fab 9O12. Assays on reconstituted PRP and PRP from fibrinogen-deficient patients revealed a fibrinogen-dependent enhancement of thrombin generation, which relied on functional GPVI. The effect of GPVI was found to depend on fibrin polymerization. A binding assay showed a specific interaction between GPVI-Fc and fibrin, inhibited by the Fab 9O12. This Fab also reduced platelet adhesion to fibrin at low (300 s(-1)) and high (1500 s(-1)) wall shear rates. Platelets adherent to fibrin displayed shape change, exposure of procoagulant phospholipids, and the formation of small clots. When hirudinated blood was perfused at 1500 s(-1) over preformed fibrin-rich clots, the Fab 9O12 decreased the recruitment of platelets by up to 85%. This study identifies GPVI as a platelet receptor for polymerized fibrin with 2 major functions: (1) amplification of thrombin generation and (2) recruitment of circulating platelets to clots. These so-far-unrecognized properties of GPVI confer on it a key role in thrombus growth and stabilization. © 2015 by The American Society of Hematology.

Arf6 controls platelet spreading and clot retraction via integrin αIIbβ3 trafficking

PubMed Central

Huang, Yunjie; Joshi, Smita; Xiang, Binggang; Kanaho, Yasunori; Li, Zhenyu; Bouchard, Beth A.; Moncman, Carole L.

2016-01-01

Platelet and megakaryocyte endocytosis is important for loading certain granule cargo (ie, fibrinogen [Fg] and vascular endothelial growth factor); however, the mechanisms of platelet endocytosis and its functional acute effects are understudied. Adenosine 5'-diphosphate–ribosylation factor 6 (Arf6) is a small guanosine triphosphate–binding protein that regulates endocytic trafficking, especially of integrins. To study platelet endocytosis, we generated platelet-specific Arf6 knockout (KO) mice. Arf6 KO platelets had less associated Fg suggesting that Arf6 affects αIIbβ3-mediated Fg uptake and/or storage. Other cargo was unaffected. To measure Fg uptake, mice were injected with biotinylated- or fluorescein isothiocyanate (FITC)–labeled Fg. Platelets from the injected Arf6 KO mice showed lower accumulation of tagged Fg, suggesting an uptake defect. Ex vivo, Arf6 KO platelets were also defective in FITC-Fg uptake and storage. Immunofluorescence analysis showed initial trafficking of FITC-Fg to a Rab4-positive compartment followed by colocalization with Rab11-positive structures, suggesting that platelets contain and use both early and recycling endosomes. Resting and activated αIIbβ3 levels, as measured by flow cytometry, were unchanged; yet, Arf6 KO platelets exhibited enhanced spreading on Fg and faster clot retraction. This was not the result of alterations in αIIbβ3 signaling, because myosin light-chain phosphorylation and Rac1/RhoA activation were unaffected. Consistent with the enhanced clot retraction and spreading, Arf6 KO mice showed no deficits in tail bleeding or FeCl3-induced carotid injury assays. Our studies present the first mouse model for defining the functions of platelet endocytosis and suggest that altered integrin trafficking may affect the efficacy of platelet function. PMID:26738539

Arf6 controls platelet spreading and clot retraction via integrin αIIbβ3 trafficking.

PubMed

Huang, Yunjie; Joshi, Smita; Xiang, Binggang; Kanaho, Yasunori; Li, Zhenyu; Bouchard, Beth A; Moncman, Carole L; Whiteheart, Sidney W

2016-03-17

Platelet and megakaryocyte endocytosis is important for loading certain granule cargo (ie, fibrinogen [Fg] and vascular endothelial growth factor); however, the mechanisms of platelet endocytosis and its functional acute effects are understudied. Adenosine 5'-diphosphate-ribosylation factor 6 (Arf6) is a small guanosine triphosphate-binding protein that regulates endocytic trafficking, especially of integrins. To study platelet endocytosis, we generated platelet-specific Arf6 knockout (KO) mice. Arf6 KO platelets had less associated Fg suggesting that Arf6 affects αIIbβ3-mediated Fg uptake and/or storage. Other cargo was unaffected. To measure Fg uptake, mice were injected with biotinylated- or fluorescein isothiocyanate (FITC)-labeled Fg. Platelets from the injected Arf6 KO mice showed lower accumulation of tagged Fg, suggesting an uptake defect. Ex vivo, Arf6 KO platelets were also defective in FITC-Fg uptake and storage. Immunofluorescence analysis showed initial trafficking of FITC-Fg to a Rab4-positive compartment followed by colocalization with Rab11-positive structures, suggesting that platelets contain and use both early and recycling endosomes. Resting and activated αIIbβ3 levels, as measured by flow cytometry, were unchanged; yet, Arf6 KO platelets exhibited enhanced spreading on Fg and faster clot retraction. This was not the result of alterations in αIIbβ3 signaling, because myosin light-chain phosphorylation and Rac1/RhoA activation were unaffected. Consistent with the enhanced clot retraction and spreading, Arf6 KO mice showed no deficits in tail bleeding or FeCl3-induced carotid injury assays. Our studies present the first mouse model for defining the functions of platelet endocytosis and suggest that altered integrin trafficking may affect the efficacy of platelet function. © 2016 by The American Society of Hematology.

NOD2 Receptor is Expressed in Platelets and Enhances Platelet Activation and Thrombosis

PubMed Central

Zhang, Si; Zhang, Shenghui; Hu, Liang; Zhai, Lili; Xue, Ruyi; Ye, Jianqin; Chen, Leilei; Cheng, Guanjun; Mruk, Jozef; Kunapuli, Satya P.; Ding, Zhongren

2015-01-01

Background Pattern recognition receptor NOD2 (nucleotide binding oligomerization domain 2) is well investigated in immunity, its expression and function in platelets has never been explored. Method and Results Using RT-PCR and Western blot we show that both human and mouse platelets express NOD2, and its agonist MDP induced NOD2 activation as evidenced by receptor dimerization. NOD2 activation potentiates platelet aggregation and secretion induced by low concentration of thrombin or collagen, as well as clot retraction. These potentiating effects of MDP were not seen in platelets from NOD2-deficient mice. Plasma from septic patients also potentiates platelet aggregation induced by thrombin or collagen NOD2-dependently. Using intravital microscopy, we found that MDP administration accelerated in vivo thrombosis in FeCl3-injured mesenteric arteriole thrombosis mouse model. Platelet depletion and transfusion experiments confirmed that NOD2 from platelets contributes to the in vivo thrombosis in mice. NOD2 activation also accelerates platelet-dependent hemostasis. We further found that platelets express RIP2 (receptor-interacting protein 2), and provided evidences suggesting that MAPK and NO/sGC/cGMP/PGK pathways downstream of RIP2 mediate the role of NOD2 in platelets. Finally, MDP stimulates proinflammatory cytokine IL-1β maturation and accumulation in human and mouse platelets NOD2-dependently. Conclusions NOD2 is expressed in platelets and functions in platelet activation and arterial thrombosis, possibly during infection. To our knowledge, this is the first study on NOD-like receptors in platelets which links thrombotic events to inflammation. PMID:25825396

Platelet-activated clotting time does not measure platelet reactivity during cardiac surgery.

PubMed

Shore-Lesserson, L; Ammar, T; DePerio, M; Vela-Cantos, F; Fisher, C; Sarier, K

1999-08-01

Platelet dysfunction is a major contributor to bleeding after cardiopulmonary bypass (CPB), yet it remains difficult to diagnose. A point-of-care monitor, the platelet-activated clotting time (PACT), measures accelerated shortening of the kaolin-activated clotting time by addition of platelet activating factor. The authors sought to evaluate the clinical utility of the PACT by conducting serial measurements of PACT during cardiac surgery and correlating postoperative measurements with blood loss. In 50 cardiac surgical patients, blood was sampled at 10 time points to measure PACT. Simultaneously, platelet reactivity was measured by the thrombin receptor agonist peptide-induced expression of P-selectin, using flow cytometry. These tests were temporally analyzed. PACT values, P-selectin expression, and other coagulation tests were analyzed for correlation with postoperative chest tube drainage. PACT and P-selectin expression were maximally reduced after protamine administration. Changes in PACT did not correlate with changes in P-selectin expression at any time interval. Total 8-h chest tube drainage did not correlate with any coagulation test at any time point except with P-selectin expression after protamine administration (r = -0.4; P = 0.03). The platelet dysfunction associated with CPB may be a result of depressed platelet reactivity, as shown by thrombin receptor activating peptide-induced P-selectin expression. Changes in PACT did not correlate with blood loss or with changes in P-selectin expression suggesting that PACT is not a specific measure of platelet reactivity.

Influence of cytochrome 2C19 allelic variants on on-treatment platelet reactivity evaluated by five different platelet function tests.

PubMed

Gremmel, Thomas; Kopp, Christoph W; Moertl, Deddo; Seidinger, Daniela; Koppensteiner, Renate; Panzer, Simon; Mannhalter, Christine; Steiner, Sabine

2012-05-01

The antiplatelet effect of clopidogrel has been linked to cytochrome P450 2C19 (CYP2C19) carrier status. The presence of loss of function and gain of function variants were found to have a gene-dose effect on clopidogrel metabolism. However, genotyping is only one aspect of predicting response to clopidogrel and several platelet function tests are available to measure platelet response. Patients and methods We studied the influence of CYP2C19 allelic variants on on-treatment platelet reactivity as assessed by light transmission aggregometry (LTA), the VerifyNow P2Y12 assay, the VASP assay, multiple electrode aggregometry (MEA), and the Impact-R in 288 patients after stenting for cardiovascular disease. Allelic variants of CYP2C19 were determined with the Infiniti® CYP450 2C19+ assay and categorized into four metabolizer states (ultrarapid, extensive, intermediate, poor). Platelet reactivity increased linearly from ultrarapid to poor metabolizers using the VerifyNow P2Y12 assay (P = 0.04), the VASP assay (P = 0.02) and the Impact-R (P = 0.04). The proportion of patients with high on-treatment residual platelet reactivity (HRPR) identified by LTA, the VerifyNow P2Y12 assay and the VASP assay increased when the metabolizer status decreased, while no such relationship could be identified for results of MEA and Impact-R. The presence of loss of function variants (*2/*2, *2-8*/wt, *2/*17) was an independent predictor of HRPR in LTA and the VASP assay while it did not reach statistical significance in the VerifyNow P2Y12 assay, MEA, and the Impact-R. Depending on the type of platelet function test differences in the association of on-treatment platelet reactivity with CYP2C19 carrier status are observed. Copyright © 2011 Elsevier Ltd. All rights reserved.

Severe platelet dysfunction in NHL patients receiving ibrutinib is absent in patients receiving acalabrutinib

PubMed Central

Bye, Alexander P.; Unsworth, Amanda J.; Desborough, Michael J.; Hildyard, Catherine A. T.; Appleby, Niamh; Bruce, David; Kriek, Neline; Nock, Sophie H.; Sage, Tanya; Hughes, Craig E.

2017-01-01

The Bruton tyrosine kinase (Btk) inhibitor ibrutinib induces platelet dysfunction and causes increased risk of bleeding. Off-target inhibition of Tec is believed to contribute to platelet dysfunction and other side effects of ibrutinib. The second-generation Btk inhibitor acalabrutinib was developed with improved specificity for Btk over Tec. We investigated platelet function in patients with non-Hodgkin lymphoma (NHL) receiving ibrutinib or acalabrutinib by aggregometry and by measuring thrombus formation on collagen under arterial shear. Both patient groups had similarly dysfunctional aggregation responses to collagen and collagen-related peptide, and comparison with mechanistic experiments in which platelets from healthy donors were treated with the Btk inhibitors suggested that both drugs inhibit platelet Btk and Tec at physiological concentrations. Only ibrutinib caused dysfunctional thrombus formation, whereas size and morphology of thrombi following acalabrutinib treatment were of normal size and morphology. We found that ibrutinib but not acalabrutinib inhibited Src family kinases, which have a critical role in platelet adhesion to collagen that is likely to underpin unstable thrombus formation observed in ibrutinib patients. We found that platelet function was enhanced by increasing levels of von Willebrand factor (VWF) and factor VIII (FVIII) ex vivo by addition of intermediate purity FVIII (Haemate P) to blood from patients, resulting in consistently larger thrombi. We conclude that acalabrutinib avoids major platelet dysfunction associated with ibrutinib therapy, and platelet function may be enhanced in patients with B-cell NHL by increasing plasma VWF and FVIII. PMID:29296914

Tyrosine phosphorylated c-Cbl regulates platelet functional responses mediated by outside-in signaling

PubMed Central

Buitrago, Lorena; Langdon, Wallace Y.

2011-01-01

c-Cbl protein functions as an E3 ligase and scaffolding protein, where 3 residues, Y700, Y731, and Y774, upon phosphorylation, have been shown to initiate several signaling cascades. In this study, we investigated the role of these phospho-tyrosine residues in the platelet functional responses after integrin engagement. We observed that c-Cbl Y700, Y731 and Y774 undergo phosphorylation upon platelet adhesion to immobilized fibrinogen, which was inhibited in the presence of PP2, a pan-src family kinase (SFK) inhibitor, suggesting that c-Cbl is phosphorylated downstream of SFKs. However, OXSI-2, a Syk inhibitor, significantly reduced c-Cbl phosphorylation at residues Y774 and Y700, without affecting Y731 phosphorylation. Interestingly, PP2 inhibited both platelet-spreading on fibrinogen as well as clot retraction, whereas OXSI-2 blocked only platelet-spreading, suggesting a differential role of these tyrosine residues. The physiologic role of c-Cbl and Y731 was studied using platelets from c-Cbl KO and c-CblYF/YF knock-in mice. c-Cbl KO and c-CblYF/YF platelets had a significantly reduced spreading over immobilized fibrinogen. Furthermore, clot retraction with c-Cbl KO and c-CblYF/YF platelets was drastically delayed. These results indicate that c-Cbl and particularly its phosphorylated residue Y731 plays an important role in platelet outside-in signaling contributing to platelet-spreading and clot retraction. PMID:21967979

Flavan-3-ol-enriched dark chocolate and white chocolate improve acute measures of platelet function in a gender-specific way--a randomized-controlled human intervention trial.

PubMed

Ostertag, Luisa M; Kroon, Paul A; Wood, Sharon; Horgan, Graham W; Cienfuegos-Jovellanos, Elena; Saha, Shikha; Duthie, Garry G; de Roos, Baukje

2013-02-01

We examined whether flavan-3-ol-enriched dark chocolate, compared with standard dark and white chocolate, beneficially affects platelet function in healthy subjects, and whether this relates to flavan-3-ol bioavailability. A total of 42 healthy subjects received an acute dose of flavan-3-ol-enriched dark, standard dark or white chocolate, in random order. Blood and urine samples were obtained just before and 2 and 6 h after consumption for measurements of platelet function, and bioavailability and excretion of flavan-3-ols. Flavan-3-ol-enriched dark chocolate significantly decreased adenosine diphosphate-induced platelet aggregation and P-selectin expression in men (all p ≤ 0.020), decreased thrombin receptor-activating peptide-induced platelet aggregation and increased thrombin receptor-activating peptide-induced fibrinogen binding in women (both p ≤ 0.041), and increased collagen/epinephrine-induced ex vivo bleeding time in men and women (p ≤ 0.042). White chocolate significantly decreased adenosine diphosphate-induced platelet P-selectin expression (p = 0.002) and increased collagen/epinephrine-induced ex vivo bleeding time (p = 0.042) in men only. Differences in efficacy by which flavan-3-ols affect platelet function were only partially explained by concentrations of flavan-3-ols and their metabolites in plasma or urine. Flavan-3-ols in dark chocolate, but also compounds in white chocolate, can improve platelet function, dependent on gender, and may thus beneficially affect atherogenesis. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Relationship between platelet and urinary 8-Iso-PGF2α levels in subjects with different degrees of NOX2 regulation.

PubMed

Carnevale, Roberto; Iuliano, Luigi; Nocella, Cristina; Bartimoccia, Simona; Trapè, Stefano; Russo, Roberta; Gentile, Maria Cristina; Cangemi, Roberto; Loffredo, Lorenzo; Pignatelli, Pasquale; Violi, Francesco

2013-06-14

Urinary 8-iso-PGF2α, a marker of oxidative stress, is influenced by the activation of NOX2. It is unclear if platelets 8-iso-PGF2α contribute to urinary 8-iso-PGF2α. In a cross-sectional study, platelet, urinary, and serum 8-iso-PGF2α were determined in subjects with downregulation (X-linked chronic granulomatous disease [X-CGD], n=25) and upregulation (type II diabetic patients [T2D], n=121) of NOX2 and 153 controls matched for sex and age. In diabetic patients (n=18), the above variables were repeated before and after 7 days treatment with 100 mg/day aspirin or 100 mg/day aspirin plus 40 mg/day atorvastatin. In vitro study was performed to see the contribution of blood cells to serum 8-iso-PGF2α. Compared with controls, X-CGD patients had lower platelet, serum, and urinary 8-iso-PGF2α values; conversely, diabetic patients had higher values of 8-iso-PGF2α compared with controls. Urinary 8-iso-PGF2α significantly correlated with both platelet and serum 8-iso-PGF2α in the 2 cohorts. A parallel increase of platelet, serum, and urinary 8-iso-PGF2α by aspirin and a parallel decrease by aspirin plus atorvastatin were detected in the interventional study. In vitro study demonstrated that platelets contribute to 37% of serum 8-iso-PGF2α and that only 13% of it is of extravascular origin. The study suggests that NOX2 contributes to the formation of 8-iso-PGF2α in both platelets and urine. The direct correlation between platelet and urinary 8-iso-PGF2α suggests that, at least partly, urinary 8-iso-PGF2α reflects platelet 8-iso-PGF2α production. Analysis of serum 8-iso-PGF2α may represent a novel tool to investigate the production of 8-iso-PGF2α by blood cells including platelets. URL: ClinicalTrials.gov. Unique Identifier: NCT01250340.

Nitric oxide decreases coagulation protein function in rabbits as assessed by thromboelastography.

PubMed

Nielsen, V G

2001-02-01

Nitric oxide (NO) is administered via infusion of donors such as nitroglycerin or in inhaled form for treatment of ischemia and pulmonary hypertension, respectively. In rabbits, the NO donor, DETANONOate, decreases whole blood clotting function as assessed by thromboelastographic variables (R, reaction time; alpha, angle; and G, a measure of clot strength). I hypothesized that DETANONOate-derived NO would adversely affect coagulation protein and platelet function. Blood obtained from ear arteries of conscious rabbits (n = 8) anticoagulated with sodium citrate. The blood was then incubated with 0 or 10mM DETANONOate for 30 min. After incubation and recalcification, thromboelastography was performed for 60 min under four conditions: 1) 0mM DETANONOate, 2) 0mM DETANONOate with platelet inhibition with cytochalasin D, 3) 10mM DETANONOate, and 4) 10mM DETANONOate with platelet inhibition. DETANONOate significantly (P < 0.05) increased R and decreased alpha and G in samples with or without platelet inhibition, compared with samples not exposed to DETANONOate. Lastly, the percentage of total G (G(T)) attributable to platelet function (G(P)) was significantly more in the absence of DETANONOate (G(P) = 92.3% +/- 1.6%; mean +/- SD) than after exposure to DETANONOate (G(P) = 90.2% +/- 2.3%). DETANONOate-derived NO significantly decreased coagulation protein function and platelet function. Coagulation protein function may be similarly affected in clinical situations involving the administration of NO or NO donors.

Platelets as delivery systems for disease treatments

PubMed Central

Shi, Qizhen; Montgomery, Robert R.

2010-01-01

Platelets are small, anucleate, discoid shaped blood cells that play a fundamental role in hemostasis. Platelets contain a large number of biologically active molecules within cytoplasmic granules that are critical to normal platelet function. Because platelets circulate in blood through out the body, release biological molecules and mediators on demand, and participate in hemostasis as well as many other pathophysiologic processes, targeting expression of proteins of interest to platelets and utilizing platelets as delivery systems for disease treatment would be a logical approach. This paper reviews the genetic therapy for inherited bleeding disorders utilizing platelets as delivery system, with a particular focus on platelet-derived FVIII for hemophilia A treatment. PMID:20619307

ROS-mediated platelet generation: a microenvironment-dependent manner for megakaryocyte proliferation, differentiation, and maturation

PubMed Central

Chen, S; Su, Y; Wang, J

2013-01-01

Platelets have an important role in the body because of their manifold functions in haemostasis, thrombosis, and inflammation. Platelets are produced by megakaryocytes (MKs) that are differentiated from haematopoietic stem cells via several consecutive stages, including MK lineage commitment, MK progenitor proliferation, MK differentiation and maturation, cell apoptosis, and platelet release. During differentiation, the cells migrate from the osteoblastic niche to the vascular niche in the bone marrow, which is accompanied by reactive oxygen species (ROS)-dependent oxidation state changes in the microenvironment, suggesting that ROS can distinctly influence platelet generation and function in a microenvironment-dependent manner. The objective of this review is to reveal the role of ROS in regulating MK proliferation, differentiation, maturation, and platelet activation, thereby providing new insight into the mechanism of platelet generation, which may lead to the development of new therapeutic agents for thrombocytopenia and/or thrombosis. PMID:23846224

The impact of night-shift work on platelet function in healthy medical staff.

PubMed

Nakao, Tomoko; Yasumoto, Atsushi; Tokuoka, Suzumi; Kita, Yoshihiro; Kawahara, Takuya; Daimon, Masao; Yatomi, Yutaka

2018-04-18

Rotating shift work has been reported to increase the risk of cardiovascular diseases. Vascular endothelial dysfunction and platelet activation are among the leading causes of thrombus formation in patients with myocardial infarction or stroke. Endothelial function has been shown to be impaired immediately after night-shift work; however, it is not known whether platelets are also activated. The aim of this study was to investigate the acute impact of night-shift work on platelet function. This observational study included 11 healthy medical staff members (seven women, median age 32 years). We examined each subject's platelet aggregation rates and the serum concentrations of eicosanoid mediators after night-shift work and on day-shift work without preceding night-shift work (baseline). Platelet aggregation did not differ from baseline levels after night-shift work. However, serum cyclooxygenase (COX)-metabolized eicosanoid mediators, particularly thromboxane (Tx) B 2 (a stable metabolite of TxA 2 and the most important marker of platelet activation), were significantly higher after the night-shift than at baseline (median 65.3 vs 180.4 ng/ml). Although platelet aggregation did not increase, there was an increase in serum COX-metabolized eicosanoid mediators such as TxB 2 in healthy medical staff after night-shift work. This platelet hypersensitivity may be one of the mechanisms underlying the significant association between night-shift work and adverse cardiovascular outcomes.

Platelet function is modified by common sequence variation in megakaryocyte super enhancers

PubMed Central

Petersen, Romina; Lambourne, John J.; Javierre, Biola M.; Grassi, Luigi; Kreuzhuber, Roman; Ruklisa, Dace; Rosa, Isabel M.; Tomé, Ana R.; Elding, Heather; van Geffen, Johanna P.; Jiang, Tao; Farrow, Samantha; Cairns, Jonathan; Al-Subaie, Abeer M.; Ashford, Sofie; Attwood, Antony; Batista, Joana; Bouman, Heleen; Burden, Frances; Choudry, Fizzah A.; Clarke, Laura; Flicek, Paul; Garner, Stephen F.; Haimel, Matthias; Kempster, Carly; Ladopoulos, Vasileios; Lenaerts, An-Sofie; Materek, Paulina M.; McKinney, Harriet; Meacham, Stuart; Mead, Daniel; Nagy, Magdolna; Penkett, Christopher J.; Rendon, Augusto; Seyres, Denis; Sun, Benjamin; Tuna, Salih; van der Weide, Marie-Elise; Wingett, Steven W.; Martens, Joost H.; Stegle, Oliver; Richardson, Sylvia; Vallier, Ludovic; Roberts, David J.; Freson, Kathleen; Wernisch, Lorenz; Stunnenberg, Hendrik G.; Danesh, John; Fraser, Peter; Soranzo, Nicole; Butterworth, Adam S.; Heemskerk, Johan W.; Turro, Ernest; Spivakov, Mikhail; Ouwehand, Willem H.; Astle, William J.; Downes, Kate; Kostadima, Myrto; Frontini, Mattia

2017-01-01

Linking non-coding genetic variants associated with the risk of diseases or disease-relevant traits to target genes is a crucial step to realize GWAS potential in the introduction of precision medicine. Here we set out to determine the mechanisms underpinning variant association with platelet quantitative traits using cell type-matched epigenomic data and promoter long-range interactions. We identify potential regulatory functions for 423 of 565 (75%) non-coding variants associated with platelet traits and we demonstrate, through ex vivo and proof of principle genome editing validation, that variants in super enhancers play an important role in controlling archetypical platelet functions. PMID:28703137

Talin-dependent integrin activation is required for fibrin clot retraction by platelets

PubMed Central

Haling, Jacob R.; Monkley, Susan J.; Critchley, David R.

2011-01-01

Talin functions both as a regulator of integrin affinity and as an important mechanical link between integrins and the cytoskeleton. Using genetic deletion of talin, we show for the first time that the capacity of talin to activate integrins is required for fibrin clot retraction by platelets. To further dissect which talin functions are required for this process, we tested clot retraction in platelets expressing a talin1(L325R) mutant that binds to integrins, but exhibits impaired integrin activation ascribable to disruption of the interaction between talin and the membrane-proximal region (MPR) in the β-integrin cytoplasmic domain. Talin-deficient and talin1(L325R) platelets were defective in retracting fibrin clots. However, the defect in clot retraction in talin1(L325R) platelets, but not talin-deficient platelets, was rescued by extrinsically activating integrins with manganese, thereby proving that integrin activation is required and showing that talin1(L325R) can form functional links to the actin cytoskeleton. PMID:20971947

Functional Comparison of Induced Pluripotent Stem Cell- and Blood-Derived GPIIbIIIa Deficient Platelets

PubMed Central

Haas, Jessica; Sandrock-Lang, Kirstin; Gärtner, Florian; Jung, Christian Billy; Zieger, Barbara; Parrotta, Elvira; Kurnik, Karin; Sinnecker, Daniel; Wanner, Gerhard; Laugwitz, Karl-Ludwig; Massberg, Steffen; Moretti, Alessandra

2015-01-01

Human induced pluripotent stem cells (hiPSCs) represent a versatile tool to model genetic diseases and are a potential source for cell transfusion therapies. However, it remains elusive to which extent patient-specific hiPSC-derived cells functionally resemble their native counterparts. Here, we generated a hiPSC model of the primary platelet disease Glanzmann thrombasthenia (GT), characterized by dysfunction of the integrin receptor GPIIbIIIa, and compared side-by-side healthy and diseased hiPSC-derived platelets with peripheral blood platelets. Both GT-hiPSC-derived platelets and their peripheral blood equivalents showed absence of membrane expression of GPIIbIIIa, a reduction of PAC-1 binding, surface spreading and adherence to fibrinogen. We demonstrated that GT-hiPSC-derived platelets recapitulate molecular and functional aspects of the disease and show comparable behavior to their native counterparts encouraging the further use of hiPSC-based disease models as well as the transition towards a clinical application. PMID:25607928

Quality requirements for veterinary hematology analyzers in small animals-a survey about veterinary experts' requirements and objective evaluation of analyzer performance based on a meta-analysis of method validation studies: bench top hematology analyzer.

PubMed

Cook, Andrea M; Moritz, Andreas; Freeman, Kathleen P; Bauer, Natali

2016-09-01

Scarce information exists about quality requirements and objective evaluation of performance of large veterinary bench top hematology analyzers. The study was aimed at comparing the observed total error (TEobs ) derived from meta-analysis of published method validation data to the total allowable error (TEa ) for veterinary hematology variables in small animals based on experts' opinions. Ideally, TEobs should be < TEa . An online survey was sent to veterinary experts in clinical pathology and small animal internal medicine for providing the maximal allowable deviation from a given result for each variable. Percent of TEa = (allowable median deviation/clinical threshold) * 100%. Second, TEobs for 3 laser-based bench top hematology analyzers (ADVIA 2120; Sysmex XT2000iV, and CellDyn 3500) was calculated based on method validation studies published between 2005 and 2013 (n = 4). The percent TEobs = 2 * CV (%) + bias (%). The CV was derived from published studies except for the ADVIA 2120 (internal data), and bias was estimated from the regression equation. A total of 41 veterinary experts (19 diplomates, 8 residents, 10 postgraduate students, 4 anonymous specialists) responded. The proposed range of TEa was wide, but generally ≤ 20%. The TEobs was < TEa for all variables and analyzers except for canine and feline HGB (high bias, low CV) and platelet counts (high bias, high CV). Overall, veterinary bench top analyzers fulfilled experts' requirements except for HGB due to method-related bias, and platelet counts due to known preanalytic/analytic issues. © 2016 American Society for Veterinary Clinical Pathology.

Global proteome analysis identifies active immunoproteasome subunits in human platelets.

PubMed

Klockenbusch, Cordula; Walsh, Geraldine M; Brown, Lyda M; Hoffman, Michael D; Ignatchenko, Vladimir; Kislinger, Thomas; Kast, Juergen

2014-12-01

The discovery of new functions for platelets, particularly in inflammation and immunity, has expanded the role of these anucleate cell fragments beyond their primary hemostatic function. Here, four in-depth human platelet proteomic data sets were generated to explore potential new functions for platelets based on their protein content and this led to the identification of 2559 high confidence proteins. During a more detailed analysis, consistently high expression of the proteasome was discovered, and the composition and function of this complex, whose role in platelets has not been thoroughly investigated, was examined. Data set mining resulted in identification of nearly all members of the 26S proteasome in one or more data sets, except the β5 subunit. However, β5i, a component of the immunoproteasome, was identified. Biochemical analyses confirmed the presence of all catalytically active subunits of the standard 20S proteasome and immunoproteasome in human platelets, including β5, which was predominantly found in its precursor form. It was demonstrated that these components were assembled into the proteasome complex and that standard proteasome as well as immunoproteasome subunits were constitutively active in platelets. These findings suggest potential new roles for platelets in the immune system. For example, the immunoproteasome may be involved in major histocompatibility complex I (MHC I) peptide generation, as the MHC I machinery was also identified in our data sets. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Megakaryocytic Smad4 Regulates Platelet Function through Syk and ROCK2 Expression.

PubMed

Wang, Yanhua; Jiang, Lirong; Mo, Xi; Lan, Yu; Yang, Xiao; Liu, Xinyi; Zhang, Jian; Zhu, Li; Liu, Junling; Wu, Xiaolin

2017-09-01

Smad4, a key transcription factor in the transforming growth factor- β signaling pathway, is involved in a variety of cell physiologic and pathologic processes. Here, we characterized megakaryocyte/platelet-specific Smad4 deficiency in mice to elucidate its effect on platelet function. We found that megakaryocyte/platelet-specific loss of Smad4 caused mild thrombocytopenia and significantly extended first occlusion time and tail bleeding time in mice. Smad4-deficient platelets showed reduced agonist-induced platelet aggregation. Further studies showed that a severe defect was seen in integrin α IIb β 3 -mediated bidirectional (inside-out and outside-in) signaling in Smad4-deficient platelets, as evidenced by reduced fibrinogen binding and α -granule secretion, suppressed platelet spreading and clot retraction. Microarray analysis showed that the expression levels of multiple genes were altered in Smad4-deficient platelets. Among these genes, spleen tyrosine kinase (Syk) and Rho-associated coiled-coil containing protein kinase 2 (ROCK2) were downregulated several times as confirmed by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Further research showed that Smad4 directly regulates ROCK2 transcription but indirectly regulates Syk. Megakaryocyte/platelet-specific Smad4 deficiency caused decreased expression levels of Syk and ROCK2 in platelets. These results suggest potential links among Smad4 deficiency, attenuated Syk, and ROCK2 expression and defective platelet activation. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Effect of serotonin on platelet function in cocaine exposed blood

PubMed Central

Ziu, Endrit; Hadden, Coedy; Li, Yicong; Lowery, Curtis Lee; Singh, Preeti; Ucer, Serra S.; Mercado, Charles P.; Gu, Howard H.; Kilic, Fusun

2014-01-01

5-hydroxytryptamine (5-HT) reuptake inhibitors counteract the pro-thrombotic effect of elevated plasma 5-HT by down-regulating the 5-HT uptake rates of platelets. Cocaine also down-regulates the platelet 5-HT uptake rates but in contrast, the platelets of cocaine-injected mice show a much higher aggregation rate than the platelets of control mice. To examine the involvement of plasma 5-HT in cocaine-mediated platelet aggregation, we studied the function of platelets isolated from wild-type and transgenic, peripheral 5-HT knock-out (TPH1-KO) mice, and cocaine-insensitive dopamine transporter knock in (DAT-KI) mice. In cocaine-injected mice compared to the control mice, the plasma 5-HT level as well as the surface level of P-selectin was elevated; in vitro platelet aggregation in the presence of type I fibrillar collagen was enhanced. However, cocaine injection lowered the 5-HT uptake rates of platelets and increased the plasma 5-HT levels of the DAT-KI mice but did not change their platelets aggregation rates further which are already hyper-reactive. Furthermore, the in vitro studies supporting these in vivo findings suggest that cocaine mimics the effect of elevated plasma 5-HT level on platelets and in 5-HT receptor- and transporter-dependent pathways in a two-step process propagates platelet aggregation by an additive effect of 5-HT and nonserotonergic catecholamine. PMID:25091505

Salivary Antigen-5/CAP Family Members Are Cu2+-dependent Antioxidant Enzymes That Scavenge O2⨪ and Inhibit Collagen-induced Platelet Aggregation and Neutrophil Oxidative Burst*

PubMed Central

Assumpção, Teresa C. F.; Ma, Dongying; Schwarz, Alexandra; Reiter, Karine; Santana, Jaime M.; Andersen, John F.; Ribeiro, José M. C.; Nardone, Glenn; Yu, Lee L.; Francischetti, Ivo M. B.

2013-01-01

The function of the antigen-5/CAP family of proteins found in the salivary gland of bloodsucking animals has remained elusive for decades. Antigen-5 members from the hematophagous insects Dipetalogaster maxima (DMAV) and Triatoma infestans (TIAV) were expressed and discovered to attenuate platelet aggregation, ATP secretion, and thromboxane A2 generation by low doses of collagen (<1 μg/ml) but no other agonists. DMAV did not interact with collagen, glycoprotein VI, or integrin α2β1. This inhibitory profile resembles the effects of antioxidants Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in platelet function. Accordingly, DMAV was found to inhibit cytochrome c reduction by O2⨪ generated by the xanthine/xanthine oxidase, implying that it exhibits antioxidant activity. Moreover, our results demonstrate that DMAV blunts the luminescence signal of O2⨪ generated by phorbol 12-myristate 13-acetate-stimulated neutrophils. Mechanistically, inductively coupled plasma mass spectrometry and fluorescence spectroscopy revealed that DMAV, like Cu,Zn-SOD, interacts with Cu2+, which provides redox potential for catalytic removal of O2⨪. Notably, surface plasmon resonance experiments (BIAcore) determined that DMAV binds sulfated glycosaminoglycans (e.g. heparin, KD ∼100 nmol/liter), as reported for extracellular SOD. Finally, fractions of the salivary gland of D. maxima with native DMAV contain Cu2+ and display metal-dependent antioxidant properties. Antigen-5/CAP emerges as novel family of Cu2+-dependent antioxidant enzymes that inhibit neutrophil oxidative burst and negatively modulate platelet aggregation by a unique salivary mechanism. PMID:23564450

Antioxidative and antiplatelet effects of aqueous inflorescence Piper betle extract.

PubMed

Lei, Daniel; Chan, Chiu-Po; Wang, Ying-Jan; Wang, Tong-Mei; Lin, Bor-Ru; Huang, Chun-Hsun; Lee, Jang-Jaer; Chen, Hsin-Ming; Jeng, Jiiang-Huei; Chang, Mei-Chi

2003-03-26

Piper betle, belonging to the Piperaceae family, is a tropical plant, and its leaf and inflorescence are popularly consumed by betel quid (BQ) chewers in Taiwan and many other South and Southeast Asian countries. However, little is known about the biochemical properties of inflorescence Piper betle (IPB) toward reactive oxygen species (ROS) and platelet functions. In the present work, aqueous IPB extract was shown to be a scavenger of H(2)O(2), superoxide radical, and hydroxyl radical with a 50% inhibitory concentration (IC(50)) of about 80, 28, and 73 microg/mL, respectively. IPB extract also prevented the hydroxyl radical induced PUC18 plasmid DNA breaks at concentrations higher than 40 microg/mL. Since ROS are crucial for platelet aggregation, we further found that IPB extract also inhibited the arachidonic acid (AA) induced and collagen-induced platelet aggregation, with an IC(50) of 207 and 335 microg/mL, respectively. IPB extract also inhibited the AA-, collagen- (>100 microg/mL of IPB), and thrombin (>250 microg/mL of IPB)-induced thromboxane B(2) (TXB(2)) production by more than 90%. However, IPB extract showed little effect on thrombin-induced aggregation. These results indicated that aqueous components of IPB are potential ROS scavengers and may prevent the platelet aggregation possibly via scavenging ROS or inhibition of TXB(2) production.

Pharmacological evaluation of aqueous extract of Althaea officinalis flower grown in Lebanon.

PubMed

Hage-Sleiman, Rouba; Mroueh, Mohamad; Daher, Costantine F

2011-03-01

Althaea officinalis Linn. (Malvaideae) flower is commonly used in folk medicine in Lebanon and neighboring countries. Although most of the studies have been conducted on the mucilage-rich roots, little is known about the flower. This study investigates the potential role of aqueous extract of Althaea officinalis flower in lipemia, gastric ulcer, inflammation, and platelet aggregation using the rat model. Blood lipid profile and liver function were assessed after 1 month of extract intake via drinking water. Anti-inflammatory activity was tested against acute and chronic inflammation induced by carrageenan and formalin, respectively. Antiulcer activity was evaluated using ethanol-induced gastric ulcer. Antiplatelet activity was investigated in vitro using the adenosine 5'-diphosphate (ADP)-induced platelet aggregation bioassay. The 50 mg/kg body weight dose resulted in significant increase in serum HDL cholesterol level with no effects on stool cholesterol and triacylglycerol. Increasing the dose to 500 mg/kg body weight caused a significant decrease in stool water content. No adverse effect on liver enzymes was observed. Significant anti-inflammatory (acute and chronic inflammation) and antiulcerogenic activities were observed at all used doses (50, 100, and 250 mg/kg body). Time-dependent inhibition of platelet aggregation was demonstrated at 500 µg/ml concentration. The aqueous extract of Althaea officinalis flower demonstrated potential benefits in lipemia, inflammation, gastric ulcer, and platelet aggregation with no visible adverse effect.

Synthetic analogues of flavonoids with improved activity against platelet activation and aggregation as novel prototypes of food supplements.

PubMed

Del Turco, Serena; Sartini, Stefania; Cigni, Giulia; Sentieri, Cassandra; Sbrana, Silverio; Battaglia, Debora; Papa, Angela; Da Settimo, Federico; La Motta, Concettina; Basta, Giuseppina

2015-05-15

We investigated the ability of quercetin and apigenin to modulate platelet activation and aggregation, and compared the observed efficacy with that displayed by their synthetic analogues 2-phenyl-4H-pyrido[1,2-a]pyrimidin-4-ones, 1-4, and 2,3-diphenyl-4H-pyrido[1,2-a]pyrimidin-4-ones, 5-7. Platelet aggregation was explored through a spectrophotometric assay on platelet-rich plasma (PRP) treated with the thromboxane A2 mimetic U46619, collagen and thrombin in presence/absence of various bioisosteres of flavonoids (12.5-25-50-100 μM). The platelet density, (mean platelet component, MPC), was measured by the Advia 120 Hematology System as a marker surrogate of platelet activation. The induced P-selectin expression, which reflects platelet degranulation/activation, was quantified by flow cytometry on PRP. Our synthetic compounds modulated significantly both platelet activation and aggregation, thus turning out to be more effective than the analogues quercetin and apigenin when tested at a concentration fully consistent with their use in vivo. Accordingly, they might be used as food supplements to increase the efficacy of natural flavonoids. Copyright © 2014 Elsevier Ltd. All rights reserved.

Overview of platelet physiology and laboratory evaluation of platelet function.

PubMed

Rodgers, G M

1999-06-01

Appropriate laboratory testing for the platelet-type bleeding disorders hinges on an adequate assessment in the history and physical examination. Patients with histories and screening laboratory results consistent with coagulation disorders (hemophilia, disseminated intravascular coagulation) are not appropriate candidates for platelet function testing. In contrast, patients with a lifelong history of platelet-type bleeding symptoms and perhaps a positive family history of bleeding would be appropriate for testing. Figure 6 depicts one strategy to evaluate these patients. Platelet morphology can easily be evaluated to screen for two uncommon qualitative platelet disorders: Bernard-Soulier syndrome (associated with giant platelets) and gray platelet syndrome, a subtype of storage pool disorder in which platelet granulation is morphologically abnormal by light microscopy. If the bleeding disorder occurred later in life (no bleeding with surgery or trauma early in life), the focus should be on acquired disorders of platelet function. For those patients thought to have an inherited disorder, testing for vWD should be done initially because approximately 1% of the population has vWD. The complete vWD panel (factor VIII coagulant activity, vWf antigen, ristocetin cofactor activity) should be performed because many patients will have abnormalities of only one particular panel component. Patients diagnosed with vWD should be classified using multimeric analysis to identify the type 1 vWD patients likely to respond to DDAVP. If vWD studies are normal, platelet aggregation testing should be performed, ensuring that no antiplatelet medications have been ingested at least 1 week before testing. If platelet aggregation tests are normal and if suspicion for an inherited disorder remains high, vWD testing should be repeated. The evaluation of thrombocytopenia may require bone marrow examination to exclude primary hematologic disorders. If future studies with thrombopoietin assays confirm preliminary results, however, the bone marrow examination of certain patients may be replaced by a thrombopoietin level.

Evaluation of flow cytometric HIT assays in relation to an IgG-Specific immunoassay and clinical outcome.

PubMed

Kerényi, Adrienne; Beke Debreceni, Ildikó; Oláh, Zsolt; Ilonczai, Péter; Bereczky, Zsuzsanna; Nagy, Béla; Muszbek, László; Kappelmayer, János

2017-09-01

Heparin-induced thrombocytopenia (HIT) is a severe side effect of heparin treatment caused by platelet activating IgG antibodies generated against the platelet factor 4 (PF4)-heparin complex. Thrombocytopenia and thrombosis are the leading clinical symptoms of HIT. The clinical pretest probability of HIT was evaluated by the 4T score system. Laboratory testing of HIT was performed by immunological detection of antibodies against PF4-heparin complex (EIA) and two functional assays. Heparin-dependent activation of donor platelets by patient plasma was detected by flow cytometry. Increased binding of Annexin-V to platelets and elevated number of platelet-derived microparticles (PMP) were the indicators of platelet activation. EIA for IgG isotype HIT antibodies was performed in 405 suspected HIT patients. Based on negative EIA results, HIT was excluded in 365 (90%) of cases. In 40 patients with positive EIA test result functional tests were performed. Platelet activating antibodies were detected in 17 cases by Annexin V binding. PMP count analysis provided nearly identical results. The probability of a positive flow cytometric assay result was higher in patients with elevated antibody titer. 71% of patients with positive EIA and functional assay had thrombosis. EIA is an important first line laboratory test in the diagnosis of HIT; however, HIT must be confirmed by a functional test. Annexin V binding and PMP assays using flow cytometry are functional HIT tests convenient in a clinical diagnostic laboratory. The positive results of functional assays may predict the onset of thrombosis. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

Characterization of the aggregation responses of camel platelets.

PubMed

Al Ghumlas, Abeer K; Gader, Abdel Galil M Abdel

2013-09-01

Despite evidence of active hemostasis, camel platelets barely respond to common aggregating agents at standard doses used for human platelet aggregation. The purpose of the study was to find out whether camel platelets can be activated by high doses or combinations of aggregation agonists, and to characterize the receptor that mediates the aggregation response to adenosine diphosphate (ADP), the most potent agonist for camel platelets known so far. Aggregation studies were performed with platelet-rich plasma (PRP) in response to multiple doses or combinations of ADP, epinephrine (EPN), collagen, and arachidonic acid (AA). Aggregation responses to ADP were performed before and after the addition of the ADP receptor (P2Y12) antagonist Clopidogrel. Camel platelets responded to ADP at doses higher than the standard dose for human platelets, and to combinations of EPN and other agonists, while no aggregation was elicited with EPN or AA alone. Clopidogrel blocked the ADP-induced aggregation responses in a dose-dependent fashion in vitro. Camel platelet aggregation can be activated by increasing the dose of some agonists such as ADP, but not AA or EPN. Irreversible aggregation of camel platelets could also be triggered by a combination of EPN and ADP, and collagen and AA. Inhibition with clopidogrel suggests that camel platelets express the ADP receptor, P2Y12. Understanding platelet function in camels will add to the understanding of platelet function in health and disease. © 2013 American Society for Veterinary Clinical Pathology.

Antiplatelet mechanism of an herbal mixture prepared from the extracts of Phyllostachys pubescens leaves and Prunus mume fruits.

PubMed

Son, Eunjung; Kim, Seung-Hyung; Yang, Won-Kyung; Kim, Dong-Seon; Cha, Jimin

2017-12-19

Bamboo (Phyllostachys pubescens) leaves and Japanese apricot (Mume fructus) fruit are traditionally recognized to be safe herbs broadly used for food and medicinal purposes in Southeast Asia. Our group previously explored their antiplatelet effects. This study was designed to confirm inhibition effects of PM21 (a 2:1 mixture of bamboo leaf extract and Japanese apricot fruit extract) on platelet aggregation and evaluate its potency to use as an herbal remedy to prevent and/or treat the diseases caused by platelet aggregation and thrombus formation. Washed platelets were prepared and platelet aggregation was induced by adding 5 μg/mL collagen. Anti-platelet effects of PM21 (75 mg/kg, 150 mg/kg, and 300 mg/kg for ex vivo and in vivo assays, and 50, 100, 200 μg/mL for in vitro assays) were evaluated. In ex vivo assays, PM21 was orally administered to rats daily after overnight fasting for 3 days and blood was collected 1 h after the final treatment. In vivo antithrombotic effect of PM21 was observed from a carrageenan induced mouse tail thrombosis model. In ex vivo assay, PM21 inhibited platelet aggregation significantly. PM21 showed a strong antithrombotic effect by reducing significantly the length of mouse tail thrombus. PM21 increased intracellular cAMP level and reduced the release of ATP, TXA 2 , and serotonin. PM21 also reduced intracellular concentration of calcium ion, fibrinogen binding to integrin α IIb β 3 , and phosphorylation of ERK2, p38, PLCγ2, and PI3 K. PM21 showed remarkable inhibitory effects on platelet aggregation and thrombus formation. Its inhibitory function seems to influence on GPVI binding to its ligand and subsequent initiation of a signaling cascade that involves activation of effector proteins and secretion of effector molecules, such as ATP, TXA 2 , serotonin, and Ca 2+ . PM21 also appears to exert its anti-platelet effect by deactivation of ERKs activation pathway as well as inhibition of fibrinogen binding to integrin α IIb β 3 .

Detection of platelet sensitivity to inhibitors of COX-1, P2Y1, and P2Y12 using a whole blood microfluidic flow assay

PubMed Central

Li, Ruizhi; Diamond, Scott L.

2014-01-01

BACKGROUND Microfluidic devices recreate the hemodynamic conditions of thrombosis. METHODS Whole blood inhibited with PPACK was treated ex vivo with inhibitors and perfused over collagen for 300 s (wall shear rate = 200 s−1) using a microfluidic flow assay. Platelet accumulation was measured in the presence of COX-1 inhibitor (aspirin, ASA), P2Y1 inhibitor (MRS 2179), P2Y12 inhibitor (2MeSAMP) or combined P2Y1 and P2Y12 inhibitors. RESULTS High dose ASA (500 μM), 2MeSAMP (100 μM), MRS 2179 (10 μM),or combined 2MeSAMP and MRS 2179 decreased total platelet accumulation by 27.5%, 75.6%, 77.7%, and 87.9% (p < 0.01), respectively. ASA reduced secondary aggregation rate between 150 and 300 s without effect on primary deposition rate on collagen from 60 to 150 s. In contrast, 2MeSAMP and MRS 2179 acted earlier and reduced primary deposition to collagen between 60 and 105 s and secondary aggregation between 105 and 300 s. RCOX and RP2Y (defined as a ratio of secondary aggregation rate to primary deposition rate) demonstrated 9 of 10 subjects had RCOX < 1 or RP2Y < 1 following ASA or 2MeSAMP addition, while 6 of 10 subjects had RP2Y < 1 following MRS 2179 addition. Combined MRS 2179 and 2MeSAMP inhibited primary platelet deposition rate and platelet secondary aggregation beyond that of each individual inhibitor. Receiver-Operator Characteristic area under the curve (AUC) indicated the robustness of RCOX and RP2Y to detect inhibition of secondary platelet aggregation by ASA, 2MeSAMP, and MRS 2179 (AUC of 0.874 0.966, and 0.889, respectively). CONCLUSIONS Microfluidic devices can detect platelet sensitivity to antiplatelet agents. The R-value can serve as a self-normalized metric of platelet function for a single blood sample. PMID:24365044

Comparison of Three Tests to Distinguish Platelet Reactivity in Patients with Renal Impairment during Dual Antiplatelet Therapy.

PubMed

Guo, Long Zhe; Kim, Moo Hyun; Kim, Tae Hyung; Park, Jong Seong; Jin, Enze; Shim, Chang Heon; Choi, Sun Young; Serebruany, Victor L

2016-01-01

Clopidogrel and aspirin combination remains a cornerstone for modern dual antiplatelet therapy (DAPT) following coronary stenting. Although monitoring is not currently recommended, certain high-risk cohorts may benefit from tailoring antiplatelet options to reduce thrombotic or/and hemorrhagic risks. Patients with diminished estimated glomerular filtration rate (eGFR) are prone to both vascular occlusions and bleeding events in whom monitoring may be especially advantageous. We compared the residual platelet reactivity assessed by 3 conventional tests during the maintenance antiplatelet therapy dependent on eGFR. Post-stenting patients (n = 701) receiving aspirin 100 mg/daily and clopidogrel 75 mg/daily were prospectively enrolled in the cross-sectional single-center study. Patients were dichotomized into 5 groups: eGFR >90, 60-89, 30-59, <30 ml/min/1.73 m2, and dialysis. Platelet reactivity by VerifyNow™, light transmittance aggregometry (LTA), and Multiplate analyzer by multiple electrode platelet aggregometry (MEA) assays together with eGFR calculations were done simultaneously at 1 month after coronary stenting. VerifyNow assay distinguished residual platelet reactivity dependent on eGFR deterioration (191 ± 72 vs. 216 ± 78 vs. 248 ± 80 vs. 264 ± 70 vs. 317 ± 96 PRU; p < 0.001). In contrast, LTA (34.3 ± 18.1 vs. 34.7 ± 18.1 vs. 38.0 ± 16.6 vs. 33.0 ± 17.3 vs. 34.1 ± 29.3%; p = 0.242), or MEA (37.2 ± 19.6 vs. 33.8 ± 18.4 vs. 38.6 ± 21.4 vs. 36.5 ± 20.5 vs. 38.3 ± 28.3 AU/min; p = 0.086) failed to triage platelet reactivity in renal patients. Agreement among assays to identify patients with impaired platelet reactivity and eGFR during antiplatelet therapy was low. The multivariable regression analyses confirmed the VerifyNow advantage, since the differences in the platelet reactivity were highly significant for all renal impairment (RI) groups. In contrast, LTA did not distinguish RI patients, and for the MEA, only RI5 (dialysis) cohort exhibit borderline significant decline of residual platelet reactivity. Among 3 assays, VerifyNow was capable to reliably triage residual platelet reactivity in post-stenting DAPT patients dependent on the gradual decline of eGFR during therapy with clopidogrel and aspirin. These data should be confirmed in a large validation longitudinal trial, and may justify future platelet activity monitoring for potential regimen/dose adjustment in high-risk patients. The clinical implications of these data are still unclear, but may give an indication as to whether or when DAPT dose adjustment will become a reality. © 2016 S. Karger AG, Basel.

The clearance mechanism of chilled blood platelets.

PubMed

Hoffmeister, Karin M; Felbinger, Thomas W; Falet, Hervé; Denis, Cécile V; Bergmeier, Wolfgang; Mayadas, Tanya N; von Andrian, Ulrich H; Wagner, Denisa D; Stossel, Thomas P; Hartwig, John H

2003-01-10

Platelet transfusion is a very common lifesaving medical procedure. Not widely known is the fact that platelets, unlike other blood cells, rapidly leave the circulation if refrigerated prior to transfusion. This peculiarity requires blood services to store platelets at room temperature, limiting platelet supplies for clinical needs. Here, we describe the mechanism of this clearance system, a longstanding mystery. Chilling platelets clusters their von Willebrand (vWf) receptors, eliciting recognition of mouse and human platelets by hepatic macrophage complement type 3 (CR3) receptors. CR3-expressing but not CR3-deficient mice exposed to cold rapidly decrease platelet counts. Cooling primes platelets for activation. We propose that platelets are thermosensors, primed at peripheral sites where most injuries occurred throughout evolution. Clearance prevents pathologic thrombosis by primed platelets. Chilled platelets bind vWf and function normally in vitro and ex vivo after transfusion into CR3-deficient mice. Therefore, GPIb modification might permit cold platelet storage.

Platelet gene therapy improves hemostatic function for integrin αIIbβ3-deficient dogs

PubMed Central

Fang, Juan; Jensen, Eric S.; Boudreaux, Mary K.; Du, Lily M.; Hawkins, Troy B.; Koukouritaki, Sevasti B.; Cornetta, Kenneth; Wilcox, David A.

2011-01-01

Activated blood platelets mediate the primary response to vascular injury. Although molecular abnormalities of platelet proteins occur infrequently, taken collectively, an inherited platelet defect accounts for a bleeding diathesis in ≈1:20,000 individuals. One rare example of a platelet disorder, Glanzmann thrombasthenia (GT), is characterized by life-long morbidity and mortality due to molecular abnormalities in a major platelet adhesion receptor, integrin αIIbβ3. Transfusion therapy is frequently inadequate because patients often generate antibodies to αIIbβ3, leading to immune-mediated destruction of healthy platelets. In the most severe cases allogeneic bone marrow transplantation has been used, yet because of the risk of the procedure it has been limited to few patients. Thus, hematopoietic stem cell gene transfer was explored as a strategy to improve platelet function within a canine model for GT. Bleeding complications necessitated the use of a mild pretransplant conditioning regimen; therefore, in vivo drug selection was used to improve engraftment of autologously transplanted cells. Approximately 5,000 αIIbβ3 receptors formed on 10% of platelets. These modest levels allowed platelets to adhere to αIIbβ3’s major ligand (fibrinogen), form aggregates, and mediate retraction of a fibrin clot. Remarkably, improved hemostatic function was evident, with ≤135-fold reduced blood loss, and improved buccal bleeding times decreased to 4 min for up to 5 y after transplant. One of four transplanted dogs developed a significant antibody response to αIIbβ3 that was attenuated effectively with transient immune suppression. These results indicate that gene therapy could become a practical approach for treating inherited platelet defects. PMID:21606353

Platelet gene therapy improves hemostatic function for integrin alphaIIbbeta3-deficient dogs.

PubMed

Fang, Juan; Jensen, Eric S; Boudreaux, Mary K; Du, Lily M; Hawkins, Troy B; Koukouritaki, Sevasti B; Cornetta, Kenneth; Wilcox, David A

2011-06-07

Activated blood platelets mediate the primary response to vascular injury. Although molecular abnormalities of platelet proteins occur infrequently, taken collectively, an inherited platelet defect accounts for a bleeding diathesis in ≈1:20,000 individuals. One rare example of a platelet disorder, Glanzmann thrombasthenia (GT), is characterized by life-long morbidity and mortality due to molecular abnormalities in a major platelet adhesion receptor, integrin αIIbβ3. Transfusion therapy is frequently inadequate because patients often generate antibodies to αIIbβ3, leading to immune-mediated destruction of healthy platelets. In the most severe cases allogeneic bone marrow transplantation has been used, yet because of the risk of the procedure it has been limited to few patients. Thus, hematopoietic stem cell gene transfer was explored as a strategy to improve platelet function within a canine model for GT. Bleeding complications necessitated the use of a mild pretransplant conditioning regimen; therefore, in vivo drug selection was used to improve engraftment of autologously transplanted cells. Approximately 5,000 αIIbβ3 receptors formed on 10% of platelets. These modest levels allowed platelets to adhere to αIIbβ3's major ligand (fibrinogen), form aggregates, and mediate retraction of a fibrin clot. Remarkably, improved hemostatic function was evident, with ≤135-fold reduced blood loss, and improved buccal bleeding times decreased to 4 min for up to 5 y after transplant. One of four transplanted dogs developed a significant antibody response to αIIbβ3 that was attenuated effectively with transient immune suppression. These results indicate that gene therapy could become a practical approach for treating inherited platelet defects.

Stratified aspartate aminotransferase-to-platelet ratio index accurately predicts survival in hepatocellular carcinoma patients undergoing curative liver resection.

PubMed

Yang, Hao-Jie; Jiang, Jing-Hang; Yang, Yu-Ting; Guo, Zhe; Li, Ji-Jia; Liu, Xuan-Han; Lu, Fei; Zeng, Feng-Hua; Ye, Jin-Song; Zhang, Ke-Lan; Chen, Neng-Zhi; Xiang, Bang-De; Li, Le-Qun

2017-03-01

The aspartate aminotransferase-to-platelet ratio index has been reported to predict prognosis of patients with hepatocellular carcinoma. This study examined the prognostic potential of stratified aspartate aminotransferase-to-platelet ratio index for hepatocellular carcinoma patients undergoing curative liver resection. A total of 661 hepatocellular carcinoma patients were retrieved and the associations between aspartate aminotransferase-to-platelet ratio index and clinicopathological variables and survivals (overall survival and disease-free survival) were analyzed. Higher aspartate aminotransferase-to-platelet ratio index quartiles were significantly associated with poorer overall survival (p = 0.002) and disease-free survival (p = 0.001). Multivariate analysis showed aspartate aminotransferase-to-platelet ratio index to be an independent risk factor for overall survival (p = 0.018) and disease-free survival (p = 0.01). Patients in the highest aspartate aminotransferase-to-platelet ratio index quartile were at 44% greater risk of death than patients in the first quartile (hazard ratio = 1.445, 95% confidence interval = 1.081 - 1.931, p = 0.013), as well as 49% greater risk of recurrence (hazard ratio = 1.49, 95% confidence interval = 1.112-1.998, p = 0.008). Subgroup analysis also showed aspartate aminotransferase-to-platelet ratio index to be an independent predictor of poor overall survival and disease-free survival in patients positive for hepatitis B surface antigen or with cirrhosis (both p < 0.05). Similar results were obtained when aspartate aminotransferase-to-platelet ratio index was analyzed as a dichotomous variable with cutoff values of 0.25 and 0.62. Elevated preoperative aspartate aminotransferase-to-platelet ratio index may be independently associated with poor overall survival and disease-free survival in hepatocellular carcinoma patients following curative resection.

Release of Phosphorylated HSP27 (HSPB1) from Platelets Is Accompanied with the Acceleration of Aggregation in Diabetic Patients

PubMed Central

Tokuda, Haruhiko; Kuroyanagi, Gen; Tsujimoto, Masanori; Enomoto, Yukiko; Matsushima-Nishiwaki, Rie; Onuma, Takashi; Kojima, Akiko; Doi, Tomoaki; Tanabe, Kumiko; Akamatsu, Shigeru; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Iwama, Toru; Tanikawa, Takahisa; Ishikawa, Kei; Kojima, Kumi; Kozawa, Osamu

2015-01-01

We investigated the relationship between HSP27 phosphorylation and collagen-stimulated activation of platelets in patients with diabetes mellitus (DM). Platelet-rich plasma was prepared from blood of type 2 DM patients. The platelet aggregation was analyzed in size of aggregates by an aggregometer using a laser scattering method. The protein phosphorylation was analyzed by Western blotting. Phosphorylated-HSP27 and PDGF-AB released from platelets were measured by ELISA. The phosphorylated-HSP27 levels at Ser-78 and Ser-82 induced by collagen were directly proportional to the platelet aggregation. Total HSP27 levels in platelets were decreased concomitantly with the phosphorylation. The released HSP27 levels were significantly correlated with the phosphorylated levels of HSP27 in the platelets stimulated by 0.3 μg/ml collagen. The low dose collagen-stimulated release of HSP27 was detected but relatively small in healthy donors. The released levels of PDGF-AB were in parallel with the levels of released HSP27. Area under the curve (AUC) of small aggregation (9-25 μm) induced by 0.3 μg/ml collagen was inversely proportional to the levels of released HSP27. AUC of large aggregation (50-70 μm) was directly proportional to the levels of released HSP27. Exogenous recombinant phosphorylated- HSP27 hardly affected the aggregation or the released levels of PDGF-AB induced by collagen. These results strongly suggest that HSP27 is released from human platelets accompanied with its phosphorylation induced by collagen, which is correlated with the acceleration of platelet aggregation in type 2 DM patients. PMID:26046355

Release of Phosphorylated HSP27 (HSPB1) from Platelets Is Accompanied with the Acceleration of Aggregation in Diabetic Patients.

PubMed

Tokuda, Haruhiko; Kuroyanagi, Gen; Tsujimoto, Masanori; Enomoto, Yukiko; Matsushima-Nishiwaki, Rie; Onuma, Takashi; Kojima, Akiko; Doi, Tomoaki; Tanabe, Kumiko; Akamatsu, Shigeru; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Iwama, Toru; Tanikawa, Takahisa; Ishikawa, Kei; Kojima, Kumi; Kozawa, Osamu

2015-01-01

We investigated the relationship between HSP27 phosphorylation and collagen-stimulated activation of platelets in patients with diabetes mellitus (DM). Platelet-rich plasma was prepared from blood of type 2 DM patients. The platelet aggregation was analyzed in size of aggregates by an aggregometer using a laser scattering method. The protein phosphorylation was analyzed by Western blotting. Phosphorylated-HSP27 and PDGF-AB released from platelets were measured by ELISA. The phosphorylated-HSP27 levels at Ser-78 and Ser-82 induced by collagen were directly proportional to the platelet aggregation. Total HSP27 levels in platelets were decreased concomitantly with the phosphorylation. The released HSP27 levels were significantly correlated with the phosphorylated levels of HSP27 in the platelets stimulated by 0.3 μg/ml collagen. The low dose collagen-stimulated release of HSP27 was detected but relatively small in healthy donors. The released levels of PDGF-AB were in parallel with the levels of released HSP27. Area under the curve (AUC) of small aggregation (9-25 μm) induced by 0.3 μg/ml collagen was inversely proportional to the levels of released HSP27. AUC of large aggregation (50-70 μm) was directly proportional to the levels of released HSP27. Exogenous recombinant phosphorylated- HSP27 hardly affected the aggregation or the released levels of PDGF-AB induced by collagen. These results strongly suggest that HSP27 is released from human platelets accompanied with its phosphorylation induced by collagen, which is correlated with the acceleration of platelet aggregation in type 2 DM patients.

Pseudothrombocytopenia or platelet clumping as a possible cause of low platelet count in patients with viral infection: a case series from single institution focusing on hepatitis A virus infection.

PubMed

Choe, W-H; Cho, Y-U; Chae, J-D; Kim, S-H

2013-02-01

Pseudothrombocytopenia (PTCP) is the phenomenon of ethylenediaminetetraacetic acid anticoagulant-activated platelet clumping, which results in artificially low platelet counts. Other investigators have reported a few cases of PTCP associated with viral infections. The objective of this study was to demonstrate the association of viral infection with PTCP. Medical records of patients with thrombocytopenia who were tested for peripheral blood smear examination between March 2009 and February 2011 were reviewed for platelet clumping and viral infection. Thrombocytopenic patients with viral infection had a higher frequency of platelet clumping than those with other diseases, which was statistically significant (13.8% vs. 6.5%, respectively: P = 0.003). Among the 18 cases where PTCP or platelet clumping was related to viral infection, hepatitis A virus infection (72.2%) was most common, followed by cytomegalovirus (11.1%) and influenza A H1N1 infections (5.6%). A third (33.3%) of the patients had platelet counts <100 × 10⁹/L. Pseudothrombocytopenia or platelet clumping should be considered in patients with acute viral infection, particularly if the platelet count is unexpectedly low, because failure to recognize PTCP may lead to unnecessary diagnostic tests and patient mismanagement. © 2012 Blackwell Publishing Ltd.

Functional expression of cysteinyl leukotriene receptors on human platelets.

PubMed

Hasegawa, Shunji; Ichiyama, Takashi; Hashimoto, Kunio; Suzuki, Yasuo; Hirano, Reiji; Fukano, Reiji; Furukawa, Susumu

2010-01-01

Normal peripheral blood leukocytes, such as basophils, eosinophils, B lymphocytes and monocytes/macrophages, have a cysteinyl leukotriene 1 (CysLT1) receptor, while the cysteinyl leukotriene 2 (CysLT2) receptor is expressed in cardiac Purkinje cells, endothelium, brain and leukocytes. However, it is unknown whether or not platelets express the CysLT1 or CysLT2 receptor. In this study we identify and characterize the biological function of the CysLT receptor of human platelets. We determined the CysLT1 or CysLT2 receptor mRNA expression in normal human platelets by RT-PCR and determined protein expression by Western blotting and flow cytometry. Moreover, we examined the effect of cysteinyl leukotrienes (CysLTs) in platelets on the induction of RANTES (Regulated on Activation, Normal T Expressed, and presumably Secreted). We also investigated whether the CysLT1 receptor antagonist pranlukast inhibits CysLT-induced RANTES release. In conclusion, we showed the functional expression of CysLT receptors on human platelets and demonstrated that CysLTs induced the release of significant amounts of RANTES, which suggests a novel role for human platelets in CysLT-mediated allergic inflammation.

Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins.

PubMed

O'Connor, Marie N; Salles, Isabelle I; Cvejic, Ana; Watkins, Nicholas A; Walker, Adam; Garner, Stephen F; Jones, Chris I; Macaulay, Iain C; Steward, Michael; Zwaginga, Jaap-Jan; Bray, Sarah L; Dudbridge, Frank; de Bono, Bernard; Goodall, Alison H; Deckmyn, Hans; Stemple, Derek L; Ouwehand, Willem H

2009-05-07

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)-based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins

PubMed Central

O'Connor, Marie N.; Salles, Isabelle I.; Cvejic, Ana; Watkins, Nicholas A.; Walker, Adam; Garner, Stephen F.; Jones, Chris I.; Macaulay, Iain C.; Steward, Michael; Zwaginga, Jaap-Jan; Bray, Sarah L.; Dudbridge, Frank; de Bono, Bernard; Goodall, Alison H.; Stemple, Derek L.; Ouwehand, Willem H.

2009-01-01

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)–based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis. PMID:19109564

New gene functions in megakaryopoiesis and platelet formation

PubMed Central

Gieger, Christian; Radhakrishnan, Aparna; Cvejic, Ana; Tang, Weihong; Porcu, Eleonora; Pistis, Giorgio; Serbanovic-Canic, Jovana; Elling, Ulrich; Goodall, Alison H.; Labrune, Yann; Lopez, Lorna M.; Mägi, Reedik; Meacham, Stuart; Okada, Yukinori; Pirastu, Nicola; Sorice, Rossella; Teumer, Alexander; Voss, Katrin; Zhang, Weihua; Ramirez-Solis, Ramiro; Bis, Joshua C.; Ellinghaus, David; Gögele, Martin; Hottenga, Jouke-Jan; Langenberg, Claudia; Kovacs, Peter; O’Reilly, Paul F.; Shin, So-Youn; Esko, Tõnu; Hartiala, Jaana; Kanoni, Stavroula; Murgia, Federico; Parsa, Afshin; Stephens, Jonathan; van der Harst, Pim; van der Schoot, C. Ellen; Allayee, Hooman; Attwood, Antony; Balkau, Beverley; Bastardot, François; Basu, Saonli; Baumeister, Sebastian E.; Biino, Ginevra; Bomba, Lorenzo; Bonnefond, Amélie; Cambien, François; Chambers, John C.; Cucca, Francesco; D’Adamo, Pio; Davies, Gail; de Boer, Rudolf A.; de Geus, Eco J. C.; Döring, Angela; Elliott, Paul; Erdmann, Jeanette; Evans, David M.; Falchi, Mario; Feng, Wei; Folsom, Aaron R.; Frazer, Ian H.; Gibson, Quince D.; Glazer, Nicole L.; Hammond, Chris; Hartikainen, Anna-Liisa; Heckbert, Susan R.; Hengstenberg, Christian; Hersch, Micha; Illig, Thomas; Loos, Ruth J. F.; Jolley, Jennifer; Khaw, Kay Tee; Kühnel, Brigitte; Kyrtsonis, Marie-Christine; Lagou, Vasiliki; Lloyd-Jones, Heather; Lumley, Thomas; Mangino, Massimo; Maschio, Andrea; Leach, Irene Mateo; McKnight, Barbara; Memari, Yasin; Mitchell, Braxton D.; Montgomery, Grant W.; Nakamura, Yusuke; Nauck, Matthias; Navis, Gerjan; Nöthlings, Ute; Nolte, Ilja M.; Porteous, David J.; Pouta, Anneli; Pramstaller, Peter P.; Pullat, Janne; Ring, Susan M.; Rotter, Jerome I.; Ruggiero, Daniela; Ruokonen, Aimo; Sala, Cinzia; Samani, Nilesh J.; Sambrook, Jennifer; Schlessinger, David; Schreiber, Stefan; Schunkert, Heribert; Scott, James; Smith, Nicholas L.; Snieder, Harold; Starr, John M.; Stumvoll, Michael; Takahashi, Atsushi; Tang, W. H. Wilson; Taylor, Kent; Tenesa, Albert; Thein, Swee Lay; Tönjes, Anke; Uda, Manuela; Ulivi, Sheila; van Veldhuisen, Dirk J.; Visscher, Peter M.; Völker, Uwe; Wichmann, H.-Erich; Wiggins, Kerri L.; Willemsen, Gonneke; Yang, Tsun-Po; Zhao, Jing Hua; Zitting, Paavo; Bradley, John R.; Dedoussis, George V.; Gasparini, Paolo; Hazen, Stanley L.; Metspalu, Andres; Pirastu, Mario; Shuldiner, Alan R.; van Pelt, L. Joost; Zwaginga, Jaap-Jan; Boomsma, Dorret I.; Deary, Ian J.; Franke, Andre; Froguel, Philippe; Ganesh, Santhi K.; Jarvelin, Marjo-Riitta; Martin, Nicholas G.; Meisinger, Christa; Psaty, Bruce M.; Spector, Timothy D.; Wareham, Nicholas J.; Akkerman, Jan-Willem N.; Ciullo, Marina; Deloukas, Panos; Greinacher, Andreas; Jupe, Steve; Kamatani, Naoyuki; Khadake, Jyoti; Kooner, Jaspal S.; Penninger, Josef; Prokopenko, Inga; Stemple, Derek; Toniolo, Daniela; Wernisch, Lorenz; Sanna, Serena; Hicks, Andrew A.; Rendon, Augusto; Ferreira, Manuel A.; Ouwehand, Willem H.; Soranzo, Nicole

2012-01-01

Platelets are the second most abundant cell type in blood and are essential for maintaining haemostasis. Their count and volume are tightly controlled within narrow physiological ranges, but there is only limited understanding of the molecular processes controlling both traits. Here we carried out a high-powered meta-analysis of genome-wide association studies (GWAS) in up to 66,867 individuals of European ancestry, followed by extensive biological and functional assessment. We identified 68 genomic loci reliably associated with platelet count and volume mapping to established and putative novel regulators of megakaryopoiesis and platelet formation. These genes show megakaryocyte-specific gene expression patterns and extensive network connectivity. Using gene silencing in Danio rerio and Drosophila melanogaster, we identified 11 of the genes as novel regulators of blood cell formation. Taken together, our findings advance understanding of novel gene functions controlling fate-determining events during megakaryopoiesis and platelet formation, providing a new example of successful translation of GWAS to function. PMID:22139419

Flow cytometric analysis of platelet cyclooxygenase-1 and -2 and surface glycoproteins in patients with immune thrombocytopenia and healthy individuals.

PubMed

Rubak, Peter; Kristensen, Steen D; Hvas, Anne-Mette

2017-06-01

Immature platelets may contain more platelet enzymes such as cyclooxygenase (COX)-1 and COX-2 than mature platelets. Patients with immune thrombocytopenia (ITP) have a higher fraction of immature platelets and can therefore be utilized as a biological model for investigating COX-1 and COX-2 platelet expression. The aims were to develop flow cytometric assays for platelet COX-1 and COX-2 and to investigate the COX-1 and COX-2 platelet expression, platelet turnover, and platelet glycoproteins in ITP patients (n = 10) compared with healthy individuals (n = 30). Platelet count and platelet turnover parameters (mean platelet volume (MPV), immature platelet fraction (IPF), and immature platelet count (IPC)) were measured by flow cytometry (Sysmex XE-5000). Platelet COX-1, COX-2, and the glycoproteins (GP)IIb, IX, Ib, Ia, and IIIa were all analyzed by flow cytometry (Navios) and expressed as median fluorescence intensity. COX analyses were performed in both whole blood and platelet rich plasma (PRP), whereas platelet glycoproteins were analyzed in whole blood only. ITP patients had significantly lower platelet count (55 × 10 9 /L) than healthy individuals (240 × 10 9 /L, p < 0.01), but a higher MPV (p = 0.03) and IPF (p < 0.01). IPC was similar for the two groups (p = 0.74). PRP had significantly lower MPV (p < 0.01) and significantly higher platelet count and IPC (both p-values <0.03) when compared with whole blood. IPF was similar for PRP and whole blood (p = 0.18). COX-1 expression was 10 times higher and COX-2 expression was 50% higher in PRP than in whole blood (p COX-1 < 0.01, p COX-2 < 0.01). Platelet COX-1 expression was higher in ITP patients than healthy individuals using whole blood (p COX-1 < 0.01) and PRP, though this was nonsignificant in PRP (p COX-1 = 0.17). In ITP patients, positive correlations were found between platelet turnover and COX-1 expression (all p-values <0.01, rho = 0.80-0.94), whereas healthy individuals showed significant though weaker correlations between platelet turnover and COX-1 and COX-2 expressions (all p-values <0.03, rho = 0.44-0.71). GPIIb, IX, and Ib expression was increased in ITP patients compared with healthy individuals (all p-values < 0.03). GPIIb, IX, Ib, and IIIa showed positive correlations with platelet turnover in ITP patients (all p-values <0.02, rho = 0.71-0.94), but weak and nonsignificant correlations in healthy individuals (all p-values >0.14, rho = 0.11-0.28). In conclusion, ITP patients expressed higher COX-1 and platelet glycoprotein levels than healthy individuals. COX-1 and platelet glycoproteins demonstrated positive correlations with platelet turnover in ITP patients. In healthy individuals, COX-1 and COX-2 expression correlated positively with platelet turnover. PRP was more sensitive compared with whole blood as regards determination of COX. Therefore, PRP is the recommended matrix for investigating COX-1 and COX-2 in platelets.

Deletion of Crry and DAF on murine platelets stimulates thrombopoiesis and increases factor H-dependent resistance of peripheral platelets to complement attack.

PubMed

Barata, Lidia; Miwa, Takashi; Sato, Sayaka; Kim, David; Mohammed, Imran; Song, Wen-Chao

2013-03-15

Complement receptor 1-related gene/protein y (Crry) and decay-accelerating factor (DAF) are two murine membrane C3 complement regulators with overlapping functions. Crry deletion is embryonically lethal whereas DAF-deficient mice are generally healthy. Crry(-/-)DAF(-/-) mice were viable on a C3(-/-) background, but platelets from such mice were rapidly destroyed when transfused into C3-sufficient mice. In this study, we used the cre-lox system to delete platelet Crry in DAF(-/-) mice and studied Crry/DAF-deficient platelet development in vivo. Rather than displaying thrombocytopenia, Pf4-Cre(+)-Crry(flox/flox) mice had normal platelet counts and their peripheral platelets were resistant to complement attack. However, chimera mice generated with Pf4-Cre(+)-Crry(flox/flox) bone marrows showed platelets from C3(-/-) but not C3(+/+) recipients to be sensitive to complement activation, suggesting that circulating platelets in Pf4-Cre(+)-Crry(flox/flox) mice were naturally selected in a complement-sufficient environment. Notably, Pf4-Cre(+)-Crry(flox/flox) mouse platelets became complement susceptible when factor H function was blocked. Examination of Pf4-Cre(+)-Crry(flox/flox) mouse bone marrows revealed exceedingly active thrombopoiesis. Thus, under in vivo conditions, Crry/DAF deficiency on platelets led to abnormal platelet turnover, but peripheral platelet count was compensated for by increased thrombopoiesis. Selective survival of Crry/DAF-deficient platelets aided by factor H protection and compensatory thrombopoiesis demonstrates the cooperation between membrane and fluid phase complement inhibitors and the body's ability to adaptively respond to complement regulator deficiencies.

Heterogeneity of antibody response to human platelet transfusion.

PubMed Central

Wu, K K; Thompson, J S; Koepke, J A; Hoak, J C; Flink, R

1976-01-01

To study the antibody response to human platelet transfusions, nine thrombocytopenia patients with bone marrow failure were given 6 U (3X10(11)) of random platelet concentrates twice a week. Before transfusion, none of the patients had preexisting antibodies detectable with lymphocytotoxicity, platelet aggregation, or capillary leukoagglutination techniques. After receiving 18-78 U of platelets, they became refractory to further transfusions of random platelets and alloantibodies were detectable. Two patterns of antibody response could be identified. In three patients, the sera were not lymphocytotoxic with a panel of standard cells in which all the known HLA antigens in the first and second series were represented at least once. Yet, they caused platelet aggregation with 30, 24, and 60%, respectively, of a donor population studied. The aggregating activities were inhibited by antihuman IgG but not by antihuman IgA or antihuman IgM antiserum. The aggregating antibodies could be absorbed out with donor platelets but not lymphocytes or granulocytes. Antibodies from two of these patients aggregated platelets of their respective siblings matched for both HLA haplotypes. Transfusion of platelets from these two siblings did not increase the platelet count while platelets obtained from aggregation-negative donors did. The sera from the remaining six patients were lymphocytotoxic with 15-100% of the panel of standard cells. They also had aggregating antibodies, which could be absorbed out by both platelets and lymphocytes, suggesting that they were HLA antibodies. These data suggest that the development of platelet-specific antibodies may play an important role in the immunological rejection of isologous platelets, and should be considered in the selection of donors for patients who are refractory to platelets from random donors. PMID:956376

Comparative cardiopulmonary toxicity of exhausts from soy-based biofuels and diesel in healthy and hypertensive rats

PubMed Central

Bass, Virginia L.; Schladweiler, Mette C.; Nyska, Abraham; Thomas, Ronald F.; Miller, Desinia B.; Krantz, Todd; King, Charly; Gilmour, M. Ian; Ledbetter, Allen D.; Richards, Judy E.; Kodavanti, Urmila P.

2016-01-01

Increased use of renewable energy sources raise concerns about health effects of new emissions. We analyzed relative cardiopulmonary health effects of exhausts from (1) 100% soy biofuel (B100), (2) 20% soy biofuel + 80% low sulfur petroleum diesel (B20), and (3) 100% petroleum diesel (B0) in rats. Normotensive Wistar–Kyoto (WKY) and spontaneously hypertensive rats were exposed to these three exhausts at 0, 50, 150 and 500 μg/m3, 4 h/day for 2 days or 4 weeks (5 days/week). In addition, WKY rats were exposed for 1 day and responses were analyzed 0 h, 1 day or 4 days later for time-course assessment. Hematological parameters, in vitro platelet aggregation, bronchoalveolar lavage fluid (BALF) markers of pulmonary injury and inflammation, ex vivo aortic ring constriction, heart and aorta mRNA markers of vasoconstriction, thrombosis and atherogenesis were analyzed. The presence of pigmented macrophages in the lung alveoli was clearly evident with all three exhausts without apparent pathology. Overall, exposure to all three exhausts produced only modest effects in most endpoints analyzed in both strains. BALF γ-glutamyl transferase (GGT) activity was the most consistent marker and was increased in both strains, primarily with B0 (B0>B100>B20). This increase was associated with only modest increases in BALF neutrophils. Small and very acute increases occurred in aorta mRNA markers of vasoconstriction and thrombosis with B100 but not B0 in WKY rats. Our comparative evaluations show modest cardiovascular and pulmonary effects at low concentrations of all exhausts: B0 causing more pulmonary injury and B100 more acute vascular effects. BALF GGT activity could serve as a sensitive biomarker of inhaled pollutants. PMID:26514782

Basic characteristics of plasma rich in growth factors (PRGF): blood cell components and biological effects.

PubMed

Nishiyama, Kazuhiko; Okudera, Toshimitsu; Watanabe, Taisuke; Isobe, Kazushige; Suzuki, Masashi; Masuki, Hideo; Okudera, Hajime; Uematsu, Kohya; Nakata, Koh; Kawase, Tomoyuki

2016-11-01

Platelet-rich plasma (PRP) is widely used in regenerative medicine because of its high concentrations of various growth factors and platelets. However, the distribution of blood cell components has not been investigated in either PRP or other PRP derivatives. In this study, we focused on plasma rich in growth factors (PRGF), a PRP derivative, and analyzed the distributions of platelets and white blood cells (WBCs). Peripheral blood samples were collected from healthy volunteers ( N  = 14) and centrifuged to prepare PRGF and PRP. Blood cells were counted using an automated hematology analyzer. The effects of PRP and PRGF preparations on cell proliferation were determined using human periosteal cells. In the PRGF preparations, both red blood cells and WBCs were almost completely eliminated, and platelets were concentrated by 2.84-fold, whereas in the PRP preparations, both platelets and WBCs were similarly concentrated by 8.79- and 5.51-fold, respectively. Platelet counts in the PRGF preparations were positively correlated with platelet counts in the whole blood samples, while the platelet concentration rate was negatively correlated with red blood cell counts in the whole blood samples. In contrast, platelet counts and concentration rates in the PRP preparations were significantly influenced by WBC counts in whole blood samples. The PRP preparations, but not the PRGF preparations, significantly suppressed cell growth at higher doses in vitro. Therefore, these results suggest that PRGF preparations can clearly be distinguished from PRP preparations by both inclusion of WBCs and dose-dependent stimulation of periosteal cell proliferation in vitro.

Differential changes in platelet VEGF, Tsp, CXCL12, and CXCL4 in patients with metastatic cancer.

PubMed

Wiesner, Tina; Bugl, Stefanie; Mayer, Frank; Hartmann, Jörg T; Kopp, Hans-Georg

2010-03-01

Data from animal studies indicate that platelets play a key role in tumor dissemination and metastasis. We therefore hypothesized that metastastic cancer patients may display a specific platelet phenotype. Percentage of activated, p-selectin positive platelets as well as platelet contents (i.e., plasma and platelet count-corrected serum levels of VEGF-A, CXCL12, CXCL4, and thrombospondin-1) were analyzed in 43 patients with newly diagnosed metastatic disease prior to treatment. Tumor patients had increased platelet counts and significantly elevated percentages of activated platelets. Moreover, the platelet content of VEGF-A in cancer patients was significantly increased compared to healthy controls, while thrombospondin-1, CXCL12 and CXCL4 were significantly decreased. Our data contain several unexpected results: firstly, CXCL12 was found in minute quantities in the serum as compared with murine studies. Secondly, CXCL4, which was found by mass spectrometry to be the single massively upregulated intraplatelet chemokine in mice after tumor xenotransplantation, was decreased in tumor patient platelets. While increased contents of VEGF-A have been attributed to platelet scavenger activity, the differential decrease of specific platelet contents may be due to differential secretion or altered megakaryopoiesis in metastatic cancer patients.

Ultrastructure and growth factor content of equine platelet-rich fibrin gels.

PubMed

Textor, Jamie A; Murphy, Kaitlin C; Leach, J Kent; Tablin, Fern

2014-04-01

To compare fiber diameter, pore area, compressive stiffness, gelation properties, and selected growth factor content of platelet-rich fibrin gels (PRFGs) and conventional fibrin gels (FGs). PRFGs and conventional FGs prepared from the blood of 10 healthy horses. Autologous fibrinogen was used to form conventional FGs. The PRFGs were formed from autologous platelet-rich plasma of various platelet concentrations (100 × 10³ platelets/μL, 250 × 10³ platelets/μL, 500 × 10³ platelets/μL, and 1,000 × 10³ platelets/μL). All gels contained an identical fibrinogen concentration (20 mg/mL). Fiber diameter and pore area were evaluated with scanning electron microscopy. Maximum gelation rate was assessed with spectrophotometry, and gel stiffness was determined by measuring the compressive modulus. Gel weights were measured serially over 14 days as an index of contraction (volume loss). Platelet-derived growth factor-BB and transforming growth factor-β1 concentrations were quantified with ELISAs. Fiber diameters were significantly larger and mean pore areas were significantly smaller in PRFGs than in conventional FGs. Gel weight decreased significantly over time, differed significantly between PRFGs and conventional FGs, and was significantly correlated with platelet concentration. Platelet-derived growth factor-BB and transforming growth factor-β1 concentrations were highest in gels and releasates derived from 1,000 × 10³ platelets/μL. The inclusion of platelets in FGs altered the architecture and increased the growth factor content of the resulting scaffold. Platelets may represent a useful means of modifying these gels for applications in veterinary and human regenerative medicine.

A Study of Platelet Inhibition, Using a 'Point of Care' Platelet Function Test, following Primary Percutaneous Coronary Intervention for ST-Elevation Myocardial Infarction [PINPOINT-PPCI].

PubMed

Johnson, Thomas W; Mumford, Andrew D; Scott, Lauren J; Mundell, Stuart; Butler, Mark; Strange, Julian W; Rogers, Chris A; Reeves, Barnaby C; Baumbach, Andreas

2015-01-01

Rapid coronary recanalization following ST-elevation myocardial infarction (STEMI) requires effective anti-platelet and anti-thrombotic therapies. This study tested the impact of door to end of procedure ('door-to-end') time and baseline platelet activity on platelet inhibition within 24hours post-STEMI. 108 patients, treated with prasugrel and procedural bivalirudin, underwent Multiplate® platelet function testing at baseline, 0, 1, 2 and 24hours post-procedure. Major adverse cardiac events (MACE), bleeding and stent thrombosis (ST) were recorded. Baseline ADP activity was high (88.3U [71.8-109.0]), procedural time and consequently bivalirudin infusion duration were short (median door-to-end time 55minutes [40-70] and infusion duration 30minutes [20-42]). Baseline ADP was observed to influence all subsequent measurements of ADP activity, whereas door-to-end time only influenced ADP immediately post-procedure. High residual platelet reactivity (HRPR ADP>46.8U) was observed in 75% of patients immediately post-procedure and persisted in 24% of patients at 2hours. Five patients suffered in-hospital MACE (4.6%). Acute ST occurred in 4 patients, all were <120mins post-procedure and had HRPR. No significant bleeding was observed. In a post-hoc analysis, pre-procedural morphine use was associated with significantly higher ADP activity following intervention. Baseline platelet function, time to STEMI treatment and opiate use all significantly influence immediate post-procedural platelet activity.

Platelet-derived growth factor inhibits platelet activation in heparinized whole blood.

PubMed

Selheim, F; Holmsen, H; Vassbotn, F S

1999-08-15

We previously have demonstrated that human platelets have functionally active platelet-derived growth factor alpha-receptors. Studies with gel-filtered platelets showed that an autocrine inhibition pathway is transduced through this tyrosine kinase receptor during platelet activation. The physiological significance of this inhibitory effect of platelet-derived growth factor on gel-filtered platelets activation is, however, not known. In the present study, we investigated whether platelet-derived growth factor inhibits platelet activation under more physiological conditions in heparinized whole blood, which represents a more physiological condition than gel-filtered platelets. Using flow cytometric assays, we demonstrate here that platelet-derived growth factor inhibits thrombin-, thrombin receptor agonist peptide SFLLRN-, and collagen-induced platelet aggregation and shedding of platelet-derived microparticles from the platelet plasma membrane during platelet aggregation in stirred heparinized whole blood. The inhibitory effect of platelet-derived growth factor was dose dependent. However, under nonaggregating conditions (no stirring), we could not demonstrate any significant effect of platelet-derived growth factor on thrombin- and thrombin receptor agonist peptide-induced platelet surface expression of P-selectin. Our results demonstrate that platelet-derived growth factor appears to be a true antithrombotic agent only under aggregating conditions in heparinized whole blood.

Effects of platelet and plasma transfusion on outcome in traumatic brain injury patients with moderate bleeding diatheses.

PubMed

Anglin, Catherine O; Spence, Jeffrey S; Warner, Matthew A; Paliotta, Christopher; Harper, Caryn; Moore, Carol; Sarode, Ravi; Madden, Christopher; Diaz-Arrastia, Ramon

2013-03-01

Object Coagulopathy and thrombocytopenia are common after traumatic brain injury (TBI), yet transfusion thresholds for mildly to moderately abnormal ranges of international normalized ratio and platelet count remain controversial. This study evaluates associations between fresh frozen plasma (FFP) and platelet transfusions with long-term functional outcome and survival in TBI patients with moderate hemostatic laboratory abnormalities. Methods This study is a retrospective review of prospectively collected data of patients with mild to severe TBI. Data include patient demographics, several initial injury severity metrics, daily laboratory values, Glasgow Outcome Score- Extended (GOSE) scores, Functional Status Examination (FSE) scores, and survival to 6 months. Correlations were evaluated between these variables and transfusion of FFP, platelets, packed red blood cells (RBCs), cryoprecipitate, recombinant factor VIIa, and albumin. Ordinal regression was performed to account for potential confounding variables to further define relationships between transfusion status and long-term outcome. By analyzing collected data, mild to moderate coagulopathy was defined as an international normalized ratio 1.4-2.0, moderate thrombocytopenia as platelet count 50 × 10(9)/L to 107 × 10(9)/L, and moderate anemia as 21%-30% hematocrit. Results In patients with mild to moderate laboratory hematological abnormalities, univariate analysis shows significant correlations between poor outcome scores and FFP, platelet, or packed RBC transfusion; the volume of FFP or packed RBCs transfused also correlated with poor outcome. Several measures of initial injury and laboratory abnormalities also correlated with poor outcome. Patient age, initial Glasgow Coma Scale score, and highest recorded serum sodium were included in the ordinal regression model using backward variable selection. In the moderate coagulopathy subgroup, patients transfused with FFP were more likely to have a lower GOSE score relative to those who did not receive a transfusion (OR 5.20 [95% CI 1.72-15.73]). Patients with moderate coagulopathy who received FFP and packed RBCs were even more likely to be have a lower GOSE score (OR 7.17 [95% CI 2.12-24.12]). Moderately anemic patients who received packed RBCs alone were more likely to have a worse long-term functional outcome as determined by GOSE and FSE scores (GOSE: OR 2.41 [95% CI 1.51-3.85]; and FSE: OR 3.27 [95% CI 2.00-5.35]). No transfusion types or combinations were noted to significantly correlate with the 6-month mortality in ordinal regression. Conclusions In TBI patients with moderate coagulopathy, FFP transfusions alone or a combination of FFP and packed RBCs were associated with poorer long-term functional outcomes as measured by the GOSE. Red blood cell transfusions were associated with poor long-term functional outcome in TBI patients with moderate anemia. Platelet transfusion in patients with moderate thrombocytopenia was not significantly associated with outcome. Although transfusion is beneficial to many patients with severe hematological abnormalities, it is not without risk, and the indications for transfusion should be carefully considered in patients with moderate hematological abnormalities.

Platelets Subvert T Cell Immunity Against Cancer via GARP-TGFβ Axis

PubMed Central

Rachidi, Saleh; Metelli, Alessandra; Riesenberg, Brian; Wu, Bill X; Nelson, Michelle H; Fugle, Caroline W; Paulos, Chrystal M; Rubinstein, Mark P; Garrett-Mayer, Elizabeth; Hennig, Mirko; Bearden, Daniel W; Yang, Yi; Liu, Bei; Li, Zihai

2017-01-01

Cancer-associated thrombocytosis has long been linked to poor clinical outcome, but the underlying mechanism is enigmatic. We hypothesized that platelets promote malignancy and resistance to therapy by dampening host immunity. We herein show that genetic targeting of platelets significantly enhances adoptive T cell therapy of cancer. An unbiased biochemical and structural biology approach established transforming growth factor β (TGFβ) and lactate as the major platelet-derived soluble factors to obliterate CD4+ and CD8+ T cell functions. Moreover, we found that platelets are the dominant source of functional TGFβ systemically as well as in the tumor microenvironment through constitutive expression of TGFβ-docking receptor Glycoprotein A Repetitions Predominant (GARP) rather than secretion of TGFβ per se. Indeed, platelet-specific deletion of GARP-encoding gene Lrrc32 blunted TGFβ activity at the tumor site and potentiated protective immunity against both melanoma and colon cancer. Finally, we found that T cell therapy of cancer can be substantially improved by concurrent treatment with readily available anti-platelet agents. We conclude that platelets constrain T cell immunity though a GARP-TGFβ axis and suggest a combination of immunotherapy and platelet inhibitors as a therapeutic strategy against cancer. PMID:28763790

Platelets Cellular and Functional Characteristics in Patients with Atrial Fibrillation: A Comprehensive Meta-Analysis and Systematic Review

PubMed Central

Weymann, Alexander; Ali-Hasan-Al-Saegh, Sadeq; Sabashnikov, Anton; Popov, Aron-Frederik; Mirhosseini, Seyed Jalil; Nombela-Franco, Luis; Testa, Luca; Lotfaliani, Mohammadreza; Zeriouh, Mohamed; Liu, Tong; Dehghan, Hamidreza; Yavuz, Senol; de Oliveira Sá, Michel Pompeu Barros; Baker, William L.; Jang, Jae-Sik; Gong, Mengqi; Benedetto, Umberto; Dohmen, Pascal M.; D’Ascenzo, Fabrizio; Deshmukh, Abhishek J.; Biondi-Zoccai, Giuseppe; Calkins, Hugh; Stone, Gregg W.

2017-01-01

Background This systematic review with meta-analysis aimed to determine the strength of evidence for evaluating the association of platelet cellular and functional characteristics including platelet count (PC), MPV, platelet distribution width (PDW), platelet factor 4, beta thromboglobulin (BTG), and p-selectin with the occurrence of atrial fibrillation (AF) and consequent stroke. Material/Methods We conducted a meta-analysis of observational studies evaluating platelet characteristics in patients with paroxysmal, persistent and permanent atrial fibrillations. A comprehensive subgroup analysis was performed to explore potential sources of heterogeneity. Results Literature search of all major databases retrieved 1,676 studies. After screening, a total of 73 studies were identified. Pooled analysis showed significant differences in PC (weighted mean difference (WMD)=−26.93 and p<0.001), MPV (WMD=0.61 and p<0.001), PDW (WMD=−0.22 and p=0.002), BTG (WMD=24.69 and p<0.001), PF4 (WMD=4.59 and p<0.001), and p-selectin (WMD=4.90 and p<0.001). Conclusions Platelets play a critical and precipitating role in the occurrence of AF. Whereas distribution width of platelets as well as factors of platelet activity was significantly greater in AF patients compared to SR patients, platelet count was significantly lower in AF patients. PMID:28302997

Functional display of platelet-binding VWF fragments on filamentous bacteriophage.

PubMed

Yee, Andrew; Tan, Fen-Lai; Ginsburg, David

2013-01-01

von Willebrand factor (VWF) tethers platelets to sites of vascular injury via interaction with the platelet surface receptor, GPIb. To further define the VWF sequences required for VWF-platelet interaction, a phage library displaying random VWF protein fragments was screened against formalin-fixed platelets. After 3 rounds of affinity selection, DNA sequencing of platelet-bound clones identified VWF peptides mapping exclusively to the A1 domain. Aligning these sequences defined a minimal, overlapping segment spanning P1254-A1461, which encompasses the C1272-C1458 cystine loop. Analysis of phage carrying a mutated A1 segment (C1272/1458A) confirmed the requirement of the cystine loop for optimal binding. Four rounds of affinity maturation of a randomly mutagenized A1 phage library identified 10 and 14 unique mutants associated with enhanced platelet binding in the presence and absence of botrocetin, respectively, with 2 mutants (S1370G and I1372V) common to both conditions. These results demonstrate the utility of filamentous phage for studying VWF protein structure-function and identify a minimal, contiguous peptide that bind to formalin-fixed platelets, confirming the importance of the VWF A1 domain with no evidence for another independently platelet-binding segment within VWF. These findings also point to key structural elements within the A1 domain that regulate VWF-platelet adhesion.

Magnetic and Contrast Properties of Labeled Platelets for Magnetomotive Optical Coherence Tomography

PubMed Central

Oldenburg, Amy L.; Gallippi, Caterina M.; Tsui, Frank; Nichols, Timothy C.; Beicker, Kellie N.; Chhetri, Raghav K.; Spivak, Dmitry; Richardson, Aaron; Fischer, Thomas H.

2010-01-01

This article introduces a new functional imaging paradigm that uses optical coherence tomography (OCT) to detect rehydrated, lyophilized platelets (RL platelets) that are in the preclinical trial stage and contain superparamagnetic iron oxides (SPIOs) approved by the U.S. Food and Drug Administration. Platelets are highly functional blood cells that detect and adhere to sites of vascular endothelial damage by forming primary hemostatic plugs. By applying magnetic gradient forces, induced nanoscale displacements (magnetomotion) of the SPIO-RL platelets are detected as optical phase shifts in OCT. In this article, we characterize the iron content and magnetic properties of SPIO-RL platelets, construct a model to predict their magnetomotion in a tissue medium, and demonstrate OCT imaging in tissue phantoms and ex vivo pig arteries. Tissue phantoms containing SPIO-RL platelets exhibited >3 dB contrast/noise ratio at ≥1.5 × 109 platelets/cm3. OCT imaging was performed on ex vivo porcine arteries after infusion of SPIO-RL platelets, and specific contrast was obtained on an artery that was surface-damaged (P < 10−6). This may enable new technologies for in vivo monitoring of the adherence of SPIO-RL platelets to sites of bleeding and vascular damage, which is broadly applicable for assessing trauma and cardiovascular diseases. PMID:20923673

Lactodifucotetraose, a human milk oligosaccharide, attenuates platelet function and inflammatory cytokine release.

PubMed

Newburg, David S; Tanritanir, Ayse C; Chakrabarti, Subrata

2016-07-01

Human milk strongly quenches inflammatory processes in vitro, and breastfed infants have lower incidence of inflammatory diseases than those fed artificially. Platelets from neonates, in contrast to those from adults, are less responsive to platelet agonists such as collagen, thrombin, ADP, and epinephrine. Breastfed infants absorb oligosaccharides intact from the human milk in their gut to the circulation. This study was to determine whether these oligosaccharides can attenuate platelet function and platelet secretion of pro-inflammatory proteins, and to identify the active component. The natural mixture of oligosaccharides from human milk and pure individual human milk oligosaccharides were tested for their ability to modulate responses of platelets isolated from human blood following exposure to thrombin, ADP, and collagen. Human milk and the natural mixture of human milk oligosaccharides inhibited platelet release of inflammatory proteins. Of the purified human milk oligosaccharides tested, only lactodifucotetraose (LDFT) significantly inhibited thrombin induced release of the pro-inflammatory proteins RANTES and sCD40L. LDFT also inhibited platelet adhesion to a collagen-coated surface, as well as platelet aggregation induced by ADP or collagen. These data indicate that LDFT may help modulate hemostasis by suppressing platelet-induced inflammatory processes in breastfed infants. This activity suggests further study of LDFT for its potential as a therapeutic agent in infants and adults.

Gap junctions and connexin hemichannels in the regulation of haemostasis and thrombosis.

PubMed

Vaiyapuri, Sakthivel; Flora, Gagan D; Gibbins, Jonathan M

2015-06-01

Platelets are involved in the maintenance of haemostasis but their inappropriate activation leads to thrombosis, a principal trigger for heart attack and ischaemic stroke. Although platelets circulate in isolation, upon activation they accumulate or aggregate together to form a thrombus, where they function in a co-ordinated manner to prevent loss of blood and control wound repair. Previous report (1) indicates that the stability and functions of a thrombus are maintained through sustained, contact-dependent signalling between platelets. Given the role of gap junctions in the co-ordination of tissue responses, it was hypothesized that gap junctions may be present within a thrombus and mediate intercellular communication between platelets. Therefore studies were performed to explore the presence and functions of connexins in platelets. In this brief review, the roles of hemichannels and gap junctions in the control of thrombosis and haemostasis and the future directions for this research will be discussed.

Essential role of protein kinase C delta in platelet signaling, alpha IIb beta 3 activation, and thromboxane A2 release.

PubMed

Yacoub, Daniel; Théorêt, Jean-François; Villeneuve, Louis; Abou-Saleh, Haissam; Mourad, Walid; Allen, Bruce G; Merhi, Yahye

2006-10-06

The protein kinase C (PKC) family is an essential signaling mediator in platelet activation and aggregation. However, the relative importance of the major platelet PKC isoforms and their downstream effectors in platelet signaling and function remain unclear. Using isolated human platelets, we report that PKCdelta, but not PKCalpha or PKCbeta, is required for collagen-induced phospholipase C-dependent signaling, activation of alpha(IIb)beta(3), and platelet aggregation. Analysis of PKCdelta phosphorylation and translocation to the membrane following activation by both collagen and thrombin indicates that it is positively regulated by alpha(IIb)beta(3) outside-in signaling. Moreover, PKCdelta triggers activation of the mitogen-activated protein kinase-kinase (MEK)/extracellular-signal regulated kinase (ERK) and the p38 MAPK signaling. This leads to the subsequent release of thromboxane A(2), which is essential for collagen-induced but not thrombin-induced platelet activation and aggregation. This study adds new insight to the role of PKCs in platelet function, where PKCdelta signaling, via the MEK/ERK and p38 MAPK pathways, is required for the secretion of thromboxane A(2).

The effectiveness of low-dose desmopressin in improving hypothermia-induced impairment of primary haemostasis under influence of aspirin - a randomized controlled trial.

PubMed

Tsui, Pui Yee; Cheung, Chi Wai; Lee, Yvonne; Leung, Susan Wai Sum; Ng, Kwok Fu Jacobus

2015-05-28

Mild hypothermia (34-35 °C) increases perioperative blood loss. Our previous studies showed that desmopressin could have in vitro beneficial effects on hypothermia-induced primary haemostasis impairment. In this study, we investigate the in vitro effects of desmopressin on hypothermia-induced primary haemostasis impairment under the influence of aspirin in healthy volunteers. Sixty healthy volunteers were randomly allocated to taking aspirin 100 mg or placebo for three days. On the sixth day blood samples were taken before and after the injection of desmopressin (1.5 microgram or 5 microgram) or normal saline subcutaneously. Measurements including Platelet Function Analyzer (PFA-100®) closure times, plasma von Willebrand Factor antigen, haemoglobin and platelet levels were made at 32 °C and 37 °C respectively. Collagen/epinephrine closure time (EPICT) was significantly prolonged by 21.13 % (95 %CI 2.34-39.74 %, p = 0.021) in aspirin group at 37 °C. While hypothermia alone prolonged both collagen/adenosine diphosphate (ADPCT) and EPICT by 17.63 % (95 %CI 13.5-20.85 %, p < 0.001) and 8.0 % (95 %CI 6.38-10.04 %, p = 0.024) respectively, addition of aspirin only further prolonged EPICT by 19.9 % (95 %CI 3.32-36.49 %, p = 0.013). In aspirin group, desmopressin 1.5 microgram and 5 microgram significantly reduced ADPCT to below baseline levels at 37 °C (p = 0.025 and <0.001 respectively), whereas reduction in EPICT was seen with desmopressin 5 microgram (p =0.008). The effect was less pronounced at 32 °C, with a significant reduction in EPICT obtained with a dosage of 5 microgram only (p = 0.011). It was shown that aspirin could further potentiate the hypothermia-induced closure time prolongations. Low dose desmopressin (1.5 microgram) reduced PFA-100® closure times towards baseline. A higher dosage (5 microgram) further reduced the closure times below baseline. Therefore low dose desmopressin (1.5 microgram) might have the potential to correct hypothermia-induced primary haemostasis impairment under the influence of aspirin during the perioperative period. ClinicalTrials.gov: NCT01382134.

Use of 8-methoxypsoralen and long-wavelength ultraviolet radiation for decontamination of platelet concentrates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, L.; Wiesehahn, G.P.; Morel, P.A.

1989-07-01

Transmission of viral diseases through blood products remains an unsolved problem in transfusion medicine. We have developed a psoralen photochemical system for decontamination of platelet concentrates in which platelets are treated with long wavelength ultraviolet radiation (UVA, 320-400 nm) in the presence of 8-methoxypsoralen (8-MOP). Bacteria, RNA viruses, and DNA viruses ranging in genome size from 1.2 x 10(6) daltons, encompassing the size range of human pathogens, were inoculated into platelet concentrates and subjected to treatment. This system inactivated 25 to 30 logs/h of bacteria Escherichia coli or Staphylococcus aureus, 6 logs/h of bacteriophage fd, 0.9 log/h of bacteriophage R17more » and 1.1 logs/h of feline leukemia virus (FeLV) in platelet concentrates maintained in standard storage bags. Platelet integrity and in vitro function before, immediately following photochemical treatment, and during prolonged storage after treatment, were evaluated by measuring: (1) extracellular pH; (2) platelet yields; (3) extracellular lactate dehydrogenase (LDH) levels; (4) platelet morphology; (5) platelet aggregation responsiveness; (6) thromboxane beta-2 (TXB-2) production; (7) dense body secretion; and (8) alpha granule secretion. These assays demonstrated that this photochemical inactivation system inactivated bacteria and viruses in platelet concentrates with minimal adverse effects on the in vitro function of platelets in comparison to untreated control concentrates maintained under current, standard blood bank conditions.« less

Early outgrowth cells versus endothelial colony forming cells functions in platelet aggregation.

PubMed

Bou Khzam, Lara; Bouchereau, Olivier; Boulahya, Rahma; Hachem, Ahmed; Zaid, Younes; Abou-Saleh, Haissam; Merhi, Yahye

2015-11-09

Endothelial progenitor cells (EPCs) have been implicated in neoangiogenesis, endothelial repair and cell-based therapies for cardiovascular diseases. We have previously shown that the recruitment of EPCs to sites of vascular lesions is facilitated by platelets where EPCs, in turn, modulate platelet function and thrombosis. However, EPCs encompass a heterogeneous population of progenitor cells that may exert different effects on platelet function. Recent evidence suggests the existence of two EPC subtypes: early outgrowth cells (EOCs) and endothelial colony-forming cells (ECFCs). We aimed at characterizing these two EPC subtypes and at identifying their role in platelet aggregation. EOCs and ECFCs were generated from human peripheral blood mononuclear cells (PBMCs) seeded in conditioned media on fibronectin and collagen, respectively. The morphological, phenotypical and functional characteristics of EOCs and ECFCs were assessed by optical and confocal laser scanning microscopes, cell surface markers expression, and Matrigel tube formation. The impact of EOCs and ECFCs on platelet aggregation was monitored in collagen-induced optical aggregometry and compared with PBMCs and human umbilical vein endothelial cells (HUVECs). The levels of the anti-platelet agents' nitric oxide (NO) and prostacyclin (PGI2) released from cultured cells as well as the expression of their respective producing enzymes NO synthases (NOS) and cyclooxygenases (COX) were also assessed. We showed that EOCs display a monocytic-like phenotype whereas ECFCs have an endothelial-like phenotype. We demonstrated that both EOCs and ECFCs and their supernatants inhibited platelet aggregation; however ECFCs were more efficient than EOCs. This could be related to the release of significantly higher amounts of NO and PGI2 from ECFCs, in comparison to EOCs. Indeed, ECFCs, like HUVECs, constitutively express the endothelial (eNOS)-and inducible (iNOS)-NOS isoforms, and COX-1 and weakly express COX-2, whereas EOCs do not constitutively express these NO and PGI2 producing enzymes. The different morphological, phenotypic and more importantly the release of the anti-aggregating agents PGI2 and NO in each EPC subtype are implicated in their respective roles in platelet function and thus, may be linked to the increased efficiency of ECFCs in inhibiting platelet aggregation as compared to EOCs.

Review of in vivo studies of dimethyl sulfoxide cryopreserved platelets.

PubMed

Slichter, Sherrill J; Jones, Melinh; Ransom, Janet; Gettinger, Irena; Jones, Mary Kay; Christoffel, Todd; Pellham, Esther; Bailey, S Lawrence; Corson, Jill; Bolgiano, Doug

2014-10-01

A literature review was conducted to assess the efficacy and safety of dimethyl sulfoxide (DMSO) cryopreserved platelets for potential military use. In vivo DMSO cryopreserved platelet studies published between 1972 and June of 2013 were reviewed. Assessed were the methods of cryopreservation, posttransfusion platelet responses, prevention or control of bleeding, and adverse events. Using the Department of Defense's preferred 6% DMSO cryopreservation method with centrifugation to remove the DMSO plasma before freezing at -65°C and no postthaw wash, mean radiolabeled platelet recoveries in 32 normal subjects were 33% ± 10% (52% ± 12% of the same subject's fresh platelet recoveries), and survivals were 7.5 ± 1.2 days (89% ± 15% of fresh platelet survivals). Using a variety of methods to freeze autologous platelets from 178 normal subjects, mean radiolabeled platelet recoveries were consistently 39% ± 9%, and survivals, 7.4 ± 1.4 days. More than 3000 cryopreserved platelet transfusions were given to 1334 patients. There were 19 hematology/oncology patient studies, and, in 9, mean 1-hour corrected count increments were 11 100 ± 3600 (range, 5700-15 800) after cryopreserved autologous platelet transfusions. In 5 studies, bleeding times improved after transfusion; in 3, there was either no improvement or a variable response. In 4 studies, there was immediate cessation of bleeding after transfusion; in 3 studies, patients being supported only with cryopreserved platelets had no bleeding. In 1 cardiopulmonary bypass study, cryopreserved platelets resulted in significantly less bleeding vs standard platelets. In 3 trauma studies, cryopreserved platelets were hemostatically effective. No significant adverse events were reported in any study. In summary, cryopreserved platelets have platelet recoveries that are about half of fresh platelets, but survivals are only minimally reduced. The platelets appear hemostatically effective and have no significant adverse events. Copyright © 2014 Elsevier Inc. All rights reserved.

Efficacy of Intra-articular Platelet-Rich Plasma Injections in Knee Osteoarthritis: A Systematic Review.

PubMed

Meheux, Carlos J; McCulloch, Patrick C; Lintner, David M; Varner, Kevin E; Harris, Joshua D

2016-03-01

To determine (1) whether platelet-rich plasma (PRP) injection significantly improves validated patient-reported outcomes in patients with symptomatic knee osteoarthritis (OA) at 6 and 12 months postinjection, (2) differences in outcomes between PRP and corticosteroid injections or viscosupplementation or placebo injections at 6 and 12 months postinjection, and (3) similarities and differences in outcomes based on the PRP formulations used in the analyzed studies. PubMed, Cochrane Central Register of Controlled Trials, SCOPUS, and Sport Discus were searched for English-language, level I evidence, human in vivo studies on the treatment of symptomatic knee OA with intra-articular PRP compared with other options, with a minimum of 6 months of follow-up. A quality assessment of all articles was performed using the Modified Coleman Methodology Score (average, 83.3/100), and outcomes were analyzed using 2-proportion z-tests. Six articles (739 patients, 817 knees, 39% males, mean age of 59.9 years, with 38 weeks average follow-up) were analyzed. All studies met minimal clinical important difference criteria and showed significant improvements in statistical and clinical outcomes, including pain, physical function, and stiffness, with PRP. All but one study showed significant differences in clinical outcomes between PRP and hyaluronic acid (HA) or PRP and placebo in pain and function. Average pretreatment Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores were 52.36 and 52.05 for the PRP and HA groups, respectively (P = .420). Mean post-treatment WOMAC scores for PRP were significantly better than for HA at 3 to 6 months (28.5 and 43.4, respectively; P = .0008) and at 6 to 12 months (22.8 and 38.1, respectively; P = .0062). None of the included studies used corticosteroids. In patients with symptomatic knee OA, PRP injection results in significant clinical improvements up to 12 months postinjection. Clinical outcomes and WOMAC scores are significantly better after PRP versus HA at 3 to 12 months postinjection. There is limited evidence for comparing leukocyte-rich versus leukocyte-poor PRP or PRP versus steroids in this study. Level I, systematic review of Level I studies. Copyright © 2016 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

Human Cancer and Platelet Interaction, a Potential Therapeutic Target.

PubMed

Wang, Shike; Li, Zhenyu; Xu, Ren

2018-04-20

Cancer patients experience a four-fold increase in thrombosis risk, indicating that cancer development and progression are associated with platelet activation. Xenograft experiments and transgenic mouse models further demonstrate that platelet activation and platelet-cancer cell interaction are crucial for cancer metastasis. Direct or indirect interaction of platelets induces cancer cell plasticity and enhances survival and extravasation of circulating cancer cells during dissemination. In vivo and in vitro experiments also demonstrate that cancer cells induce platelet aggregation, suggesting that platelet-cancer interaction is bidirectional. Therefore, understanding how platelets crosstalk with cancer cells may identify potential strategies to inhibit cancer metastasis and to reduce cancer-related thrombosis. Here, we discuss the potential function of platelets in regulating cancer progression and summarize the factors and signaling pathways that mediate the cancer cell-platelet interaction.

Decrease of oxidative phosphorylation system function in severe septic patients.

PubMed

Lorente, Leonardo; Martín, María M; López-Gallardo, Ester; Blanquer, José; Solé-Violán, Jordi; Labarta, Lorenzo; Díaz, César; Jiménez, Alejandro; Montoya, Julio; Ruiz-Pesini, Eduardo

2015-10-01

The comparison of oxidative phosphorylation system capacities between septic patients and control subjects has been scarcely analyzed and only in studies with small sample size (fewer than 40 septic patients and 40 controls). Thus, the objective of this study was to compare platelet respiratory complex IV (CIV) activity between severe septic patients and healthy individuals in a larger series (including 198 severe septic patients and 96 healthy controls). A prospective, multicenter, observational study was carried out in 6 Spanish intensive care units. We obtained blood samples from 198 severe septic patients at day 1, 4, and 8 of the severe sepsis diagnosis and 96 sex- and age-matched healthy control individuals and determined platelet CIV-specific activity. The end point of the study was 30-day mortality. Control individuals showed higher platelet CIV-specific activity (P < .001) than surviving (n = 130) or nonsurviving (n = 68) severe septic patients at day 1, 4, and 8 of severe sepsis diagnosis. The major finding of our work, involving the largest series to date of severe septic patients with data on oxidative phosphorylation system capacity, was that surviving and nonsurviving septic patients showed lower platelet CIV-specific activity during the first week of sepsis than healthy controls. Copyright © 2015 Elsevier Inc. All rights reserved.

Platelet-Released Growth Factors Modulate the Secretion of Cytokines in Synoviocytes under Inflammatory Joint Disease

PubMed Central

Rasuo, Biljana; Hock, Jennifer Vanessa Phi; Kweider, Nisreen; Fragoulis, Athanassios; Sönmez, Tolga Taha; Jahr, Holger; Pufe, Thomas; Lippross, Sebastian

2017-01-01

The etiology and pathogenesis of rheumatoid arthritis (RA) are marked by a complex interplay of various cell populations and is mediated by different signaling pathways. Traditionally, therapies have primarily focused on pain relief, reducing inflammation and the recovery of joint function. More recently, however, researchers have discussed the therapeutic efficacy of autologous platelet-rich plasma (PRP). The main objective of this work is to examine the influences of platelet-released growth factor (PRGF) on human synoviocytes under inflammatory conditions. Additionally, it is checked to which extend treatment with platelet concentrate influences the release of cytokines form synoviocytes. For this purpose, an in vitro RA model was created by stimulating the cells with the TNF-α. The release of cytokines was measured by ELISA. The cytokine gene expression was analyzed by real-time PCR. It has been observed that the stimulation concentration of 10 ng/ml TNF-α resulted in a significantly increased endogenous secretion and gene expression of IL-6 and TNF-α. The anti-inflammatory effect of PRGF could be confirmed through significant reduction of TNF-α and IL-1β. An induced inflammatory condition seems to cause PRGF to inhibit the release of proinflammatory cytokines. Further study is required to understand the exact effect mechanism of PRGF on synoviocytes. PMID:29348703

Platelet Storage Lesions: What More Do We Know Now?

PubMed

Ng, Monica Suet Ying; Tung, John-Paul; Fraser, John Francis

2018-04-17

Platelet concentrate (PC) transfusions are a lifesaving adjunct to control and prevent bleeding in cancer, hematologic, surgical, and trauma patients. Platelet concentrate availability and safety are limited by the development of platelet storage lesions (PSLs) and risk of bacterial contamination. Platelet storage lesions are a series of biochemical, structural, and functional changes that occur from blood collection to transfusion. Understanding of PSLs is key for devising interventions that prolong PC shelf life to improve PC access and wastage. This article will review advancements in clinical and mechanistic PSL research. In brief, exposure to artificial surfaces and high centrifugation forces during PC preparation initiate PSLs by causing platelet activation, fragmentation, and biochemical release. During room temperature storage, enhanced glycolysis and reduced mitochondrial function lead to glucose depletion, lactate accumulation, and product acidification. Impaired adenosine triphosphate generation reduces platelet capacity to perform energetically demanding processes such as hypotonic stress responses and activation/aggregation. Storage-induced alterations in platelet surface proteins such as thrombin receptors and glycoproteins decrease platelet aggregation. During storage, there is an accumulation of immunoactive proteins such as leukocyte-derive cytokines (tumor necrosis factor α, interleukin (IL) 1α, IL-6, IL-8) and soluble CD40 ligand which can participate in transfusion-related acute lung injury and nonhemolytic transfusion reactions. Storage-induced microparticles have been linked to enhanced platelet aggregation and immune system modulation. Clinically, stored PCs have been correlated with reduced corrected count increment, posttransfusion platelet recovery, and survival across multiple meta-analyses. Fresh PC transfusions have been associated with superior platelet function in vivo; however, these differences were abrogated after a period of circulation. There is currently insufficient evidence to discern the effect of PSLs on transfusion safety. Various bag and storage media changes have been proposed to reduce glycolysis and platelet activation during room temperature storage. Moreover, cryopreservation and cold storage have been proposed as potential methods to prolong PC shelf life by reducing platelet metabolism and bacterial proliferation. However, further work is required to elucidate and manage the PSLs specific to these storage protocols before its implementation in blood banks. Copyright © 2018 Elsevier Inc. All rights reserved.

Studies of the Effects of Perfluorocarbon Emulsions on Platelet Number and Function in Models of Critical Battlefield Injury

DTIC Science & Technology

2014-01-01

br in og en Time Fibrinogen Assay after Intravenous  Perfluorocarbon  Infusion Oxygent Hespan Control 3. Fibrinogen measurement ...1 Award Number: W81XWH-13-1-0017 TITLE: Studies of the Effects of Perfluorocarbon Emulsions on Platelet Number and Function...of Perfluorocarbon Emulsions on Platelet 5b. GRANT NUMBER W81XWH-13-1-0017 Number and Function in Models of Critical Battlefield Injury 5c

Accurate measurement of volume and shape of resting and activated blood platelets from light scattering.

PubMed

Moskalensky, Alexander E; Yurkin, Maxim A; Konokhova, Anastasiya I; Strokotov, Dmitry I; Nekrasov, Vyacheslav M; Chernyshev, Andrei V; Tsvetovskaya, Galina A; Chikova, Elena D; Maltsev, Valeri P

2013-01-01

We introduce a novel approach for determination of volume and shape of individual blood platelets modeled as an oblate spheroid from angle-resolved light scattering with flow-cytometric technique. The light-scattering profiles (LSPs) of individual platelets were measured with the scanning flow cytometer and the platelet characteristics were determined from the solution of the inverse light-scattering problem using the precomputed database of theoretical LSPs. We revealed a phenomenon of parameter compensation, which is partly explained in the framework of anomalous diffraction approximation. To overcome this problem, additional a priori information on the platelet refractive index was used. It allowed us to determine the size of each platelet with subdiffraction precision and independent of the particular value of the platelet aspect ratio. The shape (spheroidal aspect ratio) distributions of platelets showed substantial differences between native and activated by 10 μM adenosine diphosphate samples. We expect that the new approach may find use in hematological analyzers for accurate measurement of platelet volume distribution and for determination of the platelet activation efficiency.

New Sesquiterpenoids and Anti-Platelet Aggregation Constituents from the Rhizomes of Curcuma zedoaria.

PubMed

Chen, Jih-Jung; Tsai, Tung-Han; Liao, Hsiang-Ruei; Chen, Li-Chai; Kuo, Yueh-Hsiung; Sung, Ping-Jyun; Chen, Chun-Lin; Wei, Chun-Sheng

2016-10-17

Two new sesquiterpenoids-13-hydroxycurzerenone ( 1 ) and 1-oxocurzerenone ( 2 )-have been isolated from the rhizomes of Curcuma zedoaria , together with 13 known compounds ( 3 - 15 ). The structures of two new compounds were determined through spectroscopic and MS analyses. Among the isolated compounds, 13-hydroxycurzerenone ( 1 ), 1-oxocurzerenone ( 2 ), curzerenone ( 3 ), germacrone ( 4 ), curcolone ( 5 ), procurcumenol ( 6 ), ermanin ( 7 ), curcumin ( 8 ), and a mixture of stigmast-4-en-3,6-dione ( 12 ) and stigmasta-4,22-dien-3,6-dione ( 13 ) exhibited inhibition (with inhibition % in the range of 21.28%-67.58%) against collagen-induced platelet aggregation at 100 μM. Compounds 1 , 5 , 7 , 8 , and the mixture of 12 and 13 inhibited arachidonic acid (AA)-induced platelet aggregation at 100 μM with inhibition % in the range of 23.44%-95.36%.

Three-dimensional multi-scale model of deformable platelets adhesion to vessel wall in blood flow

PubMed Central

Wu, Ziheng; Xu, Zhiliang; Kim, Oleg; Alber, Mark

2014-01-01

When a blood vessel ruptures or gets inflamed, the human body responds by rapidly forming a clot to restrict the loss of blood. Platelets aggregation at the injury site of the blood vessel occurring via platelet–platelet adhesion, tethering and rolling on the injured endothelium is a critical initial step in blood clot formation. A novel three-dimensional multi-scale model is introduced and used in this paper to simulate receptor-mediated adhesion of deformable platelets at the site of vascular injury under different shear rates of blood flow. The novelty of the model is based on a new approach of coupling submodels at three biological scales crucial for the early clot formation: novel hybrid cell membrane submodel to represent physiological elastic properties of a platelet, stochastic receptor–ligand binding submodel to describe cell adhesion kinetics and lattice Boltzmann submodel for simulating blood flow. The model implementation on the GPU cluster significantly improved simulation performance. Predictive model simulations revealed that platelet deformation, interactions between platelets in the vicinity of the vessel wall as well as the number of functional GPIbα platelet receptors played significant roles in platelet adhesion to the injury site. Variation of the number of functional GPIbα platelet receptors as well as changes of platelet stiffness can represent effects of specific drugs reducing or enhancing platelet activity. Therefore, predictive simulations can improve the search for new drug targets and help to make treatment of thrombosis patient-specific. PMID:24982253

Anti-thrombotic and anti-inflammatory activities of protopine.

PubMed

Saeed, S A; Gilani, A H; Majoo, R U; Shah, B H

1997-07-01

The effects of protopine on human platelet aggregation and arachidonic acid (AA) metabolism via cyclooxygenase (COX) and lipoxygenase (LOP) enzymes were examined. Platelet aggregation induced by various platelet agonists (AA, ADP, collagen and PAF) was strongly inhibited by protopine in a concentration-related manner. The IC50 values (microM) of protopine (mean +/- SEM) against: AA; 12 +/- 2: ADP; 9 +/- 2: collagen; 16 +/- 2 and PAF; 11 +/- 1, were much less than those observed for aspirin. In addition, protopine selectively inhibited the synthesis of thromboxane A2 (TXA2) via COX pathway and had no effect on the LOP pathway in platelets. In vivo, pretreatment with protopine (50-100 mg kg-1) protected rabbits from the lethal effects of AA (2 mg kg-1) or PAF (11 micrograms kg-1) in dose-dependent fashion. Protopine (50-100 mg kg-1) also inhibited carrageenan-induced rat paw oedema with a potency of three-fold as compared to aspirin. These results are suggestive that protopine acts as a potent inhibitor of thromboxane synthesis and PAF with anti-inflammatory properties.

Compression force sensing regulates integrin αIIbβ3 adhesive function on diabetic platelets.

PubMed

Ju, Lining; McFadyen, James D; Al-Daher, Saheb; Alwis, Imala; Chen, Yunfeng; Tønnesen, Lotte L; Maiocchi, Sophie; Coulter, Brianna; Calkin, Anna C; Felner, Eric I; Cohen, Neale; Yuan, Yuping; Schoenwaelder, Simone M; Cooper, Mark E; Zhu, Cheng; Jackson, Shaun P

2018-03-14

Diabetes is associated with an exaggerated platelet thrombotic response at sites of vascular injury. Biomechanical forces regulate platelet activation, although the impact of diabetes on this process remains ill-defined. Using a biomembrane force probe (BFP), we demonstrate that compressive force activates integrin α IIb β 3 on discoid diabetic platelets, increasing its association rate with immobilized fibrinogen. This compressive force-induced integrin activation is calcium and PI 3-kinase dependent, resulting in enhanced integrin affinity maturation and exaggerated shear-dependent platelet adhesion. Analysis of discoid platelet aggregation in the mesenteric circulation of mice confirmed that diabetes leads to a marked enhancement in the formation and stability of discoid platelet aggregates, via a mechanism that is not inhibited by therapeutic doses of aspirin and clopidogrel, but is eliminated by PI 3-kinase inhibition. These studies demonstrate the existence of a compression force sensing mechanism linked to α IIb β 3 adhesive function that leads to a distinct prothrombotic phenotype in diabetes.

Agonist-induced platelet reactivity correlates with bleeding in haemato-oncological patients.

PubMed

Batman, B; van Bladel, E R; van Hamersveld, M; Pasker-de Jong, P C M; Korporaal, S J A; Urbanus, R T; Roest, M; Boven, L A; Fijnheer, R

2017-11-01

Prophylactic platelet transfusions are administered to prevent bleeding in haemato-oncological patients. However, bleeding still occurs, despite these transfusions. This practice is costly and not without risk. Better predictors of bleeding are needed, and flow cytometric evaluation of platelet function might aid the clinician in identifying patients at risk of bleeding. This evaluation can be performed within the hour and is not hampered by low platelet count. Our objective was to assess a possible correlation between bleeding and platelet function in thrombocytopenic haemato-oncological patients. Inclusion was possible for admitted haemato-oncology patients aged 18 years and above. Furthermore, an expected need for platelet transfusions was necessary. Bleeding was graded according to the WHO bleeding scale. Platelet reactivity to stimulation by either adenosine diphosphate (ADP), cross-linked collagen-related peptide (CRP-xL), PAR1- or PAR4-activating peptide (AP) was measured using flow cytometry. A total of 114 evaluations were available from 21 consecutive patients. Platelet reactivity in response to stimulation by all four studied agonists was inversely correlated with significant bleeding. Odds ratios (OR) for bleeding were 0·28 for every unit increase in median fluorescence intensity (MFI) [95% confidence interval (CI) 0·11-0·73] for ADP; 0·59 [0·40-0·87] for CRP-xL; 0·59 [0·37-0·94] for PAR1-AP; and 0·43 [0·23-0·79] for PAR4-AP. The platelet count was not correlated with bleeding (OR 0·99 [0·96-1·02]). Agonist-induced platelet reactivity was significantly correlated to bleeding. Platelet function testing could provide a basis for a personalized transfusion regimen, in which platelet transfusions are limited to those at risk of bleeding. © 2017 International Society of Blood Transfusion.

The Effect of a Simulated Commercial Flight Environment with Hypoxia and Low Humidity on Clotting, Platelet, and Endothelial Function in Participants with Type 2 Diabetes - A Cross-over Study.

PubMed

Konya, Judit; Al Qaissi, Ahmed; Sathyapalan, Thozhukat; Ajjan, Ramzi; Madden, Leigh; Naseem, Khalid M; Garrett, Andrew Thomas; Kilpatrick, Eric; Atkin, Stephen L

2018-01-01

To determine if clotting, platelet, and endothelial function were affected by simulated short-haul commercial air flight conditions (SF) in participants with type 2 diabetes (T2DM) compared to controls. 10 participants with T2DM (7 females, 3 males) and 10 controls (3 females, 7 males) completed the study. Participants were randomized to either spend 2 h in an environmental chamber at sea level conditions (temperature: 23°C, oxygen concentration 21%, humidity 45%), or subject to a simulated 2-h simulated flight (SF: temperature: 23°C, oxygen concentration 15%, humidity 15%), and crossed over 7 days later. Main outcome measures: clot formation and clot lysis parameters, functional platelet activation markers, and endothelial function measured by reactive hyperemia index (RHI) by EndoPAT and serum microparticles. Comparing baseline with SF conditions, clot maximal absorption was increased in controls (0.375 ± 0.05 vs. 0.39 ± 0.05, p  < 0.05) and participants with T2DM (0.378 ± 0.089 vs. 0.397 ± 0.089, p  < 0.01), while increased basal platelet activation for both fibrinogen binding and P-selectin expression ( p  < 0.05) was seen in participants with T2DM. Parameters of clot formation and clot lysis, stimulated platelet function (stimulated platelet response to ADP and sensitivity to prostacyclin), and endothelial function were unchanged. While SF resulted in the potential of denser clot formation with enhanced basal platelet activation in T2DM, the dynamic clotting, platelet, and endothelial markers were not affected, suggesting that short-haul commercial flying adds no additional hazard for venous thromboembolism for participants with T2DM compared to controls.

Exome-chip meta-analysis identifies association between variation in ANKRD26 and platelet aggregation.

PubMed

Chen, Ming-Huei; Yanek, Lisa R; Backman, Joshua D; Eicher, John D; Huffman, Jennifer E; Ben-Shlomo, Yoav; Beswick, Andrew D; Yerges-Armstrong, Laura M; Shuldiner, Alan R; O'Connell, Jeffrey R; Mathias, Rasika A; Becker, Diane M; Becker, Lewis C; Lewis, Joshua P; Johnson, Andrew D; Faraday, Nauder

2017-11-29

Previous genome-wide association studies (GWAS) have identified several variants associated with platelet function phenotypes; however, the proportion of variance explained by the identified variants is mostly small. Rare coding variants, particularly those with high potential for impact on protein structure/function, may have substantial impact on phenotype but are difficult to detect by GWAS. The main purpose of this study was to identify low frequency or rare variants associated with platelet function using genotype data from the Illumina HumanExome Bead Chip. Three family-based cohorts of European ancestry, including ~4,000 total subjects, comprised the discovery cohort and two independent cohorts, one of European and one of African American ancestry, were used for replication. Optical aggregometry in platelet-rich plasma was performed in all the discovery cohorts in response to adenosine diphosphate (ADP), epinephrine, and collagen. Meta-analyses were performed using both gene-based and single nucleotide variant association methods. The gene-based meta-analysis identified a significant association (P = 7.13 × 10 -7 ) between rare genetic variants in ANKRD26 and ADP-induced platelet aggregation. One of the ANKRD26 SNVs - rs191015656, encoding a threonine to isoleucine substitution predicted to alter protein structure/function, was replicated in Europeans. Aggregation increases of ~20-50% were observed in heterozygotes in all cohorts. Novel genetic signals in ABCG1 and HCP5 were also associated with platelet aggregation to ADP in meta-analyses, although only results for HCP5 could be replicated. The SNV in HCP5 intersects epigenetic signatures in CD41+ megakaryocytes suggesting a new functional role in platelet biology for HCP5. This is the first study to use gene-based association methods from SNV array genotypes to identify rare variants related to platelet function. The molecular mechanisms and pathophysiological relevance for the identified genetic associations requires further study.

[Effects of silkworm pupa oil on serum lipids level and platelet function in rats].

PubMed

Yang, Xuefeng; Huang, Lianzhen; Hu, Jianping; Li, Tao

2002-08-01

To observe the effects of silkworm pupa oil on serum lipids level and platelet function in rats, according to serum TG, TC level, 40 male Wistar rats are divided into four groups (normal control group, high fat control group, silkworm pupa oil group and silkworm pupa oil + VE group). The rats are fed different diets and six weeks later, serum lipids level and platelet function are measured. The results show that (1) Compared with high fat control group, serum TC, TG, LDL-C level, AI value, Platelet aggregability, plasma TXB2 level and T/P ratio decrease significantly while HDL-C level and 6-k-PGF1 level increase in silkworm pupa oil group; (2) Serum TC, LDL-C level, T/P ratio and platelet aggregability are significantly lower in silkworm pupa oil + VE group than in silkworm pupa oil group. It is suggested that silkworm pupa oil rich in alpha-linolenic acid can reduce serum lipids level and inhibit platelet aggregation, which is more effective with the supplementation with VE.

Human Platelets Exhibit Chemotaxis using Functional N-Formyl Peptide Receptors

DTIC Science & Technology

2005-01-01

activated phagocytes. Therefore, we examined the chemotactic migration of platelets qualita- tively by videomicroscopy . Platelets in medium were al- lowed...significantly decreased M. Czapiga et al. /Experimental Hematology 33 (2005) 73–84 79Figure 3. Videomicroscopy of human platelets in response to formyl...selected platelets during videomicroscopy from the time of the addition of fMLF (104 M in 1 µL) or PBS. Movement between markers represents 10 frames

[Thrombopenia and radial aplasia: 2 cases with platelet function and ultrastructural studies of megakaryocytes and platelets (author's transl)].

PubMed

Juhan, I; Bayle, J; Mattei, J F; Thevenieau, D; Perrimond, H; Muratore, R

1979-10-01

The authors report on two cases of congenital thrombopenia with radial aplasia. Both children display several formative abnormalities and a mild thrombopenia; hemorragic manifestations occurred in the first case only. Megacryoblastic to platelets series, as studied with electronic microscopy, show small-sized, "microcytic" and hypogranular megacaryocytes, displaying a maturative disorder (dysmegacaryocytopoiesis). In functional studies, platelets of the first patient show an imperfect nucleotidic release and do not agregate normally with ristocetin. The second case exhibits mostly a PF3 reduction. The variety of expression of the megacaryocytic-platelets disorders appears likewise in the squelettal and visceral malformations. The whole disorder could be ascribed to a pleiotropic abnormal gene with a variable expressivity.

Towards optical control of single blood platelet activation

NASA Astrophysics Data System (ADS)

Spiryova, Darya V.; Karmatskih, Oleg Yu.; Vorob'ev, Alexei Yu.; Moskalensky, Alexander E.

2018-04-01

Blood platelets play a pivotal role in blood coagulation and in other normal and pathological processes. The understanding of fundamental mechanisms underlying their functions is very important for diagnostics and treatment. Single-cell experiments are needed for this purpose, which are complicated by insufficient spatiotemporal precision of conventional activation protocols. We present an approach to trigger single platelet activation optically, without the need of reagent mixing. This is achieved using photolabile compound, which rapidly delivers epinephrine upon UV irradiation. We demonstrated the applicability of the technique to rapidly induce platelet activation for studying dynamics of activation. The presented method may give novel fundamental knowledge about platelet functions and facilitate current research of their ability to deliver drugs to tumors or vascular injury sites.

Generation of Megakaryocytes and Platelets from Human Pluripotent Stem Cells.

PubMed

Pick, Marjorie

2016-01-01

Human pluripotent stem cells (hPSC) have the potential to produce any tissue type in the body and thus represent a source of cells for regenerative medicine. Here we have shown that human platelets can be produced from embryonic or induced pluripotent stem cells in a defined culture system. We describe a serum- and feeder-free culture system that enabled the generation of megakaryocyte (Mk) progenitors and functional platelets from hPSCs. After 13 days the differentiated population included precursor cells that formed colonies containing differentiated Mks, and after 20 days these Mks were able to fragment into platelet-like particles that were functional. This protocol represents an important step towards the generation of human platelets for therapeutic use.

Quantitative evaluation of morphological changes in activated platelets in vitro using digital holographic microscopy.

PubMed

Kitamura, Yutaka; Isobe, Kazushige; Kawabata, Hideo; Tsujino, Tetsuhiro; Watanabe, Taisuke; Nakamura, Masayuki; Toyoda, Toshihisa; Okudera, Hajime; Okuda, Kazuhiro; Nakata, Koh; Kawase, Tomoyuki

2018-06-18

Platelet activation and aggregation have been conventionally evaluated using an aggregometer. However, this method is suitable for short-term but not long-term quantitative evaluation of platelet aggregation, morphological changes, and/or adhesion to specific materials. The recently developed digital holographic microscopy (DHM) has enabled the quantitative evaluation of cell size and morphology without labeling or destruction. Thus, we aim to validate its applicability in quantitatively evaluating changes in cell morphology, especially in the aggregation and spreading of activated platelets, thus modifying typical image analysis procedures to suit aggregated platelets. Freshly prepared platelet-rich plasma was washed with phosphate-buffered saline and treated with 0.1% CaCl 2 . Platelets were then fixed and subjected to DHM, scanning electron microscopy (SEM), atomic force microscopy, optical microscopy, and flow cytometry (FCM). Tightly aggregated platelets were identified as single cells. Data obtained from time-course experiments were plotted two-dimensionally according to the average optical thickness versus attachment area and divided into four regions. The majority of the control platelets, which supposedly contained small and round platelets, were distributed in the lower left region. As activation time increased, however, this population dispersed toward the upper right region. The distribution shift demonstrated by DHM was essentially consistent with data obtained from SEM and FCM. Therefore, DHM was validated as a promising device for testing platelet function given that it allows for the quantitative evaluation of activation-dependent morphological changes in platelets. DHM technology will be applicable to the quality assurance of platelet concentrates, as well as diagnosis and drug discovery related to platelet functions. Copyright © 2018 Elsevier Ltd. All rights reserved.

Adhesion of normal erythrocytes at depressed venous shear rates to activated neutrophils, activated platelets, and fibrin polymerized from plasma.

PubMed

Goel, Mukul S; Diamond, Scott L

2002-11-15

Deep vein thrombosis (DVT) is a low flow pathology often prevented by vascular compression to increase blood movement. We report new heterotypic adhesive interactions of normal erythrocytes operative at low wall shear rates (gamma(w)) below 100 s(-1). Adhesion at gamma(w) = 50 s(-1) of washed red blood cells (RBCs) to fibrinogen-adherent platelets was 4-fold less (P <.005) than to collagen-adherent platelets (279 +/- 105 RBC/mm(2)). This glycoprotein VI (GPVI)-triggered adhesion was antagonized (> 80% reduction) by soluble fibrinogen (3 mg/mL) and ethylenediaminetetraacetic acid (EDTA). RBC-platelet adhesion was reduced in half by antibodies against CD36 or GPIb, but not by antibodies against GPIIb/IIIa, von Willebrand factor (VWF), thrombospondin (TSP), P-selectin, beta(1), alpha(v), or CD47. Adhesion of washed RBCs to fibrinogen-adherent neutrophils was increased 6-fold in the presence of 20 microM N-formyl-Met-Leu-Phe to a level of 67 RBCs per 100 neutrophils after 5 minutes at 50 s(-1). RBC-neutrophil adhesion was diminished by anti-CD11b (76%), anti-RBC Landsteiner-Wiener (LW) (ICAM4; 40%), or by EDTA (> 80%), but not by soluble fibrinogen or antibodies against CD11a, CD11c, CD36, TSP, beta(1), alpha(v), or CD47. RBC adhesion to activated platelets and activated neutrophils was prevented by wall shear stress above 1 dyne/cm(2) (at 100 s(-1)). Whereas washed RBCs did not adhere to fibrin formed from purified fibrinogen, adhesion was marked when pure fibrin was precoated with TSP or when RBCs were perfused over fibrin formed from recalcified plasma. Endothelial activation and unusually low flow may be a setting prone to receptor-mediated RBC adhesion to adherent neutrophils (or platelets/fibrin), all of which may contribute to DVT.

Change of mean platelet volume values in asthmatic children as an inflammatory marker.

PubMed

Tuncel, T; Uysal, P; Hocaoglu, A B; Erge, D O; Karaman, O; Uzuner, N

2012-01-01

Asthma is the most common chronic disease of childhood in industrialised countries. T helper-2 (Th-2) cells, mast cells and eosinophils have a role in inflammation of asthma. Recently it was shown that platelets also play a role in asthma. Mean platelet volume shows platelet size and reflects platelet activation. The aim of this retrospective study is to evaluate levels of mean platelet volume in asthmatic patients during asymptomatic periods and exacerbations compared with healthy controls. The study consisted of 100 asthmatic patients (male/female: 55/45, mean age: 8.2±3.3) and 49 age and sex matched healthy children as a control group. Mean platelet volume values of asthmatic patients during asymptomatic period were 7.7±0.8fL while mean platelet volume values in asthmatics during exacerbation were 7.8±0.9fL. Comparison of mean platelet volume values of asthmatic patients and healthy controls both in acute asthmatic attack and asymptomatic period showed no difference (p>0.05). Comparison of mean platelet volume values at asthmatic attack and asymptomatic period also had no difference (p>0.05). The presence of atopy, infection, eosinophilia, elevated immunoglobulin E, and severity of acute asthmatic attack did not influence mean platelet volume values. The results of our study suggest that mean platelet volume values may not be used as a marker in bronchial asthma, although prospective studies with larger number of patients are needed to evaluate the role of mean platelet volume in asthma. Copyright © 2011 SEICAP. Published by Elsevier Espana. All rights reserved.

Identification of potential tumor-educated platelets RNA biomarkers in non-small-cell lung cancer by integrated bioinformatical analysis.

PubMed

Xue, Linlin; Xie, Li; Song, Xingguo; Song, Xianrang

2018-04-17

Platelets have emerged as key players in tumorigenesis and tumor progression. Tumor-educated platelet (TEP) RNA profile has the potential to diagnose non-small-cell lung cancer (NSCLC). The objective of this study was to identify potential TEP RNA biomarkers for the diagnosis of NSCLC and to explore the mechanisms in alternations of TEP RNA profile. The RNA-seq datasets GSE68086 and GSE89843 were downloaded from Gene Expression Omnibus DataSets (GEO DataSets). Then, the functional enrichment of the differentially expressed mRNAs was analyzed by the Database for Annotation Visualization and Integrated Discovery (DAVID). The miRNAs which regulated the differential mRNAs and the target mRNAs of miRNAs were identified by miRanda and miRDB. Then, the miRNA-mRNA regulatory network was visualized via Cytoscape software. Twenty consistently altered mRNAs (2 up-regulated and 18 down-regulated) were identified from the two GSE datasets, and they were significantly enriched in several biological processes, including transport and establishment of localization. Twenty identical miRNAs were found between exosomal miRNA-seq dataset and 229 miRNAs that regulated 20 consistently differential mRNAs in platelets. We also analyzed 13 spliceosomal mRNAs and their miRNA predictions; there were 27 common miRNAs between 206 differential exosomal miRNAs and 338 miRNAs that regulated 13 distinct spliceosomal mRNAs. This study identified 20 potential TEP RNA biomarkers in NSCLC for diagnosis by integrated bioinformatical analysis, and alternations in TEP RNA profile may be related to the post-transcriptional regulation and the splicing metabolisms of spliceosome. © 2018 Wiley Periodicals, Inc.

Role of G protein signaling in the formation of the fibrin(ogen)-integrin αIIbβ3-actin cytoskeleton complex in platelets.

PubMed

Budnik, Ivan; Shenkman, Boris; Savion, Naphtali

2016-09-01

Effective platelet function requires formation of a physical link between fibrin(ogen), integrin αIIbβ3, and cytoplasmic actin filaments. We investigated the role of the Gαq, Gαi, and Gα12/13 families of heterotrimeric GTP-binding proteins (G proteins) in the assembly of a ligand-αIIbβ3-actin cytoskeleton complex. Selective and combined activation of the G proteins was achieved by using combinations of various platelet agonists and inhibitors. Formation and stability of fibrinogen-αIIbβ3 interaction were evaluated by the extent of platelet aggregation and the rate of eptifibatide-induced platelet disaggregation; association of αIIbβ3 with the cytoskeleton was analyzed by western blot. Formation of the fibrin-αIIbβ3-actin cytoskeleton complex was evaluated by rotational thromboelastometry assay in which clot formation was induced by the mixture of reptilase and factor XIIIa. We demonstrated that involvement of heterotrimeric G proteins in the formation of the ligand-αIIbβ3-cytoskeleton complex depends on whether fibrinogen or fibrin serves as the integrin ligand. Formation of the fibrinogen-αIIbβ3-cytoskeleton complex requires combined activation of at least two G protein pathways while the maximal αIIbβ3-cytoskeleton association and the strongest αIIbβ3-fibrinogen binding supporting irreversible platelet aggregation require combined activation of all three-Gαq, Gαi, and Gα12/13-G protein families. In contrast, formation of the fibrin-αIIbβ3-cytoskeleton complex mediating clot retraction is critically dependent on the activation of the Gαi family, especially on the activation of Gαz.

Usefulness of the VerifyNow P2Y12 assay to evaluate the antiplatelet effects of ticagrelor and clopidogrel therapies.

PubMed

Jeong, Young-Hoon; Bliden, Kevin P; Antonino, Mark J; Park, Ki-Soo; Tantry, Udaya S; Gurbel, Paul A

2012-07-01

We analyzed the antiplatelet effects of different P2Y(12) receptor blockers with VerifyNow P2Y12 assay (VN-P2Y12) and light transmittance aggregometry (LTA). The point-of-care VN-P2Y12 has been used to assess the antiplatelet effects in clopidogrel-treated patients but has not been evaluated in detail in patients treated with ticagrelor. Patients were randomly assigned to either ticagrelor [180 mg loading/90 mg twice daily (n = 37)] or clopidogrel [600 mg loading/75 mg daily (n = 39)] on top of aspirin treatment, and platelet reactivity was measured serially during onset, maintenance, and offset phases. High on-treatment platelet reactivity (HPR) was defined as 5 and 20 μM adenosine diphosphate-induced maximal platelet aggregation ≥46% and ≥59%, respectively, and P2Y12 reaction units ≥235. Platelet function measured by VN-P2Y12 correlated well with LTA (.812 ≤ ρ ≤ .823, P < .001). VN-P2Y12 "BASE" values were consistent during administration of both agents. Calculated and reported percent inhibitions by VN-P2Y12 were similar (difference, -0.6%; 95% agreement limits, -22.9% to 21.6%). Platelet inhibition by VN-P2Y12 during clopidogrel and ticagrelor administrations was comparable to platelet inhibition by LTA. HPR determined by LTA and VN-P2Y12 were well matched, and the risk stratification between the two methods showed strong agreement after both therapies (κ > .7). The VerifyNow P2Y12 assay is effective in assessing the antiplatelet effects and in identifying HPR during clopidogrel or ticagrelor therapy. Copyright © 2012 Mosby, Inc. All rights reserved.

Evaluation of Dried Storage of Platelets for Transfusion: Physiologic Integrity and Hemostatic Functionality

DTIC Science & Technology

1994-10-27

paraformaidehyde in 500 mM Trehalose stored desiccated at RT, 4* C, or at -70,° C. Neither prep maintained good morphology at any temperature, and there...platelets, or para-platelets dried in Trehalose are as susceptible to loss of integrity over time as other preps. Our platelet handling techniques have

[Influence of S-nitrosoglutathione on agglutination and nitric oxide concentration in frozen platelets].

PubMed

Wu, Tao; Liu, Jing-Han; Li, Hui; Zhou, Wu; Wang, Shu-Ying

2012-04-01

The aim of this study was to investigate the influence of S-nitrosoglutathione (GSNO) on agglutination and nitric oxide (NO) concentration in frozen platelets. The agglutination of platelets was detected by using platelet agglutination apparatus, the level of NO in platelets was detected by the nitrate enzyme reduction method. The results showed that the rates of agglutination in freeze platelets and frozen platelets treated with GSNO were (35.47 ± 2.93) and (24.43 ± 3.07), which were significantly lower than that in fresh liquid platelets (63.44 ± 2.96). The level of NO concentration in frozen platelets was (22.16 ± 6.38), which was significantly lower than that in fresh liquid platelets (31.59 ± 16.88). The level of NO concentration in frozen platelets treated with GSNO was (45.64 ± 6.31), which was significantly higher than that in fresh liquid platelets (P < 0.01). It is concluded that GSNO increases the concentration of NO in frozen platelets, inhibits platelet activation and maintains platelet function, thus GSNO can be used as a frozen protective agent.

Antiplatelet effects of protopine isolated from Corydalis tubers.

PubMed

Ko, F N; Wu, T S; Lu, S T; Wu, Y C; Huang, T F; Teng, C M

1989-10-15

Protopine inhibited the aggregation and ATP release of rabbit platelets induced by ADP, arachidonic acid, PAF, collagen and ionophore A23187. Although the platelet aggregation caused by thrombin was not inhibited by protopine (100 micrograms/ml), the release reaction was partially suppressed. In rabbit platelet-rich plasma, protopine also inhibited the platelet aggregation caused by ADP, arachidonic acid, PAF and collagen. The thromboxane B2 formation of washed platelets caused by arachidonic acid, collagen, ionophore A23187 and thrombin was suppressed by protopine. Protopine inhibited the intracellular calcium increase caused by arachidonic acid in quin-2/AM loaded rabbit platelets. In the presence of indomethacin, the intracellular calcium increase caused by collagen and PAF was completely suppressed by protopine, and the intracellular calcium increase caused by thrombin was partially inhibited. The phosphoinositides breakdown caused by collagen and PAF was inhibited by protopine, but that by thrombin was not affected significantly. Protopine did not cause the elevation of cyclic AMP level of platelets. It is concluded that the antiplatelet effects of protopine is due to inhibition on thromboxane formation and phosphoinositides breakdown and then lead to the decrease of intracellular calcium concentration.

Relationship Between Platelet and Urinary 8‐Iso‐PGF2α Levels in Subjects With Different Degrees of NOX2 Regulation

PubMed Central

Carnevale, Roberto; Iuliano, Luigi; Nocella, Cristina; Bartimoccia, Simona; Trapè, Stefano; Russo, Roberta; Gentile, Maria Cristina; Cangemi, Roberto; Loffredo, Lorenzo; Pignatelli, Pasquale; Violi, Francesco

2013-01-01

Background Urinary 8‐iso‐PGF2α, a marker of oxidative stress, is influenced by the activation of NOX2. It is unclear if platelets 8‐iso‐PGF2α contribute to urinary 8‐iso‐PGF2α. Methods and Results In a cross‐sectional study, platelet, urinary, and serum 8‐iso‐PGF2α were determined in subjects with downregulation (X‐linked chronic granulomatous disease [X‐CGD], n=25) and upregulation (type II diabetic patients [T2D], n=121) of NOX2 and 153 controls matched for sex and age. In diabetic patients (n=18), the above variables were repeated before and after 7 days treatment with 100 mg/day aspirin or 100 mg/day aspirin plus 40 mg/day atorvastatin. In vitro study was performed to see the contribution of blood cells to serum 8‐iso‐PGF2α. Compared with controls, X‐CGD patients had lower platelet, serum, and urinary 8‐iso‐PGF2α values; conversely, diabetic patients had higher values of 8‐iso‐PGF2α compared with controls. Urinary 8‐iso‐PGF2α significantly correlated with both platelet and serum 8‐iso‐PGF2α in the 2 cohorts. A parallel increase of platelet, serum, and urinary 8‐iso‐PGF2α by aspirin and a parallel decrease by aspirin plus atorvastatin were detected in the interventional study. In vitro study demonstrated that platelets contribute to 37% of serum 8‐iso‐PGF2α and that only 13% of it is of extravascular origin. Conclusions The study suggests that NOX2 contributes to the formation of 8‐iso‐PGF2α in both platelets and urine. The direct correlation between platelet and urinary 8‐iso‐PGF2α suggests that, at least partly, urinary 8‐iso‐PGF2α reflects platelet 8‐iso‐PGF2α production. Analysis of serum 8‐iso‐PGF2α may represent a novel tool to investigate the production of 8‐iso‐PGF2α by blood cells including platelets. Clinical Trial Registration URL: ClinicalTrials.gov. Unique Identifier: NCT01250340. PMID:23770972

How does measurement of platelet P-selectin compare with other methods of measuring platelet function as a means of determining the effectiveness of antiplatelet therapy?

PubMed

Fox, Susan C; May, Jane A; Dovlatova, Natalia; Glenn, Jackie R; Johnson, Andrew; White, Ann E; Radhakrishnan, Ashwin; Heptinstall, Stan

2018-02-20

Measurement of P-selectin on activated platelets as a means of measuring platelet function utilizing the technology described here has the advantage of not requiring immediate access to specialist equipment and expertise. Blood samples are activated, fixed, stored, and transported to a central laboratory for flow cytometric analysis. Here we have compared P-selectin with other more traditional approaches to measuring platelet function in blood and/or platelet-rich plasma (PRP) from patients with acute coronary syndromes on treatment for at least 1 month with either aspirin and clopidogrel or aspirin with prasugrel. The comparators were light transmission aggregometry (LTA), VerifyNow and Multiplate aggregometry (for determining the effects of aspirin) and LTA, VerifyNow and Multiplate together with the BioCytex VASP phosphorylation assay (for the P2Y 12 antagonists). The P-selectin Aspirin Test revealed substantial inhibition of platelet function in all but three of 96 patients receiving aspirin with clopidogrel and in none of 51 patients receiving aspirin and prasugrel. The results were very similar to those obtained using LTA. There was only one patient with high residual platelet aggregation and low P-selectin expression. The same patients identified as "non-responders" to aspirin also presented with the highest residual platelet activity as measured using the VerifyNow system, although not quite as well separated from the other values. With the Multiplate test only one of these patients clearly stood out from the others. The results obtained using the P-selectin P2Y 12 Test in 102 patients taking aspirin and clopidogrel were similar to the more traditional approaches in that a wide scatter of results was obtained. Generally, high values seen with the P-selectin P2Y 12 Test were also high with the LTA, VerifyNow, Multiplate, and BioCytex VASP P2Y 12 Tests. Similarly, low residual platelet function using the P2Y 12 test was seen irrespective of the testing procedure used. However, there were differences in some patients. Prasugrel was always more effective than clopidogrel in inhibiting platelet function with none of 56 patients (P-selectin and VerifyNow), only 2 of 56 patients (Multiplate) and only 3 of 56 patients (Biocytex VASP) demonstrating high on-treatment residual platelet reactivity (HRPR) defined using previously published cut-off values. The exception was LTA where there were 11 of 56 patients with HRPR. It remains to be seen which experimental approach provides the most useful information regarding outcomes after adjusting therapies in treated patients.

[Experimental research on the effects of different activators on the formation of platelet-rich gel and the release of bioactive substances in human platelet-rich plasma].

PubMed

Yang, Y; Zhang, W; Cheng, B

2017-01-20

Objective: To explore the effects of calcium gluconate and thrombin on the formation of platelet-rich gel (PRG) and the release of bioactive substances in human platelet-rich plasma (PRP) and the clinical significance. Methods: Six healthy blood donors who met the inclusion criteria were recruited in our unit from May to August in 2016. Platelet samples of each donor were collected for preparation of PRP. (1) PRP in the volume of 10 mL was collected from each donor and divided into thrombin activation group (TA, added with 0.5 mL thrombin solution in dose of 100 U/mL) and calcium gluconate activation group (CGA, added with 0.5 mL calcium gluconate solution in dose of 100 g/L) according to the random number table, with 5 mL PRP in each group. Then the PRP of the two groups was activated in water bath at 37 ℃ for 1 h. The formation time of PRG was recorded, and the formation situation of PRG was observed within 1 hour of activation. After being activated for 1 h, one part of PRG was collected to observe the distribution of fibrous protein with HE staining, and another part of PRG was collected to observe platelet ultrastructure under transmission electron microscope (TEM). After being activated for 1 h, the supernatant was collected to determine the content of transforming growth factor β(1, )platelet-derived growth factor BB (PDGF-BB), vascular endothelial growth factor, basic fibroblast growth factor (bFGF), epidermal growth factor, and insulin-like growth factorⅠby enzyme-linked immunosorbent assay. (2) Another 10 mL PRP from each donor was collected and grouped as above, and the platelet suspension was obtained after two times of centrifugation and resuspension with phosphate buffered saline, respectively. And then they were treated with corresponding activator for 1 h as that in experiment (1). Nanoparticle tracking analyzer was used to detect the concentrations of microvesicles with different diameters and total microvesicles derived from platelet. Data were processed with t test. Results: (1) The formation time of PRG in group TA was (228±40) s, and the PRG volume reached the maximum at this moment. The PRG volume shrunk to the minimum after 30 minutes of activation. The formation time of PRG in group CGA was (690±71) s, and the PRG volume reached the maximum at this moment. After 55 minutes of activation, the PRG volume shrunk to the minimum. The formation time of PRG in group TA was obviously shorter than that in group CGA ( t =15.17, P <0.01). (2) HE staining showed that after 1 hour of activation, the red-stained area of fibrous protein in PRG of group TA was large and densely distributed, while that of group CGA was small and loosely distributed. TEM revealed that after 1 hour of activation, the platelets in PRG of group TA were fragmented, while lysing platelet structure, lysing α granule structure, intact α granule structure, and intact dense body structure were observed in PRG of group CGA. (3) The content of PDGF-BB released by PRP in group TA was (7.4±0.8) ng/mL, which was obviously higher than that in group CGA [(4.9±0.5) ng/mL, t =5.41, P <0.01]. The content of bFGF released by PRP in group CGA was (960±151) pg/mL, which was significantly higher than that in group TA [(384±56) pg/mL, t =8.75, P <0.01]. The content of the other 4 growth factors released by PRP in the two groups was close (with t values from 0.11 to 1.97, P values above 0.05). (4) The concentrations of total microvesicles, microvesicles with diameter more than 100 nm, and exosomes with diameter less than or equal to 100 nm derived from platelet in group CGA were (165.8±15.1)×10(8)/mL, (142.4±12.3)×10(8)/mL, and (23.4±2.9)×10(8)/mL respectively, which were significantly higher than those in group TA [(24.7±4.6)×10(8)/mL, (22.6±4.0)×10(8)/mL, and (2.1±0.7)×10(8)/mL, with t values from 17.36 to 22.66, P values below 0.01]. Conclusions: Calcium gluconate can slowly activate PRP, resulting in slowly shrunk PRG with high content of bFGF and high concentration of microvesicles, which is suitable for repairing articular cavity and sinus tract wound. Thrombin can rapidly activate PRP, resulting in quickly shrunk PRG with high content of PDGF-BB and a certain concentration of microvesicles, which is suitable for repairing acute trauma.

Effects of washed platelets vs platelet-rich plasma on the proliferation and mineralization of rat dental pulp cells.

PubMed

Zhang, L; Xie, Y H; Lin, B R

2015-08-14

We examined the effects of washed platelets (WPLTs) and platelet-rich plasma (PRP) on the proliferation and mineralization of rat dental pulp cells. Rat dental pulp cells were separated, cultured, and identified. Medium containing 1, 10, 100, or 500 mL/L PRP or WPLTs was added to 4th generation cells. The MTS method was used to determine cell proliferation. Alizarin red staining was used to observe the formation of mineralized nodules after cell mineralization and induction for 10 and 20 days under different culture conditions, and the areas of the mineralized nodules formed 20 days after induction were computed. The addition of 1, 10, and 100 mL/L WPLTs or PRP significantly promoted rat dental pulp cell proliferation (P < 0.05) whereas 500 mL/L WPLTs or PRP had no significant effect (P > 0.05). Under the same concentrations, no significant differences on cell proliferation were observed between WPLT and PRP treatments (P > 0.05 in all groups). After 10 days mineralization and culture, the 100 and 500 mL/L WPLT and PRP group positive nodule rates were significantly higher than those of the low concentration and the control groups (P < 0.05). After 20 days, the areas of the mineralized nodules formed in the 100 and 500 mL/L WPLT and PRP groups were significantly larger than those in the control group (P < 0.05). These results demonstrate that both WPLTs and PRP are equally able to significantly promote the proliferation and calcification of rat dental pulp cells under a certain range of concentrations.

The neuropeptide substance P stimulates the effector functions of platelets.

PubMed Central

Damonneville, M; Monté, D; Auriault, C; Capron, A

1990-01-01

Sensory neuropeptides, such as substance P, appear as potent mediators of various immunological reactions, and inhibit or stimulate a wide range of functions of immune inflammatory cells. Platelets were recently shown to participate as effector cells in an IgE or lymphokine-dependent killing of parasites. Substance P and its carboxy-terminal fragment SP (4-11) induce the cytotoxic activity of platelets towards the larvae of Schistosoma mansoni, respectively, by 90% and 40%, whereas the modified C terminal SP, the SP-free acid, exhibits no effect on the platelets. The neuropeptide effects occur at low doses (10(-8) M), are specific as shown by inhibition studies with a substance P antagonist, the D-SP. Binding data obtained after flow cytofluorometry with FITC-SP lead to the conclusion that SP binds specifically to about 20% of the homogenous population of platelets. Moreover, IgE could modulate the SP-dependent functions of platelets since the pre-incubation with myeloma human IgE or with AP2 monoclonal antibodies--known to inhibit the IgE-dependent killing of these cells-leads to a dramatic decrease of the SP dependent cytotoxic activity of platelets towards the larvae. These findings identify a potent mechanism for nervous system regulation of host defence responses. PMID:1696868

Prevalence of bacterial contamination in platelet concentrates at the National Center of Blood Transfusion (Mexico).

PubMed

Ibáñez-Cervantes, G; Bello-López, J M; Fernández-Sánchez, V; Domínguez-Mendoza, C A; Acevedo-Alfaro, L I

2017-06-01

Most common bacterial sepsis associated with transfusion is caused by contaminated Platelet Concentrates (PC). The screening of PC to detect bacterial contamination is obligatory in Mexico, and it is carried out in quality control programs. In Mexico, the identification and molecular characterization of bacterial contaminants to detect contamination sources have not been implemented due to high costs; however, it is an actual current need. One hundred PC were randomly selected and microbiologically analyzed. This sample size corresponds to 1% of the PC obtained by the National Center of Blood Transfusion (NCBT) in Mexico City according to the Official Mexican Standard NOM-253-SSA1-2012. Additionally, molecular biology tests were implemented in order to identify the possible contamination sources. Nine of the 100 PC analyzed (9%) showed bacterial contamination; analysis of the nucleotide sequences revealed the presence of characteristic microbiota from donor skin and soil. Diverse clonal relationship between the strains was identified in Staphylococcus epidermidis. Detection of contaminants associated with environmental and skin flora, shows the need to implement measures in the process of disinfecting skin at the site of phlebotomy and cleaning each of the areas involved in blood collection. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Hydroxysafflor yellow A of Carthamus tinctorius attenuates lung injury of aged rats exposed to gasoline engine exhaust by down-regulating platelet activation.

PubMed

Wang, Chaoyun; Wang, Chunhua; Ma, Chunlei; Huang, Qingxian; Sun, Hongliu; Zhang, Xiaomin; Bai, Xianyong

2014-02-15

Long-term inhalation of gasoline engine exhaust (GEE) increases the risk of respiratory disease. Studies have suggested involvement of platelets in the development of some lung diseases. Hydroxysafflor yellow A (HSYA), a flavonoid compound, prevents hemostasis. Therefore, we investigated its effects on GEE-induced lung injury, and role of platelets in injury. Sixty-week-old male Sprague-Dawley rats were exposed to GEE for 4h/day for 6 weeks, and then grouped as follows: control, GEE, GEE+HSYA, GEE+HSYA+GW9662, and GEE+GW9662. Arterial oxygen tension (PaO2), carbon dioxide tension (PaCO2), pH, and the PaO2/fraction of inspired oxygen ratio (PaO2/FiO2) in the blood were detected using a blood gas analyzer. Wet/dry lung weight ratio, total protein in bronchoalveolar lavage fluid (BALF), and cytokine concentrations in serum and BALF were determined. Furthermore, cyclic adenosine monophosphate (cAMP) level and expression levels of target proteins were analyzed. Platelets were counted and their state was evaluated. HSYA attenuated GEE-mediated decreases in PaO2, PaO2/FiO2, platelet cAMP level, protein kinase A (PKA) activity, and peroxisome proliferator-activated receptor γ (PPARγ) expression. HSYA also attenuated GEE-mediated increases in lung permeability, cytokine levels in serum and BALF, plasma platelet count, and ADP-mediated platelet aggregation. Moreover, it suppressed GEE-induced increases in the expression of adhesion molecules and proinflammatory cytokines in platelets and lung tissue. Therefore, HSYA is therapeutically effective for GEE-mediated lung injury and acts by enhancing PKA activity and inhibiting platelet activation. Copyright © 2013 Elsevier GmbH. All rights reserved.

Disruption of the Mouse μ-Calpain Gene Reveals an Essential Role in Platelet Function

PubMed Central

Azam, Mohammad; Andrabi, Shaida S.; Sahr, Kenneth E.; Kamath, Lakshmi; Kuliopulos, Athan; Chishti, Athar H.

2001-01-01

Conventional calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. There are two forms of conventional calpains: the μ-calpain, or calpain I, which requires micromolar calcium for half-maximal activation, and the m-calpain, or calpain II, which functions at millimolar calcium concentrations. We evaluated the functional role of the 80-kDa catalytic subunit of μ-calpain by genetic inactivation using homologous recombination in embryonic stem cells. The μ-calpain-deficient mice are viable and fertile. The complete deficiency of μ-calpain causes significant reduction in platelet aggregation and clot retraction but surprisingly the mutant mice display normal bleeding times. No detectable differences were observed in the cleavage pattern and kinetics of calpain substrates such as the β3 subunit of αIIbβ3 integrin, talin, and ABP-280 (filamin). However, μ-calpain null platelets exhibit impaired tyrosine phosphorylation of several proteins including the β3 subunit of αIIbβ3 integrin, correlating with the agonist-induced reduction in platelet aggregation. These results provide the first direct evidence that μ-calpain is essential for normal platelet function, not by affecting the cleavage of cytoskeletal proteins but by potentially regulating the state of tyrosine phosphorylation of the platelet proteins. PMID:11238954

Effects of high flavanol dark chocolate on cardiovascular function and platelet aggregation.

PubMed

Rull, Gurvinder; Mohd-Zain, Zetty N; Shiel, Julian; Lundberg, Martina H; Collier, David J; Johnston, Atholl; Warner, Timothy D; Corder, Roger

2015-08-01

Regular consumption of chocolate and cocoa products has been linked to reduced cardiovascular mortality. This study compared the effects of high flavanol dark chocolate (HFDC; 1064mg flavanols/day for 6weeks) and low flavanol dark chocolate (LFDC; 88mg flavanols/day for 6weeks) on blood pressure, heart rate, vascular function and platelet aggregation in men with pre-hypertension or mild hypertension. Vascular function was assessed by pulse wave analysis using radial artery applanation tonometry in combination with inhaled salbutamol (0.4mg) to assess changes due to endothelium-dependent vasodilatation. HFDC did not significantly reduce blood pressure compared to baseline or LFDC. Heart rate was increased by LFDC compared to baseline, but not by HFDC. Vascular responses to salbutamol tended to be greater after HFDC. Platelet aggregation induced by collagen or the thromboxane analogue U46619 was unchanged after LFDC or HFDC, whereas both chocolates reduced responses to ADP and the thrombin receptor activator peptide, SFLLRNamide (TRAP6), relative to baseline. Pre-incubation of platelets with theobromine also attenuated platelet aggregation induced by ADP or TRAP6. We conclude that consumption of HFDC confers modest improvements in cardiovascular function. Platelet aggregation is modulated by a flavanol-independent mechanism that is likely due to theobromine. Copyright © 2015 Elsevier Inc. All rights reserved.

Characterizations of the TiO2-x films synthesized by e-beam evaporation for endovascular applications

NASA Astrophysics Data System (ADS)

Lin, Zeng; Lee, In-Seop; Choi, Yoon-Jeong; Noh, In-Sup; Chung, Sung-Min

2009-02-01

Different chemical states of titanium oxide films were deposited on commercially pure Ti (CP Ti) by electron-beam evaporation at different oxygen flow rates to examine a possibility of their applications to endovascular stents. The surface morphology, chemical composition and crystal structure of the obtained titanium oxide films were analyzed by FE-SEM, XPS and XRD, respectively. As a function of the deposition parameters employed, the obtained titanium oxide films demonstrated different mixtures of anatase phase, Ti2O3 and TiO. By the formation of titanium oxide film on the CP Ti plate, the contact angle was decreased and the cellular activity of porcine aortic smooth muscle cells was increased. Post-deposition annealing was also found to be an important factor to achieve advantageous biocompatibility. When haemocompatibility was investigated by observing adhesion of blood platelets from platelet-rich plasma, less platelet adhesion was observed on titanium oxide films. These results indicated that titanium oxide film synthesized by e-beam evaporation could be applicable to coronary stents.

Binding of thrombin-activated platelets to a fibrin scaffold through α(IIb)β₃ evokes phosphatidylserine exposure on their cell surface.

PubMed

Brzoska, Tomasz; Suzuki, Yuko; Mogami, Hideo; Sano, Hideto; Urano, Tetsumei

2013-01-01

Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose phosphatidylserine (PS) and fibrin accumulate only in the center of the thrombus but not in its periphery. To address the question how exposure of platelet anionic phospholipids is regulated within the thrombus, an in-vitro experiment using diluted platelet-rich plasma was employed, in which the fibrin network was formed in the presence of platelets, and PS exposure on the platelet surface was analyzed using Confocal Laser Scanning Microscopy. Almost all platelets exposed PS after treatment with tissue factor, thrombin or ionomycin. Argatroban abrogated fibrin network formation in all samples, however, platelet PS exposure was inhibited only in tissue factor- and thrombin-treated samples but not in ionomycin-treated samples. FK633, an α(IIb)β₃ antagonist, and cytochalasin B impaired platelet binding to the fibrin scaffold and significantly reduced PS exposure evoked by thrombin. Gly-Pro-Arg-Pro amide abrogated not only fibrin network formation, but also PS exposure on platelets without suppressing platelet binding to fibrin/fibrinogen. These results suggest that outside-in signals in platelets generated by their binding to the rigid fibrin network are essential for PS exposure after thrombin treatment.

Binding of Thrombin-Activated Platelets to a Fibrin Scaffold through αIIbβ3 Evokes Phosphatidylserine Exposure on Their Cell Surface

PubMed Central

Brzoska, Tomasz; Suzuki, Yuko; Mogami, Hideo; Sano, Hideto; Urano, Tetsumei

2013-01-01

Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose phosphatidylserine (PS) and fibrin accumulate only in the center of the thrombus but not in its periphery. To address the question how exposure of platelet anionic phospholipids is regulated within the thrombus, an in-vitro experiment using diluted platelet-rich plasma was employed, in which the fibrin network was formed in the presence of platelets, and PS exposure on the platelet surface was analyzed using Confocal Laser Scanning Microscopy. Almost all platelets exposed PS after treatment with tissue factor, thrombin or ionomycin. Argatroban abrogated fibrin network formation in all samples, however, platelet PS exposure was inhibited only in tissue factor- and thrombin-treated samples but not in ionomycin-treated samples. FK633, an αIIbβ3 antagonist, and cytochalasin B impaired platelet binding to the fibrin scaffold and significantly reduced PS exposure evoked by thrombin. Gly-Pro-Arg-Pro amide abrogated not only fibrin network formation, but also PS exposure on platelets without suppressing platelet binding to fibrin/fibrinogen. These results suggest that outside-in signals in platelets generated by their binding to the rigid fibrin network are essential for PS exposure after thrombin treatment. PMID:23383331

A scalable, micropore, platelet rich plasma separation device.

PubMed

Dickson, Mary Nora; Amar, Levy; Hill, Michael; Schwartz, Joseph; Leonard, Edward F

2012-12-01

We have designed a novel, low energy platelet-rich-plasma (PRP) separator capable of producing 50 mL of PRP in 30 min, intended for military and emergency applications. Blood flows over a 3 mm length of sieve at high rates of shear. A plasma-platelet filtrate passes through the sieve's pores while erythrocytes remain. The filtrate is flowed over a second 3 mm length of smaller-pored sieve that withdraws plasma. Bulk blood volume is maintained by returning platelet-free plasma to the erythrocyte pool, enabling a nearly complete multi-pass platelet extraction. The total percentage of platelets extracted is:θ(T)=1-exp (-V(f)(T)Φ(P)/V) where V is the original plasma volume, V ( f )(T) is the total filtered volume, and ϕ ( P ) is platelet passage ratio (filtrate concentration/bulk average concentration) taken to be constant. Maximum θ(T) occurs at maximum V ( f )(T)× ϕ ( P ) Test microsieves, 3 mm long × 3 mm wide, were used. ϕ ( P ) values measured at various filtrate flow rates (20-100 uL/min) and utilizing various filter pore sizes (1.2-3.5 μm), was as high as 150 %. Maximum V ( f )(T)× ϕ ( P ) was achieved utilizing the 3.5 um filters at the highest flow rate, 100 uL/min. Erythrocyte leakages were always below 2,000/uL, far below the allowable limit stipulated by the American Association of Blood Banking. These data imply that a 13.7 cm(2) filter area is sufficient to achieve the target separation of 50 mL of platelet concentrate in 30 min. The filtration cartridge would consist of multiple microporous strips of 3 mm width arranged in parallel so that each element would see the conditions used in the prototype experiments presented here. Other microfiltration schemes suggest no method of scaling to practical levels.

Platelet-Rich-Plasma injection seems to be effective in treatment of plantar fasciitis: a case series.

PubMed

van Egmond, Jeroen C; Breugem, Stefan J M; Driessen, Marcel; Bruijn, Daniel J

2015-06-01

Plantar fasciitis is the most common cause of heel pain. Diverse non-operative treatment options are available. The purpose of this study was to determine if a single platelet-rich-plasma injection at the origin of the plantar fascia in patients with plantar fasciitis gives a functional improvement. Patients with plantar fasciitis and failed conservative treatment were included in this retrospective study. Included patients were sent four questionnaires after platelet-rich-plasma injection. Primary outcome is functional improvement, determined by foot function index in which lower scores correlates with a better foot function. A total of 61 feet in 58 patients were included. The median foot function index before treatment was 69.4 and after treatment 31.8, which is a significant decrease. In 80.3% of the patients the foot function index decreased. Therefore platelet-rich-plasma injection seems to be effective in treatment of patients with plantar fasciitis when conservative treatment failed.

Proteome changes in platelets after pathogen inactivation--an interlaboratory consensus.

PubMed

Prudent, Michel; D'Alessandro, Angelo; Cazenave, Jean-Pierre; Devine, Dana V; Gachet, Christian; Greinacher, Andreas; Lion, Niels; Schubert, Peter; Steil, Leif; Thiele, Thomas; Tissot, Jean-Daniel; Völker, Uwe; Zolla, Lello

2014-04-01

Pathogen inactivation (PI) of platelet concentrates (PCs) reduces the proliferation/replication of a large range of bacteria, viruses, and parasites as well as residual leucocytes. Pathogen-inactivated PCs were evaluated in various clinical trials showing their efficacy and safety. Today, there is some debate over the hemostatic activity of treated PCs as the overall survival of PI platelets seems to be somewhat reduced, and in vitro measurements have identified some alterations in platelet function. Although the specific lesions resulting from PI of PCs are still not fully understood, proteomic studies have revealed potential damages at the protein level. This review merges the key findings of the proteomic analyses of PCs treated by the Mirasol Pathogen Reduction Technology, the Intercept Blood System, and the Theraflex UV-C system, respectively, and discusses the potential impact on the biological functions of platelets. The complementarities of the applied proteomic approaches allow the coverage of a wide range of proteins and provide a comprehensive overview of PI-mediated protein damage. It emerges that there is a relatively weak impact of PI on the overall proteome of platelets. However, some data show that the different PI treatments lead to an acceleration of platelet storage lesions, which is in agreement with the current model of platelet storage lesion in pathogen-inactivated PCs. Overall, the impact of the PI treatment on the proteome appears to be different among the PI systems. Mirasol impacts adhesion and platelet shape change, whereas Intercept seems to impact proteins of intracellular platelet activation pathways. Theraflex influences platelet shape change and aggregation, but the data reported to date are limited. This information provides the basis to understand the impact of different PI on the molecular mechanisms of platelet function. Moreover, these data may serve as basis for future developments of PI technologies for PCs. Further studies should address the impact of both the PI and the storage duration on platelets in PCs because PI may enable the extension of the shelf life of PCs by reducing the bacterial contamination risk. Copyright © 2014 Elsevier Inc. All rights reserved.

Sinus grafting using recombinant human tissue factor, platelet-rich plasma gel, autologous bone, and anorganic bovine bone mineral xenograft: histologic analysis and case reports.

PubMed

Philippart, Pierre; Daubie, Valéry; Pochet, Roland

2005-01-01

The purpose of this study was to analyze healthy bone formation by means of histology and immunohistochemistry after grafting with a mixture of autologous ground calvarial bone, inorganic xenograft, platelet-rich plasma (PRP), and recombinant human tissue factor (rhTF). Maxillary sinus floor augmentation was performed on 3 patients by grafting with 5 to 10 mL of a paste consisting of autologous powder from calvarial bone (diameter < 1 mm), 50% v/v anorganic bovine bone mineral xenograft (PepGen P-15, a new tissue-engineered bone replacement graft material), PRP (1.8 x 10(6) platelets/mm3 plasma), and about 1 microg rhTF. Six and 10 months after grafting, bone cores were extracted for implant fixation and analyzed. Histology demonstrated a high degree of inorganic xenograft integration and natural bone regeneration. Both the xenograft and newly synthesized bone were colonized with osteocytes and surrounded by osteoblasts. Six-month-old bone cores demonstrated a ratio of synthesized bone to xenograft particles ratio of 0.5, whereas 10-month-old cores demonstrated a ratio of 2. A low degree of inflammation could also be observed using S100A8 immunohistochemistry. Autologous grafting in edentulous patients is a complex procedure; the successful substitution of synthetic analogs for ground bone is a major challenge. In this investigation, it was shown that inorganic xenograft in the harvested bone paste could be safe for patients and had high bone regeneration capacity over time. The sinus graft showed intense bone formation 6 months after grafting and a further increase in bone growth 10 months after grafting.

Unaltered Angiogenesis-Regulating Activities of Platelets in Mild Type 2 Diabetes Mellitus despite a Marked Platelet Hyperreactivity.

PubMed

Miao, Xinyan; Zhang, Wei; Huang, Zhangsen; Li, Nailin

2016-01-01

Type 2 diabetes mellitus (T2DM) is associated with platelet dysfunction and impaired angiogenesis. Aim of the study is to investigate if platelet dysfunction might hamper platelet angiogenic activities in T2DM patients. Sixteen T2DM patients and gender/age-matched non-diabetic controls were studied. Flow cytometry and endothelial colony forming cell (ECFC) tube formation on matrigel were used to assess platelet reactivity and angiogenic activity, respectively. Thrombin receptor PAR1-activating peptide (PAR1-AP) induced higher platelet P-selectin expression, and evoked more rapid and intense platelet annexin V binding in T2DM patients, seen as a more rapid increase of annexin V+ platelets (24.3±6.4% vs 12.6±3.8% in control at 2 min) and a higher elevation (30.9±5.1% vs 24.3±3.0% at 8 min). However, PAR1-AP and PAR4-AP induced similar releases of angiogenic regulators from platelets, and both stimuli evoked platelet release of platelet angiogenic regulators to similar extents in T2DM and control subjects. Thus, PAR1-stimulated platelet releasate (PAR1-PR) and PAR4-PR similarly enhanced capillary-like network/tube formation of ECFCs, and the enhancements did not differ between T2DM and control subjects. Direct supplementation of platelets to ECFCs at the ratio of 1:200 enhanced ECFC tube formation even more markedly, leading to approximately 100% increases of the total branch points of ECFC tube formation, for which the enhancements were also similar between patients and controls. In conclusion, platelets from T2DM subjects are hyperreactive. Platelet activation induced by high doses of PAR1-AP, however, results in similar releases of angiogenic regulators in mild T2DM and control subjects. Platelets from T2DM and control subjects also demonstrate similar enhancements on ECFC angiogenic activities.

Comparative gene expression profiling of in vitro differentiated megakaryocytes and erythroblasts identifies novel activatory and inhibitory platelet membrane proteins.

PubMed

Macaulay, Iain C; Tijssen, Marloes R; Thijssen-Timmer, Daphne C; Gusnanto, Arief; Steward, Michael; Burns, Philippa; Langford, Cordelia F; Ellis, Peter D; Dudbridge, Frank; Zwaginga, Jaap-Jan; Watkins, Nicholas A; van der Schoot, C Ellen; Ouwehand, Willem H

2007-04-15

To identify previously unknown platelet receptors we compared the transcriptomes of in vitro differentiated megakaryocytes (MKs) and erythroblasts (EBs). RNA was obtained from purified, biologically paired MK and EB cultures and compared using cDNA microarrays. Bioinformatical analysis of MK-up-regulated genes identified 151 transcripts encoding transmembrane domain-containing proteins. Although many of these were known platelet genes, a number of previously unidentified or poorly characterized transcripts were also detected. Many of these transcripts, including G6b, G6f, LRRC32, LAT2, and the G protein-coupled receptor SUCNR1, encode proteins with structural features or functions that suggest they may be involved in the modulation of platelet function. Immunoblotting on platelets confirmed the presence of the encoded proteins, and flow cytometric analysis confirmed the expression of G6b, G6f, and LRRC32 on the surface of platelets. Through comparative analysis of expression in platelets and other blood cells we demonstrated that G6b, G6f, and LRRC32 are restricted to the platelet lineage, whereas LAT2 and SUCNR1 were also detected in other blood cells. The identification of the succinate receptor SUCNR1 in platelets is of particular interest, because physiologically relevant concentrations of succinate were shown to potentiate the effect of low doses of a variety of platelet agonists.

Does Platelet Reactivity Predict Bleeding in Patients Needing Urgent Coronary Artery Bypass Grafting During Dual Antiplatelet Therapy?

PubMed

Mahla, Elisabeth; Prueller, Florian; Farzi, Sylvia; Pregartner, Gudrun; Raggam, Reinhard B; Beran, Elisabeth; Toller, Wolfgang; Berghold, Andrea; Tantry, Udaya S; Gurbel, Paul A

2016-12-01

Up to 15% of patients require coronary artery bypass grafting (CABG) during dual antiplatelet therapy. Available evidence suggests an association between platelet reactivity and CABG-related bleeding. However, platelet reactivity cutoffs for bleeding remain elusive. We sought to explore the association between platelet reactivity and bleeding. Patients on aspirin and a P2Y 12 receptor inhibitor within 48 hours before isolated CABG (n = 149) were enrolled in this prospective study. Blood was drawn 2 to 4 hours preoperatively and platelet reactivity assessed by light transmittance aggregometry (LTA), vasodilator-stimulated phosphoprotein (VASP) assay, Multiplate analyzer and Innovance PFA2Y. The primary endpoint was calculated red blood cell loss computed as follows: (blood volume × preoperative hematocrit × 0.91) - (blood volume × hematocrit × 0.91 on postoperative day 5) + (mL of transfused red blood cells × 0.59). Preoperative platelet reactivity was low [median (interquartile range): LTA: 20 (9-28)%; VASP-PRI: 39 (15-73)%; Multiplate adenosine phosphate test: 16 (12-22) U∗min]. Innovance PFA2Y ≥300 seconds, 72%. Median (IQR) red blood cell loss in patients in first the LTA tertile was 1,449 (1,020 to 1,754) mL compared with 1,107 (858 to 1,512) mL and 1,075 (811 to 1,269) mL in those in the second and third tertiles, respectively (p < 0.004). Bleeding Academic Research Consortium (BARC)-4 bleeding differed between tertiles (62% versus 46% versus 36%; p = 0.037). In a multivariable linear regression model, aspirin dose ≥300 mg, cardiopulmonary bypass time, EuroSCORE, and tertile distribution of platelet reactivity were significantly associated with red blood cell loss. A gradual decrease in red blood cell loss and BARC-4 bleeding occurs with increasing platelet reactivity in patients on antiplatelet therapy undergoing CABG. Our findings support current guidelines to determine time of surgery based on an objective measurement of platelet function (Platelet Inhibition and Bleeding in Patients Undergoing Emergent Cardiac Surgery; clinicaltrials.gov NCT01468597). Copyright © 2016 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

Platelets Mediate Host Defense against Staphylococcus aureus through Direct Bactericidal Activity and by Enhancing Macrophage Activities.

PubMed

Ali, Ramadan A; Wuescher, Leah M; Dona, Keith R; Worth, Randall G

2017-01-01

Platelets are the chief effector cells in hemostasis. However, recent evidence suggests they have multiple roles in host defense against infection. Reports by us and others showed that platelets functionally contribute to protection against Staphylococcus aureus infection. In the current study, the capacity of mouse platelets to participate in host defense against S. aureus infection was determined by assessing two possibilities. First, we determined the ability of platelets to kill S. aureus directly; and, second, we tested the possibility that platelets enhance macrophage phagocytosis and intracellular killing of S. aureus In this study we report evidence in support of both mechanisms. Platelets effectively killed two different strains of S. aureus. A clinical isolate of methicillin-resistant S. aureus was killed by platelets (>40% killing in 2 h) in a thrombin-dependent manner whereas a methicillin-sensitive strain was killed to equal extent but did not require thrombin. Interestingly, thrombin-stimulated platelets also significantly enhanced peritoneal macrophage phagocytosis of both methicillin-resistant S. aureus and methicillin-sensitive S. aureus by >70%, and restricted intracellular growth by >40%. Enhancement of macrophage anti-S. aureus activities is independent of contact with platelets but is mediated through releasable products, namely IL-1β. These data confirm our hypothesis that platelets participate in host defense against S. aureus both through direct killing of S. aureus and enhancing the antimicrobial function of macrophages in protection against S. aureus infection. Copyright © 2016 by The American Association of Immunologists, Inc.

Comparison of 3D cellular imaging techniques based on scanned electron probes: Serial block face SEM vs. Axial bright-field STEM tomography.

PubMed

McBride, E L; Rao, A; Zhang, G; Hoyne, J D; Calco, G N; Kuo, B C; He, Q; Prince, A A; Pokrovskaya, I D; Storrie, B; Sousa, A A; Aronova, M A; Leapman, R D

2018-06-01

Microscopies based on focused electron probes allow the cell biologist to image the 3D ultrastructure of eukaryotic cells and tissues extending over large volumes, thus providing new insight into the relationship between cellular architecture and function of organelles. Here we compare two such techniques: electron tomography in conjunction with axial bright-field scanning transmission electron microscopy (BF-STEM), and serial block face scanning electron microscopy (SBF-SEM). The advantages and limitations of each technique are illustrated by their application to determining the 3D ultrastructure of human blood platelets, by considering specimen geometry, specimen preparation, beam damage and image processing methods. Many features of the complex membranes composing the platelet organelles can be determined from both approaches, although STEM tomography offers a higher ∼3 nm isotropic pixel size, compared with ∼5 nm for SBF-SEM in the plane of the block face and ∼30 nm in the perpendicular direction. In this regard, we demonstrate that STEM tomography is advantageous for visualizing the platelet canalicular system, which consists of an interconnected network of narrow (∼50-100 nm) membranous cisternae. In contrast, SBF-SEM enables visualization of complete platelets, each of which extends ∼2 µm in minimum dimension, whereas BF-STEM tomography can typically only visualize approximately half of the platelet volume due to a rapid non-linear loss of signal in specimens of thickness greater than ∼1.5 µm. We also show that the limitations of each approach can be ameliorated by combining 3D and 2D measurements using a stereological approach. Copyright © 2018. Published by Elsevier Inc.

[Study on the difference of blood coagulation function in patients with traumatic brain injury in plain and plateau area].

PubMed

Sun, J; Tian, Y; Jiang, R C; Dong, X L; Wang, Y; Wu, W B; Wu, K X; Zhang, J N

2016-10-25

Objective: In this study, we tested platelet count (PC), prothrombin time (PT), activated partial thromboplastin time (APTT), and other indicators of coagulation function, and revealed their difference in patients with traumatic brain injury (TBI) between plain and plateau area. Base on the results, we may provide research basis for the therapy of TBI associated coagulopathy in different areas. Methods: 151 TBI patients from Tianjin Medical University General Hospital, and 74 from People's Hospital of Tibet Autonomous Region in the period from Dec 2013 to Dec 2015 were enrolled.Coagulation function, including PC, platelet distribution width (PDW), mean platelet volume (MPV), platelet - large cell ratio (P- LCR), PT, APTT, fibrinogen (FIB), and D- Dimer were tested within 8 h. The difference in patients with TBI between plain and plateau areas were compared and analyzed. Results: Compared with plain area, the PC of patients with TBI in plateau area is lower [(168±49)×10 9 /L vs (196±72)×10 9 /L, P <0.05], while PT and APTT were extended [(13.5±1.3) s vs (12.0±4.0) s, (38±4) s vs(27±6) s, P <0.01]. On the other hand, FIB increases [(3.1±1.2) g/L vs (2.6±1.0) g/L, P <0.01] and D-Dimer decreases [(3.1±3.3) μg/L vs (4.7±3.6) μg/L, P <0.01] in plateau area compared with plain area. Conclusion: Due to the people of plateau area living in hypoxia state, the coagulation function is activated for a long time.Once TBI happens, the platelets and coagulation factors may be excessive consumption, resulting in hypocoagulable state and high risk of rebleeding, while the fibrinolysis system in patients with TBI of plateau area is not activated obviously.Therefore, it should give full consideration to these differences in the treatment of patients with TBI in plateau area, instead of directly copying the standard therapy of the people in plain area.The treatment recommendations should primarily supplement coagulation materials, and antifibrinolytics may unlikely have the therapy effect.

Microfluidics for simultaneous quantification of platelet adhesion and blood viscosity

PubMed Central

Yeom, Eunseop; Park, Jun Hong; Kang, Yang Jun; Lee, Sang Joon

2016-01-01

Platelet functions, including adhesion, activation, and aggregation have an influence on thrombosis and the progression of atherosclerosis. In the present study, a new microfluidic-based method is proposed to estimate platelet adhesion and blood viscosity simultaneously. Blood sample flows into an H-shaped microfluidic device with a peristaltic pump. Since platelet aggregation may be initiated by the compression of rotors inside the peristaltic pump, platelet aggregates may adhere to the H-shaped channel. Through correlation mapping, which visualizes decorrelation of the streaming blood flow, the area of adhered platelets (APlatelet) can be estimated without labeling platelets. The platelet function is estimated by determining the representative index IA·T based on APlatelet and contact time. Blood viscosity is measured by monitoring the flow conditions in the one side channel of the H-shaped device. Based on the relation between interfacial width (W) and pressure ratio of sample flows to the reference, blood sample viscosity (μ) can be estimated by measuring W. Biophysical parameters (IA·T, μ) are compared for normal and diabetic rats using an ex vivo extracorporeal model. This microfluidic-based method can be used for evaluating variations in the platelet adhesion and blood viscosity of animal models with cardiovascular diseases under ex vivo conditions. PMID:27118101

Impact of acute exacerbations on platelet reactivity in chronic obstructive pulmonary disease patients.

PubMed

Muñoz-Esquerre, Mariana; Ferreiro, José Luis; Huertas, Daniel; Marcano, Ana Lucrecia; López-Sánchez, Marta; Roura, Gerard; Gómez-Hospital, Joan Antoni; Dorca, Jordi; Cequier, Angel; Santos, Salud

2018-01-01

A higher risk of atherothrombotic cardiovascular events, which are platelet-driven processes, has been described during acute exacerbations of chronic obstructive pulmonary disease (AECOPD). However, the relevance of platelet reactivity during AECOPD and whether this is affected by antiplatelet agents are not fully elucidated to date. This study aimed to evaluate whether platelet reactivity is augmented during an exacerbation in COPD patients with and without antiplatelet therapy and its association with systemic inflammatory parameters. Prospective, observational, ex vivo investigation was conducted in consecutive patients suffering an exacerbation of COPD. Platelet reactivity was assessed during AECOPD and at stable state. Platelet function assays included: 1) vasodilator-stimulated phosphoprotein assay expressed as P2Y 12 reactivity index (PRI), 2) multiple electrode aggregometry and 3) optical aggregometry. Systemic inflammatory parameters such as leukocyte count, interleukin-6 and fibrinogen were also assessed. Higher platelet reactivity was observed during AECOPD compared to stability measured by vasodilator-stimulated phosphoprotein (PRI: 75.2%±1.9% vs 68.8%±2.4%, p =0.001). This augmented platelet aggregability was also observed in the subset of patients on antiplatelet therapy (PRI: 72.8%±3.1% vs 61.7%±7.5%, p =0.071). Consistent findings were observed with all other platelet function tests. Patients with greater enhancement of inflammatory markers during AECOPD were more likely to present a higher increase in platelet reactivity. Platelet reactivity is increased during AECOPD, which may contribute to the augmented cardiovascular risk of these patients. Additionally, the increase in platelet reactivity might be associated with an increment in inflammatory markers during exacerbations.

Scalable Generation of Universal Platelets from Human Induced Pluripotent Stem Cells

PubMed Central

Feng, Qiang; Shabrani, Namrata; Thon, Jonathan N.; Huo, Hongguang; Thiel, Austin; Machlus, Kellie R.; Kim, Kyungho; Brooks, Julie; Li, Feng; Luo, Chenmei; Kimbrel, Erin A.; Wang, Jiwu; Kim, Kwang-Soo; Italiano, Joseph; Cho, Jaehyung; Lu, Shi-Jiang; Lanza, Robert

2014-01-01

Summary Human induced pluripotent stem cells (iPSCs) provide a potentially replenishable source for the production of transfusable platelets. Here, we describe a method to generate megakaryocytes (MKs) and functional platelets from iPSCs in a scalable manner under serum/feeder-free conditions. The method also permits the cryopreservation of MK progenitors, enabling a rapid “surge” capacity when large numbers of platelets are needed. Ultrastructural/morphological analyses show no major differences between iPSC platelets and human blood platelets. iPSC platelets form aggregates, lamellipodia, and filopodia after activation and circulate in macrophage-depleted animals and incorporate into developing mouse thrombi in a manner identical to human platelets. By knocking out the β2-microglobulin gene, we have generated platelets that are negative for the major histocompatibility antigens. The scalable generation of HLA-ABC-negative platelets from a renewable cell source represents an important step toward generating universal platelets for transfusion as well as a potential strategy for the management of platelet refractoriness. PMID:25418726

Scalable generation of universal platelets from human induced pluripotent stem cells.

PubMed

Feng, Qiang; Shabrani, Namrata; Thon, Jonathan N; Huo, Hongguang; Thiel, Austin; Machlus, Kellie R; Kim, Kyungho; Brooks, Julie; Li, Feng; Luo, Chenmei; Kimbrel, Erin A; Wang, Jiwu; Kim, Kwang-Soo; Italiano, Joseph; Cho, Jaehyung; Lu, Shi-Jiang; Lanza, Robert

2014-11-11

Human induced pluripotent stem cells (iPSCs) provide a potentially replenishable source for the production of transfusable platelets. Here, we describe a method to generate megakaryocytes (MKs) and functional platelets from iPSCs in a scalable manner under serum/feeder-free conditions. The method also permits the cryopreservation of MK progenitors, enabling a rapid "surge" capacity when large numbers of platelets are needed. Ultrastructural/morphological analyses show no major differences between iPSC platelets and human blood platelets. iPSC platelets form aggregates, lamellipodia, and filopodia after activation and circulate in macrophage-depleted animals and incorporate into developing mouse thrombi in a manner identical to human platelets. By knocking out the β2-microglobulin gene, we have generated platelets that are negative for the major histocompatibility antigens. The scalable generation of HLA-ABC-negative platelets from a renewable cell source represents an important step toward generating universal platelets for transfusion as well as a potential strategy for the management of platelet refractoriness.

Equol is more active than soy isoflavone itself to compete for binding to thromboxane A(2) receptor in human platelets.

PubMed

Muñoz, Yenny; Garrido, Argelia; Valladares, Luis

2009-03-01

Several dietary intervention studies examining the health effect of soy isoflavones allude to the importance of equol in establishing the cardiovascular response to soy protein. Although, the specific mechanism by which this action occurs has not been established. The aim of this study was to investigate the inhibitory effect of soy-isoflavones and the metabolite of daidzein, equol, on agonist-induced platelet responses dependent on thromboxane A(2) (TxA(2)) receptor. Competitive radioligand binding assay was used to screen for affinity of these compounds to the TxA(2) receptor. The effect of equol on platelet activation, evaluate through of release of the ATP, by analogs of TxA(2) was analyzed. The effect of equol on platelet aggregation was investigated with ADP, U46619 (a TxA(2) mimic) and the calcium ionophore A23187. The data showed that aglycone isoflavones and equol bind to TxA(2) receptor in the micromol/L range, whereas their glucoside derivates had very low binding activity for this receptor. Under equilibrium conditions, the following order of the relative affinity in inhibiting [(3)H]-SQ29585 binding was: equol>genistein>daidzein>glycitein>genistin, daidzin, glycitin. Equol interaction was reversible and competitive for labeled-SQ29548 with not apparent decrease in the number of TxA(2) binding sites. In addition, from platelet activation studies, equol effectively inhibited ATP secretion elicited by the TxA(2) analog U46619. On the other hand, equol inhibited the platelet aggregation induced by U46619 and A23187, while it failed to inhibit that induced by ADP. The aglycone isoflavones from soy, and particularly equol, have been found to have biological effects attributable to thromboxane A(2) receptor antagonism. These findings may help elucidate how dietary isoflavone modulate platelet function and explain why soy-rich foods are claimed to have beneficial effects in the prevention of thrombotic events.

CXC chemokine ligand 4 (Cxcl4) is a platelet-derived mediator of experimental liver fibrosis.

PubMed

Zaldivar, Mirko Moreno; Pauels, Katrin; von Hundelshausen, Philipp; Berres, Marie-Luise; Schmitz, Petra; Bornemann, Jörg; Kowalska, M Anna; Gassler, Nikolaus; Streetz, Konrad L; Weiskirchen, Ralf; Trautwein, Christian; Weber, Christian; Wasmuth, Hermann E

2010-04-01

Liver fibrosis is a major cause of morbidity and mortality worldwide. Platelets are involved in liver damage, but the underlying molecular mechanisms remain elusive. Here, we investigate the platelet-derived chemokine (C-X-C motif) ligand 4 (CXCL4) as a molecular mediator of fibrotic liver damage. Serum concentrations and intrahepatic messenger RNA of CXCL4 were measured in patients with chronic liver diseases and mice after toxic liver injury. Platelet aggregation in early fibrosis was determined by electron microscopy in patients and by immunohistochemistry in mice. Cxcl4(-/-) and wild-type mice were subjected to two models of chronic liver injury (CCl(4) and thioacetamide). The fibrotic phenotype was analyzed by histological, biochemical, and molecular analyses. Intrahepatic infiltration of immune cells was investigated by fluorescence-activated cell sorting, and stellate cells were stimulated with recombinant Cxcl4 in vitro. The results showed that patients with advanced hepatitis C virus-induced fibrosis or nonalcoholic steatohepatitis had increased serum levels and intrahepatic CXCL4 messenger RNA concentrations. Platelets were found directly adjacent to collagen fibrils. The CCl(4) and thioacetamide treatment led to an increase of hepatic Cxcl4 levels, platelet activation, and aggregation in early fibrosis in mice. Accordingly, genetic deletion of Cxcl4 in mice significantly reduced histological and biochemical liver damage in vivo, which was accompanied by changes in the expression of fibrosis-related genes (Timp-1 [tissue inhibitor of matrix metalloproteinase 1], Mmp9 [matrix metalloproteinase 9], Tgf-beta [transforming growth factor beta], IL10 [interleukin 10]). Functionally, Cxcl4(-/-) mice showed a strongly decreased infiltration of neutrophils (Ly6G) and CD8(+) T cells into the liver. In vitro, recombinant murine Cxcl4 stimulated the proliferation, chemotaxis, and chemokine expression of hepatic stellate cells. The results underscore an important role of platelets in chronic liver damage and imply a new target for antifibrotic therapies.

Evidence for shear-mediated Ca2+ entry through mechanosensitive cation channels in human platelets and a megakaryocytic cell line.

PubMed

Ilkan, Zeki; Wright, Joy R; Goodall, Alison H; Gibbins, Jonathan M; Jones, Chris I; Mahaut-Smith, Martyn P

2017-06-02

The role of mechanosensitive (MS) Ca 2+ -permeable ion channels in platelets is unclear, despite the importance of shear stress in platelet function and life-threatening thrombus formation. We therefore sought to investigate the expression and functional relevance of MS channels in human platelets. The effect of shear stress on Ca 2+ entry in human platelets and Meg-01 megakaryocytic cells loaded with Fluo-3 was examined by confocal microscopy. Cells were attached to glass coverslips within flow chambers that allowed applications of physiological and pathological shear stress. Arterial shear (1002.6 s -1 ) induced a sustained increase in [Ca 2+ ] i in Meg-01 cells and enhanced the frequency of repetitive Ca 2+ transients by 80% in platelets. These Ca 2+ increases were abrogated by the MS channel inhibitor Grammostola spatulata mechanotoxin 4 (GsMTx-4) or by chelation of extracellular Ca 2+ Thrombus formation was studied on collagen-coated surfaces using DiOC 6 -stained platelets. In addition, [Ca 2+ ] i and functional responses of washed platelet suspensions were studied with Fura-2 and light transmission aggregometry, respectively. Thrombus size was reduced 50% by GsMTx-4, independently of P2X1 receptors. In contrast, GsMTx-4 had no effect on collagen-induced aggregation or on Ca 2+ influx via TRPC6 or Orai1 channels and caused only a minor inhibition of P2X1-dependent Ca 2+ entry. The Piezo1 agonist, Yoda1, potentiated shear-dependent platelet Ca 2+ transients by 170%. Piezo1 mRNA transcripts and protein were detected with quantitative RT-PCR and Western blotting, respectively, in both platelets and Meg-01 cells. We conclude that platelets and Meg-01 cells express the MS cation channel Piezo1, which may contribute to Ca 2+ entry and thrombus formation under arterial shear. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The predictive value of multiple electrode platelet aggregometry for postoperative bleeding complications in patients undergoing coronary artery bypass graft surgery

PubMed Central

Woźniak, Karolina; Hryniewiecki, Tomasz; Kruk, Mariusz; Różański, Jacek; Kuśmierczyk, Mariusz

2016-01-01

Introduction Postoperative bleeding is one of the most serious complications of cardiac surgery and requires transfusion of blood or blood products. Acetylsalicylic acid (ASA) and clopidogrel (CLO) are the two most commonly used antiplatelet agents; when used in combination (i.e., as dual antiplatelet therapy [DAPT]), they exert a synergistic effect. Dual antiplatelet therapy, however, significantly increases the risk of postoperative bleeding. The effect of antiplatelet therapy can be monitored by platelet aggregation testing. One of the most commonly methods used for assessing platelet reactivity is multiple electrode aggregometry (MEA) which can be performed with the use of Multiplate analyzer. Although the method has long been used in interventional cardiology to assess the effect of antiplatelet therapy, it is not available at cardiac surgery departments as a standard diagnostic procedure. The aim of the study was to establish the frequency of bleeding complications following coronary artery bypass graft (CABG) surgery in patients on single antiplatelet therapy (SAPT) and patients on DAPT and to determine the usefulness of routine measurement of platelet responsiveness before CABG surgery in patients receiving antiplatelet therapy. Material and methods A consecutive cohort of 200 patients referred for elective surgical treatment of stable coronary artery disease was enrolled (100 consecutive patients on SAPT [ASA 75 mg/day] and 100 consecutive patients on DAPT [ASA 75 mg/day + CLO 75 mg/day]). All subjects continued their antiplatelet therapy until the day before surgery. For each subject, platelet aggregation testing in the form of an ASPI test and an ADP test was performed on the Multiplate analyzer. Each subject underwent coronary artery bypass grafting surgery. For the primary and secondary endpoints in our study we adopted the definition provided in ‘Standardised Bleeding Definitions for Cardiovascular Clinical Trials: A Consensus Report from the Bleeding Academic Research Consortium’ (‘Circulation’, 2011) for BARC type 4 bleeding (i.e. CABG-related bleeding). Results An ROC curve was constructed for the ASPI test and ADP test for a total of 200 patients. No significant correlations were demonstrated between the ASPI test results and either the primary endpoint or the secondary endpoints. A correlation was found between the ADP test results and the composite primary endpoint and each of the secondary endpoints. The primary endpoint of major postoperative bleeding occurred in 16 subjects. From the ROC curve, we established the optimal cut-off value for the ADP test of 26 U at sensitivity of 72%, specificity of 69%, positive predictive value of 69.90%, and negative predictive value of 71.13%. Conclusions In patients on antiplatelet therapy, an ADP test result of < 26 U is strongly predictive of serious bleeding complications after CABG surgery. The MEA ADP test allows to identify the group of patients at an increased risk of postoperative bleeding. PMID:27212971

Platelets subvert T cell immunity against cancer via GARP-TGFβ axis.

PubMed

Rachidi, Saleh; Metelli, Alessandra; Riesenberg, Brian; Wu, Bill X; Nelson, Michelle H; Wallace, Caroline; Paulos, Chrystal M; Rubinstein, Mark P; Garrett-Mayer, Elizabeth; Hennig, Mirko; Bearden, Daniel W; Yang, Yi; Liu, Bei; Li, Zihai

2017-05-05

Cancer-associated thrombocytosis has long been linked to poor clinical outcome, but the underlying mechanism is enigmatic. We hypothesized that platelets promote malignancy and resistance to therapy by dampening host immunity. We show that genetic targeting of platelets enhances adoptive T cell therapy of cancer. An unbiased biochemical and structural biology approach established transforming growth factor β (TGFβ) and lactate as major platelet-derived soluble factors to obliterate CD4 + and CD8 + T cell functions. Moreover, we found that platelets are the dominant source of functional TGFβ systemically as well as in the tumor microenvironment through constitutive expression of the TGFβ-docking receptor glycoprotein A repetitions predominant (GARP) rather than secretion of TGFβ per se. Platelet-specific deletion of the GARP-encoding gene Lrrc32 blunted TGFβ activity at the tumor site and potentiated protective immunity against both melanoma and colon cancer. Last, this study shows that T cell therapy of cancer can be substantially improved by concurrent treatment with readily available antiplatelet agents. We conclude that platelets constrain T cell immunity through a GARP-TGFβ axis and suggest a combination of immunotherapy and platelet inhibitors as a therapeutic strategy against cancer. Copyright © 2017, American Association for the Advancement of Science.

Comparison of the inhibitory effects of cilostazol, acetylsalicylic acid and ticlopidine on platelet functions ex vivo. Randomized, double-blind cross-over study.

PubMed

Ikeda, Y; Kikuchi, M; Murakami, H; Satoh, K; Murata, M; Watanabe, K; Ando, Y

1987-05-01

A randomized double-blind cross-over study was conducted to determine the inhibitory effects of acetylsalicylic acid (ASA), ticlopidine (TP) and cilostazol (OPC-13013; in the following briefly called CS), a new antithrombotic agent on platelet functions ex vivo. Nine patients with cerebral thrombosis were enrolled in this study. Patients were given each of the three drugs for one week in a complete cross-over design according to a randomization schedule, followed by a wash-out period with a placebo for one week. It was found that CS and TP significantly inhibited platelet aggregation induced by ADP. Collagen- and arachidonic acid-induced platelet aggregation was all inhibited by CS, TP and ASA. Duncan's multiple range test to compare the anti-platelet effects of the three drugs revealed that: CS greater than ASA and TP greater than ASA in inhibiting ADP-induced platelet aggregation and CS greater than TP and ASA greater than TP in inhibiting arachidonic acid-induced platelet aggregation. These results may suggest that CS is superior to ASA and TP in inhibiting platelet aggregation ex vivo.

Basic characteristics of plasma rich in growth factors (PRGF): blood cell components and biological effects

PubMed Central

Nishiyama, Kazuhiko; Okudera, Toshimitsu; Watanabe, Taisuke; Isobe, Kazushige; Suzuki, Masashi; Masuki, Hideo; Okudera, Hajime; Uematsu, Kohya; Nakata, Koh

2016-01-01

Abstract Platelet‐rich plasma (PRP) is widely used in regenerative medicine because of its high concentrations of various growth factors and platelets. However, the distribution of blood cell components has not been investigated in either PRP or other PRP derivatives. In this study, we focused on plasma rich in growth factors (PRGF), a PRP derivative, and analyzed the distributions of platelets and white blood cells (WBCs). Peripheral blood samples were collected from healthy volunteers (N = 14) and centrifuged to prepare PRGF and PRP. Blood cells were counted using an automated hematology analyzer. The effects of PRP and PRGF preparations on cell proliferation were determined using human periosteal cells. In the PRGF preparations, both red blood cells and WBCs were almost completely eliminated, and platelets were concentrated by 2.84‐fold, whereas in the PRP preparations, both platelets and WBCs were similarly concentrated by 8.79‐ and 5.51‐fold, respectively. Platelet counts in the PRGF preparations were positively correlated with platelet counts in the whole blood samples, while the platelet concentration rate was negatively correlated with red blood cell counts in the whole blood samples. In contrast, platelet counts and concentration rates in the PRP preparations were significantly influenced by WBC counts in whole blood samples. The PRP preparations, but not the PRGF preparations, significantly suppressed cell growth at higher doses in vitro. Therefore, these results suggest that PRGF preparations can clearly be distinguished from PRP preparations by both inclusion of WBCs and dose‐dependent stimulation of periosteal cell proliferation in vitro. PMID:29744155

Assessment of canine autologous platelet-rich plasma produced with a commercial centrifugation and platelet recovery kit.

PubMed

Frye, Chris W; Enders, Andrew; Brooks, Marjory B; Struble, Angela M; Wakshlag, Joseph J

2016-01-01

To characterize the cellular composition (platelets, erythrocytes, and leukocytes) and confirm reproducibility of platelet enrichment, as well as determine the platelet activation status in the final product of a commercial platelet-rich plasma kit using canine blood. Venous blood from 20 sedated client-owned dogs was used to prepare platelet-rich plasma (PRP) from a commercial kit. Complete blood counts were performed to determine erythrocyte, leukocyte, and platelet numbers in both whole blood (WB) and resultant PRP. The WB and PRP samples from jugular (fast collection) and cephalic (slow collection) venipuncture were also compared. P-selectin externalization was measured in WB and PRP samples from 15 of 20 dogs. This commercial kit produced an average percent recovery in platelets of 64.7 ± 17.4; erythrocytes of 3.7 ± 0.8, and leukocytes of 31.6 ± 10.0. Neutrophil, monocyte, and lymphocyte percent recovery was 19.6 ± 7.2, 44.89 ± 19.8, and 57.5 ± 10.6, respectively. The recovery of platelets from jugular venipuncture (59.7 ± 13.6%) was lower than from cephalic recovery (68.8 ± 19.1%). The mean percent P-Selectin externalization for WB, PRP, and PRP with thrombin was 25.5 ± 30.9, 4.5 ± 6.4, and 90.6 ± 4.4 respectively. Cellular reproducibility of this kit was confirmed and platelets were concentrated within autologous serum. Additionally, measurements of P-selectin externalization showed that platelets are inactive in PRP unless stimulated to degranulate.

The C-terminal CGHC motif of protein disulfide isomerase supports thrombosis

PubMed Central

Zhou, Junsong; Wu, Yi; Wang, Lu; Rauova, Lubica; Hayes, Vincent M.; Poncz, Mortimer; Essex, David W.

2015-01-01

Protein disulfide isomerase (PDI) has two distinct CGHC redox-active sites; however, the contribution of these sites during different physiologic reactions, including thrombosis, is unknown. Here, we evaluated the role of PDI and redox-active sites of PDI in thrombosis by generating mice with blood cells and vessel wall cells lacking PDI (Mx1-Cre Pdifl/fl mice) and transgenic mice harboring PDI that lacks a functional C-terminal CGHC motif [PDI(ss-oo) mice]. Both mouse models showed decreased fibrin deposition and platelet accumulation in laser-induced cremaster arteriole injury, and PDI(ss-oo) mice had attenuated platelet accumulation in FeCl3-induced mesenteric arterial injury. These defects were rescued by infusion of recombinant PDI containing only a functional C-terminal CGHC motif [PDI(oo-ss)]. PDI infusion restored fibrin formation, but not platelet accumulation, in eptifibatide-treated wild-type mice, suggesting a direct role of PDI in coagulation. In vitro aggregation of platelets from PDI(ss-oo) mice and PDI-null platelets was reduced; however, this defect was rescued by recombinant PDI(oo-ss). In human platelets, recombinant PDI(ss-oo) inhibited aggregation, while recombinant PDI(oo-ss) potentiated aggregation. Platelet secretion assays demonstrated that the C-terminal CGHC motif of PDI is important for P-selectin expression and ATP secretion through a non-αIIbβ3 substrate. In summary, our results indicate that the C-terminal CGHC motif of PDI is important for platelet function and coagulation. PMID:26529254

Effect of diazepam and clonazepam on the function of isolated rat platelet and neutrophil.

PubMed

Rajtar, Grazyna; Zółkowska, Dorota; Kleinrok, Zdzisław

2002-04-01

Benzodiazepine binding sites distinct from the GABA-receptor-chloride-complex in the central nervous system have been recognized in many peripheral tissues, but their physiological role remains unexplained. Our study was undertaken to examine the effects of diazepam, clonazepam, and PK 11195, a peripheral benzodiazepine receptor antagonist, on the functional and biochemical responses of platelets and neutrophils stimulated by different physiological agonists. The experiments were conducted on isolated washed rat platelets activated by arachidonic acid (AA), adenosine 5'-diphosphate (ADP), or thrombin and on isolated rat neutrophils activated by a chemotactic peptide, formyl methionyl leucyl phenylalanine (fMLP). The results showed that neither diazepam nor clonazepam nor PK 11195 alone augmented the response of resting platelets or modified neutrophil response, but diazepam and clonazepam in a concentration-dependent manner inhibited thrombin, ADP or AA-stimulated platelet aggregation and the thrombin-induced increase in free intracellular Ca2+. Both drugs also exerted an inhibitory effect on reactive oxygen species (ROS) produced by fMLP-stimulated neutrophils. However, diazepam was about 10 times more effective than clonazepam. PK11195 did not influence platelet and neutrophil function stimulated by agonists, but reversed the inhibitory action of both benzodiazepines on platelet activation and ROS production. The results indicated that in vitro diazepam, and in a much smaller degree clonazepam, may down-regulate platelet activation and release of some proinflammatory mediators by stimulated neutrophils. These effects are probably exerted by a specific benzodiazepine binding sites.

Dose response of surfactants to attenuate gas embolism related platelet aggregation

NASA Astrophysics Data System (ADS)

Eckmann, David M.; Eckmann, Yonaton Y.; Tomczyk, Nancy

2014-03-01

Intravascular gas embolism promotes blood clot formation, cellular activation, and adhesion events, particularly with platelets. Populating the interface with surfactants is a chemical-based intervention to reduce injury from gas embolism. We studied platelet activation and platelet aggregation, prominent adverse responses to blood contact with bubbles. We examined dose-response relationships for two chemically distinct surfactants to attenuate the rise in platelet function stimulated by exposure to microbubbles. Significant reduction in platelet aggregation and platelet activation occurred with increasing concentration of the surfactants, indicating presence of a saturable system. A population balance model for platelet aggregation in the presence of embolism bubbles and surfactants was developed. Monte Carlo simulations for platelet aggregation were performed. Results agree qualitatively with experimental findings. Surfactant dose-dependent reductions in platelet activation and aggregation indicate inhibition of the gas/liquid interface's ability to stimulate cellular activation mechanically.

Platelet response heterogeneity in thrombus formation.

PubMed

Munnix, Imke C A; Cosemans, Judith M E M; Auger, Jocelyn M; Heemskerk, Johan W M

2009-12-01

Vascular injury leads to formation of a structured thrombus as a consequence of platelet activation and aggregation, thrombin and fibrin formation, and trapping of leukocytes and red cells. This review summarises current evidence for heterogeneity of platelet responses and functions in the thrombus-forming process. Environmental factors contribute to response heterogeneity, as the platelets in a thrombus adhere to different substrates, and sense specific (ant)agonists and rheological conditions. Contraction of platelets and interaction with fibrin and other blood cells cause further response variation. On the other hand, response heterogeneity can also be due to intrinsic differences between platelets in age and in receptor and signalling proteins. As a result, at least three subpopulations of platelets are formed in a thrombus: aggregating platelets with (reversible) integrin activation, procoagulant (coated) platelets exposing phosphatidylserine and binding coagulation factors, and contracting platelets with cell-cell contacts. This recognition of thrombus heterogeneity has implications for the use and development of antiplatelet medication.

Shiga Toxin 2 and Lipopolysaccharide Induce Human Microvascular Endothelial Cells To Release Chemokines and Factors That Stimulate Platelet Function

PubMed Central

Guessous, Fadila; Marcinkiewicz, Marek; Polanowska-Grabowska, Renata; Kongkhum, Sudawadee; Heatherly, Daniel; Obrig, Tom; Gear, Adrian R. L.

2005-01-01

Shiga toxins (Stxs) produced by Shigella dysenteriae type 1 and enterohemorrhagic Escherichia coli are the most common cause of hemolytic-uremic syndrome (HUS). It is well established that vascular endothelial cells, mainly those located in the renal microvasculature, are targets for Stxs. The aim of the present research was to evaluate whether E. coli-derived Shiga toxin 2 (Stx2) incubated with human microvascular endothelial cells (HMEC-1) induces release of chemokines and other factors that might stimulate platelet function. HMEC-1 were exposed for 24 h in vitro to Stx2, lipopolysaccharide (LPS), or the Stx2-LPS combination, and chemokine production was assessed by immunoassay. More interleukin-8 was released than stromal cell-derived factor 1α (SDF-1α) or SDF-1β and RANTES. The Stx2-LPS combination potentiated chemokine release, but Stx2 alone caused more release of SDF-1α at 24 h than LPS or Stx2-LPS did. In the presence of low ADP levels, HMEC-1 supernatants activated platelet function assessed by classical aggregometry, single-particle counting, granule secretion, P-selectin exposure, and the formation of platelet-monocyte aggregates. Supernatants from HMEC-1 exposed only to Stx2 exhibited enhanced exposure of platelet P-selectin and platelet-THP-1 cell interactions. Blockade of platelet cyclooxygenase by indomethacin prevented functional activation. The chemokine RANTES enhanced platelet aggregation induced by SDF-1α, macrophage-derived chemokine, or thymus and activation-regulated chemokine in the presence of very low ADP levels. These data support the hypothesis that microvascular endothelial cells exposed to E. coli O157:H7-derived Stx2 and LPS release chemokines and other factors, which when combined with low levels of primary agonists, such as ADP, cause platelet activation and promote the renal thrombosis associated with HUS. PMID:16299328

Expression and functionality of Toll-like receptor 3 in the megakaryocytic lineage

PubMed Central

D’Atri, L. P.; Etulain, J.; Rivadeneyra, L.; Lapponi, M. J.; Centurion, M.; Cheng, K.; Yin, H.; Schattner, M.

2015-01-01

Summary Background In addition to their key role in hemostasis, platelets and megakaryocytes also regulate immune and inflammatory responses, in part through their expression of Toll-like receptors (TLRs). Among the TLRs, TLR3 recognizes double-stranded (ds) RNA associated with viral infection. Thrombocytopenia is a frequent complication of viral infection. However, the expression and functionality of TLR3 in megakaryocytes and platelets is not yet well understood. Objective To study the expression and functionality of TLR3 in the megakaryocytic lineage. Methods and Results RT-PCR, flow cytometric, and immunofluorescence assays showed that TLR3 is expressed in CD34+ cells, megakaryocytes, and platelets. Immunoblotting assays showed that stimulation of megakaryocytes with two synthetic agonists of TLR3, Poly(I:C) and Poly(A:U), activated the NF-κB, PI3K/Akt, ERK1/2, and p38 pathways. TLR3-megakaryocyte activation resulted in reduced platelet production in vitro and IFN-β release through the PI3K/Akt and NF-κB signaling pathways. TLR3 ligands potentiated the aggregation mediated by classical platelet agonists. This effect was also observed for ATP release, but not for P-selectin or CD40L membrane exposure, indicating that TLR3 activation was not involved in alpha granule release. In addition, TLR3 agonists induced activation of the NF-κB, PI3K/Akt, and ERK1/2 pathways in platelets. Reduction of platelet production and platelet fibrinogen binding mediated by Poly(I:C) or Poly(A:U) were prevented by the presence of an inhibitor of TLR3/dsRNA complex. Conclusions Our findings indicate that functional TLR3 is expressed in CD34+ cells, megakaryocytes, and platelets, and suggest a potential role for this receptor in the megakaryo/thrombopoiesis alterations that occur in viral infections. PMID:25594115

Platelet granule release is associated with reactive oxygen species generation during platelet storage: A direct link between platelet pro-inflammatory and oxidation states.

PubMed

Ghasemzadeh, Mehran; Hosseini, Ehteramolsadat

2017-08-01

Upon platelet stimulation with agonists, reactive oxygen species (ROS) generation enhances platelet activation and granule release. Whether ROS generation during platelet storage could be directly correlated with the expression of proinflammatory molecules and granule release has been investigated in this study. PRP-platelet concentrates were subjected to flowcytometry analysis to assess the expression of platelet activation marker, P-selectin and CD40L during storage. Intracellular ROS generation was also detected in platelet by flowcytometry using dihydrorhodamine (DHR) 123. Through the dual staining, ROS production was analyzed in either P-selectin positive or negative populations. ROS formation in platelet population was significantly increased by either TRAP (a potent agonist that induces granule release) or PMA (a classic inducer of ROS generation), while the effects of each agonists on P-selectin expression and ROS generation in platelets were comparable. Platelet storage was also associated with the increasing levels of ROS (day 0 vs. day 5; p<0.001) while this increasing pattern was directly correlated with the either expressed P-selectin or CD40L. In addition, in 5 day-stored platelets, samples with ROS levels above 40% showed significantly higher levels of P-selectin and CD40L expression. P-selectin negative population of platelet did not show significant amount of ROS. Our data demonstrated decreased levels of important platelet pro-inflammatory molecules in stored platelets with lower levels of intraplatelet ROS. However, whether quenching of ROS generation during platelet storage can attenuate adverse transfusion reactions raised by platelet pro-inflammatory status is required to be further studied. Copyright © 2017 Elsevier Ltd. All rights reserved.

Possible roles of platelet-derived microparticles in atherosclerosis.

PubMed

Wang, Zhi-Ting; Wang, Zi; Hu, Yan-Wei

2016-05-01

Platelets and platelet-derived microparticles (PMPs) play important roles in cardiovascular diseases, especially atherosclerosis. Continued research has revealed that PMPs have numerous functions in atherosclerosis, not only in thrombosis formation, but also by induction of inflammation. PMPs also induce formation of foam cells. Recent evidence strongly indicates a significant role of PMPs in atherosclerosis. Here, current research on the function of PMPs in atherosclerosis is reviewed. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Method of preparing copper-dendritic composite alloys for mechanical reduction

DOEpatents

Verhoeven, John D.; Gibson, Edwin D.; Schmidt, Frederick A.; Spitzig, William A.

1988-01-01

Copper-dendritic composite alloys are prepared for mechanical reduction to increase tensile strength by dispersing molten droplets of the composite alloy into an inert gas; solidifying the droplets in the form of minute spheres or platelets; and compacting a mass of the spheres or platelets into an integrated body. The spheres preferably have diameters of from 50 to 2000 .mu.m, and the platelets thicknesses of 100 to 2000 .mu.m. The resulting spheres or platelets will contain ultra-fine dendrites which produce higher strengths on mechanical reduction of the bodies formed therefrom, or comparable strengths at lower reduction values. The method is applicable to alloys of copper with vanadium, niobium, tantalum, chromium, molybdenum, tungsten, iron and cobalt.

Method of preparing copper-dendritic composite alloys for mechanical reduction

DOEpatents

Verhoeven, J.D.; Gibson, E.D.; Schmidt, F.A.; Spitzig, W.A.

1988-09-13

Copper-dendritic composite alloys are prepared for mechanical reduction to increase tensile strength by dispersing molten droplets of the composite alloy into an inert gas; solidifying the droplets in the form of minute spheres or platelets; and compacting a mass of the spheres or platelets into an integrated body. The spheres preferably have diameters of from 50 to 2,000 [mu]m, and the platelets thicknesses of 100 to 2,000 [mu]m. The resulting spheres or platelets will contain ultra-fine dendrites which produce higher strengths on mechanical reduction of the bodies formed therefrom, or comparable strengths at lower reduction values. The method is applicable to alloys of copper with vanadium, niobium, tantalum, chromium, molybdenum, tungsten, iron and cobalt. 3 figs.

Platelets and their interactions with other immune cells

PubMed Central

Lam, Fong W.; Vijayan, K. Vinod; Rumbaut, Rolando E.

2015-01-01

Platelets are anucleate blood cells, long known to be critically involved in hemostasis and thrombosis. In addition to their role in blood clots, increasing evidence reveals significant roles for platelets in inflammation and immunity. However, the notion that platelets represent immune cells is not broadly recognized in the field of Physiology. This manuscript reviews the role of platelets in inflammation and immune responses, and highlights their interactions with other immune cells, including examples of major functional consequences of these interactions. PMID:26140718

Hexamoll DINCH plasticised PVC containers for the storage of platelets

PubMed Central

Nair, C. S. Bhaskaran; Vidya, R.; Ashalatha, P. M.

2011-01-01

Introduction: Containers for the storage of platelets are made using polyvinyl chloride plasticised with di, (2-ethyl hexyl) phthalate, n-butyryl, tri (n-hexyl) citrate and tri (2-ethyl hexyl) mellitate or using special poly olefins without plasticiser. Of these, the first two have disadvantages such as plasticiser leaching and impairment of platelet function. Polyolefin bags cannot be HF welded or steam sterilized. Mellitate plasticised bags can store platelets well for five days but they are not completely phthalate free. Research and Development: We have developed a new generation of containers made of PVC plasticised with the non DEHP, non aromatic plasticiser,1,2- Cyclohexanedicarboxylic acid, diisononyl ester (Hexamoll DINCH) which can store platelets without loss of function for at least six days. Observation: The present studies show that DINCH plasticised PVC bags (TPL-167) are well suited for the storage of platelet concentrates for more than five days. Conclusion: The present studies show that the PVC plasticised with the non phthalate, non aromatic, non toxic plasticiser DINCH is a viable alternative to other existing containers for the storage of platelets for more than five days. PMID:21572709

Platelets: versatile effector cells in hemostasis, inflammation, and the immune continuum

PubMed Central

Vieira-de-Abreu, Adriana; Campbell, Robert A.; Weyrich, Andrew S.

2015-01-01

Platelets are chief effector cells in hemostasis. In addition, however, their specializations include activities and intercellular interactions that make them key effectors in inflammation and in the continuum of innate and adaptive immunity. This review focuses on the immune features of human platelets and platelets from experimental animals and on interactions between inflammatory, immune, and hemostatic activities of these anucleate but complex and versatile cells. The experimental findings and evidence for physiologic immune functions include previously unrecognized biologic characteristics of platelets and are paralleled by new evidence for unique roles of platelets in inflammatory, immune, and thrombotic diseases. PMID:21818701