Label-free detection of aggregated platelets in blood by machine-learning-aided optofluidic time-stretch microscopy.

PubMed

Jiang, Yiyue; Lei, Cheng; Yasumoto, Atsushi; Kobayashi, Hirofumi; Aisaka, Yuri; Ito, Takuro; Guo, Baoshan; Nitta, Nao; Kutsuna, Natsumaro; Ozeki, Yasuyuki; Nakagawa, Atsuhiro; Yatomi, Yutaka; Goda, Keisuke

2017-07-11

According to WHO, about 10 million new cases of thrombotic disorders are diagnosed worldwide every year. Thrombotic disorders, including atherothrombosis (the leading cause of death in the US and Europe), are induced by occlusion of blood vessels, due to the formation of blood clots in which aggregated platelets play an important role. The presence of aggregated platelets in blood may be related to atherothrombosis (especially acute myocardial infarction) and is, hence, useful as a potential biomarker for the disease. However, conventional high-throughput blood analysers fail to accurately identify aggregated platelets in blood. Here we present an in vitro on-chip assay for label-free, single-cell image-based detection of aggregated platelets in human blood. This assay builds on a combination of optofluidic time-stretch microscopy on a microfluidic chip operating at a high throughput of 10 000 blood cells per second with machine learning, enabling morphology-based identification and enumeration of aggregated platelets in a short period of time. By performing cell classification with machine learning, we differentiate aggregated platelets from single platelets and white blood cells with a high specificity and sensitivity of 96.6% for both. Our results indicate that the assay is potentially promising as predictive diagnosis and therapeutic monitoring of thrombotic disorders in clinical settings.

A Human Platelet Calcium Calculator Trained by Pairwise Agonist Scanning

PubMed Central

Lee, Mei Yan; Diamond, Scott L.

2015-01-01

Since platelet intracellular calcium mobilization [Ca(t)]i controls granule release, cyclooxygenase-1 and integrin activation, and phosphatidylserine exposure, blood clotting simulations require prediction of platelet [Ca(t)]i in response to combinatorial agonists. Pairwise Agonist Scanning (PAS) deployed all single and pairwise combinations of six agonists (ADP, convulxin, thrombin, U46619, iloprost and GSNO used at 0.1, 1, and 10xEC50; 154 conditions including a null condition) to stimulate platelet P2Y1/P2Y12 GPVI, PAR1/PAR4, TP, IP receptors, and guanylate cyclase, respectively, in Factor Xa-inhibited (250 nM apixaban), diluted platelet rich plasma that had been loaded with the calcium dye Fluo-4 NW. PAS of 10 healthy donors provided [Ca(t)]i data for training 10 neural networks (NN, 2-layer/12-nodes) per donor. Trinary stimulations were then conducted at all 0.1x and 1xEC50 doses (160 conditions) as was a sampling of 45 higher ordered combinations (four to six agonists). The NN-ensemble average was a calcium calculator that accurately predicted [Ca (t)]i beyond the single and binary training set for trinary stimulations (R = 0.924). The 160 trinary synergy scores, a normalized metric of signaling crosstalk, were also well predicted (R = 0.850) as were the calcium dynamics (R = 0.871) and high-dimensional synergy scores (R = 0.695) for the 45 higher ordered conditions. The calculator even predicted sequential addition experiments (n = 54 conditions, R = 0.921). NN-ensemble is a fast calcium calculator, ideal for multiscale clotting simulations that include spatiotemporal concentrations of ADP, collagen, thrombin, thromboxane, prostacyclin, and nitric oxide. PMID:25723389

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).

5-HT receptor probe (/sup 3/H)8-OH-DPAT labels the 5-HT transporter in human platelets

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

Ieni, J.R.; Meyerson, L.R.

1988-01-01

The present study characterizes a serotonin (5-HT) binding site on human platelet membranes, using (/sup 3/H)8-OH-DPAT as the radioligand. (/sup 3/H)8-OH-DPAT binds specifically and saturably to a site on human platelet membranes with an average K/sub D/ of 43 nM and B/sub max/ of 1078 fmol/mg protein. Determinations of IC/sub 50/ values for various serotonergic characterizing agents in platelets for displacement of (/sup 3/H)8-OH-DPAT were performed. The pharmacological inhibitory profile of the platelet 8-OH-DPAT site is not consistent with profiles reported for brain. 8-OH-DPAT does not inhibit (/sup 3/H) imipramine binding, however, it does inhibit (/sup 3/H)5-HT uptake in humanmore » platelets near 5-HT's K/sub m/ value (IC/sub 50/ = 2-4 ..mu..M). These results suggest that the human platelet site labelled by (/sub 3/H)8-OH-DPAT is pharmocologically different from the neuronal site and probably is a component of the 5-HT transporter. 32 references, 1 figure, 4 tables.« less

Distribution and dosimetry of In-111 labeled leukocytes and platelets in humans

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

Goodwin, D.A.; Finston, R.A.; Smith, S.I.

1981-06-01

The distribution of In-111 labeled leukocytes and platelets was studied by whole body gamma camera imaging in patients. Images were made approximately one hour and 24 hours after IV injection, and stored in digital form in computer memory. Estimates of the quantitative organ distribution were made from the geometric mean of the anterior and posterior region of interest counts after suitable background subtraction. Nearly quantitative retention of cell activity was observed with little or no excretion seen in either gut or kidneys. Mixed leukocytes distributed in spleen, liver and bone marrow in decreasing order of concentration, similar at both times,more » with a transient lung uptake noted at the one hour time only. The dose from 0.5 mCi In-111-WBC's was: liver, 1.4 rad; spleen, 8.5 rad; marrow, 2.3 rad. Lymphocytes had similar distribution with the addition of inguinal and cervical lymph nodes. The dose from 0.5 mCi In-111-lymphocytes was: liver, 0.8 rad; spleen, 6.7 rad; marrow and lymphatic tissue, 1.4 rad. Platelets distributed primarily in the blood pool with most of the remainder concentrating in the spleen, with a small amount in the penis. The dose from 0.5 mCi of In-111-platelets was: liver, 3.2 rad; spleen, 8.6 rad; and whole body, 0.3 rad.« less

Platelet kinetics and biodistribution in canine endotoxemia

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

Sostman, H.D.; Zoghbi, S.S.; Smith, G.J.

Kinetics and magnitudes of changes in indium-labeled platelet biodistribution were studied in dogs given E. coli endotoxin. Marked, reversible, dose-dependent shifts of platelets from blood to lung and apparently irreversible shifts to liver were demonstrated. These were contemporaneous with alterations in blood gases and in pulmonary and systemic hemodynamics. Morphologic studies revealed atelectasis, sequestration of leukocytes and platelets in the lungs, and mild interstitial pulmonary edema. This study provides in vivo quantification of labeled platelet response to a specific stimulus, and illustrates a method that could be applied to more extensive study of blood element participation in acute lung injury.

Lea blood group antigen on human platelets.

PubMed

Dunstan, R A; Simpson, M B; Rosse, W F

1985-01-01

One- and two-stage radioligand assays were used to determine if human platelets possess the Lea antigen. Goat IgG anti-Lea antibody was purified by multiple adsorptions with Le(a-b-) human red blood cells, followed by affinity chromatography with synthetic Lea substance and labeling with 125I. Human IgG anti-Lea antibody was used either in a two stage radioassay with 125I-labeled mouse monoclonal IgG anti-human IgG as the second antibody or, alternatively, purified by Staph protein A chromatography, labeled with 125I, and used in a one-stage radioassay. Platelets from donors of appropriate red blood cell phenotypes were incubated with the antisera, centrifuged through phthalate esters, and assayed in a gamma scintillation counter. Dose response and saturation curve analysis demonstrate the presence of Lewis a antigen on platelets from Lea+ donors. Furthermore, platelets from an Le(a-b-) donor incubated in Le (a+b-) plasma adsorb Lea antigen in a similar manner to red blood cells. The clinical significance of these antigens in platelet transfusion remains undefined.

Aspirin inhibition of platelet deposition at angioplasty sites: demonstration by platelet scintigraphy

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

Cuningham, D.A.; Kumar, B.; Siegel, B.A.

In-111 platelet scintigraphy was used to evaluate the effects of prior aspirin administration on the accumulation of In-111-labeled autologous platelets at sites of arterial injury resulting from iliac, femoral, or popliteal transluminal angioplasty in a nonrandomized study of 17 men. The degree of platelet localization at angioplasty sites was significantly less in nine men who had received aspirin in varying doses within the 4 days before angioplasty than in eight men who had not received aspirin for at least two weeks. The results suggest that aspirin treatment before angioplasty limits the early platelet deposition at the angioplasty site in men.

Left ventricular thrombi: in vivo detection by indium-111 platelet imaging and two dimensional echocardiography

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

Stratton, J.R.; Ritchie, J.L.; Hamilton, G.W.

1981-04-01

Indium-111 platelet imaging, which can identify sites of active intravascular platelet deposition, and two dimensional echocardiography, which can identify intracardiac masses, can both be used to detect left ventricular thrombi noninvasively. We compared these techniques in 44 men at risk for thrombi from remote transmural myocardial infarction (31 patients) or cardiomyopathy (13 patients). All 44 patients underwent platelet imaging; 35 underwent echocardiography. On platelet imaging nine patients had thrombi and onehad a possible thrombus. Of these 10 studies, none were positive at 2 hours, 5 were positive at 24 hours and all were positive 48 or 72 hours after plateletmore » labeling. Nine of these patients underwent echocardiography, and all had an intraventricular mass. The findings on platelet scanning were negative in six patients who had positive (four patients) or equivocally positive (two patients) findings on echocardiography. All patients with thrombi detected by either noninvasive method had transmural anterior myocardial infarction with ventricular aneurysm. Of the seven patients who underwent cardiac surgery or autopsy, three had thrombi. Platelet imaging failed to identify one thrombus in a patient in whom imaging was performed only at 24 hours after labeling. There were no false positive platelet images in this group. Five of these seven patients (two with throbi, three without) underwent echocardiography; in all cases the echocardiographic findings agreed with the pathologic findings. Both platelet imaging and echocardiography detect ventricular thrombi. Platelet imaging may detect only the most hematologically active thrombi. Both techniques may help define patients at risk of embolization and may be useful for in vivo assessment of antithrombotic drugs.« less

Discrimination between platelet-mediated and coagulation-mediated mechanisms in a model of complex thrombus formation in vivo

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

Cadroy, Y.; Horbett, T.A.; Hanson, S.R.

1989-04-01

To study mechanisms of complex thrombus formation in vivo, and to compare the relative antithrombotic effects of anticoagulants and antiplatelet agents, a model was developed in baboons. Segments of collagen-coated tubing followed by two sequentially placed expansion chambers exhibiting disturbed flow patterns were exposed to native blood under laminar flow conditions. The device was incorporated for 1 hour into an exteriorized arteriovenous shunt in baboons under controlled blood flow (20 ml/min). Morphologic evaluation by scanning electron microscopy showed that thrombi associated with collagen were relatively rich in platelets but thrombi in the chambers were rich in fibrin and red cells.more » Deposition of indium 111-labeled platelets was continuously measured with a scintillation camera. Platelet deposition increased in a linear (collagen-coated segment) or exponential (chambers 1 and 2) fashion over time, with values after 40 minutes averaging 24.1 +/- 3.3 x 10(8) platelets (collagen segment), 16.7 +/- 3.4 x 10(8) platelets (chamber 1), and 8.4 +/- 2.4 x 10(8) platelets (chamber 2). Total fibrinogen deposition after 40 minutes was determined by using iodine 125-labeled baboon fibrinogen and averaged 0.58 +/- 0.14 mg in the collagen segment, 1.51 +/- 0.27 mg in chamber 1, and 0.95 +/- 0.25 mg in chamber 2. Plasma levels of beta-thromboglobulin (beta TG), platelet-factor 4 (PF4), and fibrinopeptide A (FPA) increased fourfold to fivefold after 60 minutes of blood exposure to the thrombotic device. Platelet deposition onto the collagen segment, chamber 1, and chamber 2 was linearly dependent on the circulating platelet count. Platelet accumulation in chamber 1 and chamber 2 was also dependent on the presence of the proximal collagen segment.« less

Physiopathology of blood platelets and development of platelets substitutes. Progress report, August 1, 1976--October 31, 1977. [/sup 51/Cr

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

Baldini, M G

1977-07-31

Progress is reported on the following research projects: the effect of estrogen on platelet aggregability and thrombus formation; the antithrombotic effect of platelet inhibiting agents in a bench model of artificial kidney; the arrest of hemorrhage in severely alloimmunized thrombocytopenic patients; and in vivo elution of /sup 51/Cr from labeled platelets induced by antibody. (HLW)

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.

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

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

Clinical use of indium-111 labeled blood products

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

Loken, M.K.; Clay, M.E.; Carpenter, R.T.

1985-12-01

Following the introduction of In-111 oxine as a label for blood cells by McAffee and Thakur in 1976, these procedures have become increasingly important in the practice of nuclear medicine. Of particular interest are studies involving the use of labeled leukocytes for the detection of focal infection. The clinical utility of labeled platelets is less well developed, although the use of platelets to detect the formation of thrombi in blood vessels and on vascular grafts and prostheses is gaining prominence. This report summarizes the techniques presently employed at the University of Minnesota for the labeling of blood products, and theirmore » clinical use. Consideration also is given to the desired expertise and cost factors involved in the labeling of leukocytes and platelets.43 references.« less

Flow cytometric and radioisotopic determinations of platelet survival time in normal cats and feline leukemia virus-infected cats

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

Jacobs, R.M.; Boyce, J.T.; Kociba, G.J.

This study demonstrates the potential usefulness of a flow cytometric technique to measure platelet survival time in cats utilizing autologous platelets labeled in vitro with fluorescein isothiocyanate (FITC). When compared with a 51Cr method, no significant differences in estimated survival times were found. Both the 51Cr and FITC-labeling procedures induced similar changes in platelet shape and collagen-induced aggregation. Platelets labeled with FITC had significantly greater volumes compared with those of glutaraldehyde-fixed platelets. These changes were primarily related to the platelet centrifugation and washing procedures rather than the labels themselves. This novel technique potentially has wide applicability to cell circulation timemore » studies as flow cytometry equipment becomes more readily available. Problems with the technique are discussed. In a preliminary study of the platelet survival time in feline leukemia virus (FeLV)-infected cats, two of three cats had significantly reduced survival times using both flow cytometric and radioisotopic methods. These data suggest increased platelet turnover in FeLV-infected cats.« less

The Use of Spinning-Disk Confocal Microscopy for the Intravital Analysis of Platelet Dynamics in Response to Systemic and Local Inflammation

PubMed Central

Jenne, Craig N.; Wong, Connie H. Y.; Petri, Björn; Kubes, Paul

2011-01-01

Platelets are central players in inflammation and are an important component of the innate immune response. The ability to visualize platelets within the live host is essential to understanding their role in these processes. Past approaches have involved adoptive transfer of labelled platelets, non-specific dyes, or the use of fluorescent antibodies to tag platelets in vivo. Often, these techniques result in either the activation of the platelet, or blockade of specific platelet receptors. In this report, we describe two new methods for intravital visualization of platelet biology, intravenous administration of labelled anti-CD49b, which labels all platelets, and CD41-YFP transgenic mice, in which a percentage of platelets express YFP. Both approaches label endogenous platelets and allow for their visualization using spinning-disk confocal fluorescent microscopy. Following LPS-induced inflammation, we were able to measure a significant increase in both the number and size of platelet aggregates observed within the vasculature of a number of different tissues. Real-time observation of these platelet aggregates reveals them to be large, dynamic structures that are continually expanding and sloughing-off into circulation. Using these techniques, we describe for the first time, platelet recruitment to, and behaviour within numerous tissues of the mouse, both under control conditions and following LPS induced inflammation. PMID:21949865

Platelet Counts in Insoluble Platelet-Rich Fibrin Clots: A Direct Method for Accurate Determination.

PubMed

Kitamura, Yutaka; Watanabe, Taisuke; Nakamura, Masayuki; Isobe, Kazushige; Kawabata, Hideo; Uematsu, Kohya; Okuda, Kazuhiro; Nakata, Koh; Tanaka, Takaaki; Kawase, Tomoyuki

2018-01-01

Platelet-rich fibrin (PRF) clots have been used in regenerative dentistry most often, with the assumption that growth factor levels are concentrated in proportion to the platelet concentration. Platelet counts in PRF are generally determined indirectly by platelet counting in other liquid fractions. This study shows a method for direct estimation of platelet counts in PRF. To validate this method by determination of the recovery rate, whole-blood samples were obtained with an anticoagulant from healthy donors, and platelet-rich plasma (PRP) fractions were clotted with CaCl 2 by centrifugation and digested with tissue-plasminogen activator. Platelet counts were estimated before clotting and after digestion using an automatic hemocytometer. The method was then tested on PRF clots. The quality of platelets was examined by scanning electron microscopy and flow cytometry. In PRP-derived fibrin matrices, the recovery rate of platelets and white blood cells was 91.6 and 74.6%, respectively, after 24 h of digestion. In PRF clots associated with small and large red thrombi, platelet counts were 92.6 and 67.2% of the respective total platelet counts. These findings suggest that our direct method is sufficient for estimating the number of platelets trapped in an insoluble fibrin matrix and for determining that platelets are distributed in PRF clots and red thrombi roughly in proportion to their individual volumes. Therefore, we propose this direct digestion method for more accurate estimation of platelet counts in most types of platelet-enriched fibrin matrix.

Platelet Counts in Insoluble Platelet-Rich Fibrin Clots: A Direct Method for Accurate Determination

PubMed Central

Kitamura, Yutaka; Watanabe, Taisuke; Nakamura, Masayuki; Isobe, Kazushige; Kawabata, Hideo; Uematsu, Kohya; Okuda, Kazuhiro; Nakata, Koh; Tanaka, Takaaki; Kawase, Tomoyuki

2018-01-01

Platelet-rich fibrin (PRF) clots have been used in regenerative dentistry most often, with the assumption that growth factor levels are concentrated in proportion to the platelet concentration. Platelet counts in PRF are generally determined indirectly by platelet counting in other liquid fractions. This study shows a method for direct estimation of platelet counts in PRF. To validate this method by determination of the recovery rate, whole-blood samples were obtained with an anticoagulant from healthy donors, and platelet-rich plasma (PRP) fractions were clotted with CaCl2 by centrifugation and digested with tissue-plasminogen activator. Platelet counts were estimated before clotting and after digestion using an automatic hemocytometer. The method was then tested on PRF clots. The quality of platelets was examined by scanning electron microscopy and flow cytometry. In PRP-derived fibrin matrices, the recovery rate of platelets and white blood cells was 91.6 and 74.6%, respectively, after 24 h of digestion. In PRF clots associated with small and large red thrombi, platelet counts were 92.6 and 67.2% of the respective total platelet counts. These findings suggest that our direct method is sufficient for estimating the number of platelets trapped in an insoluble fibrin matrix and for determining that platelets are distributed in PRF clots and red thrombi roughly in proportion to their individual volumes. Therefore, we propose this direct digestion method for more accurate estimation of platelet counts in most types of platelet-enriched fibrin matrix. PMID:29450197

Specific binding of magnetic nanoparticle probes to platelets in whole blood detected by magnetorelaxometry

NASA Astrophysics Data System (ADS)

Eberbeck, Dietmar; Wiekhorst, Frank; Steinhoff, Uwe; Schwarz, Kay Oliver; Kummrow, Andreas; Kammel, Martin; Neukammer, Jörg; Trahms, Lutz

2009-05-01

The binding of monoclonal antibodies labelled with magnetic nanoparticles to CD61 surface proteins expressed by platelets in whole blood samples was measured by magnetorelaxometry. This technique is sensitive to immobilization of the magnetic labels upon binding. Control experiments with previous saturation of the epitopes on the platelet surfaces demonstrated the specificity of the binding. The kinetics of the antibody antigen reaction is accessible with a temporal resolution of 12 s. The minimal detectable platelet concentration is about 2000 μL -1 (sample volume 150 μL). The proportionality of the magnetic relaxation amplitude to the number of bound labels allows a quantification of the antibody binding capacity.

Necrotic platelets provide a procoagulant surface during thrombosis

PubMed Central

Hua, Vu Minh; Abeynaike, Latasha; Glaros, Elias; Campbell, Heather; Pasalic, Leonardo; Chen, Vivien M. Y.

2015-01-01

A subpopulation of platelets fulfills a procoagulant role in hemostasis and thrombosis by enabling the thrombin burst required for fibrin formation and clot stability at the site of vascular injury. Excess procoagulant activity is linked with pathological thrombosis. The identity of the procoagulant platelet has been elusive. The cell death marker 4-[N-(S-glutathionylacetyl)amino]phenylarsonous acid (GSAO) rapidly enters a subpopulation of agonist-stimulated platelets via an organic anion-transporting polypeptide and is retained in the cytosol through covalent reaction with protein dithiols. Labeling with GSAO, together with exposure of P-selectin, distinguishes necrotic from apoptotic platelets and correlates with procoagulant potential. GSAO+ platelets form in occluding murine thrombi after ferric chloride injury and are attenuated with megakaryocyte-directed deletion of the cyclophilin D gene. These platelets form a procoagulant surface, supporting fibrin formation, and reduction in GSAO+ platelets is associated with reduction in platelet thrombus size and fibrin formation. Analysis of platelets from human subjects receiving aspirin therapy indicates that these procoagulant platelets form despite aspirin therapy, but are attenuated by inhibition of the necrosis pathway. These findings indicate that the major subpopulation of platelets involved in fibrin formation are formed via regulated necrosis involving cyclophilin D, and that they may be targeted independent of platelet activation. PMID:26474813

A novel platelet concentrate: titanium-prepared platelet-rich fibrin.

PubMed

Tunalı, Mustafa; Özdemir, Hakan; Küçükodacı, Zafer; Akman, Serhan; Yaprak, Emre; Toker, Hülya; Fıratlı, Erhan

2014-01-01

We developed a new product called titanium-prepared platelet-rich fibrin (T-PRF). The T-PRF method is based on the hypothesis that titanium may be more effective in activating platelets than the silica activators used with glass tubes in Chouckroun's leukocyte- and platelet-rich fibrin (L-PRF) method. In this study, we aimed to define the structural characteristics of T-PRF and compare it with L-PRF. Blood samples were collected from 10 healthy male volunteers. The blood samples were drawn using a syringe. Nine milliliters was transferred to a dry glass tube, and 9 mL was transferred to a titanium tube. Half of each clot (i.e., the blood that was clotted using T-PRF or L-PRF) was processed with a scanning electron microscope (SEM). The other half of each clot was processed for fluorescence microscopy analysis and light microscopy analysis. The T-PRF samples seemed to have a highly organized network with continuous integrity compared to the other L-PRF samples. Histomorphometric analysis showed that T-PRF fibrin network covers larger area than L-PRF fibrin network; also fibrin seemed thicker in the T-PRF samples. This is the first human study to define T-PRF as an autogenous leukocyte- and platelet-rich fibrin product. The platelet activation by titanium seems to offer some high characteristics to T-PRF.

Platelet compatibility of magnesium alloys.

PubMed

Yahata, Chie; Mochizuki, Akira

2017-09-01

Lately, Mg alloys have been investigated as a new class of biomaterials owing to their excellent biodegradability and biocompatibility. It has previously been reported that the in vitro compatibility of a Mg alloy containing aluminum and zinc (AZ) alloy with the blood coagulation system is excellent due to Mg 2+ ions eluting from the alloy. In this study, the compatibility of the AZ alloy with platelets was evaluated by scanning electron microscopy (SEM) and flow cytometry. In the flow cytometry analysis, the platelets were stained using PAC-1 and P-selectin antibodies. SEM images and PAC-1 analyses showed no negative effects on the platelets, whereas P-selectin analysis showed marked platelet activation. To understand these contradictory results, the amount of β-thromboglobulin (β-TG) released from the platelets was investigated. From that investigation, it was concluded that platelets are markedly activated by the alloys. In addition to clarifying divergent results depending on the analysis method used, the effects of Mg 2+ ions and pH on platelet activation were studied. These results show that platelet activation is caused by an increase in pH at the alloy surface owing to the erosion of the alloy. Copyright © 2017 Elsevier B.V. All rights reserved.

Depressed reticuloendothelial clearance of platelets in rats after trauma.

PubMed

Kaplan, J E; Moon, D G; Minnear, F L; Saba, T M

1984-02-01

Platelet microembolization may contribute to microcirculatory and organ damage following trauma and shock. It is hypothesized that posttraumatic reticuloendothelial depression predisposes to such microembolization by failure to clear altered platelets from the circulation. The present study evaluated the short-term (1 h) clearance and organ localization of radiolabeled homologous damaged platelets in normal rats and in rats following sublethal Noble-Collip drum trauma. Platelets were collected in citrated platelet-rich plasma from normal rats and labeled with 51Cr in citrated saline. Platelets were altered by repeated centrifugation in protein-free medium. These platelets differed functionally and morphologically from normal platelets. Disappearance of iv injected damaged platelets conformed to a two-compartment exponential clearance. Velocity of clearance in the rapid compartment correlated with hepatic platelet localization, whereas velocity of clearance in the second compartment correlated with splenic platelet localization. Clearance rate of the rapid compartment was depressed at 1 h after trauma and elevated at 24 h. These changes were associated with a decrease in hepatic platelet localization at 1 h and an increase above normal at 24 h. Splenic platelet localization was decreased by 3 h following trauma. Pulmonary platelet localization was increased at all times following trauma. It is concluded that the posttrauma state is associated with a defect in the reticuloendothelial system clearance of altered platelets, which may augment embolization of platelets in the lung.

Effect of aspirin and ticlopidine on platelet deposition in carotid atherosclerosis: assessment by indium-111 platelet scintigraphy

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

Isaka, Y.; Kimura, K.; Etani, H.

The antiplatelet effects of aspirin and ticlopidine were studied by a dual-tracer method, using indium-111 labeled platelets and technetium-99m human serum albumin, in a group of 12 patients with suspected ischemic cerebrovascular disease. The magnitude of platelet accumulation at the carotid bifurcation was expressed as the ratio of radioactivity of indium-111 platelets deposited on the vascular wall to those circulating in the blood-pool (PAI, platelet accumulation index), 48 hr after injection of labeled platelets. PAI values were measured before (baseline studies) and after the antithrombotic therapies (aspirin studies: 325 mg bid for 22.3 +/- 1.3 days, ticlopidine studies: 100 mgmore » tid for 21.8 +/- 2.1 days). At the baseline, the mean PAI value at 24 carotid bifurcations in the patient group was 15.7 +/- 15.3% (mean +/- S.D.) compared to -4.3 +/- 9.1 at 24 carotid bifurcations in 12 normal subjects (p less than 0.01). We defined the upper limit for a normal PAI (%) value to be +13.9, namely the mean PAI plus 2 SD for the carotid bifurcation in normal subjects and used this value for semiquantitative analysis. At the baseline, significant elevation of PAI (more than 13.9%; positive scintigram) was observed at 12 of 24 vessels, while 12 other regions were negative (less than 13.9%). In the lesions with positive scintigraphic results at the baseline, the mean PAI (%) value from the baseline, aspirin and ticlopidine studies was 29.5 +/- 7.0, 11.2 +/- 8.5 (p less than 0.01 versus baseline) and 21.4 +/- 21.3 (not significant from baseline), respectively.« less

Correlative fluorescence and scanning transmission electron microscopy of quantum dot-labeled proteins on whole cells in liquid.

PubMed

Peckys, Diana B; Bandmann, Vera; de Jonge, Niels

2014-01-01

Correlative fluorescence microscopy combined with scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, STEM can be accomplished in two ways. The microchip with the labeled cells and one microchip with a spacer are assembled into a special microfluidic device and imaged with dedicated high-voltage STEM. Alternatively, thin edges of cells can be studied with environmental scanning electron microscopy with a STEM detector, by placing a microchip with cells in a cooled wet environment. © 2014 Elsevier Inc. All rights reserved.

Pathogen Reduced, Plasmalyte Extended Stored Platelets

DTIC Science & Technology

2017-10-01

products of very different signal strengths . In May 2017 we modified the protocol to replace the chromium label with a second indium label of fresh...failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO...determine the efficacy of using a pathogen inactivation technique (Mirasol) coupled with a platelet additive solution (PAS) to extend the life of stored

Fibrin monomer increases platelet adherence to tumor cells in a flowing system: a possible role in metastasis?

PubMed

Biggerstaff, J P; Seth, N B; Meyer, T V; Amirkhosravi, A; Francis, J L

1998-12-15

Considerable evidence exists linking hemostasis and malignancy. Platelet adhesion to tumor cells has been implicated in the metastatic process. Plasma fibrinogen (Fg) and fibrin (Fn) monomer, increased in cancer, may play a role in tumor biology. Binding of Fn monomer to tumor cells and its effect on platelet-tumor cell adhesion in a flowing system were studied. Fn monomer was produced by adding thrombin (1 micro/mL) to FXIII- and plasminogen-free Fg in the presence of Gly-Pro-Arg-Pro (GPRP) amide. Fn monomer binding to live A375 cells was visualized by confocal laser scanning microscopy (CLSM). Adherent cells were perfused for 1h with Fn monomer, washed and stained in situ with anti-human Fn (American Biogenetic Sciences, Inc.) followed by goat anti-mouse IgG(FITC). Platelet adherence to Fn monomer treated A375 cells was performed under flow conditions by passing platelets (5x10(4)/microl 0.25 mL/min; labeled with the carbocyanine dye DiI) over the tumor cells for 30 min. CLSM images were obtained after washing. There was considerable binding of Fn monomer, but not Fg alone. Platelets adhered relatively weakly to untreated A375 cells and this was not significantly affected by pre-treatment of the tumor cells with fibrinogen or thrombin. However, pre-treatment with Fn monomer resulted in extensive platelet binding to tumor cells, suggesting that coagulation activation and the subsequent increase in circulating Fn monomer may enhance platelet adhesion to circulating tumor cells and thereby facilitate metastatic spread.

Semantic Labelling of Road Furniture in Mobile Laser Scanning Data

NASA Astrophysics Data System (ADS)

Li, F.; Oude Elberink, S.; Vosselman, G.

2017-09-01

Road furniture semantic labelling is vital for large scale mapping and autonomous driving systems. Much research has been investigated on road furniture interpretation in both 2D images and 3D point clouds. Precise interpretation of road furniture in mobile laser scanning data still remains unexplored. In this paper, a novel method is proposed to interpret road furniture based on their logical relations and functionalities. Our work represents the most detailed interpretation of road furniture in mobile laser scanning data. 93.3 % of poles are correctly extracted and all of them are correctly recognised. 94.3 % of street light heads are detected and 76.9 % of them are correctly identified. Despite errors arising from the recognition of other components, our framework provides a promising solution to automatically map road furniture at a detailed level in urban environments.

Effects of a garlic-derived principle (ajoene) on aggregation and arachidonic acid metabolism in human blood platelets.

PubMed

Srivastava, K C; Tyagi, O D

1993-08-01

When garlic cloves are chopped or crushed several dialkyl thiosulfinates are rapidly formed by the action of the enzyme alliin lyase or alliinase (EC 4.4.1.4) on S(+)-alkyl-L-cysteine sulfoxides. Allicin (diallyl thiosulfinate or allyl 2-propene thiosulfinate) is the dominant thiosulfinate released. A variety of sulfur containing compounds are formed from allicin and other thiosulfinates depending on the way in which garlic is handled. One such compound identified recently is ajoene which has been reported to possess antithrombotic properties. We present here data on the antiplatelet properties of ajoene together with its effects on the metabolism of arachidonic acid (AA) in intact platelets. Thus, ajoene was found to inhibit platelet aggregation induced by AA, adrenaline, collagen, adenosine diphosphate (ADP) and calcium ionophore A23187; the nature of the inhibition was irreversible. In washed platelets stimulated by labelled arachidonate, ajoene inhibited the formation of thromboxane A2; 12-lipoxygenase product(s) were reduced at higher ajoene concentrations. This garlic-derived substance inhibited the incorporation of labelled AA into platelet phospholipids at higher concentration. In labelled platelets, on stimulation with either calcium ionophore A23187 or collagen, reduced amounts of thromboxane and 12-HETE (12-hydroxyeicosatetraenoic acid) were produced in ajoene-treated platelets compared to control platelets. This substance had no effect on the deacylation of platelet phospholipids. The results suggest that at least one of the mechanisms by which ajoene shows antiplatelet effects could be related to altered metabolism of AA.

Binding of /sup 125/I-labeled endotoxin to bovine, canine, and equine platelets and endotoxin-induced agglutination of canine platelets

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

Meyers, K.M.; Boehme, M.; Inbar, O.

Endotoxin from Escherichia coli O127:B8, Salmonella abortus-equi and S minnesota induced clumping of some canine platelets (PLT) at a final endotoxin concentration of 1 microgram/ml. Endotoxin-induced clumping of canine PLT was independent of PLT energy-requiring processes, because clumping was observed with canine PLT incubated with 2-deoxy-D-glucose and antimycin A. The PLT responded to adenosine diphosphate before, but not after, incubation with the metabolic inhibitors. Endotoxin induced a slight and inconsistant clumping of bovine and equine PLT at high (mg/ml) endotoxin concentration. High-affinity binding sites could not be demonstrated on canine, bovine, and equine PLT, using /sup 125/I-labeled E coli O127:B8more » endotoxin. Nonspecific binding was observed and appeared to be due primarily to an extraneous coat on the PLT surface that was removed by gel filtration. The endotoxin that was bound to PLT did not appear to modify PLT function. An attempt to identify plasma proteins that bound physiologically relevant amounts of endotoxin was not successful. The significance of the endotoxin-induced clumping or lack of it on the pathophysiology of endotoxemia is discussed.« less

PLATELET ADHESION TO POLYURETHANE UREA UNDER PULSATILE FLOW CONDITIONS

PubMed Central

Navitsky, Michael A.; Taylor, Joshua O.; Smith, Alexander B.; Slattery, Margaret J.; Deutsch, Steven; Siedlecki, Christopher A.; Manning, Keefe B.

2014-01-01

Platelet adhesion to a polyurethane urea surface is a precursor to thrombus formation within blood-contacting cardiovascular devices, and platelets have been found to adhere strongly to polyurethane surfaces below a shear rate of approximately 500 s−1. The aim of the current work is to determine platelet adhesion properties to the polyurethane urea surface as a function of time varying shear exposure. A rotating disk system is used to study the influence of steady and pulsatile flow conditions (e.g. cardiac inflow and sawtooth waveforms) for platelet adhesion to the biomaterial surface. All experiments retain the same root mean square angular rotation velocity (29.63 rad/s) and waveform period. The disk is rotated in platelet rich bovine plasma for two hours with adhesion quantified by confocal microscopy measurements of immunofluorescently labeled bovine platelets. Platelet adhesion under pulsating flow is found to exponentially decay with increasing shear rate. Adhesion levels are found to depend upon peak platelet flux and shear rate regardless of rotational waveform. In combination with flow measurements, these results may be useful for predicting regions susceptible to thrombus formation within ventricular assist devices. PMID:24721222

Imaging analyses of coagulation-dependent initiation of fibrinolysis on activated platelets and its modification by thrombin-activatable fibrinolysis inhibitor.

PubMed

Brzoska, Tomasz; Suzuki, Yuko; Sano, Hideto; Suzuki, Seiichirou; Tomczyk, Martyna; Tanaka, Hiroki; Urano, Tetsumei

2017-04-03

Using intravital confocal microscopy, we observed previously that the process of platelet phosphatidylserine (PS) exposure, fibrin formation and lysine binding site-dependent plasminogen (plg) accumulation took place only in the centre of thrombi, not at their periphery. These findings prompted us to analyse the spatiotemporal regulatory mechanisms underlying coagulation and fibrinolysis. We analysed the fibrin network formation and the subsequent lysis in an in vitro experiment using diluted platelet-rich plasma supplemented with fluorescently labelled coagulation and fibrinolytic factors, using confocal laser scanning microscopy. The structure of the fibrin network formed by supplemented tissue factor was uneven and denser at the sites of coagulation initiation regions (CIRs) on PS-exposed platelets. When tissue-type plasminogen activator (tPA; 7.5 nM) was supplemented, labelled plg (50 nM) as well as tPA accumulated at CIRs, from where fibrinolysis started and gradually expanded to the peripheries. The lysis time at CIRs and their peripheries (50 µm from the CIR) were 27.9 ± 6.6 and 44.4 ± 9.7 minutes (mean ± SD, n=50 from five independent experiments) after the addition of tissue factor, respectively. Recombinant human soluble thrombomodulin (TMα; 2.0 nM) attenuated the CIR-dependent plg accumulation and strongly delayed fibrinolysis at CIRs. A carboxypeptidase inhibitor dose-dependently enhanced the CIR-dependent fibrinolysis initiation, and at 20 µM it completely abrogated the TMα-induced delay of fibrinolysis. Our findings are the first to directly present crosstalk between coagulation and fibrinolysis, which takes place on activated platelets' surface and is further controlled by thrombin-activatable fibrinolysis inhibitor (TAFI).

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.

Inheritance pattern of platelet membrane fluidity in Alzheimer disease.

PubMed Central

Chakravarti, A; Slaugenhaupt, S A; Zubenko, G S

1989-01-01

The fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene in labeled platelet membranes, an index of membrane fluidity, is a stable, familial trait that is associated with a clinically distinct subtype of Alzheimer disease. Complex segregation analysis of this continuous variable was performed on 95 members of 14 pedigrees identified through probands who had autopsy-confirmed or clinically diagnosed Alzheimer disease. The results suggest that platelet membrane fluidity is controlled by a single genetic locus, PMF, with two alleles that have additive effects. The PMF locus appears to explain approximately 80% of the total variation in platelet membrane fluidity within the families of patients with Alzheimer disease. PMID:2729275

Noninvasive radioisotopic technique for detection of platelet deposition in mitral valve prostheses and quantitation of visceral microembolism in dogs

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

Dewanjee, M.K.; Fuster, V.; Rao, S.A.

1983-05-01

A noninvasive technique has been developed in the dog model for imaging, with a gamma camera, the platelet deposition on Bjoerk-Shiley mitral valve prostheses early postoperatively. At 25 hours after implantation of the prosthesis and 24 hours after intravenous administration of 400 to 500 microCi of platelets labeled with indium-111, the platelet deposition in the sewing ring and perivalvular cardiac tissue can be clearly delineated in a scintiphotograph. An in vitro technique was also developed for quantitation of visceral microemboli in brain, lungs, kidneys, and other tissues. Biodistribution of the labeled platelets was quantitated, and the tissue/blood radioactivity ratio wasmore » determined in 22 dogs in four groups: unoperated normal dogs, sham-operated dogs, prosthesis-implanted dogs, and prosthesis-implanted dogs treated with dipyridamole before and aspirin and dipyridamole immediately after operation. Fifteen to 20% of total platelets were consumed as a consequence of the surgical procedure. On quantitation, we found that platelet deposition on the components of the prostheses was significantly reduced in prosthesis-implanted animals treated with dipyridamole and aspirin when compared with prosthesis-implanted, untreated dogs. All prosthesis-implanted animals considered together had a twofold to fourfold increase in tissue/blood radioactivity ratio in comparison with unoperated and sham-operated animals, an indication that the viscera work as filters and trap platelet microemboli that are presumably produced in the region of the mitral valve prostheses. In the dog model, indium-111-labeled platelets thus provide a sensitive marker for noninvasive imaging of platelet deposition on mechanical mitral valve prostheses, in vitro evaluation of platelet microembolism in viscera, in vitro quantitation of surgical consumption of platelets, and evaluation of platelet-inhibitor drugs.« less

Platelet indirect radioactive Coombs test. Its utilization for PLA1 grouping.

PubMed

Soulier, J P; Patereau, C; Drouet, J

1975-01-01

A platelet indirect radioactive Coombs test (PIRC) has been described. The technique for purification and labelling the antiglobulin has been precised. This test allowed the typing of platelets in the PLA system by using an absorbed serum from a mother of a thrombocytopenic child. Six other families of neonatal thrombocytopenias were tested. In three of them, the mothers were found PLA1 negative (PLA2, PLA2). Among a panel of 93 platelets, two (0.022) were found PLA1, negative. This PIRC test has many advantages compared to other tests such as platelet complement fixation, assay for blocking antibodies or antiglobulin consumption: it gives objective and quantitative results and is highly reproducible; anticomplementary serum may be tested.

Thiols in the alphaIIbbeta3 integrin are necessary for platelet aggregation.

PubMed

Manickam, Nagaraj; Sun, Xiuhua; Hakala, Kevin W; Weintraub, Susan T; Essex, David W

2008-07-01

Sulfhydryl groups of platelet surface proteins are important in platelet aggregation. While p-chloromercuribenzene sulphonate (pCMBS) has been used in most studies on platelet surface thiols, the specific thiol-proteins that pCMBS reacts with to inhibit aggregation have not been well defined. Since the thiol-containing P2Y(12) ADP receptor is involved in most types of platelet aggregation, we used the ADP scavenger apyrase and the P2Y(12) receptor antagonist 2-MeSAMP to examine thiol-dependent reactions in the absence of contributions from this receptor. We provide evidence for a non-P2Y(12) thiol-dependent reaction near the final alphaIIbbeta3-dependent events of aggregation. We then used 3-(N-maleimidylpropionyl)biocytin (MPB) and pCMBS to study thiols in alphaIIbbeta3. As previously reported, disruption of the receptor was required to obtain labelling of thiols with MPB. Specificity of labelling for thiols in the alphaIIb and beta3 subunits was confirmed by identification of the purified proteins by mass spectrometry and by inhibition of labelling with 5,5'-dithiobis-(2-nitrobenzoic acid). In contrast to MPB, pCMBS preferentially reacted with thiols in alphaIIbbeta3 and blocked aggregation under physiological conditions. Similarly, pCMBS preferentially inhibited signalling-independent activation of alphaIIbbeta3 by Mn(2+). Our results suggest that the thiols in alphaIIbbeta3 that are blocked by pCMBS are important in the activation of this integrin.

An Evaluation of the Accuracy of the Subtraction Method Used for Determining Platelet Counts in Advanced Platelet-Rich Fibrin and Concentrated Growth Factor Preparations

PubMed Central

Watanabe, Taisuke; Isobe, Kazushige; Suzuki, Taiji; Kawabata, Hideo; Nakamura, Masayuki; Tsukioka, Tsuneyuki; Okudera, Toshimitsu; Okudera, Hajime; Uematsu, Kohya; Okuda, Kazuhiro; Nakata, Koh; Kawase, Tomoyuki

2017-01-01

Platelet concentrates should be quality-assured of purity and identity prior to clinical use. Unlike for the liquid form of platelet-rich plasma, platelet counts cannot be directly determined in solid fibrin clots and are instead calculated by subtracting the counts in other liquid or semi-clotted fractions from those in whole blood samples. Having long suspected the validity of this method, we herein examined the possible loss of platelets in the preparation process. Blood samples collected from healthy male donors were immediately centrifuged for advanced platelet-rich fibrin (A-PRF) and concentrated growth factors (CGF) according to recommended centrifugal protocols. Blood cells in liquid and semi-clotted fractions were directly counted. Platelets aggregated on clot surfaces were observed by scanning electron microscopy. A higher centrifugal force increased the numbers of platelets and platelet aggregates in the liquid red blood cell fraction and the semi-clotted red thrombus in the presence and absence of the anticoagulant, respectively. Nevertheless, the calculated platelet counts in A-PRF/CGF preparations were much higher than expected, rendering the currently accepted subtraction method inaccurate for determining platelet counts in fibrin clots. To ensure the quality of solid types of platelet concentrates chairside in a timely manner, a simple and accurate platelet-counting method should be developed immediately. PMID:29563413

Human plasma fibrinogen adsorption and platelet adhesion to polystyrene.

PubMed

Tsai, W B; Grunkemeier, J M; Horbett, T A

1999-02-01

The purpose of this study was to further investigate the role of fibrinogen adsorbed from plasma in mediating platelet adhesion to polymeric biomaterials. Polystyrene was used as a model hydrophobic polymer; i.e., we expected that the role of fibrinogen in platelet adhesion to polystyrene would be representative of other hydrophobic polymers. Platelet adhesion was compared to both the amount and conformation of adsorbed fibrinogen. The strategy was to compare platelet adhesion to surfaces preadsorbed with normal, afibrinogenemic, and fibrinogen-replenished afibrinogenemic plasmas. Platelet adhesion was determined by the lactate dehydrogenase (LDH) method, which was found to be closely correlated with adhesion of 111In-labeled platelets. Fibrinogen adsorption from afibrinogenemic plasma to polystyrene (Immulon I(R)) was low and <10 ng/cm2. Platelet adhesion was absent on surfaces preadsorbed with afibrinogenemic plasma when the residual fibrinogen was low enough (<60 microg/mL). Platelet adhesion was restored on polystyrene preadsorbed with fibrinogen-replenished afibrinogenemic plasma. Addition of even small, subnormal concentrations of fibrinogen to afibrinogenemic plasma greatly increased platelet adhesion. In addition, surface-bound fibrinogen's ability to mediate platelet adhesion was different, depending on the plasma concentration from which fibrinogen was adsorbed. These differences correlated with changes in the binding of a monoclonal antibody that binds to the Aalpha chain RGDS (572-575), suggesting alteration in the conformation or orientation of the adsorbed fibrinogen. Platelet adhesion to polystyrene preadsorbed with blood plasma thus appears to be a strongly bivariate function of adsorbed fibrinogen, responsive to both low amounts and altered states of the adsorbed molecule. Copyright 1999 John Wiley & Sons, Inc.

Acetylglyceryl ether phosphorylcholine (AGEPC; platelet-activating factor)-induced stimulation of rabbit platelets: correlation between phosphatidic acid level, 45Ca2+ uptake, and (3H)serotonin secretion

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

Shukla, S.D.; Hanahan, D.J.

1984-08-01

When 32P-labeled rabbits platelet were incubated with 5 X 10(-10) M 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), either in the presence or absence (0.1 mM EGTA) of added Ca2+, there was a three- to five-fold increase in the (32P)phosphatidic acid (PA) pool within 15 to 20 s. This event was followed by a gradual decrease in the (32P)PA level to near basal level in 5 min. AGEPC effected this change in (32P)PA in a characteristic dose- and time-dependent manner. Polar head group analogs of AGEPC, such as AGEDME and AGEMME, also effected an increase in PA labeling at levels comparable to those previously reportedmore » for their activity toward rabbit platelets. Other analogs, i.e., lysoGEPC and the enantiomer, sn-1-AGEPC, which are inactive toward rabbit platelets, also showed no effect on the level of (32P)PA. The finding that the PA level in rabbit platelets could be manipulated by the addition of AGEPC, without any added Ca2+, provided an excellent model system for establishing a correlation between the uptake of Ca2+, serotonin release, and PA level. Thus, PA must be regarded as a sensitive indicator of a reaction mechanism important to the platelet response to AGEPC, and could be the focal point in promoting calcium uptake during the stimulation process.« less

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

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

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.

Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System.

PubMed

Poon, Candice C; Ebacher, Vincent; Liu, Katherine; Yong, Voon Wee; Kelly, John James Patrick

2018-05-03

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. Unfortunately, many researchers spend large amounts of time and resources developing and optimizing workflows that are only relevant to their own experiments. In this article, we describe a protocol that can be used by those with access to a widefield high-content analysis system (WHCAS) to image any slide-mounted tissue, with options for customization within pre-built modules found in the associated software. Not originally intended for slide scanning, the steps detailed in this article make it possible to acquire slide scanning images in the WHCAS which can be imported into the associated software. In this example, the automated segmentation of brain tumor slides is demonstrated, but the automated segmentation of any fluorescently-labeled nuclear or cytoplasmic marker is possible. Furthermore, there are a variety of other quantitative software modules including assays for protein localization/translocation, cellular proliferation/viability/apoptosis, and angiogenesis that can be run. This technique will save researchers time and effort and create an automated protocol for slide analysis.

Space and Time Resolved Detection of Platelet Activation and von Willebrand Factor Conformational Changes in Deep Suspensions.

PubMed

Biasetti, Jacopo; Sampath, Kaushik; Cortez, Angel; Azhir, Alaleh; Gilad, Assaf A; Kickler, Thomas S; Obser, Tobias; Ruggeri, Zaverio M; Katz, Joseph

2017-01-01

Tracking cells and proteins' phenotypic changes in deep suspensions is critical for the direct imaging of blood-related phenomena in in vitro replica of cardiovascular systems and blood-handling devices. This paper introduces fluorescence imaging techniques for space and time resolved detection of platelet activation, von Willebrand factor (VWF) conformational changes, and VWF-platelet interaction in deep suspensions. Labeled VWF, platelets, and VWF-platelet strands are suspended in deep cuvettes, illuminated, and imaged with a high-sensitivity EM-CCD camera, allowing detection using an exposure time of 1 ms. In-house postprocessing algorithms identify and track the moving signals. Recombinant VWF-eGFP (rVWF-eGFP) and VWF labeled with an FITC-conjugated polyclonal antibody are employed. Anti-P-Selectin FITC-conjugated antibodies and the calcium-sensitive probe Indo-1 are used to detect activated platelets. A positive correlation between the mean number of platelets detected per image and the percentage of activated platelets determined through flow cytometry is obtained, validating the technique. An increase in the number of rVWF-eGFP signals upon exposure to shear stress demonstrates the technique's ability to detect breakup of self-aggregates. VWF globular and unfolded conformations and self-aggregation are also observed. The ability to track the size and shape of VWF-platelet strands in space and time provides means to detect pro- and antithrombotic processes.

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

Blood platelet kinetics and platelet transfusion.

PubMed

Aster, Richard H

2013-11-01

The discovery of citrate anticoagulant in the 1920s and the development of plastic packs for blood collection in the 1960s laid the groundwork for platelet transfusion therapy on a scale not previously possible. A major limitation, however, was the finding that platelet concentrates prepared from blood anticoagulated with citrate were unsuitable for transfusion because of platelet clumping. We found that this could be prevented by simply reducing the pH of platelet-rich plasma to about 6.5 prior to centrifugation. We used this approach to characterize platelet kinetics and sites of platelet sequestration in normal and pathologic states and to define the influence of variables such as anticoagulant and ABO incompatibility on post-transfusion platelet recovery. The "acidification" approach enabled much wider use of platelet transfusion therapy until alternative means of producing concentrates suitable for transfusion became available.

Drug-Free Platelets Can Act as Seeds for Aggregate Formation During Antiplatelet Therapy

PubMed Central

Hoefer, Thomas; Armstrong, Paul C.; Finsterbusch, Michaela; Chan, Melissa V.; Kirkby, Nicholas S.

2015-01-01

Objective— Reduced antiplatelet drug efficacy occurs in conditions of increased platelet turnover, associated with increased proportions of drug-free, that is, uninhibited, platelets. Here, we detail mechanisms by which drug-free platelets promote platelet aggregation in the face of standard antiplatelet therapy. Approach and Results— To model standard antiplatelet therapy, platelets were treated in vitro with aspirin, the P2Y12 receptor blocker prasugrel active metabolite, or aspirin plus prasugrel active metabolite. Different proportions of uninhibited platelets were then introduced. Light transmission aggregometry analysis demonstrated clear positive associations between proportions of drug-free platelets and percentage platelet aggregation in response to a range of platelet agonists. Using differential platelet labeling coupled with advanced flow cytometry and confocal imaging we found aggregates formed in mixtures of aspirin-inhibited platelets together with drug-free platelets were characterized by intermingled platelet populations. This distribution is in accordance with the ability of drug-free platelets to generate thromboxane A2 and so drive secondary platelet activation. Conversely, aggregates formed in mixtures of prasugrel active metabolite–inhibited or aspirin plus prasugrel active metabolite–inhibited platelets together with drug-free platelets were characterized by distinct cores of drug-free platelets. This distribution is consistent with the ability of drug-free platelets to respond to the secondary activator ADP. Conclusions— These experiments are the first to image the interactions of inhibited and uninhibited platelets in the formation of platelet aggregates. They demonstrate that a general population of platelets can contain subpopulations that respond strikingly differently to overall stimulation of the population and so act as the seed for platelet aggregation. PMID:26272940

Effect of Blood Shear Forces on Platelet Mediated Thrombosis Inside Arterial Stenosis.

NASA Astrophysics Data System (ADS)

Maalej, Nabil

Shear induced activation of platelets plays a major role in the onset of thrombosis in atherosclerotic arteries. Blood hemodynamics and its effect on platelet kinetics has been studied mainly in in vitro and in ex vivo experiments. We designed new in vivo methods to study blood hemodynamic effects on platelet kinetics in canine stenosed carotid arteries. A carotid artery-jugular vein anastomotic shunt was produced. Intimal damage and controlled variations in the degree of stenosis were produced on the artery. An inflatable cuff was placed around the jugular vein to control vascular resistance. An electromagnetic flowmeter was used to measure blood flow. Doppler ultrasound crystals were used to measure the velocity profiles inside and distal to the stenosis. Stenosis geometry was obtained using digital subtraction angiography and quantitative arteriography. Using these measurements we calculated the wall shear stress using the finite difference solution of the Navier-Stokes equations. To study platelet kinetics, autologous platelets were labeled with Indium Oxine and injected IV. A collimated Nal gamma counter was placed over the stenosis to detect radio-labeled platelet accumulation as platelet mediated thrombi formed in the stenosis. The radioactive count rate increased in an inverse parallel fashion to the decline in flow rate during thrombus formation. The platelet accumulation increased with the increase of percent stenosis and was maximal at the narrow portion of the stenosis. Acute thrombus formation leading to arterial occlusion was only observed for stenosis higher than 70 +/- 5%. Platelet accumulation rate was not significant until the pressure gradient across the stenosis exceeded 40 +/- 10 mmHg. Totally occlusive thrombus formation was only observed for shear stresses greater than a critical value of 100 +/- 10 Pa. Beyond this critical value acute platelet thrombus formation increased exponentially with shear. Increased shear stresses were found to

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

Combination of intracellular staining of retrogradely labeled neurons and anterograde fluorescent tracing: use of the confocal laser scanning microscope.

PubMed

Shi, C; Cassell, M D

1993-04-01

This report describes a combined retrograde tracing, intracellular injection and anterograde fluorescence labeling method using the application of confocal laser scanning microscopy. By simultaneously viewing the morphology of identified projection neurons and the distribution of anterogradely labeled fibers and terminals, this approach allows accurate characterization of the anatomical relationships between these two elements. To demonstrate this approach, the retrograde tracer Fast Blue was injected into the bed nucleus of stria terminalis (BNST) and the anterograde tracer tetramethylrhodamine-conjugated dextran was injected into the insular cortex in adult rats. After one week survival time, the brains were fixed and sectioned on a vibratome. Individual BNST projecting neurons identified in the amygdaloid complex on 120 microns thick sections were intracellularly injected with Lucifer Yellow under visual control and analyzed with confocal laser scanning microscopy. The results demonstrate that images from very thin optical sections can clearly show potential synaptic contacts between anterograde labeling and intracellularly labeled projecting neurons. Stacked images from optical sections show, in very great detail, the morphology of projection neurons in three-dimensions. Compared to other methodological combinations, the present method provides a more simple and efficient means to trace three successive components of a putative neuron chain.

Effects of Platelet-Poor Plasma, Platelet-Rich Plasma, and Platelet-Rich Fibrin on Healing of Extraction Sockets with Buccal Dehiscence in Dogs

PubMed Central

Hatakeyama, Ichiro; Takahashi, Yukinobu; Omura, Ken

2014-01-01

Alveolar bone resorption generally occurs during healing after tooth extraction. This study aimed to evaluate the effects of platelet-poor plasma (PPP), platelet-rich plasma (PRP), and platelet-rich fibrin (PRF) on healing in a ridge-augmentation model of the canine socket with dehiscence of the buccal wall. The third mandibular premolars of 12 beagle dogs were extracted and a 3 mm buccal dehiscence from the alveolar crest to the buccal wall of the extraction socket was created. These sockets were then divided into four groups on the basis of the material used to fill the sockets: PPP, PRP, PRF, and control (no graft material) groups. Results were evaluated at 4 and 8 weeks after surgery. The ultrastructural morphology and constructs of each blood product were studied by a scanning electron microscope (SEM) or calculating concentrations of platelets, fibrinogen, platelet-derived growth factor, and transforming growth factor-β. A total of five microcomputed tomography images of specimens were selected for measurement, and the area occupied by the newly formed bone as well as the horizontal bone width were measured. Moreover, decalcified tissue specimens from each defect were analyzed histologically. The median area of new bone at 4 and 8 weeks and median horizontal bone width at 8 weeks were the highest in the PPP group. However, bone maturation in the PRF and the PRP groups was more progressed than that in the PPP and control groups. By SEM findings, the PRF group showed a more highly condensed fibrin fiber network that was regularly arranged when compared with the PPP and PRP groups. The growth factors released from platelets in PRP indicated higher concentrations than that in PRF. Under more severe conditions for bone formation, as in this experiment, the growth factors released from platelets had a negative effect on bone formation. This study showed that PPP is an effective material for the preservation of sockets with buccal dehiscence. PMID:24098948

Effects of platelet-poor plasma, platelet-rich plasma, and platelet-rich fibrin on healing of extraction sockets with buccal dehiscence in dogs.

PubMed

Hatakeyama, Ichiro; Marukawa, Eriko; Takahashi, Yukinobu; Omura, Ken

2014-02-01

Alveolar bone resorption generally occurs during healing after tooth extraction. This study aimed to evaluate the effects of platelet-poor plasma (PPP), platelet-rich plasma (PRP), and platelet-rich fibrin (PRF) on healing in a ridge-augmentation model of the canine socket with dehiscence of the buccal wall. The third mandibular premolars of 12 beagle dogs were extracted and a 3 mm buccal dehiscence from the alveolar crest to the buccal wall of the extraction socket was created. These sockets were then divided into four groups on the basis of the material used to fill the sockets: PPP, PRP, PRF, and control (no graft material) groups. Results were evaluated at 4 and 8 weeks after surgery. The ultrastructural morphology and constructs of each blood product were studied by a scanning electron microscope (SEM) or calculating concentrations of platelets, fibrinogen, platelet-derived growth factor, and transforming growth factor-β. A total of five microcomputed tomography images of specimens were selected for measurement, and the area occupied by the newly formed bone as well as the horizontal bone width were measured. Moreover, decalcified tissue specimens from each defect were analyzed histologically. The median area of new bone at 4 and 8 weeks and median horizontal bone width at 8 weeks were the highest in the PPP group. However, bone maturation in the PRF and the PRP groups was more progressed than that in the PPP and control groups. By SEM findings, the PRF group showed a more highly condensed fibrin fiber network that was regularly arranged when compared with the PPP and PRP groups. The growth factors released from platelets in PRP indicated higher concentrations than that in PRF. Under more severe conditions for bone formation, as in this experiment, the growth factors released from platelets had a negative effect on bone formation. This study showed that PPP is an effective material for the preservation of sockets with buccal dehiscence.

Storage of platelets: effects associated with high platelet content in platelet storage containers.

PubMed

Gulliksson, Hans; Sandgren, Per; Sjödin, Agneta; Hultenby, Kjell

2012-04-01

A major problem associated with platelet storage containers is that some platelet units show a dramatic fall in pH, especially above certain platelet contents. The aim of this study was a detailed investigation of the different in vitro effects occurring when the maximum storage capacity of a platelet container is exceeded as compared to normal storage. Buffy coats were combined in large-volume containers to create primary pools to be split into two equal aliquots for the preparation of platelets (450-520×10(9) platelets/unit) in SSP+ for 7-day storage in two containers (test and reference) with different platelet storage capacity (n=8). Exceeding the maximum storage capacity of the test platelet storage container resulted in immediate negative effects on platelet metabolism and energy supply, but also delayed effects on platelet function, activation and disintegration. Our study gives a very clear indication of the effects in different phases associated with exceeding the maximum storage capacity of platelet containers but throw little additional light on the mechanism initiating those negative effects. The problem appears to be complex and further studies in different media using different storage containers will be needed to understand the mechanisms involved.

Interaction of human platelets with laminin and identification of the 67 kDa laminin receptor on platelets.

PubMed Central

Tandon, N N; Holland, E A; Kralisz, U; Kleinman, H K; Robey, F A; Jamieson, G A

1991-01-01

A microtitre adhesion assay has been developed to define parameters affecting the adherence of washed platelets to laminin. Adherence was optimally supported by Mg2+ and was inhibited by Ca2+ and by anti-laminin Fab fragments, but significant adhesion (75-90% of control) was found both in heparinized plasma containing physiological levels of bivalent cations and in plasma anti-coagulated with EGTA. Adherence was unaffected by platelet activation with ADP but was decreased by 50% by treatment with alpha-thrombin (1 unit/ml, 5 min). Adherence was unaffected by monospecific polyclonal antibodies to glycoprotein (GP) Ib and GPIV, and was normal with platelets from two patients with Glanzmann's thrombasthaenia, indicating that GPIb, the GPIIb/IIIa complex and GPIV are not involved in platelet-laminin interaction. Affinity chromatography of Triton-solubilized membranes on laminin-Sepharose followed by elution with 0.2 M-glycine/HCl (pH 2.85) identified a major band with a molecular mass of 67 kDa in the reduced and of 53 kDa in the unreduced form. This protein gave a positive reaction on Western blotting with a monospecific polyclonal antibody raised against the high-affinity laminin receptor isolated from human breast carcinoma tissue. The adhesion of platelets to laminin was inhibited by two monoclonal IgM antibodies specific to the LR-1 domain of the 67 kDa receptor. The binding protein was surface-oriented, as shown by flow cytofluorimetry and by the fact that it could be iodinated in intact platelets, but it was not labelled by the periodate-borotritide procedure, suggesting that it did not contain terminal sialic acid. The laminin-derived peptides Tyr-Ile-Gly-Ser-Arg and Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg-NH2, which constitute a complementary binding domain in laminin for the 67 kDa receptor, themselves supported platelet adhesion, bound to the receptor and inhibited the adhesion of platelets to laminin. In addition, Fab fragments of anti

Platelet adhesion on phosphorus-incorporated tetrahedral amorphous carbon films

NASA Astrophysics Data System (ADS)

Liu, Aiping; Zhu, Jiaqi; Liu, Meng; Dai, Zhifei; Han, Xiao; Han, Jiecai

2008-11-01

The haemocompatibility of phosphorus-incorporated tetrahedral amorphous carbon (ta-C:P) films, synthesized by filtered cathodic vacuum arc technique with PH 3 as the dopant source, was assessed by in vitro platelet adhesion tests. Results based on scanning electron microscopy and contact angle measurements reveal that phosphorus incorporation improves the wettability and blood compatibility of ta-C film. Our studies may provide a novel approach for the design and synthesis of doped ta-C films to repel platelet adhesion and reduce thrombosis risk.

Centripetal myosin redistribution in thrombin-stimulated platelets. Relationship to platelet Factor 4 secretion.

PubMed

Painter, R G; Ginsberg, M H

1984-11-01

We have examined the F-actin and myosin distribution in resting and thrombin-activated platelets by double label immunofluorescence microscopy. In resting, discoid platelets, F-actin and myosin staining was distributed in a diffuse pattern throughout the interior of the cell with slight accentuation at the cell periphery. In contrast, platelet factor 4 antigen (PF4) was more centrally localized in a fine punctate distribution which is consistent with its localization in alpha-granules. Within 5 sec after thrombin stimulation both F-actin and myosin staining were increased at the periphery of the now spherical platelets. Subsequently, a myosin-containing spherical structure decreased in diameter closely surrounding a phase-dense central zone. In contrast, F-actin staining continued to be accentuated at the cell periphery and was prominent in filopodia and blebs. As previously shown, PF4 staining was localized after 30 sec within large intracellular masses that corresponded to closed vacuolar structures at the ultrastructural level. Morphometric analysis of electron micrographs showed that formation of these vacuolar structures kinetically paralleled alpha-granule disappearance and preceded PF4 release. These PF4-containing structures translocated to the cell periphery after 1-3 min, where they appeared to fuse with the plasma membrane. Ultrastructural analysis of thin sections showed that the myosin-rich spherical structure spatially and temporally correlated with a band of microfilaments that closely surrounded the organelle-rich central zone of the cell. Morphometric analysis of these micrographs showed that the absolute volume of this central zone decreased with time after thrombin addition, showing a significant change after 15 sec and reaching a maximum value after 3-5 min. Changes in the volume of this compartment kinetically preceded PF4 release. On the basis of these data, we propose that an actomyosin contractile force is generated which centripetally

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

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

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.

Imaging and Elastometry of Blood Clots Using Magnetomotive Optical Coherence Tomography and Labeled Platelets

PubMed Central

Oldenburg, Amy L.; Wu, Gongting; Spivak, Dmitry; Tsui, Frank; Wolberg, Alisa S.; Fischer, Thomas H.

2013-01-01

Improved methods for imaging and assessment of vascular defects are needed for directing treatment of cardiovascular pathologies. In this paper, we employ magnetomotive optical coherence tomography (MMOCT) as a platform both to detect and to measure the elasticity of blood clots. Detection is enabled through the use of rehydrated, lyophilized platelets loaded with superparamagnetic iron oxides (SPIO-RL platelets) that are functional infusion agents that adhere to sites of vascular endothelial damage. Evidence suggests that the sensitivity for detection is improved over threefold by magnetic interactions between SPIOs inside RL platelets. Using the same MMOCT system, we show how elastometry of simulated clots, using resonant acoustic spectroscopy, is correlated with the fibrin content of the clot. Both methods are based upon magnetic actuation and phase-sensitive optical monitoring of nanoscale displacements using MMOCT, underscoring its utility as a broad-based platform to detect and measure the molecular structure and composition of blood clots. PMID:23833549

Imaging and Elastometry of Blood Clots Using Magnetomotive Optical Coherence Tomography and Labeled Platelets.

PubMed

Oldenburg, Amy L; Wu, Gongting; Spivak, Dmitry; Tsui, Frank; Wolberg, Alisa S; Fischer, Thomas H

2011-07-21

Improved methods for imaging and assessment of vascular defects are needed for directing treatment of cardiovascular pathologies. In this paper, we employ magnetomotive optical coherence tomography (MMOCT) as a platform both to detect and to measure the elasticity of blood clots. Detection is enabled through the use of rehydrated, lyophilized platelets loaded with superparamagnetic iron oxides (SPIO-RL platelets) that are functional infusion agents that adhere to sites of vascular endothelial damage. Evidence suggests that the sensitivity for detection is improved over threefold by magnetic interactions between SPIOs inside RL platelets. Using the same MMOCT system, we show how elastometry of simulated clots, using resonant acoustic spectroscopy, is correlated with the fibrin content of the clot. Both methods are based upon magnetic actuation and phase-sensitive optical monitoring of nanoscale displacements using MMOCT, underscoring its utility as a broad-based platform to detect and measure the molecular structure and composition of blood clots.

Prophylactic platelet transfusion plus supportive care versus supportive care alone in adults with dengue and thrombocytopenia: a multicentre, open-label, randomised, superiority trial.

PubMed

Lye, David C; Archuleta, Sophia; Syed-Omar, Sharifah F; Low, Jenny G; Oh, Helen M; Wei, Yuan; Fisher, Dale; Ponnampalavanar, Sasheela S L; Wijaya, Limin; Lee, Linda K; Ooi, Eng-Eong; Kamarulzaman, Adeeba; Lum, Lucy C; Tambyah, Paul A; Leo, Yee-Sin

2017-04-22

Dengue is the commonest vector-borne infection worldwide. It is often associated with thrombocytopenia, and prophylactic platelet transfusion is widely used despite the dearth of robust evidence. We aimed to assess the efficacy and safety of prophylactic platelet transfusion in the prevention of bleeding in adults with dengue and thrombocytopenia. We did an open-label, randomised, superiority trial in five hospitals in Singapore and Malaysia. We recruited patients aged at least 21 years who had laboratory-confirmed dengue (confirmed or probable) and thrombocytopenia (≤20 000 platelets per μL), without persistent mild bleeding or any severe bleeding. Patients were assigned (1:1), with randomly permuted block sizes of four or six and stratified by centre, to receive prophylactic platelet transfusion in addition to supportive care (transfusion group) or supportive care alone (control group). In the transfusion group, 4 units of pooled platelets were given each day when platelet count was 20 000 per μL or lower; supportive care consisted of bed rest, fluid therapy, and fever and pain medications. The primary endpoint was clinical bleeding (excluding petechiae) by study day 7 or hospital discharge (whichever was earlier), analysed by intention to treat. Safety outcomes were analysed according to the actual treatment received. This study was registered with ClinicalTrials.gov, number NCT01030211, and is completed. Between April 29, 2010, and Dec 9, 2014, we randomly assigned 372 patients to the transfusion group (n=188) or the control group (n=184). The intention-to-treat analysis included 187 patients in the transfusion group (one patient was withdrawn immediately) and 182 in the control group (one was withdrawn immediately and one did not have confirmed or probable dengue). Clinical bleeding by day 7 or hospital discharge occurred in 40 (21%) patients in the transfusion group and 48 (26%) patients in the control group (risk difference -4·98% [95% CI -15·08 to

Blood platelet adhesion to protein studied by on-line acoustic wave sensor.

PubMed

Cavic, B A; Freedman, J; Morel, Z; Mody, M; Rand, M L; Stone, D C; Thompson, M

2001-03-01

The attachment of blood platelets to the surface of bare and protein-coated thickness-shear mode acoustic wave devices operating in a flow-through configuration has been studied. Platelets in washed from bind to the gold electrodes of such sensors, but the resulting frequency shifts are far less than predicted by the conventional mass-based model of device operation. Adherence to albumin and various types of collagen can be produced by on-line introduction of protein or by a pre-coating strategy. Differences in attachment of platelets to collagen types I and IV and the Horm variety can be detected. Platelets attached to collagen yield an interesting delayed, but reversible signal on exposure to a flowing medium of low pH. Scanning electron microscopy of sensor surfaces at various time points in this experiment reveals that originally intact platelets are eventually destroyed by the high acidity of the medium. The reversible frequency is attributed to the presence of removable platelet granular components at the sensor-liquid interface.

A comprehensive proteomics study on platelet concentrates: Platelet proteome, storage time and Mirasol pathogen reduction technology.

PubMed

Salunkhe, Vishal; De Cuyper, Iris M; Papadopoulos, Petros; van der Meer, Pieter F; Daal, Brunette B; Villa-Fajardo, María; de Korte, Dirk; van den Berg, Timo K; Gutiérrez, Laura

2018-03-19

Platelet concentrates (PCs) represent a blood transfusion product with a major concern for safety as their storage temperature (20-24°C) allows bacterial growth, and their maximum storage time period (less than a week) precludes complete microbiological testing. Pathogen inactivation technologies (PITs) provide an additional layer of safety to the blood transfusion products from known and unknown pathogens such as bacteria, viruses, and parasites. In this context, PITs, such as Mirasol Pathogen Reduction Technology (PRT), have been developed and are implemented in many countries. However, several studies have shown in vitro that Mirasol PRT induces a certain level of platelet shape change, hyperactivation, basal degranulation, and increased oxidative damage during storage. It has been suggested that Mirasol PRT might accelerate what has been described as the platelet storage lesion (PSL), but supportive molecular signatures have not been obtained. We aimed at dissecting the influence of both variables, that is, Mirasol PRT and storage time, at the proteome level. We present comprehensive proteomics data analysis of Control PCs and PCs treated with Mirasol PRT at storage days 1, 2, 6, and 8. Our workflow was set to perform proteomics analysis using a gel-free and label-free quantification (LFQ) approach. Semi-quantification was based on LFQ signal intensities of identified proteins using MaxQuant/Perseus software platform. Data are available via ProteomeXchange with identifier PXD008119. We identified marginal differences between Mirasol PRT and Control PCs during storage. However, those significant changes at the proteome level were specifically related to the functional aspects previously described to affect platelets upon Mirasol PRT. In addition, the effect of Mirasol PRT on the platelet proteome appeared not to be exclusively due to an accelerated or enhanced PSL. In summary, semi-quantitative proteomics allows to discern between proteome changes due to

Plasminogen associates with phosphatidylserine-exposing platelets and contributes to thrombus lysis under flow

PubMed Central

Whyte, Claire S.; Swieringa, Frauke; Mastenbroek, Tom G.; Lionikiene, Ausra S.; Lancé, Marcus D.; van der Meijden, Paola E. J.; Heemskerk, Johan W. M.

2015-01-01

The interaction of plasminogen with platelets and their localization during thrombus formation and fibrinolysis under flow are not defined. Using a novel model of whole blood thrombi, formed under flow, we examine dose-dependent fibrinolysis using fluorescence microscopy. Fibrinolysis was dependent upon flow and the balance between fibrin formation and plasminogen activation, with tissue plasminogen activator-mediated lysis being more efficient than urokinase plasminogen activator-mediated lysis. Fluorescently labeled plasminogen radiates from platelet aggregates at the base of thrombi, primarily in association with fibrin. Hirudin attenuates, but does not abolish plasminogen binding, denoting the importance of fibrin. Flow cytometry revealed that stimulation of platelets with thrombin/convulxin significantly increased the plasminogen signal associated with phosphatidylserine (PS)-exposing platelets. Binding was attenuated by tirofiban and Gly-Pro-Arg-Pro amide, confirming a role for fibrin in amplifying plasminogen binding to PS-exposing platelets. Confocal microscopy revealed direct binding of plasminogen and fibrinogen to different platelet subpopulations. Binding of plasminogen and fibrinogen co-localized with PAC-1 in the center of spread platelets. In contrast, PS-exposing platelets were PAC-1 negative, and bound plasminogen and fibrinogen in a protruding “cap.” These data show that different subpopulations of platelets harbor plasminogen by diverse mechanisms and provide an essential scaffold for the accumulation of fibrinolytic proteins that mediate fibrinolysis under flow. PMID:25712989

Platelet-derived-growth-factor-induced signalling in human platelets: phosphoinositide-3-kinase-dependent inhibition of platelet activation.

PubMed Central

Selheim, F; Fukami, M H; Holmsen, H; Vassbotn, F S

2000-01-01

Human platelets release platelet-derived growth factor (PDGF) from alpha-granules during platelet activation. We have previously shown that platelets have PDGF alpha-receptors, a transmembrane tyrosine kinase that takes part in negative feedback regulation during platelet activation. Here we have described a study of PDGF-induced tyrosine phosphorylation of platelet substrates and phosphoinositide 3-kinase (PI-3K) activity in collagen-stimulated platelets. By immunoblotting with phosphotyrosine antibodies of collagen-activated platelets we found that PDGF increased the phosphorylation of several platelet substrates, e.g. pp140, pp120 and pp85. PDGF inhibited collagen-induced platelet activation in the presence of inhibitors of autocrine stimulation, thus blocking the pure collagen-induced signal transduction. PDGF enhanced the collagen-induced formation of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) as measured by HPLC. Wortmannin and LY294002, two unrelated inhibitors of PI-3K, were used to investigate the role of PI-3K in PDGF-induced platelet signalling. Incubation of platelets with wortmannin and LY294002 blocked the formation of three phosphorylated inositides as well as the inhibitory effect of PDGF on collagen-induced platelet activation. We conclude that the inhibitory effect of PDGF on platelet activation is PI-3K dependent. This is the first demonstration of a negative regulatory function of 3-phosphorylated inositides in platelets. PMID:10947961

Platelet-derived-growth-factor-induced signalling in human platelets: phosphoinositide-3-kinase-dependent inhibition of platelet activation.

PubMed

Selheim, F; Fukami, M H; Holmsen, H; Vassbotn, F S

2000-09-01

Human platelets release platelet-derived growth factor (PDGF) from alpha-granules during platelet activation. We have previously shown that platelets have PDGF alpha-receptors, a transmembrane tyrosine kinase that takes part in negative feedback regulation during platelet activation. Here we have described a study of PDGF-induced tyrosine phosphorylation of platelet substrates and phosphoinositide 3-kinase (PI-3K) activity in collagen-stimulated platelets. By immunoblotting with phosphotyrosine antibodies of collagen-activated platelets we found that PDGF increased the phosphorylation of several platelet substrates, e.g. pp140, pp120 and pp85. PDGF inhibited collagen-induced platelet activation in the presence of inhibitors of autocrine stimulation, thus blocking the pure collagen-induced signal transduction. PDGF enhanced the collagen-induced formation of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) as measured by HPLC. Wortmannin and LY294002, two unrelated inhibitors of PI-3K, were used to investigate the role of PI-3K in PDGF-induced platelet signalling. Incubation of platelets with wortmannin and LY294002 blocked the formation of three phosphorylated inositides as well as the inhibitory effect of PDGF on collagen-induced platelet activation. We conclude that the inhibitory effect of PDGF on platelet activation is PI-3K dependent. This is the first demonstration of a negative regulatory function of 3-phosphorylated inositides in platelets.

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.

Microfluidic measurement for blood flow and platelet adhesion around a stenotic channel: Effects of tile size on the detection of platelet adhesion in a correlation map

PubMed Central

Jung, Sung Yong; Yeom, Eunseop

2017-01-01

Platelet aggregation affects the surrounding blood flow and usually occurs where a blood vessel is narrowed as a result of atherosclerosis. The relationship between blood flow and platelet aggregation is not yet fully understood. This study proposes a microfluidic method to measure the velocity and platelet aggregation simultaneously by combining the micro-particle image velocimetry technique and a correlation mapping method. The blood flow and platelet adhesion procedure in a stenotic micro-channel with 90% severity were observed for a relatively long period of 4 min. In order to investigate the effect of tile size on the detection of platelet adhesion, 2D correlation coefficients were evaluated with binary images obtained by manual labeling and the correlation mapping method with different sizes of the square tile ranging from 3 to 50 pixels. The maximum 2D correlation coefficient occurred with the optimum tile size of 5 × 5 pixels. Since the blood flow and platelet aggregation are mutually influenced by each other, blood flow and platelet adhesion were continuously varied. When there was no platelet adhesion (t = 0 min), typical blood flow is observed. The blood flow passes through the whole channel smoothly, and jet-like flow occurs in the post-stenosis region. However, the flow pattern changes when platelet adhesion starts at the stenosis apex and after the stenosis. These adhesions induce narrow high velocity regions to become wider over a range of area from upstream to downstream of the stenosis. Separated jet-like flows with two high velocity regions are also created. The changes in flow patterns may alter the patterns of platelet adhesion. As the area of the plate adhesion increases, the platelets plug the micro-channel and there is only a small amount of blood flow, finally. The microfluidic method could provide new insights for better understanding of the interactions between platelet aggregation and blood flow in various physiological

Microfluidic measurement for blood flow and platelet adhesion around a stenotic channel: Effects of tile size on the detection of platelet adhesion in a correlation map.

PubMed

Jung, Sung Yong; Yeom, Eunseop

2017-03-01

Platelet aggregation affects the surrounding blood flow and usually occurs where a blood vessel is narrowed as a result of atherosclerosis. The relationship between blood flow and platelet aggregation is not yet fully understood. This study proposes a microfluidic method to measure the velocity and platelet aggregation simultaneously by combining the micro-particle image velocimetry technique and a correlation mapping method. The blood flow and platelet adhesion procedure in a stenotic micro-channel with 90% severity were observed for a relatively long period of 4 min. In order to investigate the effect of tile size on the detection of platelet adhesion, 2D correlation coefficients were evaluated with binary images obtained by manual labeling and the correlation mapping method with different sizes of the square tile ranging from 3 to 50 pixels. The maximum 2D correlation coefficient occurred with the optimum tile size of 5 × 5 pixels. Since the blood flow and platelet aggregation are mutually influenced by each other, blood flow and platelet adhesion were continuously varied. When there was no platelet adhesion (t = 0 min), typical blood flow is observed. The blood flow passes through the whole channel smoothly, and jet-like flow occurs in the post-stenosis region. However, the flow pattern changes when platelet adhesion starts at the stenosis apex and after the stenosis. These adhesions induce narrow high velocity regions to become wider over a range of area from upstream to downstream of the stenosis. Separated jet-like flows with two high velocity regions are also created. The changes in flow patterns may alter the patterns of platelet adhesion. As the area of the plate adhesion increases, the platelets plug the micro-channel and there is only a small amount of blood flow, finally. The microfluidic method could provide new insights for better understanding of the interactions between platelet aggregation and blood flow in various physiological

Effects of antithrombotic drugs in patients with left ventricular thrombi: assessment with indium-111 platelet imaging and two-dimensional echocardiography

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

Stratton, J.R.; Ritchie, J.L.

Patients with left ventricular thrombi not caused by recent myocardial infarction were prospectively studied by indium-111 platelet imaging and two-dimensional echocardiography to determine the reproducibility of these techniques and the short-term effects of sulfinpyrazone (200 mg four times daily), aspirin (325 mg three times daily) plus dipyridamole (75 mg three times daily), and full-dose warfarin. At baseline, all patients underwent indium-111 platelet imaging and echocardiography, and the results were positive for thrombus. In six patients on no antithrombotic drug therapy, repeat platelet scans and echocardiographic studies at 6.0 +/- 3.3 weeks remained positive and were unchanged. In seven patients studiedmore » on sulfinpyrazone, three platelet scans became negative, two became equivocal, and two were unchanged; the presence and size of thrombus was constant by echocardiography in all seven patients. Of the six patients studied on aspirin plus dipyridamole, one platelet scan became negative, those of three became equivocal, and two were unchanged; all echocardiographic findings remained positive, but one patient had decreased thrombus size. Among four warfarin-treated patients, three had resolution of platelet deposition and one was unchanged; by echocardiography, thrombus resolved in one patient, was decreased in size in one, and was unchanged in two. We conclude that, in the absence of antithrombotic drug therapy, platelet imaging and echocardiographic findings are stable in patients with left ventricular thrombi not caused by recent myocardial infarction. Sulfinpyrazone, aspirin plus dipyridamole, and warfarin all interrupt platelet deposition in some patients with chronic left ventricular thrombi.« less

Mechanism of inhibition of cyclo-oxygenase in human blood platelets by carbamate insecticides.

PubMed Central

Krug, H F; Hamm, U; Berndt, J

1988-01-01

Carbamates are a widely used class of insecticides and herbicides. They were tested for their ability to affect human blood platelet aggregation and arachidonic acid metabolism in platelets. (1) The herbicides of the carbamate type have no, or only little, influence up to a concentration of 100 microM; the carbamate insecticides, however, inhibit both aggregation and arachidonic acid metabolism in a dose- and time-dependent manner. (2) Carbaryl, the most effective compound, inhibits platelet aggregation and cyclo-oxygenase activity completely at 10 microM. The liberation of arachidonic acid from phospholipids and the lipoxygenase pathway are not affected, whereas the products of the cyclo-oxygenase pathway are drastically decreased. (3) By using [14C]carbaryl labelled in the carbamyl or in the ring moiety, it could be proved that the carbamyl residue binds covalently to platelet proteins. In contrast with acetylsalicylic acid, which acetylates only one protein, carbaryl carbamylates a multitude of platelet proteins. (4) One of the carbamylated proteins was found to be the platelet cyclo-oxygenase, indicating that carbaryl resembles in this respect acetylsalicylic acid, which is known to inhibit this enzyme specifically by acetylation. Images Fig. 4. PMID:3128272

21 CFR 606.121 - Container label.

Code of Federal Regulations, 2013 CFR

2013-04-01

... prepared in a system that might compromise sterility, the hour of expiration. (ii) If Source Plasma... collection date for each unit in the pool. (5) For Whole Blood, Plasma, Platelets, and partial units of Red... the container label for Source Plasma is not required to list the negative results of serological...

21 CFR 606.121 - Container label.

Code of Federal Regulations, 2014 CFR

2014-04-01

... prepared in a system that might compromise sterility, the hour of expiration. (ii) If Source Plasma... collection date for each unit in the pool. (5) For Whole Blood, Plasma, Platelets, and partial units of Red... the container label for Source Plasma is not required to list the negative results of serological...

Platelet glycoproteins associated with aspirin-treatment upon platelet activation

PubMed Central

Shah, Punit; Yang, Weiming; Sun, Shisheng; Pasay, Jered; Faraday, Nauder; Zhang, Hui

2017-01-01

Platelet glycoproteins are known to play central roles in hemostasis and vascular integrity and have pathologic roles in vascular occlusive diseases such as myocardial infarction and stroke. Characterizing glycoproteins within and secreted by platelets can provide insight into the mechanisms that underlie vascular pathologies and the therapeutic benefits or failure of anti-platelet agents. To study the impact of aspirin, which is commonly prescribed for primary and secondary cardiovascular prevention, on the platelet glycoproteome, we evaluated washed platelets from ten donors. The platelet glycoproteome, was studied using an iTRAQ in resting and stimulated states and with and without aspirin treatment. Using solid phase extraction of glycosite-containing peptides (SPEG), we were able to identify 799 unique N-linked glycosylation sites (glycosites) in platelets, representing the largest and the most comprehensive analysis to date. We were able to identity a number of glycoproteins impacted by aspirin treatment, which we validated using global proteomics analysis of platelets and their secreted proteins. In our analyses, metallopeptidase inhibitor 1 (TIMP1) was the single most significantly affected glycoprotein by aspirin treatment. ELISA assays confirmed proteomic results and validated our strategy. Functional analysis demonstrated that TIMP1 levels were highly correlated with platelet reactivity in vitro, with a correlation coefficient of −0.5. The release of TIMP1 from platelets, which was previously unknown to be affected by aspirin treatment, may play important roles in hemostasis and/or vascular integrity. If validated, our findings may be useful for developing assays that assess platelet response to aspirin or other anti-platelet therapies. PMID:27452734

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.

Platelet-TLR7 mediates host survival and platelet count during viral infection in the absence of platelet-dependent thrombosis

PubMed Central

Koupenova, Milka; Vitseva, Olga; MacKay, Christopher R.; Beaulieu, Lea M.; Benjamin, Emelia J.; Mick, Eric; Kurt-Jones, Evelyn A.; Ravid, Katya

2014-01-01

Viral infections have been associated with reduced platelet counts, the biological significance of which has remained elusive. Here, we show that infection with encephalomyocarditis virus (EMCV) rapidly reduces platelet count, and this response is attributed to platelet Toll-like receptor 7 (TLR7). Platelet-TLR7 stimulation mediates formation of large platelet-neutrophil aggregates, both in mouse and human blood. Intriguingly, this process results in internalization of platelet CD41-fragments by neutrophils, as assessed biochemically and visualized by microscopy, with no influence on platelet prothrombotic properties. The mechanism includes TLR7-mediated platelet granule release, translocation of P-selectin to the cell surface, and a consequent increase in platelet-neutrophil adhesion. Viral infection of platelet-depleted mice also led to increased mortality. Transfusion of wild-type, TLR7-expressing platelets into TLR7-deficient mice caused a drop in platelet count and increased survival post EMCV infection. Thus, this study identifies a new link between platelets and their response to single-stranded RNA viruses that involves activation of TLR7. Finally, platelet-TLR7 stimulation is independent of thrombosis and has implications to the host immune response and survival. PMID:24755410

Incorporation of a circulating protein into megakaryocyte and platelet granules

NASA Technical Reports Server (NTRS)

Handagama, P. J.; George, J. N.; Shuman, M. A.; McEver, R. P.; Bainton, D. F.

1987-01-01

To determine whether or not proteins circulating in plasma can be incorporated into megakaryocytes and platelets, horseradish peroxidase (HRP) was injected intravenously into guinea pigs and these cells were examined for its uptake by electron microscopy and cytochemistry. Enriched samples of megakaryocytes enabled ultrastructural analysis of large numbers of these rare cells. In megakaryocytes, 50% of alpha granules contained HRP between 75 min and 7 hr after injection. At 24 hr, 25% of the megakaryocyte granules were peroxidase-positive, less were positive by 48 hr, and there were none at 4 days. Thus, the findings demonstrate that a circulating protein can be endocytosed by megakaryocytes and rapidly packaged into alpha granules. Platelet granules also contain HRP by 7 hr after injection, and they can secrete it in response to thrombin. Unfortunately, our present studies do not allow us to distinguish between direct endocytosis by the platelet and/or shedding of new platelets from recently labeled megakaryocytes. It is concluded that while some alpha granule proteins are synthesized by megakaryocytes, others may be acquired from plasma by endocytosis. In addition to providing evidence that some of the proteins of alpha granules may be of exogenous origin, this study has allowed the definition of a pathway whereby plasma proteins may be temporarily sequestered in megakaryocytes before reentering the circulation in platelets.

Three-dimensional structure and cytokine distribution of platelet-rich fibrin.

PubMed

Bai, Meng-Yi; Wang, Ching-Wei; Wang, Jyun-Yi; Lin, Ming-Fang; Chan, Wing P

2017-02-01

Previous reports have revealed that several cytokines (including platelet-derived growth factor-BB, transforming growth factors-β1 and insulin-like growth factor-1) can enhance the rate of bone formation and synthesis of extracellular matrix in orthopaedics or periodontology. This study aimed to determine the concentration of cytokines within platelet-rich fibrin microstructures and investigate whether there are differences in the different portions of platelet-rich fibrin, which has implications for proper clinical use of platelet-rich fibrin gel. Whole blood was obtained from six New Zealand rabbits (male, 7 to 39 weeks old, weight 2.7-4 kg); it was then centrifuged for preparation of platelet-rich fibrin gels and harvest of plasma. The resultant platelet-rich fibrin gels were used for cytokine determination, histological analyses and scanning electron microscopy. All plasmas obtained were subject to the same cytokine determination assays for the purpose of comparison. Cytokines platelet-derived growth factor-BB and transforming growth factor-β1 formed concentration gradients from high at the red blood cell end of the platelet-rich fibrin gel (p=1.88×10-5) to low at the plasma end (p=0.19). Insulin-like growth factor-1 concentrations were similar at the red blood cell and plasma ends. The porosities of the platelet-rich fibrin samples taken in sequence from the red blood cell end to the plasma end were 6.5% ± 4.9%, 24.8% ± 7.5%, 30.3% ± 8.5%, 41.4% ± 12.3%, and 40.3% ± 11.7%, respectively, showing a gradual decrease in the compactness of the platelet-rich fibrin network. Cytokine concentrations are positively associated with platelet-rich fibrin microstructure and portion in a rabbit model. As platelet-rich fibrin is the main entity currently used in regenerative medicine, assessing cytokine concentration and the most valuable portion of PRF gels is essential and recommended to all physicians.

Phase transitions during compression and decompression of clots from platelet-poor plasma, platelet-rich plasma and whole blood.

PubMed

Liang, Xiaojun; Chernysh, Irina; Purohit, Prashant K; Weisel, John W

2017-09-15

Blood clots are required to stem bleeding and are subject to a variety of stresses, but they can also block blood vessels and cause heart attacks and ischemic strokes. We measured the compressive response of human platelet-poor plasma (PPP) clots, platelet-rich plasma (PRP) clots and whole blood clots and correlated these measurements with confocal and scanning electron microscopy to track changes in clot structure. Stress-strain curves revealed four characteristic regions, for compression-decompression: (1) linear elastic region; (2) upper plateau or softening region; (3) non-linear elastic region or re-stretching of the network; (4) lower plateau in which dissociation of some newly made connections occurs. Our experiments revealed that compression proceeds by the passage of a phase boundary through the clot separating rarefied and densified phases. This observation motivates a model of fibrin mechanics based on the continuum theory of phase transitions, which accounts for the pre-stress caused by platelets, the adhesion of fibrin fibers in the densified phase, the compression of red blood cells (RBCs), and the pumping of liquids through the clot during compression/decompression. Our experiments and theory provide insights into the mechanical behavior of blood clots that could have implications clinically and in the design of fibrin-based biomaterials. The objective of this paper is to measure and mathematically model the compression behavior of various human blood clots. We show by a combination of confocal and scanning electron microscopy that compression proceeds by the passage of a front through the sample that separates a densified region of the clot from a rarefied region, and that the compression/decompression response is reversible with hysteresis. These observations form the basis of a model for the compression response of clots based on the continuum theory of phase transitions. Our studies may reveal how clot rheology under large compression in vivo due

Platelet and not erythrocyte microparticles are procoagulant in transfused thalassaemia major patients.

PubMed

Agouti, Imane; Cointe, Sylvie; Robert, Stéphane; Judicone, Coralie; Loundou, Anderson; Driss, Fathi; Brisson, Alain; Steschenko, Dominique; Rose, Christian; Pondarré, Corinne; Bernit, Emmanuelle; Badens, Catherine; Dignat-George, Françoise; Lacroix, Romaric; Thuret, Isabelle

2015-11-01

The level of circulating platelet-, erythrocyte-, leucocyte- and endothelial-derived microparticles detected by high-sensitivity flow cytometry was investigated in 37 β-thalassaemia major patients receiving a regular transfusion regimen. The phospholipid procoagulant potential of the circulating microparticles and the microparticle-dependent tissue factor activity were evaluated. A high level of circulating erythrocyte- and platelet-microparticles was found. In contrast, the number of endothelial microparticles was within the normal range. Platelet microparticles were significantly higher in splenectomized than in non-splenectomized patients, independent of platelet count (P < 0·001). Multivariate analysis indicated that phospholipid-dependent procoagulant activity was influenced by both splenectomy (P = 0·001) and platelet microparticle level (P < 0·001). Erythrocyte microparticles were not related to splenectomy, appear to be devoid of proper procoagulant activity and no relationship between their production and haemolysis, dyserythropoiesis or oxidative stress markers could be established. Intra-microparticle labelling with anti-HbF antibodies showed that they originate only partially (median of 28%) from thalassaemic erythropoiesis. In conclusion, when β-thalassaemia major patients are intensively transfused, the procoagulant activity associated with thalassaemic erythrocyte microparticles is probably diluted by transfusions. In contrast, platelet microparticles, being both more elevated and more procoagulant, especially after splenectomy, may contribute to the residual thrombotic risk reported in splenectomized multi-transfused β-thalassaemia major patients. © 2015 John Wiley & Sons Ltd.

A quantitative ELISA procedure for the measurement of membrane-bound platelet-associated IgG (PAIgG).

PubMed

Lynch, D M; Lynch, J M; Howe, S E

1985-03-01

A quantitative ELISA assay for the measurement of in vivo bound platelet-associated IgG (PAIgG) using intact patient platelets is presented. The assay requires quantitation and standardization of the number of platelets bound to microtiter plate wells and an absorbance curve using quantitated IgG standards. Platelet-bound IgG was measured using an F(ab')2 peroxidase labeled anti-human IgG and o-phenylenediamine dihydrochloride (OPD) as the substrate. Using this assay, PAIgG for normal individuals was 2.8 +/- 1.6 fg/platelet (mean +/- 1 SD; n = 30). Increased levels were found in 28 of 30 patients with clinical autoimmune thrombocytopenia (ATP) with a range of 7.0-80 fg/platelet. Normal PAIgG levels were found in 26 of 30 patients with nonimmune thrombocytopenia. In the sample population studied, the PAIgG assay showed a sensitivity of 93%, specificity of 90%, a positive predictive value of 0.90, and a negative predictive value of 0.93. The procedure is highly reproducible (CV = 6.8%) and useful in evaluating patients with suspected immune mediated thrombocytopenia.

Platelet-Specific Chemokines Contribute to the Pathogenesis of Acute Lung Injury.

PubMed

Bdeir, Khalil; Gollomp, Kandace; Stasiak, Marta; Mei, Junjie; Papiewska-Pajak, Izabela; Zhao, Guohua; Worthen, G Scott; Cines, Douglas B; Poncz, Mortimer; Kowalska, M Anna

2017-02-01

Platelets and neutrophils contribute to the development of acute lung injury (ALI). However, the mechanism by which platelets make this contribution is incompletely understood. We investigated whether the two most abundant platelet chemokines, CXCL7, which induces neutrophil chemotaxis and activation, and CXCL4, which does neither, mediate ALI through complementary pathogenic pathways. To examine the role of platelet-derived chemokines in the pathogenesis of ALI using Cxcl7 -/- and Cxcl4 -/- knockout mice and mice that express human CXCL7 or CXCL4, we measured levels of chemokines in these mice. ALI was then induced by acid aspiration, and the severity of injury was evaluated by histology and by the presence of neutrophils and protein in the bronchoalveolar lavage fluid. Pulmonary vascular permeability was studied in vivo by measuring extravasation of fluorescently labeled dextran. Murine CXCL7, both recombinant and native protein released from platelets, can be N-terminally processed by cathepsin G to yield a biologically active CXCL7 fragment. Although Cxcl7 -/- mice are protected from lung injury through the preservation of endothelial/epithelial barrier function combined with impaired neutrophils transmigration, Cxcl4 -/- mice are protected through improved barrier function without affecting neutrophils transmigration to the airways. Sensitivity to ALI is restored by transgenic expression of CXCL7 or CXCL4. Platelet-derived CXCL7 and CXCL4 contribute to the pathogenesis of ALI through complementary effects on neutrophil chemotaxis and through activation and vascular permeability.

Evidence that the platelet plasma membrane does not contain a (Ca2+ + Mg2+)-dependent ATPase.

PubMed

Steiner, B; Lüscher, E F

1985-09-10

The present study was designed to determine the subcellular distribution of the platelet (Ca2+ + Mg2+)-ATPase. Human platelets were surface labeled by the periodate-boro[3H]hydride method. Plasma membrane vesicles were then isolated to a purity of approx. 90% by a procedure utilizing wheat germ agglutinin affinity chromatography. These membranes were found to be 2.6-fold enriched in surface glycoproteins compared to an unfractionated vesicle fraction and almost 7-fold enriched compared to intact platelets. In contrast, the isolated plasma membranes showed a decreased specific activity of the (Ca2+ + Mg2+)-ATPase compared to the unfractionated vesicle fraction. This decrease in specific activity was found to be similar to that of an endoplasmic reticulum marker, glucose-6-phosphatase, and to that of a platelet inner membrane marker, phospholipase A2. We conclude, therefore, that the (Ca2+ + Mg2+)-ATPase is not located in the platelet plasma membrane but is restricted to membranes of intracellular origin.

Insomnia, platelet serotonin and platelet monoamine oxidase in chronic alcoholism.

PubMed

Nenadic Sviglin, Korona; Nedic, Gordana; Nikolac, Matea; Mustapic, Maja; Muck-Seler, Dorotea; Borovecki, Fran; Pivac, Nela

2011-08-18

Insomnia is a common sleep disorder frequently occurring in chronic alcoholic patients. Neurobiological basis of insomnia, as well as of alcoholism, is associated with disrupted functions of the main neurotransmitter systems, including the serotonin (5-hydroxytryptamine, 5-HT) system. Blood platelets are considered a limited peripheral model for the central 5-HT neurons, since both platelets and central 5-HT synaptosomes have similar dynamics of 5-HT. Platelet 5-HT concentration and platelet monoamine oxidase type B (MAO-B) are assumed to represent biomarkers for particular symptoms and behaviors in psychiatric disorders. The hypothesis of this study was that platelet 5-HT concentration and platelet MAO-B activity will be altered in chronic alcoholic patients with insomnia compared to comparable values in patients without insomnia. The study included 498 subjects: 395 male and 103 female medication-free patients with alcohol dependence and 502 healthy control subjects: 325 men and 177 women. The effects of early, middle and late insomnia (evaluated using the Hamilton Depression Rating Scale), as well as sex, age and smoking on platelet 5-HT concentration and platelet MAO-B activity were evaluated using one-way ANOVA and multiple regression analysis by the stepwise method. Platelet 5-HT concentration, but not platelet MAO-B activity, was significantly reduced in alcoholic patients with insomnia compared to patients without insomnia. Multiple regression analysis revealed that platelet 5-HT concentration was affected by middle insomnia, smoking and sex, while platelet MAO activity was affected only by sex and age. The present and previous data suggest that platelet 5-HT concentration might be used, after controlling for sex and smoking, as a biomarker for insomnia in alcoholism, PTSD and in rotating shift workers. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Effects of Nd:YAG laser-heated metal cap on human platelets in vitro

NASA Astrophysics Data System (ADS)

Liu, Xia; Guo, You-chi

1993-03-01

Human platelet-rich plasma (PRP) was irradiated in vitro with a fiberoptic Nd:YAG laser-heated metal cap to study its effects on platelets. The energy of the laser was 5 and 10 watts with an irradiation time of 0, 3, 6, and 9 seconds and 14 watts with an irradiation time of 0, 3, 4, and 5 seconds, respectively. The irradiated PRPs were analyzed for platelet count, aggregation reaction, thromboxane (TX)B2 measurement and electron microscopy. Various degrees of decrease in platelet count were observed in all groups. Except the 5Wx3S group, the other groups showed an increase in the maximum aggregation rate of platelets, which corresponded to the enhancement of TXB2 formation. It was also demonstrated by a transmission electron microscopy in 10Wx3S, 10Wx6S, 10Wx9S, 14Wx3S, 14Wx4S, and 14Wx5S energy groups that alpha- and dense-particles in irradiated platelets became sparse in number or even disappeared, less electron density, irregularity in size and shape, and a tendency for these particles to cluster around platelet membranes and open canalicular systems, which dilated apparently. Furthermore, scanning electron microscopy depicted the appearance of short and thick pseudopods on the surfaces of some irradiated platelets and an increase in the axis rate in most of the irradiated platelets.

Does the Drug Facts Label for nonprescription drugs meet its design objectives? A new procedure for assessing label effectiveness

PubMed Central

Ryan, Michael P; Costello-White, Reagan N

2017-01-01

We demonstrate an expanded procedure for assessing drug-label comprehension. Innovations include a pretest of drug preconceptions, verbal ability and label attentiveness measures, a label-scanning task, a free-recall test, category-clustering measures, and preconception-change scores. In total, 55 female and 39 male undergraduates read a facsimile Drug Facts Label for aspirin, a Cohesive-Prose Label, or a Scrambled-Prose Label. The Drug Facts Label outperformed the Scrambled-Prose Label, but not the Cohesive-Prose Label, in scanning effectiveness. The Drug Facts Label was no better than the Cohesive-Prose Label or the Scrambled-Prose Label in promoting attentiveness, recall and organization of drug facts, or misconception refutation. Discussion focuses on the need for refutational labels based on a sequence-of-events text schema. PMID:29379613

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.

[Platelet allo-antibodies identification strategies for preventing and managing platelet refractoriness].

PubMed

Basire, A; Picard, C

2014-11-01

Platelet refractoriness is a serious complication for patients receiving recurrent platelet transfusions, which can be explained by non-immune and immune causes. Human Leukocyte Antigens (HLA) allo-immunization, especially against HLA class I, is the major cause for immune platelet refractoriness. To a lesser extent, allo-antibodies against specific Human Platelet Antigen (HPA) are also involved. Pregnancy, transplantation and previous transfusions can lead to allo-immune reaction against platelet antigens. After transfusion, platelet count is decreased by accelerated platelet destruction related to antibodies fixation on incompatible platelet antigens. New laboratory tests for allo-antibodies identification were developed to improve sensibility and specificity, especially with the LUMINEX(®) technology. The good use and interpretation of these antibodies assays can improve strategies for platelet refractoriness prevention and management with a patient adapted response. Compatible platelets units can be selected according to their identity with recipient typing or immune compatibility regarding HLA or HPA antibodies or HLA epitope compatibility. Prospective studies are needed to further confirm the clinical benefit of new allo-antibodies identification methods and consensus strategies for immune platelet refractoriness management. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Inhibition of rabbit platelet activation in vitro by antagonists of platelet-activating factor (PAF)

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

Cox, C.P.; Wood, K.L.

1986-03-05

The authors used washed, (/sup 3/H)serotonin-labeled rabbit platelets to study the in vitro aggregation and secretion responses induced by graded doses of PAF in the presence or absence of specific antagonists of PAF. These antagonists included CV-3988, L-652,731, triazolam and alprazolam. Platelets were pretreated with either an antagonist or the appropriate diluent for 60 sec prior to the addition of PAF (2 x 10/sup -10/ to 2 x 10/sup -7/ M). Aggregation was monitored continuously and recorded as the height of the aggregation tracing at 60 sec post-PAF. Secretion of (/sup 3/H)-serotonin was measured in a sample of the plateletsmore » removed at 60 sec post-PAF. When 2 x 10/sup -10/ M PAF was used as the stimulus, the concentration of antagonist needed for 50% inhibition (IC/sub 50/) of secretion was obtained at 0.05 ..mu..M, 0.15 ..mu..M, 0.6 ..mu..M and 2.5 ..mu..M, respectively, for L-652,731, CV-3988, triazolam and alprazolam. The corresponding IC/sub 50/ for aggregation was obtained at 0.2 ..mu..M, 0.1 ..mu..M, 1.5 ..mu..M and 6.5 ..mu..M, respectively. The inhibitory effects of these antagonists could be overcome by increasing the dose of PAF used. Although all of the antagonists were capable of completely inhibiting platelet aggregation and secretion, L-652,731 was the most potent PAF antagonist on a molar basis.« less

Doxorubicin-loaded platelets conjugated with anti-CD22 mAbs: a novel targeted delivery system for lymphoma treatment with cardiopulmonary avoidance.

PubMed

Xu, Peipei; Zuo, Huaqin; Zhou, Rongfu; Wang, Fan; Liu, Xu; Ouyang, Jian; Chen, Bing

2017-08-29

B-cell lymphoma accounts for approximately 85% of all adult non-Hodgkin's lymphoma cases. Doxorubicin (DOX) is an indispensable drug for the treatment of non-Hodgkin's lymphoma. However, DOX causes severe cardiotoxicity, which limits its use in conventional treatment strategies. In this study, we developed a novel drug delivery system for lymphoma treatment: DOX-loaded platelets that were conjugated with anti-CD22 monoclonal antibodies (mAbs) (DOX-platelet-CD22). Platelets are bio- and immune-compatible drug carriers that can prolong the circulation time of drugs. Anti-CD22 mAb-labeled platelets can precisely deliver DOX to tumor cells. Our in vitro and in vivo experiments showed the enhanced antitumor activity and attenuated cardiotoxicity of DOX when delivered as DOX-platelet-CD22. Compared with other delivery systems, the uptake of DOX-platelet-CD22 by macrophage-like cells decreased. Moreover, DOX-platelet-CD22 showed platelet properties, such as tumor cell-induced platelet aggregation. Therefore, targeted chemotherapy that is mediated by DOX-platelet-CD22 is a promising option for lymphoma treatment.

IMMUNOREACTIONS INVOLVING PLATELETS

PubMed Central

Shulman, N. Raphael

1958-01-01

Quantitative aspects of platelet agglutination and inhibition of clot retraction by the antibody of quinidine purpura were described. The reactions appeared to depend on formation of types of antibody-quinidine-platelet complexes which could fix complement but complement was not necessary for these reactions. Complement fixation was at least 10 times more sensitive than platelet agglutination or inhibition of clot retraction for measurement and detection of antibody activity. Although it has been considered that antibodies of drug purpura act as platelet lysins in the presence of complement and that direct lysis of platelets accounts for development of thrombocytopenia in drug purpura, the present study suggests that attachment of antibody produces a change in platelets which is manifested in vitro only by increased susceptibility to non-specific factors which can alter the stability of platelets in the absence of antibody. The attachment of antibody to platelets in vivo may only indirectly affect platelet survival. In contrast to human platelets, dog, rabbit, and guinea pig platelets, and normal or trypsin-treated human red cells did not agglutinate, fix complement, or adsorb antibody; and intact human endothelial cells did not fix complement or adsorb antibody. Rhesus monkey platelets were not agglutinated by the antibody but did adsorb antibody and fix complement although their activity in these reactions differed quantitatively from that of human platelets. Cinchonine could be substituted for quinidine in agglutination and inhibition of clot retraction reactions but quinine and cinchonidine could not. Attempts to cause passive anaphylaxis in guinea pigs with the antibody of quinidine purpura were not successful. PMID:13525580

Platelet-Rich Plasma and Platelet Gel: A Review

PubMed Central

Everts, Peter A.M.; Knape, Johannes T.A.; Weibrich, Gernot; Schönberger, Jacques P.A.M.; Hoffmann, Johannes; Overdevest, Eddy P.; Box, Henk A.M.; van Zundert, André

2006-01-01

Abstract: Strategies to reduce blood loss and transfusion of allogeneic blood products during surgical procedures are important in modern times. The most important and well-known autologous techniques are preoperative autologous predonation, hemodilution, perioperative red cell salvage, postoperative wound blood autotransfusion, and pharmacologic modulation of the hemostatic process. At present, new developments in the preparation of preoperative autologous blood component therapy by whole blood platelet-rich plasma (PRP) and platelet-poor plasma (PPP) sequestration have evolved. This technique has been proven to reduce the number of allogeneic blood transfusions during open heart surgery and orthopedic operations. Moreover, platelet gel and fibrin sealant derived from PRP and PPP mixed with thrombin, respectively, can be exogenously applied to tissues to promote wound healing, bone growth, and tissue sealing. However, to our disappointment, not many well-designed scientific studies are available, and many anecdotic stories exist, whereas questions remain to be answered. We therefore decided to study perioperative blood management in more detail with emphasis on the application and production of autologous platelet gel and the use of fibrin sealant. This review addresses a large variety of aspects relevant to platelets, platelet-rich plasma, and the application of platelet gel. In addition, an overview of recent animal and human studies is presented. PMID:16921694

Role of leukocytes and platelets in acute myocardial infarction

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

Bednar, M.M.

1986-01-01

Myocardial ischemia initiates an inflammatory-like response in which invading neutrophils exacerbate the degree of injury. The effects of nafazatrom, a new antithrombotic agent, on leukocyte function in vitro and in vivo were related to its ability to salvage ischemic myocardium in an occulsion-reperfusion model of myocardial injury in the anesthetized dogs. Measurements of the neutrophil-specific myeloperoxidase enzyme in ischemic myocardium indicate that the smaller infarct size in dogs treated with nafazatrom is accompanied by a diminished leukocyte infiltration. The results obtained with nafazatrom emphasize the important role of the neutrophil in ischemia-induced myocardial damage. The possibility that myocardial ischemia-induced plateletmore » deposition was secondary to a neutrophil-mediated event was assessed by the injection of PGI{sub 2}-washed autologous {sup 111}indium-labeled platelets and measuring the amount of radioactivity in different regions of the heart following a 90 min. occlusion of the left anterior descending coronary artery followed by reperfusion for periods up to 5 hrs. Neutropenia, induced with specific sheep anti-dog neutrophil antiserum, significantly reduced platelet accumulation in the ischemic myocardium following 5 hrs. reperfusion and abolished the transmural platelet distribution. These results suggest that myocardial platelet deposition is secondary to a neutrophil-mediated event in this occlusion-reperfusion model of myocardial injury.« less

Primary porcine Kupffer cell phagocytosis of human platelets involves the CD18 receptor.

PubMed

Chihara, Ray K; Paris, Leela L; Reyes, Luz M; Sidner, Richard A; Estrada, Jose L; Downey, Susan M; Wang, Zheng-Yu; Tector, A Joseph; Burlak, Christopher

2011-10-15

Hepatic failure has been treated successfully with clinical extracorporeal perfusions of porcine livers. However, dog-to-pig and pig-to-baboon liver xenotransplant models have resulted in severe bleeding secondary to liver xenograft-induced thrombocytopenia. Kupffer cells (KC) are abundant phagocytic cells in the liver. KC express the CD11b/CD18 receptor, which has been implicated in chilled platelet binding and phagocytosis through interaction with platelet surface proteins and carbohydrates. We sought to identify the role of KC CD18 in liver xenograft-induced thrombocytopenia. Primary pig KC were characterized by flow cytometry, immunoblots, and quantitative polymerase chain reaction. Pig KC were used in inhibition assays with fluorescently labeled human platelets. The CD18 receptor was targeted for siRNA knockdown. Domestic and α1,3-galactosyltransferase double knockout porcine KC cultures were approximately 92% positive for CD18 as detected by quantitative polymerase chain reaction and flow cytometry. Use of CD18 blocking antibodies resulted in reduction of human platelet binding and phagocytosis. Additionally, asialofetuin, not fetuin, inhibited platelet phagocytosis suggesting the involvement of an oligosaccharide-binding site. Furthermore, reduced CD18 expression by siRNA resulted in decreased human platelet binding. Our data suggest that primary pig KC bind and phagocytose human platelets with involvement of CD18. Further understanding and modification of CD18 expression in pigs may result in a liver xenograft with reduced thrombocytopenic effects, which could be used as a bridge to allogeneic liver transplantation.

[Platelet function in acute myeloid leukemia. II. Aggregation of isolated platelets].

PubMed

Zawilska, K; Komarnicki, M; Mańka, B

1978-01-01

In 22 patients with acute myeloid leukaemia (17 cases of myeloblastic leukaemia, 4 cases of myelomonocytic leukaemia and 1 case of undifferentiated-cell leukaemia) platelets were isolated from the plasma by the method of Nicholls and Hampton as modified by Levy-Toledano by centrifugation in albumin gradient. The aim of platelet isolation was their "concentration" in cases of thrombocytopenia to values making possible aggregation tests, and platelet separation from the influence of plasma factors. Then aggregation of isolated platelets caused by ADP was studied. In 16 out of 22 patients a fall of aggregation was observed, with the mean values of aggregation rate and intensity were significantly lower. Parallelly done determinations of aggregating activity released from the platelets by thrombin showed lower values as compared with platelets from healthy subjects. In might be thought, in this connection, that the demonstrated reduction of isolated platelets is associated with a diminution of the nucleotide pool or disturbances of the platelet release reaction. The disturbances of the platelet release reaction. The disturbances of aggregation of isolated platelets and reduction of the aggregating activity were most pronounced in acute myelomonocytic leukaemia.

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

Two efficient label-equivalence-based connected-component labeling algorithms for 3-D binary images.

PubMed

He, Lifeng; Chao, Yuyan; Suzuki, Kenji

2011-08-01

Whenever one wants to distinguish, recognize, and/or measure objects (connected components) in binary images, labeling is required. This paper presents two efficient label-equivalence-based connected-component labeling algorithms for 3-D binary images. One is voxel based and the other is run based. For the voxel-based one, we present an efficient method of deciding the order for checking voxels in the mask. For the run-based one, instead of assigning each foreground voxel, we assign each run a provisional label. Moreover, we use run data to label foreground voxels without scanning any background voxel in the second scan. Experimental results have demonstrated that our voxel-based algorithm is efficient for 3-D binary images with complicated connected components, that our run-based one is efficient for those with simple connected components, and that both are much more efficient than conventional 3-D labeling algorithms.

Inhibitory Effect of Flavonolignans on the P2Y12 Pathway in Blood Platelets.

PubMed

Bijak, Michal; Szelenberger, Rafal; Dziedzic, Angela; Saluk-Bijak, Joanna

2018-02-10

Adenosine diphosphate (ADP) is the major platelet agonist, which is important in the shape changes, stability, and growth of the thrombus. Platelet activation by ADP is associated with the G protein-coupled receptors P2Y1 and P2Y12. The pharmacologic blockade of the P2Y12 receptor significantly reduces the risk of peripheral artery disease, myocardial infarction, ischemic stroke, and vascular death. Recent studies demonstrated the inhibition of ADP-induced blood platelet activation by three major compounds of the flavonolignans group: silybin, silychristin, and silydianin. For this reason, the aim of the current work was to verify the effects of silybin, silychristin, and silydianin on ADP-induced physiological platelets responses, as well as mechanisms of P2Y12-dependent intracellular signal transduction. We evaluated the effect of tested flavonolignans on ADP-induced blood platelets' aggregation in platelet-rich plasma (PRP) (using light transmission aggregometry), adhesion to fibrinogen (using the static method), and the secretion of PF-4 (using the ELISA method). Additionally, using the double labeled flow cytometry method, we estimated platelet vasodilator-stimulated phosphoprotein (VASP) phosphorylation. We demonstrated a dose-dependent reduction of blood platelets' ability to perform ADP-induced aggregation, adhere to fibrinogen, and secrete PF-4 in samples treated with flavonolignans. Additionally, we observed that all of the tested flavonolignans were able to increase VASP phosphorylation in blood platelets samples, which is correlated with P2Y12 receptor inhibition. All of these analyses show that silychristin and silybin have the strongest inhibitory effect on blood platelet activation by ADP, while silydianin also inhibits the ADP pathway, but to a lesser extent. The results obtained in this study clearly demonstrate that silybin, silychristin, and silydianin have inhibitory properties against the P2Y12 receptor and block ADP-induced blood platelet

Does the liquid method of electret forming influence the adhesion of blood platelets?

PubMed

Lowkis, B; Szymanowicz, M

1995-01-01

This work presents the results of the effect of the electric charge on the adhesion of blood platelets. All experiments were carried out on polyethylene foil. The liquid method was used to form electrets. The evaluation of the electret effect influence on the adhesion of blood platelets was made on the basis of the observation of the electret surface after the contact with fresh citrate human blood group O Rh+ in an electron scanning microscope. Experimental results confirmed the essential influence of the electric charge on the process of adhesion of blood platelets. It was noticed that the preliminary aging of electrets decreases the density of the surface charge and improves the athrombogenic characteristics of polyethylene foil.

Platelet-Specific Chemokines Contribute to the Pathogenesis of Acute Lung Injury

PubMed Central

Bdeir, Khalil; Gollomp, Kandace; Stasiak, Marta; Mei, Junjie; Papiewska-Pajak, Izabela; Zhao, Guohua; Worthen, G. Scott; Cines, Douglas B.; Poncz, Mortimer

2017-01-01

Platelets and neutrophils contribute to the development of acute lung injury (ALI). However, the mechanism by which platelets make this contribution is incompletely understood. We investigated whether the two most abundant platelet chemokines, CXCL7, which induces neutrophil chemotaxis and activation, and CXCL4, which does neither, mediate ALI through complementary pathogenic pathways. To examine the role of platelet-derived chemokines in the pathogenesis of ALI using Cxcl7−/− and Cxcl4−/− knockout mice and mice that express human CXCL7 or CXCL4, we measured levels of chemokines in these mice. ALI was then induced by acid aspiration, and the severity of injury was evaluated by histology and by the presence of neutrophils and protein in the bronchoalveolar lavage fluid. Pulmonary vascular permeability was studied in vivo by measuring extravasation of fluorescently labeled dextran. Murine CXCL7, both recombinant and native protein released from platelets, can be N-terminally processed by cathepsin G to yield a biologically active CXCL7 fragment. Although Cxcl7−/− mice are protected from lung injury through the preservation of endothelial/epithelial barrier function combined with impaired neutrophils transmigration, Cxcl4−/− mice are protected through improved barrier function without affecting neutrophils transmigration to the airways. Sensitivity to ALI is restored by transgenic expression of CXCL7 or CXCL4. Platelet-derived CXCL7 and CXCL4 contribute to the pathogenesis of ALI through complementary effects on neutrophil chemotaxis and through activation and vascular permeability. PMID:27755915

Dynamic adhesion of eryptotic erythrocytes to immobilized platelets via platelet phosphatidylserine receptors.

PubMed

Walker, Britta; Towhid, Syeda T; Schmid, Evi; Hoffmann, Sascha M; Abed, Majed; Münzer, Patrick; Vogel, Sebastian; Neis, Felix; Brucker, Sara; Gawaz, Meinrad; Borst, Oliver; Lang, Florian

2014-02-01

Glucose depletion of erythrocytes triggers suicidal erythrocyte death or eryptosis, which leads to cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptotic erythrocytes adhere to endothelial cells by a mechanism involving phosphatidylserine at the erythrocyte surface and CXCL16 as well as CD36 at the endothelial cell membrane. Nothing has hitherto been known about an interaction between eryptotic erythrocytes and platelets, the decisive cells in primary hemostasis and major players in thrombotic vascular occlusion. The present study thus explored whether and how glucose-depleted erythrocytes adhere to platelets. To this end, adhesion of phosphatidylserine-exposing erythrocytes to platelets under flow conditions was examined in a flow chamber model at arterial shear rates. Platelets were immobilized on collagen and further stimulated with adenosine diphosphate (ADP, 10 μM) or thrombin (0.1 U/ml). As a result, a 48-h glucose depletion triggered phosphatidylserine translocation to the erythrocyte surface and augmented the adhesion of erythrocytes to immobilized platelets, an effect significantly increased upon platelet stimulation. Adherence of erythrocytes to platelets was blunted by coating of erythrocytic phosphatidylserine with annexin V or by neutralization of platelet phosphatidylserine receptors CXCL16 and CD36 with respective antibodies. In conclusion, glucose-depleted erythrocytes adhere to platelets. The adhesive properties of platelets are augmented by platelet activation. Erythrocyte adhesion to immobilized platelets requires phosphatidylserine at the erythrocyte surface and CXCL16 as well as CD36 expression on platelets. Thus platelet-mediated erythrocyte adhesion may foster thromboocclusive complications in diseases with stimulated phosphatidylserine exposure of erythrocytes.

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

Differences in levels of platelet-derived microparticles in platelet components prepared using the platelet rich plasma, buffy coat, and apheresis procedures.

PubMed

Noulsri, Egarit; Udomwinijsilp, Prapaporn; Lerdwana, Surada; Chongkolwatana, Viroje; Permpikul, Parichart

2017-04-01

There has been an increased interest in platelet-derived microparticles (PMPs) in transfusion medicine. Little is known about PMP status during the preparation of platelet concentrates for transfusion. The aim of this study is to compare the PMP levels in platelet components prepared using the buffy coat (BC), platelet-rich plasma platelet concentrate (PRP-PC), and apheresis (AP) processes. Platelet components were prepared using the PRP-PC and BC processes. Apheresis platelets were prepared using the Trima Accel and Amicus instruments. The samples were incubated with annexin A5-FITC, CD41-PE, and CD62P-APC. At day 1 after processing, the PMPs and activated platelets were determined using flow cytometry. Both the percentage and number of PMPs were higher in platelet components prepared using the Amicus instrument (2.6±1.8, 32802±19036 particles/μL) than in platelet components prepared using the Trima Accel instrument (0.5±0.4, 7568±5298 particles/μL), BC (1.2±0.6, 12,920±6426 particles/μL), and PRP-PC (0.9±0.6, 10731±5514 particles/μL). Both the percentage and number of activated platelets were higher in platelet components prepared using the Amicus instrument (33.2±13.9, 427553±196965 cells/μL) than in platelet components prepared using the Trima Accel instrument (16.2±6.1, 211209±87706 cells/μL), BC (12.9±3.2, 140624±41003 cells/μL), and PRP-PC (21.1±6.3, 265210±86257 cells/μL). The study suggests high variability of PMPs and activated platelets in platelet components prepared using different processes. This result may be important in validating the instruments involved in platelet blood collection and processing. Copyright © 2016 Elsevier Ltd. All rights reserved.

The effect of sibutramine on platelet morphology of Spraque-Dawley rats fed a high energy diet.

PubMed

Oberholzer, Hester Magdalena; Van Der Schoor, Ciska; Pretorius, Etheresia

2013-06-01

The aim of this study was to investigate the effect of Sibutramine on platelet ultrastructure and discuss the morphological observations in relation to known physiological effects of the compound. Six-week-old, female Spraque-Dawley rats were used in this study. The animals were placed on a high energy diet after which sibutramine administration followed. Blood was drawn on the day of termination and platelet rich plasma was obtained to prepare plasma smears for analysis. Scanning electron microscopy was used to investigate the ultrastructure of the platelets. Platelets of the Sibutramine-treated animals showed smooth surface with limited pseudopodia formation when compared with that of the control animals. Higher magnification of the platelet surface showed membrane tears and swelling, typically seen in necrotic cells. It can therefore be concluded from these results that Sibutramine alters the membrane morphology of platelets to that typical of necrotic cells. Copyright © 2013 Wiley Periodicals, Inc.

Efficient organ localization using multi-label convolutional neural networks in thorax-abdomen CT scans

NASA Astrophysics Data System (ADS)

Efrain Humpire-Mamani, Gabriel; Arindra Adiyoso Setio, Arnaud; van Ginneken, Bram; Jacobs, Colin

2018-04-01

Automatic localization of organs and other structures in medical images is an important preprocessing step that can improve and speed up other algorithms such as organ segmentation, lesion detection, and registration. This work presents an efficient method for simultaneous localization of multiple structures in 3D thorax-abdomen CT scans. Our approach predicts the location of multiple structures using a single multi-label convolutional neural network for each orthogonal view. Each network takes extra slices around the current slice as input to provide extra context. A sigmoid layer is used to perform multi-label classification. The output of the three networks is subsequently combined to compute a 3D bounding box for each structure. We used our approach to locate 11 structures of interest. The neural network was trained and evaluated on a large set of 1884 thorax-abdomen CT scans from patients undergoing oncological workup. Reference bounding boxes were annotated by human observers. The performance of our method was evaluated by computing the wall distance to the reference bounding boxes. The bounding boxes annotated by the first human observer were used as the reference standard for the test set. Using the best configuration, we obtained an average wall distance of 3.20~+/-~7.33 mm in the test set. The second human observer achieved 1.23~+/-~3.39 mm. For all structures, the results were better than those reported in previously published studies. In conclusion, we proposed an efficient method for the accurate localization of multiple organs. Our method uses multiple slices as input to provide more context around the slice under analysis, and we have shown that this improves performance. This method can easily be adapted to handle more organs.

Heterogeneous distribution of antigens on human platelets demonstrated by fluorescence flow cytometry.

PubMed

Dunstan, R A; Simpson, M B

1985-12-01

We have used fluorescence flow cytometry to analyse cell-to-cell variability in the density of platelet ABH, Ii, Lewis, P, P1A1, Bak,a and HLA class I antigens. Human IgG and IgM antibodies were used in a two-stage assay with goat FITC-conjugated antihuman IgG (H&L) antibody as the label, followed by single cell analysis of 10 000 platelets per sample using a 256-channel fluorescence flow cytometer (Becton-Dickinson FACS Analyser). Computer analysis of fluorescence intensity histograms for mean and peak channel and coefficient of variation shows that the degree of heterogeneity in platelet antigen density varies with each particular blood group. The broad fluorescence distribution curves with oligosaccharide antigens (CVs: A = 53, B = 40, I = 44, Lea = 40, P = 40) indicate that these antigens possess a greater variability in the number of sites per cell compared to the more homogeneous distribution of P1,A1 BaK,a and HLA (CVs: P1A1 = 24, HLA = 30). These findings may partly account for the mechanism by which transfusion of ABO-incompatible platelets results in a biphasic survival curve, with a period of early rapid removal of those platelets with a high density of antigen sites, followed by a relatively normal survival curve for those platelets that possess only a few or no antigen sites. In contrast, P1A1 and HLA sites are less variable in number from one platelet to another in a given donor, and immune-mediated removal would be more likely to approximate a single exponential curve.

Phylogenetic analysis of platelet-derived growth factor by radio- receptor assay

PubMed Central

1982-01-01

Competition between 125I-labeled platelet-derived growth factor (PDGF) and unlabeled PDGF forms the basis of a specific "radio-receptor assay" for quantifying PDGF in clotted blood serum. Human clotted blood serum contains 15 ng/ml of PDGF by radio-receptor assay; this corresponds to a PDGF content of approximately 7.5 x 10(-5) pg per circulating platelet, a figure which is corroborated by purification data. Clotted blood sera from mammals, lower vertebrates and marine invertebrates were screened for homologues of human PDGF by radio-receptor assay. All tested specimens from phylum Chordata contain a mitogenic agent that competes with human PDGF for receptor binding. Sera from tunicates down on the chordate line of evolution and sera from all tested animals on the arthropod line of development were negative. The phylogenetic distribution of PDGF homologue does not correlate with platelet distribution since platelets and their precursor cell--the bone marrow megacaryocyte--are unique to the mammalian hematopoietic system. One anatomical feature appearing coordinately with PDGF on the vertebrate line of development is a pressurized circulatory system. The coincidental appearance of these features may lend support to the hypothesis that PDGF plays a role in maintenance and repair of the vascular lining in vivo. PMID:7142300

Platelet collection efficiencies of three different platelet-rich plasma preparation systems.

PubMed

Aydin, Fatma; Pancar Yuksel, Esra; Albayrak, Davut

2015-06-01

Different systems have been used for the preparation of platelet-rich plasma (PRP), but platelet collection efficiencies of these systems are not clear. To evaluate the platelet collection efficiencies of three different PRP preparation systems. Blood samples were obtained from the same 16 volunteers for each system. The samples were centrifuged and PRP was prepared by three systems. The ratio of the total number of platelets in PRP to the total number of platelets of the venous blood sample of the patient expressed in percentage was named as platelet collection efficiency and calculated for each system. Mean platelet collection efficiencies were 66.6 (min: 56.9, max: 76.9), 58.3 (min: 27.3, max: 102.8), 50.8 (min: 27.2, max: 73) for top and bottom bag system, system using citrated tube, and the system using tube with Ficoll and cell extraction kit, respectively. Statistically significant difference was found only between the platelet collection efficiencies of systems using the tube with ficoll and cell extraction kit and the top and bottom bag system (p = 0.002). All three systems could be used for PRP preparation, but top and bottom bag system offers a slight advantage over the system using Ficoll and cell extraction kit regarding the platelet collection efficiency.

Exploratory studies of extended storage of apheresis platelets in a platelet additive solution (PAS)

PubMed Central

Corson, Jill; Jones, Mary Kay; Christoffel, Todd; Pellham, Esther; Bailey, S. Lawrence; Bolgiano, Doug

2014-01-01

To evaluate the poststorage viability of apheresis platelets stored for up to 18 days in 80% platelet additive solution (PAS)/20% plasma, 117 healthy subjects donated platelets using the Haemonetics MCS+, COBE Spectra (Spectra), or Trima Accel (Trima) systems. Control platelets from the same subjects were compared with their stored test PAS platelets by radiolabeling their stored and control platelets with either 51chromium or 111indium. Trima platelets met Food and Drug Administration poststorage platelet viability criteria for only 7 days vs almost 13 days for Haemonetics platelets; ie, platelet recoveries after these storage times averaged 44 ± 3% vs 49 ± 3% and survivals were 5.4 ± 0.3 vs 4.6 ± 0.3 days, respectively. The differences in storage duration are likely related to both the collection system and the storage bag. The Spectra and Trima platelets were hyperconcentrated during collection, and PAS was added, whereas the Haemonetics platelets were elutriated with PAS, which may have resulted in less collection injury. When Spectra and Trima platelets were stored in Haemonetics’ bags, poststorage viability was significantly improved. Platelet viability is better maintained in vitro than in vivo, allowing substantial increases in platelet storage times. However, implementation will require resolution of potential bacterial overgrowth during storage. PMID:24258816

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.

LDL oxidation by platelets propagates platelet activation via an oxidative stress-mediated mechanism.

PubMed

Carnevale, Roberto; Bartimoccia, Simona; Nocella, Cristina; Di Santo, Serena; Loffredo, Lorenzo; Illuminati, Giulio; Lombardi, Elisabetta; Boz, Valentina; Del Ben, Maria; De Marco, Luigi; Pignatelli, Pasquale; Violi, Francesco

2014-11-01

Platelets generate oxidized LDL (ox-LDL) via NOX2-derived oxidative stress. We investigated if once generated by activated platelets ox-LDL can propagate platelet activation. Experiments were performed in platelets from healthy subjects (HS), hyper-cholesterolemic patients and patients with NOX2 hereditary deficiency. Agonist-stimulated platelets from HS added with LDL were associated with a dose-dependent increase of reactive oxidant species and ox-LDL. Agonist-stimulated platelets from HS added with a fixed dose of LDL (57.14 μmol/L) or added with homogenized human atherosclerotic plaque showed enhanced ox-LDL formation (approximately +50% and +30% respectively), which was lowered by a NOX2 inhibitor (approximately -35% and -25% respectively). Compared to HS, ox-LDL production was more pronounced in agonist-stimulated platelet rich plasma (PRP) from hyper-cholesterolemic patients but was almost absent in PRP from NOX2-deficient patients. Platelet aggregation and 8-iso-PGF2α-ΙΙΙ formation increased in LDL-treated washed platelets (+42% and +53% respectively) and PRP (+31% and +53% respectively). Also, LDL enhanced platelet-dependent thrombosis at arterial shear rate (+33%) but did not affect platelet activation in NOX2-deficient patients. Platelet activation by LDL was significantly inhibited by CD36 or LOX1 blocking peptides, two ox-LDL receptor antagonists, or by a NOX2 inhibitor. LDL-added platelets showed increased p38MAPK (+59%) and PKC (+51%) phosphorylation, p47(phox) translocation to platelet membrane (+34%) and NOX2 activation (+30%), which were inhibited by ox-LDL receptor antagonists. Platelets oxidize LDL, which in turn amplify platelet activation via specific ox-LDL receptors; both effects are mediated by NOX2 activation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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 ...

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.

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

Phosphatidylserine-mediated platelet clearance by endothelium decreases platelet aggregates and procoagulant activity in sepsis.

PubMed

Ma, Ruishuang; Xie, Rui; Yu, Chengyuan; Si, Yu; Wu, Xiaoming; Zhao, Lu; Yao, Zhipeng; Fang, Shaohong; Chen, He; Novakovic, Valerie; Gao, Chunyan; Kou, Junjie; Bi, Yayan; Thatte, Hemant S; Yu, Bo; Yang, Shufen; Zhou, Jin; Shi, Jialan

2017-07-10

The mechanisms that eliminate activated platelets in inflammation-induced disseminated intravascular coagulation (DIC) in micro-capillary circulation are poorly understood. This study explored an alternate pathway for platelet disposal mediated by endothelial cells (ECs) through phosphatidylserine (PS) and examined the effect of platelet clearance on procoagulant activity (PCA) in sepsis. Platelets in septic patients demonstrated increased levels of surface activation markers and apoptotic vesicle formation, and also formed aggregates with leukocytes. Activated platelets adhered were and ultimately digested by ECs in vivo and in vitro. Blocking PS on platelets or αvβ3 integrin on ECs attenuated platelet clearance resulting in increased platelet count in a mouse model of sepsis. Furthermore, platelet removal by ECs resulted in a corresponding decrease in platelet-leukocyte complex formation and markedly reduced generation of factor Xa and thrombin on platelets. Pretreatment with lactadherin significantly increased phagocytosis of platelets by approximately 2-fold, diminished PCA by 70%, prolonged coagulation time, and attenuated fibrin formation by 50%. Our results suggest that PS-mediated clearance of activated platelets by the endothelium results in an anti-inflammatory, anticoagulant, and antithrombotic effect that contribute to maintaining platelet homeostasis during acute inflammation. These results suggest a new therapeutic target for impeding the development of DIC.

In vivo evaluation of titanium-prepared platelet-rich fibrin (T-PRF): a new platelet concentrate.

PubMed

Tunalı, Mustafa; Özdemir, Hakan; Küçükodacı, Zafer; Akman, Serhan; Fıratlı, Erhan

2013-07-01

We have developed a new, titanium-prepared, platelet-rich fibrin (T-PRF) together with the protocol for forming it, which is based on the hypothesis that titanium tubes may be more effective at activating platelets than the glass tubes used by Chouckroun in his platelet-rich fibrin (PRF) method. The aim of this study was to find a suitable animal model in which to evaluate the method and to investigate the efficacy of T-PRF for wound healing. Blood samples from 6 rabbits were used to confirm the protocol for formation of T-PRF. We evaluated T-PRF or T-PRF-like clots morphologically using scanning electron microscopy (EM). Blood samples from 5 rabbits were used to develop an experiment in which to evaluate the effects of T-PRF on wound healing. The mucoperiosteal flaps were filled with autologous T-PRF membranes from the vestibule in the anterior mandibular regions. Samples collected from the surgical sites were stained with haematoxylin and eosin. We found a mature fibrin network in T-PRF clots that had been centrifuged for 15 min at 3500 rpm and, 15 days after placement of the membrane, we found newly-forming connective tissue and islets of bony tissue in the T-PRF membrane. These results show that T-PRF could induce the formation of new bone with new connective tissue in a rabbit model of wound healing within 30 days of treatment. Published by Elsevier Ltd.

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.

Site-Specific Targeting of Platelet-Rich Plasma via Superparamagnetic Nanoparticles

PubMed Central

Talaie, Tara; Pratt, Stephen J.P.; Vanegas, Camilo; Xu, Su; Henn, R. Frank; Yarowsky, Paul; Lovering, Richard M.

2015-01-01

Background: Muscle strains are one of the most common injuries treated by physicians. Standard conservative therapy for acute muscle strains usually involves short-term rest, ice, and nonsteroidal anti-inflammatory medications, but there is no clear consensus regarding treatments to accelerate recovery. Recently, clinical use of platelet-rich plasma (PRP) has gained momentum as an option for therapy and is appealing for many reasons, most notably because it provides growth factors in physiological proportions and it is autologous, safe, easily accessible, and potentially beneficial. Local delivery of PRP to injured muscles can hasten recovery of function. However, specific targeting of PRP to sites of tissue damage in vivo is a major challenge that can limit its efficacy. Hypothesis: Location of PRP delivery can be monitored and controlled in vivo with noninvasive tools. Study Design: Controlled laboratory study. Methods: Superparamagnetic iron oxide nanoparticles (SPIONs) can be visualized by both magnetic resonance imaging (MRI) (in vivo) and fluorescence microscopy (after tissue harvesting). PRP was labeled with SPIONs and administered by intramuscular injections of SPION-containing platelets. MRI was used to monitor the ability to manipulate and retain the location of PRP in vivo by placement of an external magnet. Platelets were isolated from whole blood and incubated with SPIONs. Following SPION incubation with PRP, a magnetic field was used to manipulate platelet location in culture dishes. In vivo, the tibialis anterior (TA) muscles of anesthetized Sprague-Dawley rats were injected with SPION-containing platelets, and MRI was used to track platelet position with and without a magnet worn over the TA muscles for 4 days. Results: The method used to isolate PRP yielded a high concentration (almost 4-fold increase) of platelets. In vitro experiments showed that the platelets successfully took up SPIONs and then rapidly responded to an applied magnetic field

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

A multicolor flow cytometric assay for measurement of platelet-derived microparticles.

PubMed

Mobarrez, Fariborz; Antovic, Jovan; Egberg, Nils; Hansson, Mona; Jörneskog, Gun; Hultenby, Kjell; Wallén, Håkan

2010-03-01

Flow cytometry (FCM) is the most commonly used method for detection of platelet-derived microparticles (PDMPs), but it is poorly standardized and mainly used for "bedside" analyses in fresh samples. If PDMPs could be analyzed in previously frozen samples it would increase the usefulness of the method. However, cell membrane fragments from contaminating cells created during freezing/thawing may cause artifacts and disturb measurements. PDMPs were labeled with monoclonal antibodies directed against CD42a and CD62P, or CD42a and CD142. The PDMP gate was determined using forward scatter (FSC) and CD42a expression. The mean fluorescence intensities (MFIs) of CD62P or CD142 positive particles were translated into MESF -values (Molecules of Equivalent Soluble Fluorochrome) using a standard curve. FITC-labeled phalloidin (which binds to intracellular actin) was used to detect destroyed cells/cell fragments. Phalloidin-positive particles were significantly more common in supernatants of frozen/thawed platelet rich and platelet poor plasma samples compared with supernatants of platelet free plasma. High-speed centrifugation was then used to obtain PDMP samples with low contamination of cell fragments. Electron microscopy showed that these samples contained numerous round stained particles with cellular membranes of a size of 100-700 nm. Reproducibility experiments using plasma samples from healthy individuals showed that the coefficients of variation (CVs) of MESF values of CD62P and CD142 (both intra- and interassay) were <10%, and the variation between two cytometers in two different laboratories was <5%. We also found that PDMP expression of CD142 (i.e. tissue factor [TF]) and CD62P (i.e P-selectin) was around two times higher in samples from type 1-diabetes patients compared with those from healthy controls (p<0.001). The use of MESF values to quantify PDMP expression of P-selectin and TF yields reproducible data and enables comparison of data between laboratories. If

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

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.

Effectiveness of Platelet-rich Plasma Injection for Rotator Cuff Tendinopathy: A Prospective Open-label Study.

PubMed

Scarpone, Michael; Rabago, David; Snell, Edward; Demeo, Patrick; Ruppert, Kristine; Pritchard, Perry; Arbogast, Gennie; Wilson, John J; Balzano, John F

2013-03-01

Assess platelet rich plasma (PRP) injection for rotator cuff tendinopathy (RCT). Prospective open label study with 1-year follow-up. Participants recruited from an outpatient sports medicine clinic had clinically and magnetic resonance image (MRI)-demonstrated RCT refractory to physical therapy and corticosteroid injection. They received one ultrasound-guided injection of 3.0 mL of 1% xylocaine followed by 3.5 mL of PRP at the lesion and surrounding tendon. 0-10 visual analog scale (VAS; baseline, 8, 12, and 52 weeks). functional shoulder tests assessing rotator cuff strength and endurance (at baseline and 8 and 12 weeks), MRI severity (1-5 points [at baseline and 4 and 8 weeks]), and patient satisfaction (52 weeks). Eighteen participants with 19 assessed shoulders reported VAS pain score improvement from 7.5 ± 0.3 points to 0.5 ± 0.3 points by week 12 and 0.4 ± 0.2 (P = .0001) points at week 52. Functional outcomes significantly improved; the largest effect was seen in the external rotation test: 33.5 ± 5.7 seconds to 62.6 ± 7.2 seconds at week 12 (P = .0001). MRI appearance improved by 1 to 3 points in 16 of 18 assessed shoulders. Seventeen participants were "completely satisfied" (12) or "satisfied" (5). One participant was "unsatisfied." A single ultrasound-guided, intralesional injection of PRP resulted in safe, significant, sustained improvement of pain, function, and MRI outcomes in participants with refractory RCT. Randomized multidisciplinary effectiveness trials that add ultrasound and validated clinical outcome measures are needed to further assess PRP for RCT.

[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).

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.

Comparison of ultrastructural and nanomechanical signature of platelets from acute myocardial infarction and platelet activation

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

Li, Aiqun; Chen, Jianwei; Liang, Zhi-Hong

Acute myocardial infarction (AMI) initiation and progression follow complex molecular and structural changes in the nanoarchitecture of platelets. However, it remains poorly understood how the transformation from health to AMI alters the ultrastructural and biomechanical properties of platelets within the platelet activation microenvironment. Here, we show using an atomic force microscope (AFM) that platelet samples, including living human platelets from the healthy and AMI patient, activated platelets from collagen-stimulated model, show distinct ultrastructural imaging and stiffness profiles. Correlative morphology obtained on AMI platelets and collagen-activated platelets display distinct pseudopodia structure and nanoclusters on membrane. In contrast to normal platelets, AMImore » platelets have a stiffer distribution resulting from complicated pathogenesis, with a prominent high-stiffness peak representative of platelet activation using AFM-based force spectroscopy. Similar findings are seen in specific stages of platelet activation in collagen-stimulated model. Further evidence obtained from different force measurement region with activated platelets shows that platelet migration is correlated to the more elasticity of pseudopodia while high stiffness at the center region. Overall, ultrastructural and nanomechanical profiling by AFM provides quantitative indicators in the clinical diagnostics of AMI with mechanobiological significance.« less

Accumulation of intimal platelets in cerebral arteries following experimental subarachnoid hemorrhage in cats

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

Haining, J.L.; Clower, B.R.; Honma, Y.

From 2 hours to 23 days following experimental subarachnoid hemorrhage, the accumulation of indium-111-labeled platelets on the intimal surface of the middle cerebral artery was studied in 23 cats. Subarachnoid hemorrhage was produced by transorbital rupture of the right middle cerebral artery. Of the 23 cats, 17 exhibited right middle cerebral artery/left middle cerebral artery radioactivity ratios of greater than 1.25. When these results were compared with those of 12 control cats, 0.001 less than p less than 0.005 (chi2 test). Thus, the results from the control and experimental groups are significantly different and indicate early (after 2 hours) preferentialmore » accumulation of intimal platelets in the ruptured right middle cerebral artery compared with the unruptured left middle cerebral artery and new platelet deposition continuing for up to 23 days. However, the experimental group did not reveal a clear pattern for platelet accumulation following subarachnoid hemorrhage. There was no simple correlation between the magnitude of the radioactivity ratios and the time after hemorrhage when the cats were killed although the ratios for 2 hours to 7 days seemed greater than those for 8 to 23 days. Assuming the pivotal role of platelets in the angiopathy of subarachnoid hemorrhage, the administration of antiplatelet agents as soon as possible following its occurrence may be of value.« less

Secreted Immunomodulatory Proteins of Staphylococcus aureus Activate Platelets and Induce Platelet Aggregation.

PubMed

Binsker, Ulrike; Palankar, Raghavendra; Wesche, Jan; Kohler, Thomas P; Prucha, Josephine; Burchhardt, Gerhard; Rohde, Manfred; Schmidt, Frank; Bröker, Barbara M; Mamat, Uwe; Pané-Farré, Jan; Graf, Anica; Ebner, Patrick; Greinacher, Andreas; Hammerschmidt, Sven

2018-04-01

Staphylococcus aureus can cause bloodstream infections associated with infective endocarditis (IE) and disseminated intravascular coagulopathy (DIC). Both complications involve platelets. In view of an increasing number of antibiotic-resistant strains, new approaches to control systemic S. aureus infection are gaining importance. Using a repertoire of 52 recombinant S. aureus proteins in flow cytometry-based platelet activation and aggregation assays, we identified, in addition to the extracellular adherence protein Eap, three secreted staphylococcal proteins as novel platelet activating proteins. Eap and the chemotaxis inhibitory protein of S. aureus (CHIPS), the formyl peptide receptor-like 1 inhibitory protein (FLIPr) and the major autolysin Atl induced P-selectin expression in washed platelets and platelet-rich plasma. Similarly, AtlA, CHIPS and Eap induced platelet aggregation in whole blood. Fluorescence microscopy illustrated that P-selectin expression is associated with calcium mobilization and re-organization of the platelet actin cytoskeleton. Characterization of the functionally active domains of the major autolysin AtlA and Eap indicates that the amidase domain of Atl and the tandem repeats 3 and 4 of Eap are crucial for platelet activation. These results provide new insights in S. aureus protein interactions with platelets and identify secreted proteins as potential treatment targets in case of antibiotic-resistant S. aureus infection. Schattauer GmbH Stuttgart.

Splenic release of platelets contributes to increased circulating platelet size and inflammation after myocardial infarction.

PubMed

Gao, Xiao-Ming; Moore, Xiao-Lei; Liu, Yang; Wang, Xin-Yu; Han, Li-Ping; Su, Yidan; Tsai, Alan; Xu, Qi; Zhang, Ming; Lambert, Gavin W; Kiriazis, Helen; Gao, Wei; Dart, Anthony M; Du, Xiao-Jun

2016-07-01

Acute myocardial infarction (AMI) is characterized by a rapid increase in circulating platelet size but the mechanism for this is unclear. Large platelets are hyperactive and associated with adverse clinical outcomes. We determined mean platelet volume (MPV) and platelet-monocyte conjugation (PMC) using blood samples from patients, and blood and the spleen from mice with AMI. We further measured changes in platelet size, PMC, cardiac and splenic contents of platelets and leucocyte infiltration into the mouse heart. In AMI patients, circulating MPV and PMC increased at 1-3 h post-MI and MPV returned to reference levels within 24 h after admission. In mice with MI, increases in platelet size and PMC became evident within 12 h and were sustained up to 72 h. Splenic platelets are bigger than circulating platelets in normal or infarct mice. At 24 h post-MI, splenic platelet storage was halved whereas cardiac platelets increased by 4-fold. Splenectomy attenuated all changes observed in the blood, reduced leucocyte and platelet accumulation in the infarct myocardium, limited infarct size and alleviated cardiac dilatation and dysfunction. AMI-induced elevated circulating levels of adenosine diphosphate and catecholamines in both human and the mouse, which may trigger splenic platelet release. Pharmacological inhibition of angiotensin-converting enzyme, β1-adrenergic receptor or platelet P2Y12 receptor reduced platelet abundance in the murine infarct myocardium albeit having diverse effects on platelet size and PMC. In conclusion, AMI evokes release of splenic platelets, which contributes to the increase in platelet size and PMC and facilitates myocardial accumulation of platelets and leucocytes, thereby promoting post-infarct inflammation. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

Platelet adhesion in hypertension: application of a novel assay of platelet adhesion.

PubMed

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

2005-01-01

The increased risk of thromboembolism in hypertension may be related to a prothrombotic or hypercoagulable state, with abnormalities in haemostasis and platelet function. To investigate the role of platelets in the pathogenesis of thrombosis in hypertension, we applied a novel new assay to detect and quantify the degree of platelet adhesion to a defined coagulation molecule. Platelet-rich plasma (PRP) and citrated plasma (CP) were obtained from 50 patients with hypertension (25 treated, and 25 untreated) and 30 healthy controls. A suspension of 2 x 10(7) platelets were incubated for one hour in microtitre plates pre-coated with 5mg/mL fibrinogen. The supernatant was carefully aspirated, lysed with 5% tween and stored at -70 degrees C as supernatant platelet lysate (SPL). The wells were carefully washed with saline and bound platelets lysed as before, and stored at -70 degrees C as bound-platelet lysate (BPL). Soluble P-selectin (sP-sel) was determined in CP, SPL and BPL by enzyme-linked immunosorbent assay (ELISA). Patients with hypertension (both treated and previously untreated) had increased platelet adhesion, as determined by increased lysate sP-sel (P=0.002) in BPL, with no change in SPL (P=0.5) compared to healthy controls. There was no significant difference between treated and previously untreated hypertensives. Platelets from patients with hypertension display increased adhesion to an important coagulation factor (fibrinogen). This may, in part, account for the increased risk of thrombosis seen in these patients.

Solvothermally Synthesized Sb2Te3 Platelets Show Unexpected Optical Contrasts in Mid-Infrared Near-Field Scanning Microscopy.

PubMed

Hauer, Benedikt; Saltzmann, Tobias; Simon, Ulrich; Taubner, Thomas

2015-05-13

We report nanoscale-resolved optical investigations on the local material properties of Sb2Te3 hexagonal platelets grown by solvothermal synthesis. Using mid-infrared near-field microscopy, we find a highly symmetric pattern, which is correlated to a growth spiral and which extends over the entire platelet. As the origin of the optical contrast, we identify domains with different densities of charge carriers. On Sb2Te3 samples grown by other means, we did not find a comparable domain structure.

Apheresis platelet concentrates contain platelet-derived and endothelial cell-derived microparticles.

PubMed

Rank, A; Nieuwland, R; Liebhardt, S; Iberer, M; Grützner, S; Toth, B; Pihusch, R

2011-02-01

Microparticles (MP) are membrane vesicles with thrombogenic and immunomodulatory properties. We determined MP subgroups from resting platelets, activated platelets and endothelial cells in donors and apheresis platelet concentrates (PC). MP were double stained with annexin V and CD61 (platelet-derived MP; PMP), P-selectin or CD63 (MP from activated platelets) and CD144 plus E-selectin (endothelial cell-derived MP; EMP) and detected by flow cytometry in platelet donors (n=36) and apheresis PC (n=11; Trima™). PC contained MP, mainly from resting platelets [93% (90-95)], and minor fractions of PMP from activated platelets [P-selectin(+) or CD63(+); 4·8% (3·2-7·7) and 2·6% (2·0-4·0)]. Compared to donors, levels of annexin V+ MP, PMP, P-selectin(+) and CD63(+) MP were 1·7-, 2·3-, 8·6- and 3·1-fold higher in PC (all P<0·05). During storage (1-5 days), levels of annexin V+ MP and PMP did not increase, although small increases in the fraction of P-selectin(+) or CD63(+) MP occurred (both P<0·05). PC also contained EMP, which were 2·6- to 3·7-fold enriched in PC compared to donors (P<0·05). Transfusion of apheresis PC also results in transfusion of HLA-carrying PMP and EMP. This might counteract the aim of reducing transfused HLA load by leucodepletion. The increases in PMP exposing P-selectin or CD63 reflect mild platelet activation during storage. We conclude that in leucodepleted platelet apheresis using fluidized particle bed technology, MP are harvested mainly from the donor by apheresis. Improvement in apheresis technology might reduce MP load. © 2010 The Author(s). Vox Sanguinis © 2010 International Society of Blood Transfusion.

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.

Effect of heparin bonding on catheter-induced fibrin formation and platelet activation.

PubMed

Nichols, A B; Owen, J; Grossman, B A; Marcella, J J; Fleisher, L N; Lee, M M

1984-11-01

Pathologic and experimental evidence indicates that platelet activation and fibrin formation contribute to the pathogenesis of angina pectoris, coronary vasospasm and myocardial infarction. Detection of localized intravascular platelet activation and fibrin formation in vivo by selective blood sampling requires catheters that do not induce coagulation ex vivo. We studied the effect of heparin bonding of catheter surfaces on activation of the coagulation system by cardiovascular catheters. Woven Dacron, polyvinylchloride, and polyurethane catheters were tested and compared with identical catheters with heparin-bonded surfaces in 47 patients undergoing percutaneous cardiac catheterization. Platelet activation was measured by radioimmunoassay of plasma platelet factor 4 (PF4), beta-thromboglobulin (BTG), and thromboxane B2 (TXB2) in blood samples withdrawn through catheters, and fibrin formation was assessed by determination of fibrinopeptide A (FPA) levels. In blood samples collected through conventional catheters, FPA, PF4, BTG, and TXB2 levels were markedly elevated; blood sampling through heparin-bonded catheters had no significant effect on FPA, PF4, BTG, or TXB2 levels. Scanning electron microscopy disclosed extensive platelet aggregates and fibrin strands adherent to the surface of conventional catheters but not to heparin-bonded catheter surfaces. This study demonstrates that (1) collection of blood samples through cardiovascular catheters causes artifactual elevation of FPA, PF4, BTG, and TXB2 levels, and (2) heparin-bonded catheter surfaces effectively prevent catheter-induced platelet alpha-granule release and fibrin formation on catheter surfaces. Heparin-bonded catheters will facilitate investigation of the role of intravascular coagulation in coronary artery disease by eliminating catheter-induced fibrin formation and platelet activation.

Classification of platelet concentrates: from pure platelet-rich plasma (P-PRP) to leucocyte- and platelet-rich fibrin (L-PRF).

PubMed

Dohan Ehrenfest, David M; Rasmusson, Lars; Albrektsson, Tomas

2009-03-01

The topical use of platelet concentrates is recent and its efficiency remains controversial. Several techniques for platelet concentrates are available; however, their applications have been confusing because each method leads to a different product with different biology and potential uses. Here, we present classification of the different platelet concentrates into four categories, depending on their leucocyte and fibrin content: pure platelet-rich plasma (P-PRP), such as cell separator PRP, Vivostat PRF or Anitua's PRGF; leucocyte- and platelet-rich plasma (L-PRP), such as Curasan, Regen, Plateltex, SmartPReP, PCCS, Magellan or GPS PRP; pure plaletet-rich fibrin (P-PRF), such as Fibrinet; and leucocyte- and platelet-rich fibrin (L-PRF), such as Choukroun's PRF. This classification should help to elucidate successes and failures that have occurred so far, as well as providing an objective approach for the further development of these techniques.

Platelet-rich plasma and platelet gel preparation using Plateltex.

PubMed

Mazzucco, L; Balbo, V; Cattana, E; Borzini, P

2008-04-01

The platelet gel is made by embedding concentrate platelets within a semisolid (gel) network of polymerized fibrin. It is believed that this blood component will be used more and more in the treatment of several clinical conditions and as an adjunctive material in tissue engineering. Several systems are available to produce platelet-rich plasma (PRP) for topical therapy. Recently, a new system became commercially available, Plateltex. Here we report the technical performance of this system in comparison with the performance of other commercially available systems: PRGF, PRP-Landesber, Curasan, PCCS, Harvest, Vivostat, Regen and Fibrinet. Both the PRP and the gel were prepared according to the manufacturer's directions. The blood samples of 20 donors were used. The yield, the efficiency, and the amount of platelet-derived growth factor AB (PDGF-AB), transforming growth factor beta, vascular endothelial growth factor and fibroblast growth factor were measured in the resulting PRP. The feature of the batroxobin-induced gelation was evaluated. The yield, the collection efficiency and the growth factor content of Plateltex were comparable to those of most of the other available systems. The gelation time was not dependent on the fibrinogen concentration; however, it was strongly influenced by the contact surface area of the container where the clotting reaction took place (P < 0.0001). Plateltex provided platelet recovery, collection efficiency and PDGF-AB availability close to those provided by other systems marketed with the same intended use. Batroxobin, the enzyme provided to induce gelation, acts differently from thrombin, which is used by most other systems. Platelets treated with thrombin become activated; they release their growth factors quickly. Furthermore, thrombin-platelet interaction is a physiological mechanism that hastens the clot-retraction rate. On the contrary, platelets treated with batroxobin do not become activated; they are passively entrapped

EXTENDED STORAGE OF BUFFY-COAT PLATELET CONCENTRATES IN PLASMA OR A PLATELET ADDITIVE SOLUTION

PubMed Central

Slichter, Sherrill J.; Bolgiano, Doug; Corson, Jill; Jones, Mary Kay; Christoffel, Todd; Bailey, S. Lawrence; Pellham, Esther

2014-01-01

Background Platelet concentrates prepared from whole blood in the U.S. are made using the platelet-rich-plasma (PRP) method. The platelet concentrates must be made within 8 hours of blood collection and stored for only 5 days. In Europe and Canada, platelet concentrates are made using the buffy-coat (BC) method from whole blood held overnight at 22°C and storage times may be up to 7 days. Our studies were designed to determine how long BC platelets can be stored in plasma or Plasmalyte while meeting the FDA’s post-storage viability criteria. Study Design, Materials, And Methods Normal subjects donated whole blood that was stored at 22°C for 22 ± 2 hours prior to preparation of BC platelets. Platelets were stored for 5 to 8 days in either plasma or Plasmalyte concentrations of 65% or 80%. Radiolabeled autologous stored versus fresh platelet recoveries and survivals were assessed as well as post-storage in vitro assays. Results BC platelets stored in either plasma or 65% Plasmalyte met FDA post-storage platelet recovery criteria for 7 days but survivals for only 6 days, while storage in 80% Plasmalyte gave very poor results. Both stored platelet recoveries and survivals correlated with the same donor’s fresh results, but the correlation was much stronger between recoveries than survivals. In vitro measures of extent of shape change, morphology score, and pH best predicted post-storage platelet recoveries, while annexin V binding best predicted platelet survivals. Conclusion BC platelets stored in either plasma or 65% Plasmalyte meet FDA’s post-storage viability criteria for 6 days. PMID:24673482

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.

An enzyme-linked immunosorbent assay for the quantification of serum platelet-bindable IgG.

PubMed

Howe, S E; Lynch, D M; Lynch, J M

1984-01-01

An enzyme-linked immunosorbent assay (ELISA) using F(ab')2 peroxidase-labeled antihuman immunoglobulin and o-phenylenediamine dihydrochloride (OPD) as a substrate was developed to measure serum platelet bindable IgG (S-PBIgG). The assay was made quantitative by standardizing the number of normal "target" platelets bound to microtiter plate wells, and by incorporating quantitated IgG standards with each microtiter plate tested to prepare a standard calibration curve. By this method, S-PBIgG for normal individuals was 3.4 +/- 1.6 fg per platelet (mean +/- 1 SD; n = 40). Increased S-PBIgG levels were detected in 36 of 40 patients with clinical autoimmune thrombocytopenia (ATP), ranging from 7.0 to 85 fg per platelet. Normal S-PBIgG levels were found in 34 of 40 patients with nonimmune thrombocytopenia. This method showed a sensitivity of 90 percent, specificity of 85 percent, and in the sample population studied, a positive predictive value of 0.86 and a negative predictive value of 0.90. This assay is highly reproducible (coefficient of variation was 6.8%) and appears useful in the evaluation of patients with suspected immune-mediated thrombocytopenia.

Systematic Comparison of Label-Free, Metabolic Labeling, and Isobaric Chemical Labeling for Quantitative Proteomics on LTQ Orbitrap Velos

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

Li, Zhou; Adams, Rachel M; Chourey, Karuna

2012-01-01

A variety of quantitative proteomics methods have been developed, including label-free, metabolic labeling, and isobaric chemical labeling using iTRAQ or TMT. Here, these methods were compared in terms of the depth of proteome coverage, quantification accuracy, precision, and reproducibility using a high-performance hybrid mass spectrometer, LTQ Orbitrap Velos. Our results show that (1) the spectral counting method provides the deepest proteome coverage for identification, but its quantification performance is worse than labeling-based approaches, especially the quantification reproducibility; (2) metabolic labeling and isobaric chemical labeling are capable of accurate, precise, and reproducible quantification and provide deep proteome coverage for quantification. Isobaricmore » chemical labeling surpasses metabolic labeling in terms of quantification precision and reproducibility; (3) iTRAQ and TMT perform similarly in all aspects compared in the current study using a CID-HCD dual scan configuration. Based on the unique advantages of each method, we provide guidance for selection of the appropriate method for a quantitative proteomics study.« less

Responsiveness of platelets during storage studied with flow cytometry--formation of platelet subpopulations and LAMP-1 as new markers for the platelet storage lesion.

PubMed

Södergren, A L; Tynngård, N; Berlin, G; Ramström, S

2016-02-01

Storage lesions may prevent transfused platelets to respond to agonists and arrest bleeding. The aim of this study was to evaluate and quantify the capacity of platelet activation during storage using flow cytometry and new markers of platelet activation. Activation responses of platelets prepared by apheresis were measured on days 1, 5, 7 and 12. In addition, comparisons were made for platelet concentrates stored until swirling was affected. Lysosome-associated membrane protein-1 (LAMP-1), P-selectin and phosphatidylserine (PS) exposure were assessed by flow cytometry on platelets in different subpopulations in resting state or following stimulation with platelet agonists (cross-linked collagen-related peptide (CRP-XL), PAR1- and PAR4-activating peptides). The ability to form subpopulations upon activation was significantly decreased already at day 5 for some agonist combinations. The agonist-induced exposure of PS and LAMP-1 also gradually decreased with time. Spontaneous exposure of P-selectin and PS increased with time, while spontaneous LAMP-1 exposure was unchanged. In addition, agonist-induced LAMP-1 expression clearly discriminated platelet concentrates with reduced swirling from those with retained swirling. This suggests that LAMP-1 could be a good marker to capture changes in activation capacity in stored platelets. The platelet activation potential seen as LAMP-1 exposure and fragmentation into platelet subpopulations is potential sensitive markers for the platelet storage lesion. © 2015 International Society of Blood Transfusion.

Mean Platelet Volume (MPV), Platelet Distribution Width (PDW), Platelet Count and Plateletcrit (PCT) as predictors of in-hospital paediatric mortality: a case-control Study.

PubMed

Golwala, Zainab Mohammedi; Shah, Hardik; Gupta, Neeraj; Sreenivas, V; Puliyel, Jacob M

2016-06-01

Thrombocytopenia has been shown to predict mortality. We hypothesize that platelet indices may be more useful prognostic indicators. Our study subjects were children one month to 14 years old admitted to our hospital. To determine whether platelet count, plateletcrit (PCT), mean platelet volume (MPV) and platelet distribution width (PDW) and their ratios can predict mortality in hospitalised children. Children who died during hospital stay were the cases. Controls were age matched children admitted contemporaneously. The first blood sample after admission was used for analysis. Receiver operating characteristic (ROC) curve was used to identify the best threshold for measured variables and the ratios studied. Multiple regression analysis was done to identify independent predictors of mortality. Forty cases and forty controls were studied. Platelet count, PCT and the ratios of MPV/Platelet count, MPV/PCT, PDW/Platelet count, PDW/PCT and MPV × PDW/Platelet count × PCT were significantly different among children who survived compared to those who died. On multiple regression analysis the ratio of MPV/PCT, PDW/Platelet count and MPV/Platelet count were risk factors for mortality with an odds ratio of 4.31(95% CI, 1.69-10.99), 3.86 (95% CI, 1.53-9.75), 3.45 (95% CI, 1.38-8.64) respectively. In 67% of the patients who died MPV/PCT ratio was above 41.8 and PDW/Platelet count was above 3.86. In 65% of patients who died MPV/Platelet count was above 3.45. The MPV/PCT, PDW/Platelet count and MPV/Platelet count, in the first sample after admission in this case control study were predictors of mortality and could predict 65% to 67% of deaths accurately.

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

Antimicrobial effect of platelet-rich plasma and platelet-rich fibrin.

PubMed

Badade, Pallavi S; Mahale, Swapna A; Panjwani, Alisha A; Vaidya, Prutha D; Warang, Ayushya D

2016-01-01

Platelet concentrates have been extensively used in a variety of medical fields to promote soft- and hard-tissue regeneration. The significance behind their use lies in the abundance of growth factors (GFs) in platelets α-granules that promote wound healing. Other than releasing a pool of GFs upon activation, platelets also have many features that indicate their role in the anti-infective host defense. The aim of this study is to evaluate the antimicrobial activities of platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) against periodontal disease-associated bacteria. Blood samples were obtained from ten adult male patients. PRP and PRF were procured using centrifugation. The antimicrobial activity of PRP and PRF was evaluated by microbial culturing using bacterial strains of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. P. gingivalis and A. actinomycetemcomitans were inhibited by PRP but not by PRF. PRP is a potentially useful substance in the fight against periodontal pathogens. This might represent a valuable property in adjunct to the enhancement of tissue regeneration.

Comparative Effects of Platelet-Rich Plasma, Platelet Lysate, and Fetal Calf Serum on Mesenchymal Stem Cells.

PubMed

Lykov, A P; Bondarenko, N A; Surovtseva, M A; Kim, I I; Poveshchenko, O V; Pokushalov, E A; Konenkov, V I

2017-10-01

We studied the effects of human platelet-rich plasma and platelet lysate on proliferation, migration, and colony-forming properties of rat mesenchymal stem cells. Platelet-rich plasma and platelet lysate stimulated the proliferation, migration, and colony formation of mesenchymal stem cells. A real-time study showed that platelet-rich plasma produces the most potent stimulatory effect, while both platelet-rich plasma and platelet lysate stimulated migration of cells.

Platelet receptor polymorphisms do not influence Staphylococcus aureus–platelet interactions or infective endocarditis

PubMed Central

Daga, Shruti; Shepherd, James G.; Callaghan, J. Garreth S.; Hung, Rachel K.Y.; Dawson, Dana K.; Padfield, Gareth J.; Hey, Shi Y.; Cartwright, Robyn A.; Newby, David E.; Fitzgerald, J. Ross

2011-01-01

Cardiac vegetations result from bacterium–platelet adherence, activation and aggregation, and are associated with increased morbidity and mortality in infective endocarditis. The GPIIb/IIIa and FcγRIIa platelet receptors play a central role in platelet adhesion, activation and aggregation induced by endocarditis pathogens such as Staphylococcus aureus, but the influence of known polymorphisms of these receptors on the pathogenesis of infective endocarditis is unknown. We determined the GPIIIa platelet antigen PlA1/A2 and FcγRIIa H131R genotype of healthy volunteers (n = 160) and patients with infective endocarditis (n = 40), and investigated the influence of these polymorphisms on clinical outcome in infective endocarditis and S. aureus–platelet interactions in vitro. Platelet receptor genotype did not correlate with development of infective endocarditis, vegetation characteristics on echocardiogram or the composite clinical end-point of embolism, heart failure, need for surgery or mortality (P > 0.05 for all), even though patients with the GPIIIa PlA1/A1 genotype had increased in vivo platelet activation (P = 0.001). Furthermore, neither GPIIIa PlA1/A2 nor FcγRIIa H131R genotype influenced S. aureus-induced platelet adhesion, activation or aggregation in vitro (P > 0.05). Taken together, our data suggest that the GPIIIa and FcγRIIa platelet receptor polymorphisms do not influence S. aureus–platelet interactions in vitro or the clinical course of infective endocarditis. PMID:21044892

Platelet aggregation but not activation and degranulation during the acute post-ischemic reperfusion phase in livers with no underlying disease

PubMed Central

van Golen, Rowan F.; Stevens, Katarzyna M.; Colarusso, Pina; Jaeschke, Hartmut; Heger, Michal

2016-01-01

Background Platelets and P-selectin (CD62P) play an unequivocal role in the pathology of hepatic ischemia/reperfusion (I/R) injury. Inhibition or knock-out of P-selectin or immunodepletion of platelets results in amelioration of post-ischemic inflammation, reduced hepatocellular damage, and improved survival. However, P-selectin expression on platelets and endothelial cells, which concurs with platelet activation, has never been clearly demonstrated in I/R-subjected livers. Aims To determine whether platelets become activated and degranulate in the acute phase of liver I/R and whether the platelets interact with neutrophils. Methods Hepatic I/R was induced in male C57BL/6J mice (N = 12) using 37.5-min ischemia time. Platelets, endothelial cells, and neutrophils were fluorescently labeled by systemic administration of non-blocking antibodies. Cell kinetics were monitored by intravital spinning disk confocal microscopy during 90 min of reperfusion. Image analysis and quantification was performed with dedicated software. Results Platelets adhered to sinusoids more extensively in post-ischemic livers compared to livers not subjected to I/R and formed aggregates, which occurred directly after ischemia. Platelets and endothelial cells did not express P-selectin in post-ischemic livers. There was no interaction between platelets and neutrophils. Conclusions Platelets aggregate but do not become activated and do not degranulate in post-ischemic livers. There is no platelet-neutrophil interplay during the early reperfusion phase in a moderate model of hepatic I/R injury. The mechanisms underlying the biological effects of platelets and P-selectin in this setting warrant further investigation. Relevance for patients I/R in surgical liver patients may compromise outcome due to post-ischemic oxidative stress and sterile inflammation. Both processes are mediated in part by platelets. Understanding platelet function during I/R is key to developing effective interventions for I

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

PubMed Central

Parry, Robert C.; Wang, Qingqing; Gillespie, Kevin P.; Saillant, Noelle N.; Sims, Carrie; Mesaros, Clementina; Snyder, Nathaniel W.; Blair, Ian A.

2016-01-01

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism. PMID:27077278

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

Alternatives to allogeneic platelet transfusion.

PubMed

Desborough, Michael J R; Smethurst, Peter A; Estcourt, Lise J; Stanworth, Simon J

2016-11-01

Allogeneic platelet transfusions are widely used for the prevention and treatment of bleeding in thrombocytopenia. Recent evidence suggests platelet transfusions have limited efficacy and are associated with uncertain immunomodulatory risks and concerns about viral or bacterial transmission. Alternatives to transfusion are a well-recognised tenet of Patient Blood Management, but there has been less focus on different strategies to reduce bleeding risk by comparison to platelet transfusion. Direct alternatives to platelet transfusion include agents to stimulate endogenous platelet production (thrombopoietin mimetics), optimising platelet adhesion to endothelium by treating anaemia or increasing von Willebrand factor levels (desmopressin), increasing formation of cross-linked fibrinogen (activated recombinant factor VII, fibrinogen concentrate or recombinant factor XIII), decreasing fibrinolysis (tranexamic acid or epsilon aminocaproic acid) or using artificial or modified platelets (cryopreserved platelets, lyophilised platelets, haemostatic particles, liposomes, engineered nanoparticles or infusible platelet membranes). The evidence base to support the use of these alternatives is variable, but an area of active research. Much of the current randomised controlled trial focus is on evaluation of the use of thrombopoietin mimetics and anti-fibrinolytics. It is also recognised that one alternative strategy to platelet transfusion is choosing not to transfuse at all. © 2016 John Wiley & Sons Ltd.

Normal platelet function in platelet concentrates requires non-platelet cells: a comparative in vitro evaluation of leucocyte-rich (type 1a) and leucocyte-poor (type 3b) platelet concentrates

PubMed Central

Parrish, William R; Roides, Breana; Hwang, Julia; Mafilios, Michael; Story, Brooks; Bhattacharyya, Samir

2016-01-01

Background Therapeutic success of platelet-rich plasma (PRP) may vary based on the composition and preparation method. The objective of this study was to evaluate the cellular components of platelet concentrates produced by a leucocyte-rich (LR-PRP) and a leucocyte-poor PRP systems (LP-PRP). Methods Parameters evaluated included platelet recovery, platelet concentration, red blood cell (RBC) and white blood cell (WBC) composition, platelet growth factor release and stimulation of human tendon cell proliferation in vitro. Results Platelet recoveries were 52% for LP-PRP and 89% for LR-PRP. LR-PRP demonstrated greater reproducibility with a 4.2% coefficient of variation (CV) compared with 19.4% for LP-PRP (p<0.001). LR-PRP demonstrated a greater increase in platelet concentration (7.9-fold) than LP-PRP (2.2-fold; p<0.001). LP-PRP showed 5.0-fold reductions in WBCs, while LR-PRP showed a 4.0-fold increase (p<0.001). LP-PRP reduced RBCs to a haematocrit of 0.25, while LR-PRP reduced haematocrit to 11.8. LP-PRP did not coagulate robustly on reactivation with CaCl2, and released significantly lower levels of epidermal growth factor (EGF) and transforming growth factor β1 (TGF-β1) than whole blood (p<0.03). LP-PRP also did not stimulate tendon cell proliferation greater than whole blood. In contrast, LR-PRP showed increases in each growth factor on activation with CaCl2 (p<0.01) and stimulated greater proliferation (p<0.05) compared with whole blood. Forced activation of LP-PRP with exogenous thrombin rescued the coagulation deficiency and induced greater growth factor release than comparable whole blood (p<0.03). Conclusions These data suggest that non-platelet cellular components in platelet concentrates are important for proper platelet function, including thrombin generation, growth factor release and clot retraction. PMID:27900155

Normal platelet function in platelet concentrates requires non-platelet cells: a comparative in vitro evaluation of leucocyte-rich (type 1a) and leucocyte-poor (type 3b) platelet concentrates.

PubMed

Parrish, William R; Roides, Breana; Hwang, Julia; Mafilios, Michael; Story, Brooks; Bhattacharyya, Samir

2016-01-01

Therapeutic success of platelet-rich plasma (PRP) may vary based on the composition and preparation method. The objective of this study was to evaluate the cellular components of platelet concentrates produced by a leucocyte-rich (LR-PRP) and a leucocyte-poor PRP systems (LP-PRP). Parameters evaluated included platelet recovery, platelet concentration, red blood cell (RBC) and white blood cell (WBC) composition, platelet growth factor release and stimulation of human tendon cell proliferation in vitro. Platelet recoveries were 52% for LP-PRP and 89% for LR-PRP. LR-PRP demonstrated greater reproducibility with a 4.2% coefficient of variation (CV) compared with 19.4% for LP-PRP (p<0.001). LR-PRP demonstrated a greater increase in platelet concentration (7.9-fold) than LP-PRP (2.2-fold; p<0.001). LP-PRP showed 5.0-fold reductions in WBCs, while LR-PRP showed a 4.0-fold increase (p<0.001). LP-PRP reduced RBCs to a haematocrit of 0.25, while LR-PRP reduced haematocrit to 11.8. LP-PRP did not coagulate robustly on reactivation with CaCl 2 , and released significantly lower levels of epidermal growth factor (EGF) and transforming growth factor β1 (TGF-β1) than whole blood (p<0.03). LP-PRP also did not stimulate tendon cell proliferation greater than whole blood. In contrast, LR-PRP showed increases in each growth factor on activation with CaCl 2 (p<0.01) and stimulated greater proliferation (p<0.05) compared with whole blood. Forced activation of LP-PRP with exogenous thrombin rescued the coagulation deficiency and induced greater growth factor release than comparable whole blood (p<0.03). These data suggest that non-platelet cellular components in platelet concentrates are important for proper platelet function, including thrombin generation, growth factor release and clot retraction.

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

Platelet lipidomics: a modern day perspective on lipid discovery and characterization in platelets

PubMed Central

O’Donnell, Valerie B; Murphy, Robert C.; Watson, Steve P

2014-01-01

Lipids are diverse families of biomolecules that perform essential structural and signaling roles in platelets. Their formation and metabolism is tightly controlled by enzymes and signal transduction pathways, and their dysregulation leads to significant defects in platelet function and disease. Platelet activation is associated with significant changes to membrane lipids, and formation of diverse bioactive lipids that play essential roles in hemostasis. In recent years, new generation mass spectrometry analysis of lipids (termed “lipidomics”) has begun to alter our understanding of how these molecules participate in key cellular processes. While, the application of lipidomics to platelet biology is still in its infancy, seminal earlier studies have shaped our knowledge of how lipids regulate key aspects of platelet biology, including aggregation, shape change, coagulation and degranulation, as well as how lipids generated by platelets influence other cells, such as leukocytes and the vascular wall, and thus how they regulate hemostasis, vascular integrity and inflammation, as well as contribute to pathologies including arterial/deep vein thrombosis and atherosclerosis. This review will provide a brief historical perspective on the characterization of lipids in platelets, then an overview of the new generation lipidomic approaches, their recent application to platelet biology, and future perspectives for research in this area. The major platelet-regulatory lipid families, their formation, metabolism, and their role in health and disease, will be summarized. PMID:24677238

Short-term exposure of platelets to glucose impairs inhibition of platelet aggregation by cyclooxygenase inhibitors.

PubMed

Kobzar, Gennadi; Mardla, Vilja; Samel, Nigulas

2011-01-01

Aspirin treatment reduces cardiovascular events and deaths in high-risk non-diabetic patients, but not in patients suffering from diabetes. In these patients, hyperglycemia has been found to cause reduced platelet sensitivity to aspirin. It is supposed that long-term exposure of platelets to glucose leads to non-enzymatic glycosylation and impairs aspirin inhibition of platelet aggregation. On the other hand, short-term exposure of platelets to glucose also attenuates the effect of aspirin on platelets. The aim of the present work was to analyse the effect of short-term exposure of glucose on the inhibition of platelet aggregation by aspirin and other cyclooxygenase (COX) inhibitors. Already a 15 min exposure of platelets to glucose impaired aspirin inhibition of the platelet aggregation induced by collagen, thrombin, adenosine diphosphate (ADP), and arachidonic acid (AA). Aspirin inhibition of platelet aggregation in platelet-rich plasma (PRP) was attenuated by 5.6, 11.2, 16.8, and 22.4 mM of glucose in a concentration-dependent way. The same effect was observed with indomethacin and acetaminophen used as cyclooxygenase inhibitors instead of aspirin. N-methyl-L-arginine, an inhibitor of nitric oxide synthase, prevented the effect of glucose on aspirin, indomethacin and acetaminophen inhibition of platelet aggregation. Other monosaccharides, for example fructose and galactose, impaired aspirin inhibition as did glucose. Lactic acid (0.1, 0.2, 0.4, 0.8 mM), the end product of anaerobic glycolysis in platelets, impaired the inhibition of platelet aggregation with aspirin in a concentration-dependent way but did not affect indomethacin. It is suggested that lactic acid might be a mediator of the effect of glucose on aspirin inhibition in platelets.

Damaging effects of Clostridium perfringens delta toxin on blood platelets and their relevance to ganglioside GM2.

PubMed

Jolivet-Reynaud, C; Launay, J M; Alouf, J E

1988-04-01

The lytic effect of Clostridium perfringens delta toxin was investigated on goat, human, rabbit, and guinea pig platelets. In contrast to erythrocytes from the latter three species, which are insensitive to the toxin, the platelets were equally lysed by the same amount of toxin. These results suggest the presence of GM2 or GM2-like ganglioside(s) as a specific recognition site of the toxin on platelet plasmic membrane as previously established for sensitive erythrocytes. Plasmic membrane damage of human platelets was evidenced by the release of entrapped alpha-[14C]aminoisobutyric acid used as a cytoplasmic marker. The specific binding of hemolytically active 125I-delta toxin by human and rabbit platelets was practically identical, dose dependent, and inhibitable by GM2. Labeled toxin was also bound by various subcellular organelles separated from rabbit platelets except the 5-hydroxytryptamine (5-HT)-containing dense bodies, suggesting the absence or inaccessibility of GM2 on the surface of the latter organelles. This result correlates with the low amounts of 5-[3H]HT liberated after platelet challenge with delta toxin whereas this mediator was massively liberated upon lysis by the sulfhydryl-activated toxin alveolysin. The levels of M and P forms of phenol sulfotransferase (PST), involved in 5-HT catabolism, were determined in human platelet lysates after challenge with delta toxin, alveolysin, and other disruptive treatments. The low PST-M activities detected after lysis by delta toxin suggest that this isoenzyme is very likely associated to dense bodies in contrast to PST-P which is cytoplasmic. Platelet lysis by the toxin allows easy separation of these organelles.

Piperine Inhibits the Activities of Platelet Cytosolic Phospholipase A2 and Thromboxane A2 Synthase without Affecting Cyclooxygenase-1 Activity: Different Mechanisms of Action Are Involved in the Inhibition of Platelet Aggregation and Macrophage Inflammatory Response

PubMed Central

Son, Dong Ju; Akiba, Satoshi; Hong, Jin Tae; Yun, Yeo Pyo; Hwang, Seock Yeon; Park, Young Hyun; Lee, Sung Eun

2014-01-01

PURPOSE: Piperine, a major alkaloid of black pepper (Piper nigrum) and long pepper (Piper longum), was shown to have anti-inflammatory activity through the suppression of cyclooxygenase (COX)-2 gene expression and enzyme activity. It is also reported to exhibit anti-platelet activity, but the mechanism underlying this action remains unknown. In this study, we investigated a putative anti-platelet aggregation mechanism involving arachidonic acid (AA) metabolism and how this compares with the mechanism by which it inhibits macrophage inflammatory responses; METHODS: Rabbit platelets and murine macrophage RAW264.7 cells were treated with piperine, and the effect of piperine on the activity of AA-metabolizing enzymes, including cytosolic phospholipase A2 (cPLA2), COX-1, COX-2, and thromboxane A2 (TXA2) synthase, as well as its effect on AA liberation from the plasma membrane components, were assessed using isotopic labeling methods and enzyme immunoassay kit; RESULTS: Piperine significantly suppressed AA liberation by attenuating cPLA2 activity in collagen-stimulated platelets. It also significantly inhibited the activity of TXA2 synthase, but not of COX-1, in platelets. These results suggest that piperine inhibits platelet aggregation by attenuating cPLA2 and TXA2 synthase activities, rather than through the inhibition of COX-1 activity. On the other hand, piperine significantly inhibited lipopolysaccharide-induced generation of prostaglandin (PG)E2 and PGD2 in RAW264.7 cells by suppressing the activity of COX-2, without effect on cPLA2; CONCLUSION: Our findings indicate that piperine inhibits platelet aggregation and macrophage inflammatory response by different mechanisms. PMID:25153972

Effects of abacavir administration on structural and functional markers of platelet activation.

PubMed

Trevillyan, Janine M; Arthur, Jane F; Jing, Jing; Andrews, Robert K; Gardiner, Elizabeth E; Hoy, Jennifer F

2015-11-01

Current abacavir exposure has been reported to be associated with cardiovascular disease. Changes in platelet reactivity could plausibly explain the clinically observed pattern of association. To determine if platelet reactivity changed following abacavir exposure and whether this effect was reversible on cessation of the drug. In an open-label, interventional study abacavir, 600 mg daily, was added to a suppressive antiretroviral regimen in 20 adult HIV-positive men. Platelet function, estimated by the phosphorylated vasodilator-stimulated phosphoprotein (P-VASP) assay and through measurement of the expression and shedding of platelet-specific receptors, was assessed at baseline, following 15 days of abacavir and at completion of a 28-day washout period. The VASP-index decreased significantly from 79.1% [interquartile range (IQR) 47.8-87.6] to 32.6% (IQR -11.5-51.0) following 15 days of abacavir administration (P = 0.010), and returned to baseline levels following the washout period (day 43 =76.3%; IQR 40.7-92.3). There was no change in resting (prostaglandin E1 alone) P-VASP but a slight increase in P-VASP within stimulated platelets (prostaglandin E1 and adenosine diphosphate). Integrin β3 levels decreased significantly [208.5 ng/ml (IQR 177.0-231.1) to 177.5 ng/ml (IQR 151.7-205) P < 0.001] and there was a nonsignificant trend towards decreased soluble glycoprotein VI levels [baseline; 72.5 ng/ml (95% CI 58.3-81.5) vs. day 15; 45.0 ng/ml (95% CI 33.0-98.2) P = 0.79]. Abacavir led to reversible changes in platelet function and structure. The clinical implications of these changes are uncertain; they may represent negative feedback mechanisms in response to an abacavir-associated prothrombotic state.

Introducing cymantrene labels into scattering scanning near-field infrared microscopy.

PubMed

Kopf, Ilona; N'Dongo, Harmel W Peindy; Ballout, Fouad; Schatzschneider, Ulrich; Bründermann, Erik; Havenith, Martina

2012-11-07

In this paper we investigate metal-organic compounds as infrared (IR) active labels by scattering scanning near-field infrared microscopy (IR s-SNOM, often also abbreviated as s-SNIM) with a lateral resolution of 90 × 90 nm(2). Tailor-made IR spectroscopic probes based on cymantrene (CpMn(CO)(3) with Cp = η(5)-C(5)H(5)) conjugated to a cysteine-modified pseudoneurotensin (pNT-Cys-OH) peptide were prepared by automated microwave-assisted solid phase peptide synthesis (SPPS) and characterized by HPLC, ESI-MS and IR. Well-defined patterned self-assembled monolayers on a gold surface were prepared by microcontact printing of 1-octadecanethiol (ODT) followed by additional incubation in ethanolic solution of the cymantrene-peptide derivative. The self-assembled monolayers have been evidenced by infrared reflection absorption spectroscopy (IRRAS) and AFM. CO laser source radiation was tuned (1944, 1900, 1798 and 1658 cm(-1)) for imaging contrast with good matching correlation between spectroscopic and topographic patterns at specific characteristic metal carbonyl and amide bands (1944 cm(-1) (λ = 5.14 μm) and 1658 cm(-1) (λ = 6.03 μm)). Cymantrene probes provide an attractive method to tag a unique spectroscopic feature on any bio(macro)molecule. Introducing such probes into super-resolution IR s-SNOM will enable molecular tracking and distribution studies even in complex biological systems.

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.

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

Prothrombin Activation by Platelet-associated Prothrombinase Proceeds through the Prethrombin-2 Pathway via a Concerted Mechanism*

PubMed Central

Haynes, Laura M.; Bouchard, Beth A.; Tracy, Paula B.; Mann, Kenneth G.

2012-01-01

The protease α-thrombin is a key enzyme of the coagulation process as it is at the cross-roads of both the pro- and anti-coagulant pathways. The main source of α-thrombin in vivo is the activation of prothrombin by the prothrombinase complex assembled on either an activated cell membrane or cell fragment, the most relevant of which is the activated platelet surface. When prothrombinase is assembled on synthetic phospholipid vesicles, prothrombin activation proceeds with an initial cleavage at Arg-320 yielding the catalytically active, yet effectively anticoagulant intermediate meizothrombin, which is released from the enzyme complex ∼30–40% of the time. Prothrombinase assembled on the surface of activated platelets has been shown to proceed through the inactive intermediate prethrombin-2 via an initial cleavage at Arg-271 followed by cleavage at Arg-320. The current work tests whether or not platelet-associated prothrombinase proceeds via a concerted mechanism through a study of prothrombinase assembly and function on collagen-adhered, thrombin-activated, washed human platelets in a flow chamber. Prothrombinase assembly was demonstrated through visualization of bound factor Xa by confocal microscopy using a fluorophore-labeled anti-factor Xa antibody, which demonstrated the presence of distinct platelet subpopulations capable of binding factor Xa. When prothrombin activation was monitored at a typical venous shear rate over preassembled platelet-associated prothrombinase neither potential intermediate, meizothrombin or prethrombin-2, was observed in the effluent. Collectively, these findings suggest that platelet-associated prothrombinase activates prothrombin via an efficient concerted mechanism in which neither intermediate is released. PMID:22989889

Current concepts in platelet transfusion

PubMed Central

Mohanty, Dipika

2009-01-01

This is the era of component therapy. Therefore there is a need for rational use of platelet concentrate. Lot of knowledge has been added recently in the field of platelet specially about the platelet rich plasma and its application in clinical practice. The current review focuses on improvement in preparation of platelet rich plasma, the procedure to make the same more safe and its rational use. Furthermore newer aspects of platelet concentrate use in surgical practice and for regenerative medicine has also been discussed. It also covers some progress and hurdles in preparation of platelet substitutes. PMID:20041092

Comparative analysis of human ex vivo–generated platelets vs megakaryocyte-generated platelets in mice: a cautionary tale

PubMed Central

Wang, Yuhuan; Hayes, Vincent; Jarocha, Danuta; Sim, Xiuli; Harper, Dawn C.; Fuentes, Rudy; Sullivan, Spencer K.; Gadue, Paul; Chou, Stella T.; Torok-Storb, Beverly J.; Marks, Michael S.; French, Deborah L.

2015-01-01

Thrombopoiesis is the process by which megakaryocytes release platelets that circulate as uniform small, disc-shaped anucleate cytoplasmic fragments with critical roles in hemostasis and related biology. The exact mechanism of thrombopoiesis and the maturation pathways of platelets released into the circulation remain incompletely understood. We showed that ex vivo–generated murine megakaryocytes infused into mice release platelets within the pulmonary vasculature. Here we now show that infused human megakaryocytes also release platelets within the lungs of recipient mice. In addition, we observed a population of platelet-like particles (PLPs) in the infusate, which include platelets released during ex vivo growth conditions. By comparing these 2 platelet populations to human donor platelets, we found marked differences: platelets derived from infused megakaryocytes closely resembled infused donor platelets in morphology, size, and function. On the other hand, the PLP was a mixture of nonplatelet cellular fragments and nonuniform-sized, preactivated platelets mostly lacking surface CD42b that were rapidly cleared by macrophages. These data raise a cautionary note for the clinical use of human platelets released under standard ex vivo conditions. In contrast, human platelets released by intrapulmonary-entrapped megakaryocytes appear more physiologic in nature and nearly comparable to donor platelets for clinical application. PMID:25852052

Elucidation of flow-mediated tumour cell-induced platelet aggregation using an ultrasound standing wave trap.

PubMed

Bazou, D; Santos-Martinez, M J; Medina, C; Radomski, M W

2011-04-01

Tumour cells activate and aggregate platelets [tumour cell-induced platelet aggregation (TCIPA)] and this process plays an important role in the successful metastasis of cancer cells. To date, most studies on TCIPA have been conducted under no-flow conditions. In this study, we have investigated TCIPA in real time under flow conditions, using an ultrasound standing wave trap that allows formation and levitation of cancer cell clusters in suspension, thus mimicking the conditions generated by flowing blood. Using 59M adenocarcinoma and HT1080 fibrosarcoma cells and human platelets, cancer cell cluster-platelet aggregates were imaged in real time using epi-fluorescence microscopy (F-actin) and investigated in detail using confocal microscopy (matrix metalloproteinase-2-GPIIb/IIIa co-localization) and scanning electron and helium-ion microscopy (<1 nm resolution). The release of gelatinases from aggregates was studied using zymography. We found that platelet activation and aggregation takes place on the surface of cancer cells (TCIPA), leading to time-dependent disruption of cancer cell clusters. Pharmacological modulation of TCIPA revealed that EDTA, prostacyclin, o-phenanthroline and apyrase significantly down-regulated TCIPA and, in turn, delayed cell cluster disruption, However, EGTA and aspirin were ineffective. Pharmacological inhibition of TCIPA correlated with the down-regulation of platelet activation as shown by flow-cytometry assay of platelet P-selectin. Our results show for the first time, that during TCIPA, platelet activation disrupts cancer cell clusters and this can contribute to metastasis. Thus, selective targeting of platelet aggregate-cancer cell clusters may be an important strategy to control metastasis. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

Elucidation of flow-mediated tumour cell-induced platelet aggregation using an ultrasound standing wave trap

PubMed Central

Bazou, D; Santos-Martinez, MJ; Medina, C; Radomski, MW

2011-01-01

BACKGROUND AND PURPOSE Tumour cells activate and aggregate platelets [tumour cell-induced platelet aggregation (TCIPA)] and this process plays an important role in the successful metastasis of cancer cells. To date, most studies on TCIPA have been conducted under no-flow conditions. In this study, we have investigated TCIPA in real time under flow conditions, using an ultrasound standing wave trap that allows formation and levitation of cancer cell clusters in suspension, thus mimicking the conditions generated by flowing blood. EXPERIMENTAL APPROACH Using 59M adenocarcinoma and HT1080 fibrosarcoma cells and human platelets, cancer cell cluster–platelet aggregates were imaged in real time using epi-fluorescence microscopy (F-actin) and investigated in detail using confocal microscopy (matrix metalloproteinase-2-GPIIb/IIIa co-localization) and scanning electron and helium-ion microscopy (<1 nm resolution). The release of gelatinases from aggregates was studied using zymography. KEY RESULTS We found that platelet activation and aggregation takes place on the surface of cancer cells (TCIPA), leading to time-dependent disruption of cancer cell clusters. Pharmacological modulation of TCIPA revealed that EDTA, prostacyclin, o-phenanthroline and apyrase significantly down-regulated TCIPA and, in turn, delayed cell cluster disruption, However, EGTA and aspirin were ineffective. Pharmacological inhibition of TCIPA correlated with the down-regulation of platelet activation as shown by flow-cytometry assay of platelet P-selectin. CONCLUSION AND IMPLICATIONS Our results show for the first time, that during TCIPA, platelet activation disrupts cancer cell clusters and this can contribute to metastasis. Thus, selective targeting of platelet aggregate–cancer cell clusters may be an important strategy to control metastasis. PMID:21182493

Semi-synthetic preparation of 1-O-(1'-/sup 14/C)hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) using plant cell cultures

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

Weber, N.; Mangold, H.K.

1985-04-01

Incubation of photomixotrophic cell suspension cultures of rape (Brassica napus) and heterotrophic cell suspension cultures of soya (Glycine max) with 1-O-(1'-/sup 14/C)hexadecyl-sn-glycerol or rac-1-O-(1'-/sup 14/C)hexadecylglycerol leads in high yield (up to 78%) to labeled 1-O-hexadecyl-2-acyl-sn-glycero-3-phosphocholines. Alkaline hydrolysis of the choline glycerophospholipids yields pure 1-O-(1'-/sup 14/C)hexadecyl-sn-glycero-3-phosphocholine. 1-O-(1'-14C)Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) is obtained by acetylating the lyso compound. The semi-synthetic preparation described leads to labeled platelet activating factor in an overall yield of 50-60% without loss of specific activity.

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.

Increasing platelet concentrations in leukocyte-reduced platelet-rich plasma decrease collagen gene synthesis in tendons.

PubMed

Boswell, Stacie G; Schnabel, Lauren V; Mohammed, Hussni O; Sundman, Emily A; Minas, Tom; Fortier, Lisa A

2014-01-01

Platelet-rich plasma (PRP) is used for the treatment of tendinopathy. There are numerous PRP preparations, and the optimal combination of platelets and leukocytes is not known. Within leukocyte-reduced PRP (lrPRP), there is a plateau effect of platelet concentration, with increasing platelet concentrations being detrimental to extracellular matrix synthesis. Controlled laboratory study. Different formulations of lrPRP with respect to the platelet:leukocyte ratio were generated from venous blood of 8 horses. Explants of the superficial digital flexor tendon were cultured in lrPRP products for 96 hours. Platelet-derived growth factor-BB (PDGF-BB), tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), and interleukin-1β (IL-1β) concentrations were determined in the media by enzyme-linked immunosorbent assay. Gene expression in tendon tissue for collagen type I and III (COL1A1 and COL3A1, respectively), matrix metalloproteinase-3 and -13 (MMP-3 and MMP-13, respectively), cartilage oligomeric matrix protein (COMP), and IL-1β was determined. Data were divided into 3 groups of lrPRP based on the ratio of platelets:leukocytes and evaluated to determine the effect of platelet concentration. Complete blood counts verified leukocyte reduction and platelet enrichment in all PRP preparations. In the lrPRP preparation, the anabolic growth factors PDGF-BB and TGF-β1 were increased with increasing platelet concentrations, and the catabolic cytokine IL-1β was decreased with increasing platelet concentrations. Increasing the platelet concentration resulted in a significant reduction in COL1A1 and COL3A1 synthesis in tendons. Increasing the platelet concentration within lrPRP preparations results in the delivery of more anabolic growth factors and less proinflammatory cytokines, but the biological effect on tendons is diminished metabolism as indicated by a decrease in the synthesis of both COL1A1 and COL3A1. Together, this information suggests that

Prophylactic platelet transfusions prior to surgery for people with a low platelet count

PubMed Central

Estcourt, Lise J; Malouf, Reem; Doree, Carolyn; Trivella, Marialena; Hopewell, Sally; Birchall, Janet

2017-01-01

This is a protocol for a Cochrane Review (Intervention). The objectives are as follows: To determine the clinical effectiveness and safety of prophylactic platelet transfusions prior to surgery for people with a low platelet count or platelet dysfunction (inherited or acquired). PMID:29151812

Comparison of Immature Platelet Count to Established Predictors of Platelet Reactivity During Thienopyridine Therapy.

PubMed

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

2016-07-19

Previous data suggest that reticulated platelets significantly affect antiplatelet response to thienopyridines. It is unknown whether parameters describing reticulated platelets can predict antiplatelet response to thienopyridines. The authors sought to determine the extent to which parameters describing reticulated platelets can predict antiplatelet response to thienopyridine loading compared with established predictors. This study randomized 300 patients undergoing elective coronary stenting to loading with clopidogrel 600 mg, prasugrel 30 mg, or prasugrel 60 mg. Adenosine diphosphate (ADP)-induced platelet reactivity was assessed by impedance aggregometry before loading (intrinsic platelet reactivity) and again on day 1 after loading. Multiple parameters of reticulated platelets were assessed by automated whole blood flow cytometry: absolute immature platelet count (IPC), immature platelet fraction, and highly fluorescent immature platelet fraction. Each parameter of reticulated platelets correlated significantly with ADP-induced platelet reactivity (p < 0.01 for all 3 parameters). In a multivariable model including all 3 parameters, only IPC remained a significant predictor of platelet reactivity (p < 0.001). In models adjusting each of the 3 parameters for known predictors of on-treatment platelet reactivity including cytochrome P450 2C19 (CYP2C19) polymorphisms, age, body mass index, diabetes, and intrinsic platelet reactivity, only IPC prevailed as an independent predictor (p = 0.001). In this model, IPC was the strongest predictor of on-treatment platelet reactivity followed by intrinsic platelet reactivity. IPC is the strongest independent platelet count-derived predictor of antiplatelet response to thienopyridine treatment. Given its easy availability, together with its even stronger association with on-treatment platelet reactivity compared with known predictors, including the CYP2C19*2 polymorphism, IPC may become the preferred predictor of

Platelets promote osteosarcoma cell growth through activation of the platelet-derived growth factor receptor-Akt signaling axis.

PubMed

Takagi, Satoshi; Takemoto, Ai; Takami, Miho; Oh-Hara, Tomoko; Fujita, Naoya

2014-08-01

The interactions of tumor cells with platelets contribute to the progression of tumor malignancy, and the expression levels of platelet aggregation-inducing factors positively correlate with the metastatic potential of osteosarcoma cells. However, it is unclear how tumor-platelet interaction contributes to the proliferation of osteosarcomas. We report here that osteosarcoma-platelet interactions induce the release of platelet-derived growth factor (PDGF) from platelets, which promotes the proliferation of osteosarcomas. Co-culture of platelets with MG63 or HOS osteosarcoma cells, which could induce platelet aggregation, enhanced the proliferation of each cell line in vitro. Analysis of phospho-antibody arrays revealed that co-culture of MG63 cells with platelets induced the phosphorylation of platelet derived growth factor receptor (PDGFR) and Akt. The addition of supernatants of osteosarcoma-platelet reactants also increased the growth of MG63 and HOS cells as well as the level of phosphorylated-PDGFR and -Akt. Sunitinib or LY294002, but not erlotinib, significantly inhibited the platelet-induced proliferation of osteosarcoma cells, indicating that PDGF released from platelets plays an important role in the proliferation of osteosarcomas by activating the PDGFR and then Akt. Our results suggest that inhibitors that specifically target osteosarcoma-platelet interactions may eradicate osteosarcomas. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Platelets rich plasma versus minoxidil 5% in treatment of alopecia areata: A trichoscopic evaluation.

PubMed

El Taieb, Moustafa A; Ibrahim, Hassan; Nada, Essam A; Seif Al-Din, Mai

2017-01-01

Alopecia areata is a common cause of nonscarring alopecia that occurs in a patchy, confluent, or diffuse pattern. Dermoscopy is a noninvasive technique for the clinical diagnosis of many skin diseases. Topical minoxidil solution 5% and platelet rich plasma are important modalities used in treatment of alopecia areata. We aimed to evaluate the efficacy of PRP versus topical minoxidil 5% in the treatment of AA by clinical evaluation and trichoscopic examination. Ninety patients were allocated into three groups; the first was treated with topical minoxidil 5% solution, the second with platelets rich plasma injections, and the third with placebo. Diagnosis and follow up were done by serial digital camera photography of lesions and dermoscopic scan before and every 1 month after treatment for 3 months. Patients treated with minoxidil 5% and platelets rich plasma both have significant hair growth than placebo (p < .05). Patients treated with platelets rich plasma had an earlier response in the form of hair regrowth, reduction in short vellus hair and dystrophic hair unlike patients treated with minoxidil and control (p < .05). In conclusion, platelets rich plasma is more effective in the treatment of alopecia areata than topical minoxidil 5% as evaluated by clinical and trichoscopic examination. © 2016 Wiley Periodicals, Inc.

Block-Based Connected-Component Labeling Algorithm Using Binary Decision Trees

PubMed Central

Chang, Wan-Yu; Chiu, Chung-Cheng; Yang, Jia-Horng

2015-01-01

In this paper, we propose a fast labeling algorithm based on block-based concepts. Because the number of memory access points directly affects the time consumption of the labeling algorithms, the aim of the proposed algorithm is to minimize neighborhood operations. Our algorithm utilizes a block-based view and correlates a raster scan to select the necessary pixels generated by a block-based scan mask. We analyze the advantages of a sequential raster scan for the block-based scan mask, and integrate the block-connected relationships using two different procedures with binary decision trees to reduce unnecessary memory access. This greatly simplifies the pixel locations of the block-based scan mask. Furthermore, our algorithm significantly reduces the number of leaf nodes and depth levels required in the binary decision tree. We analyze the labeling performance of the proposed algorithm alongside that of other labeling algorithms using high-resolution images and foreground images. The experimental results from synthetic and real image datasets demonstrate that the proposed algorithm is faster than other methods. PMID:26393597

Line-scanning, stage scanning confocal microscope

NASA Astrophysics Data System (ADS)

Carucci, John A.; Stevenson, Mary; Gareau, Daniel

2016-03-01

We created a line-scanning, stage scanning confocal microscope as part of a new procedure: video assisted micrographic surgery (VAMS). The need for rapid pathological assessment of the tissue on the surface of skin excisions very large since there are 3.5 million new skin cancers diagnosed annually in the United States. The new design presented here is a confocal microscope without any scanning optics. Instead, a line is focused in space and the sample, which is flattened, is physically translated such that the line scans across its face in a direction perpendicular to the line its self. The line is 6mm long and the stage is capable of scanning 50 mm, hence the field of view is quite large. The theoretical diffraction-limited resolution is 0.7um lateral and 3.7um axial. However, in this preliminary report, we present initial results that are a factor of 5-7 poorer in resolution. The results are encouraging because they demonstrate that the linear array detector measures sufficient signal from fluorescently labeled tissue and also demonstrate the large field of view achievable with VAMS.

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.

Doxorubicin-loaded platelets conjugated with anti-CD22 mAbs: a novel targeted delivery system for lymphoma treatment with cardiopulmonary avoidance

PubMed Central

Zhou, Rongfu; Wang, Fan; Liu, Xu; Ouyang, Jian; Chen, Bing

2017-01-01

B-cell lymphoma accounts for approximately 85% of all adult non-Hodgkin's lymphoma cases. Doxorubicin (DOX) is an indispensable drug for the treatment of non-Hodgkin's lymphoma. However, DOX causes severe cardiotoxicity, which limits its use in conventional treatment strategies. In this study, we developed a novel drug delivery system for lymphoma treatment: DOX-loaded platelets that were conjugated with anti-CD22 monoclonal antibodies (mAbs) (DOX–platelet–CD22). Platelets are bio- and immune-compatible drug carriers that can prolong the circulation time of drugs. Anti-CD22 mAb-labeled platelets can precisely deliver DOX to tumor cells. Our in vitro and in vivo experiments showed the enhanced antitumor activity and attenuated cardiotoxicity of DOX when delivered as DOX–platelet–CD22. Compared with other delivery systems, the uptake of DOX–platelet–CD22 by macrophage-like cells decreased. Moreover, DOX–platelet–CD22 showed platelet properties, such as tumor cell-induced platelet aggregation. Therefore, targeted chemotherapy that is mediated by DOX–platelet–CD22 is a promising option for lymphoma treatment. PMID:28938559

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.

Platelets and atherogenesis: Platelet anti-aggregation activity and endothelial protection from tomatoes (Solanum lycopersicum L.)

PubMed Central

PALOMO, IVÁN; FUENTES, EDUARDO; PADRÓ, TERESA; BADIMON, LINA

2012-01-01

In recent years, it has been shown that platelets are not only involved in the arterial thrombotic process, but also that they play an active role in the inflammatory process of atherogenesis from the beginning. The interaction between platelets and endothelial cells occurs in two manners: activated platelets unite with intact endothelial cells, or platelets in resting adhere to activated endothelium. In this context, inhibition of the platelet function (adhesion/aggregation) could contribute to the prevention of atherothrombosis, the leading cause of cardiovascular morbidity. This can be achieved with antiplatelet agents. However, at the public health level, the level of primary prevention, a healthy diet has also been shown to exert beneficial effects. Among those elements of a healthy diet, the consumption of tomatoes (Solanum lycopersicum L.) stands out for its effect on platelet anti-aggregation activity and endothelial protection, which may be beneficial for cardiovascular health. This article briefly discusses the involvement of platelets in atherogenesis and the possible mechanisms of action provided by tomatoes for platelet anti-aggregation activity and endothelial protection. PMID:22969932

Confocal laser scanning microscopic photoconversion: a new method to stabilize fluorescently labeled cellular elements for electron microscopic analysis.

PubMed

Colello, Raymond J; Tozer, Jordan; Henderson, Scott C

2012-01-01

Photoconversion, the method by which a fluorescent dye is transformed into a stable, osmiophilic product that can be visualized by electron microscopy, is the most widely used method to enable the ultrastructural analysis of fluorescently labeled cellular structures. Nevertheless, the conventional method of photoconversion using widefield fluorescence microscopy requires long reaction times and results in low-resolution cell targeting. Accordingly, we have developed a photoconversion method that ameliorates these limitations by adapting confocal laser scanning microscopy to the procedure. We have found that this method greatly reduces photoconversion times, as compared to conventional wide field microscopy. Moreover, region-of-interest scanning capabilities of a confocal microscope facilitate the targeting of the photoconversion process to individual cellular or subcellular elements within a fluorescent field. This reduces the area of the cell exposed to light energy, thereby reducing the ultrastructural damage common to this process when widefield microscopes are employed. © 2012 by John Wiley & Sons, Inc.

The role of platelets during reproduction.

PubMed

Isermann, Berend; Nawroth, Peter P

2006-01-01

The availability of mice with defined defects within the hemostatic system enabled researchers to identify a role the coagulation system for embryonic and placental development. However, the role of platelets during development has only recently been experimentally addressed, giving some insight into potential functions of platelets during development. Thus, a quantitative embryonic platelet defect (severe thrombopenia secondary to NF-E2 deficiency) is associated with an embryonic growth retardation and reduced vascularisation of the placenta. Maternal platelet deficiency is associated with placental hemorrhage, which, however, does not impair embryonic or maternal survival. In vitro studies established that platelets or platelet conditioned medium regulate the invasive properties of human extravillous trophoblast cells and induce a phenotypical switch of trophoblast cells. These data imply that platelets are of relevance during placentation. Conversely, platelets and the formation of platelet-fibrin aggregates are dispensable for the development of the embryo proper, establishing that the lethal phenotypes observed in some embryo slacking coagulation regulators does not result from an inability to form platelet-fibrin aggregates, but likely reflects altered protease dependent signaling during vascular development.

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.

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

Behavior of platelets stained by 5,6-CF-encapsulated PEGylated liposomes after laser irradiation of vessel wall: an in-vivo model for studying site-selective delivery of diagnostic or therapeutic agents

NASA Astrophysics Data System (ADS)

Mordon, Serge R.; Begu, Sylvie; Buys, Bruno; Tourne-Peteilh, Corine; Devoisselle, Jean-Marie

2001-05-01

Vascular endothelium serves as an extensive interface between circulating blood and various tissues and organs of the body. As such, it offers an accessible target for blood-borne pharmacological and genetic manipulations that can mediate both local and systemic effects. Thus, targeting of liposomes to activated vascular endothelial cells may provide a strategy for site-selective delivery in the vascular system with broad therapeutic applicability. This study aimed to evaluate an intravital fluorescence imaging technique to visualize in-situ and in real-time the activation of platelets after staining by 5,6-CF- encapsulated PEGylated liposomes injected intravenously. The study was performed on skin by using a dorsal skin-fold chamber implanted in golden hamsters using intravital microscopy. The skin micro circulation was observed with an intravital microscope (using x25 and x40 magnification) fitted with a Xenon light source and an epi-fluorescence assembly. An ultra-high sensitivity video-camera mounted on the microscope projected the image onto a monitor, and the images were recorded for play-back analysis with a digital video cassette recorder. An inflammatory response was induced by an Argon laser emitting at 514.5nm. The 80micrometers laser beam was focused on a vessel and its position was controlled with the microscope imaging system, it was possible to see individual platelets flowing in blood vessels. As liposomes were labeled with a fluorescent probe which was hydrophilic (located in the aqueous phase), the fluorescence of platelets was due only to the uptake of liposomes. After laser irradiation, platelets activation at sites of vascular injury was obtained. Tethering, translocation of some platelets inside the irradiated zone were clearly seen. At last, detachment and extravasation of platelets were observed. A perivascular fluorescence confirmed that platelets migrated across the basal lamina into the dermal connective tissue. In conclusion, staining of

Effects of isotretinoin on the platelet counts and the mean platelet volume in patients with acne vulgaris.

PubMed

Ataseven, Arzu; Ugur Bilgin, Aynur

2014-01-01

Aim. The aim of this study was to evaluate the platelet counts and the mean platelet volume in patients who received isotretinoin for the treatment of acne vulgaris. Method. A total of 110 patients were included in this retrospective study. Complete blood count parameters were recorded prior to and three-months following the treatment. Results. Both platelet counts and the mean platelet volume were significantly decreased following the treatment. No significant differences were noted on the levels of hemoglobin, hematocrit, and white blood cell count. Conclusion. Platelet counts and mean platelet volume significantly decreased following isotretinoin treatment. Since the decrease of platelet counts and the mean platelet volume was seen concomitantly, it is concluded that the effect of isotretinoin was through the suppression of bone marrow.

Synergism between thrombin and adrenaline (epinephrine) in human platelets. Marked potentiation of inositol phospholipid metabolism.

PubMed Central

Steen, V M; Tysnes, O B; Holmsen, H

1988-01-01

We have studied synergism between adrenaline (epinephrine) and low concentrations of thrombin in gel-filtered human platelets prelabelled with [32P]Pi. Suspensions of platelets, which did not contain added fibrinogen, were incubated at 37 degrees C to measure changes in the levels of 32P-labelled phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and phosphatidate (PA), aggregation and dense-granule secretion after stimulation. Adrenaline alone (3.5-4.0 microM) did not cause a change in any parameter (phosphoinositide metabolism, aggregation and dense-granule secretion), but markedly enhanced the thrombin-induced responses over a narrow range of thrombin concentrations (0.03-0.08 units/ml). The thrombin-induced hydrolysis of inositol phospholipids by phospholipase C, which was measured as the formation of [32P]PA, was potentiated by adrenaline, as was the increase in the levels of [32P]PIP2 and [32P]PIP. The presence of adrenaline caused a shift to the left for the thrombin-induced changes in the phosphoinositide metabolism, without affecting the maximal levels of 32P-labelled compounds obtained. A similar shift by adrenaline in the dose-response relationship was previously demonstrated for thrombin-induced aggregation and dense-granule secretion. Also, the narrow range of concentrations of thrombin over which adrenaline potentiates thrombin-induced platelet responses is the same for changes in phosphoinositide metabolism and physiological responses (aggregation and dense-granule secretion). Our observations clearly indicate that adrenaline directly or indirectly influences thrombin-induced changes in phosphoinositide metabolism. PMID:2845924

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.

Technetium-99m labeled red blood cells in the evaluation of hemangiosarcoma

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

Joseph, U.A.; Jhingran, S.G.

Imaging with Tc-99m labeled red blood cells (RBC) is increasingly being used in the detection of acute gastro-intestinal bleeding, especially in patients with intermittent bleeding. A patient is presented in whom the labeled RBC scan was helpful in the incidental discovery of a previously unsuspected probable angiosarcoma of the right femur and adjacent soft tissues of the right hip due to the blood pool or blush effect of the labeled cells. The labeled RBC scan also identified extravasation due to active gastrointestinal bleeding from a previously unknown angiosarcoma of the ascending colon. Thus, the Tc-99m labeled RBC scan was usefulmore » in simultaneously detecting extravasation and blood pool effect at two remote tumor sites in the same patient.« less

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.

High-frequency (13.56-MHz) and ultrahigh-frequency (915-MHz) radio identification systems do not affect platelet activation and functions.

PubMed

Rogowska, Anna; Chabowska, Anna Małgorzata; Lipska, Alina; Boczkowska-Radziwon, Barbara; Bujno, Magdalena; Rusak, Tomasz; Dziemianczuk, Mateusz; Radziwon, Piotr

2016-05-01

In radiofrequency identification (RFID) systems used in labeling of blood components, blood cells are subjected to the direct influence of electromagnetic waves throughout the storage period. The aim of this study was to prove the safety of storage of platelet concentrates (PCs) in containers labeled with RFID tags. Ten pooled PCs obtained from 12 buffy coats each suspended in additive solution were divided into three separate containers that were assigned to three groups: control, PCs labeled with ultrahigh frequency (UHF) range tags and exposed to 915-MHz radio waves, and PCs labeled with high-frequency (HF) range tags and exposed to 13.56-MHz radio waves. PCs were stored at 20 to 24°C for 7 days. In vitro tests of platelet (PLT) function were performed on the first, fifth, and seventh days of storage. There were no significant differences in pH; hypotonic shock resistance; surface expression of CD62P, CD42a, or CD63; release of PLT-derived microparticles; PLT aggregation; and number of PLTs between PCs stored at a constant exposure to radio waves of two different frequencies and the control group on the first, fifth, and seventh days of storage. The results of the study indicate no impact of electromagnetic radiation generated in HF and UHF RFID systems and constant contact with the tags on the quality of stored PCs. © 2016 AABB.

Antibody-mediated platelet phagocytosis by human macrophages is inhibited by siRNA specific for sequences in the SH2 tyrosine kinase, Syk.

PubMed

Lu, Ying; Wang, Weiming; Mao, Huiming; Hu, Hai; Wu, Yanling; Chen, Bing-Guan; Liu, Zhongmin

2011-01-01

Immune thrombocytopenia depends upon Fc receptor-mediated phagocytosis that involves signaling through the SH2 tyrosine kinase, Syk. We designed small interfering (siRNA) sequences complementary to Syk coding regions to decrease the expression of Syk in the human macrophage cell line, THP-1. To evaluate the functional effect of siRNA on phagocytosis, we developed a new in vitro assay for antibody-mediated platelet ingestion by THP-1 cells. Incubation of THP-1 cells at 37°C with fluorescence-labeled platelets and anti-platelet antibody promoted ingestion of platelets that could be quantitated by flow cytometry. Transfection of THP-1 cells with Syk-specific siRNA resulted in a reduction in the amount of FcγRII-associated Syk protein. Coincident with decreased Syk expression, we observed inhibition of antibody-mediated platelet ingestion. These results confirm a key role for Syk in antibody-mediated phagocytosis and suggest Syk-specific siRNA as a possible therapeutic candidate for immune thrombocytopenia. Copyright © 2011 Elsevier Inc. All rights reserved.

Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting

NASA Astrophysics Data System (ADS)

Zhu, Yuanjun; Li, Ruyi; Lin, Yuan; Shui, Mengyang; Liu, Xiaoyan; Chen, Huan; Wang, Yinye

2016-07-01

Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbβ3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbβ3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency.

Quantitative Glycoproteomic Analysis Identifies Platelet-Induced Increase of Monocyte Adhesion via the Up-Regulation of Very Late Antigen 5.

PubMed

Huang, Jiqing; Kast, Juergen

2015-08-07

Physiological stimuli, such as thrombin, or pathological stimuli, such as lysophosphatidic acid (LPA), activate platelets circulating in blood. Once activated, platelets bind to monocytes via P-selectin-PSGL-1 interactions but also release the stored contents of their granules. These platelet releasates, in addition to direct platelet binding, activate monocytes and facilitate their recruitment to atherosclerotic sites. Consequently, understanding the changes platelet releasates induce in monocyte membrane proteins is critical. We studied the glyco-proteome changes of THP-1 monocytic cells affected by LPA- or thrombin-induced platelet releasates. We employed lectin affinity chromatography combined with filter aided sample preparation to achieve high glyco- and membrane protein and protein sequence coverage. Using stable isotope labeling by amino acids in cell culture, we quantified 1715 proteins, including 852 membrane and 500 glycoproteins, identifying the up-regulation of multiple proteins involved in monocyte extracellular matrix binding and transendothelial migration. Flow cytometry indicated expression changes of integrin α5, integrin β1, PECAM-1, and PSGL-1. The observed increase in monocyte adhesion to fibronectin was determined to be mediated by the up-regulation of very late antigen 5 via a P-selectin-PSGL-1 independent mechanism. This novel aspect could be validated on CD14+ human primary monocytes, highlighting the benefits of the improved enrichment method regarding high membrane protein coverage and reliable quantification.

Platelets Inhibit Migration of Canine Osteosarcoma Cells.

PubMed

Bulla, S C; Badial, P R; Silva, R C; Lunsford, K; Bulla, C

2017-01-01

The interaction between platelets and tumour cells is important for tumour growth and metastasis. Thrombocytopenia or antiplatelet treatment negatively impact on cancer metastasis, demonstrating potentially important roles for platelets in tumour progression. To our knowledge, there is no information regarding the role of platelets in cancer progression in dogs. This study was designed to test whether canine platelets affected the migratory behaviour of three canine osteosarcoma cell lines and to give insights of molecular mechanisms. Intact platelets, platelet lysate and platelet releasate inhibited the migration of canine osteosarcoma cell lines. Addition of blood leucocytes to the platelet samples did not alter the inhibitory effect on migration. Platelet treatment also significantly downregulated the transcriptional levels of SNAI2 and TWIST1 genes. The interaction between canine platelets or molecules released during platelet activation and these tumour cell lines inhibits their migration, which suggests that canine platelets might antagonize metastasis of canine osteosarcoma. This effect is probably due to, at least in part, downregulation of genes related to epithelial-mesenchymal transition. Copyright © 2016. Published by Elsevier Ltd.

Clot lysis time in platelet-rich plasma: method assessment, comparison with assays in platelet-free and platelet-poor plasmas, and response to tranexamic acid.

PubMed

Panes, Olga; Padilla, Oslando; Matus, Valeria; Sáez, Claudia G; Berkovits, Alejandro; Pereira, Jaime; Mezzano, Diego

2012-01-01

Fibrinolysis dysfunctions cause bleeding or predisposition to thrombosis. Platelets contain several factors of the fibrinolytic system, which could up or down regulate this process. However, the temporal relationship and relative contributions of plasma and platelet components in clot lysis are mostly unknown. We developed a clot lysis time (CLT) assay in platelet-rich plasma (PRP-CLT, with and without stimulation) and compared it to a similar one in platelet-free plasma (PFP) and to another previously reported test in platelet-poor plasma (PPP). We also studied the differential effects of a single dose of tranexamic acid (TXA) on these tests in healthy subjects. PFP- and PPP-CLT were significantly shorter than PRP-CLT, and the three assays were highly correlated (p < 0.0001). PFP- and PPP-, but more significantly PRP-CLT, were positively correlated with age and plasma PAI-1, von Willebrand factor, fibrinogen, LDL-cholesterol, and triglycerides (p < 0.001). All these CLT assays had no significant correlations with platelet aggregation/secretion, platelet counts, and pro-coagulant tests to explore factor X activation by platelets, PRP clotting time, and thrombin generation in PRP. Among all the studied variables, PFP-CLT was independently associated with plasma PAI-1, LDL-cholesterol, and triglycerides and, additionally, stimulated PRP-CLT was also independently associated with plasma fibrinogen. A single 1 g dose of TXA strikingly prolonged all three CLTs, but in contrast to the results without the drug, the lysis times were substantially shorter in non-stimulated or stimulated PRP than in PFP and PPP. This standardized PRP-CLT may become a useful tool to study the role of platelets in clot resistance and lysis. Our results suggest that initially, the platelets enmeshed in the clot slow down the fibrinolysis process. However, the increased clot resistance to lysis induced by TXA is overcome earlier in platelet-rich clots than in PFP or PPP clots. This is

Rationale and design of platelet transfusions in haematopoietic stem cell transplantation: the PATH pilot study.

PubMed

Tay, Jason; Allan, David; Beattie, Sara; Bredeson, Christopher; Fergusson, Dean; Maze, Dawn; Sabloff, Mitchell; Thavorn, Kednapa; Tinmouth, Alan

2016-10-24

In patients with transient thrombocytopenia being treated with high-dose chemotherapy followed by stem cell rescue-haematopoietic stem cell transplantation (HSCT), prophylactic transfusions are standard therapy to prevent bleeding. However, a recent multicentre trial suggests that prophylactic platelet transfusions in HSCT may not be necessary. Additionally, the potential overuse of platelet products places a burden on a scarce healthcare resource. Moreover, the benefit of prophylactic platelet transfusions to prevent clinically relevant haemorrhage is debatable. Current randomised data compare different thresholds for administering prophylactic platelets or prophylactic versus therapeutic platelet transfusions. An alternative strategy involves prescribing prophylactic antifibrinolytic agents such as tranexamic acid to prevent bleeding. This report describes the design of an open-labelled randomised pilot study comparing the prophylactic use of oral tranexamic acid with platelet transfusions in the setting of autologous HSCT. In 3-5 centres, 100 patients undergoing autologous HSCT will be randomly assigned to either a prophylactic tranexamic acid or prophylactic platelets bleeding prevention strategy-based daily platelet values up to 30 days post-transplant. The study will be stratified by centre and type of transplant. The primary goal is to demonstrate study feasibility while collecting clinical outcomes on (1) WHO and Bleeding Severity Measurement Scale (BSMS), (2) transplant-related mortality, (3) quality of life, (4) length of hospital stay, (5) intensive care unit admission rates, (6) Bearman toxicity scores, (7) incidence of infections, (8) transfusion requirements, (9) adverse reactions and (10) economic analyses. This study is funded by a peer-reviewed grant from the Canadian Institutes of Health Research (201 503) and is registered on Clinicaltrials.gov NCT02650791. It has been approved by the Ottawa Health Science Network Research Ethics Board. Study

Fish Oil Supplementation in Humans: Effects on Platelet Responses, Phospholipid Composition and Metabolism.

NASA Astrophysics Data System (ADS)

Skeaff, Clark Murray

Platelets are believed to play a significant role in the development of occlusive vascular diseases. Epidemiological reports have correlated the high intake of marine foods, rich in omega3 fatty acids, with diminished platelet responses and a low incidence of arterial thrombosis and myocardial infarction. The activation of platelet responses is mediated by the accelerated metabolism of membrane phospholipid; therefore, it was of interest to examine, in human volunteers, the effect of a dietary fish oil concentrate (MaxEPA), enriched in omega 3 polyunsaturated fatty acids, on platelet aggregation and phospholipid composition/metabolism. For the complete separation of cellular phospholipids, a one-dimensional thin-layer chromatography system using silica-gel pre-coated glass plates was developed. The solvent system consisted of CHCl_3/CH_3OH/CH _3COOH/H_2O (50/37.5/3.5/2.0, by vol), required approximately 90-120 minutes for full phospholipid separation, and was highly reproducible even under conditions of variable humidity and temperature. The consumption of a fish oil concentrate (MaxEPA) for 6 weeks (3.6 g of 20:5omega 3 and 2.4 g of 22:6omega3 per day) diminished both the collagen- and platelet activating factor-induced maximum aggregation responses in washed human platelet suspensions by 50.1% and 27.2%, respectively, as compared to initial unsupplemented baseline responses. Thrombin -induced aggregation remained unchanged. Thrombin stimulation of intact human platelets produced a significant decrease in the mass of phosphatidylinositol in plasma membrane. In platelets pre-labelled with (2-^3H) glycerol and stimulated with either thrombin or low-dose collagen, the loss of (^3H) phosphatidylinositol did not differ between those subjects consuming olive oil or fish oil. Likewise, the thrombin-stimulated accumulation of diacylglycerol, an activator of protein kinase C, was unaffected by fish oil consumption. The ratio of collagen -induced increase in radioactivity

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.

Platelet factor XIII increases the fibrinolytic resistance of platelet-rich clots by accelerating the crosslinking of alpha 2-antiplasmin to fibrin

NASA Technical Reports Server (NTRS)

Reed, G. L.; Matsueda, G. R.; Haber, E.

1992-01-01

Platelet clots resist fibrinolysis by plasminogen activators. We hypothesized that platelet factor XIII may enhance the fibrinolytic resistance of platelet-rich clots by catalyzing the crosslinking of alpha 2-antiplasmin (alpha 2AP) to fibrin. Analysis of plasma clot structure by polyacrylamide gel electrophoresis and immunoblotting revealed accelerated alpha 2AP-fibrin crosslinking in platelet-rich compared with platelet-depleted plasma clots. A similar study of clots formed with purified fibrinogen (depleted of factor XIII activity), isolated platelets, and specific factor XIII inhibitors indicated that this accelerated crosslinking was due to the catalytic activity of platelet factor XIII. Moreover, when washed platelets were aggregated by thrombin, there was evidence of platelet factor XIII-mediated crosslinking between platelet alpha 2AP and platelet fibrin(ogen). Specific inhibition (by a monoclonal antibody) of the alpha 2AP associated with washed platelet aggregates accelerated the fibrinolysis of the platelet aggregate. Thus in platelet-rich plasma clots, and in thrombin-induced platelet aggregates, platelet factor XIII actively formed alpha 2AP-fibrin crosslinks, which appeared to enhance the resistance of platelet-rich clots to fibrinolysis.

Biosynthesis and processing of platelet GPIIb-IIIa in human megakaryocytes.

PubMed

Duperray, A; Berthier, R; Chagnon, E; Ryckewaert, J J; Ginsberg, M; Plow, E; Marguerie, G

1987-06-01

Platelet membrane glycoprotein IIb-IIIa forms a calcium-dependent heterodimer and constitutes the fibrinogen receptor on stimulated platelets. GPIIb is a two-chain protein containing disulfide-linked alpha and beta subunits. GPIIIa is a single chain protein. These proteins are synthesized in the bone marrow by megakaryocytes, but the study of their synthesis has been hampered by the difficulty in obtaining enriched population of megakaryocytes in large numbers. To examine the biosynthesis and processing of GPIIb-IIIa, purified human megakaryocytes were isolated from liquid cultures of cryopreserved leukocytes stem cell concentrates from patients with chronic myelogenous leukemia. Immunoprecipitation of [35S]methionine pulse-chase-labeled cell extracts by antibodies specific for the alpha or beta subunits of GPIIb indicated that GPIIb was derived from a precursor of Mr 130,000 that contains the alpha and beta subunits. This precursor was converted to GPIIb with a half-life of 4-5 h. No precursor form of GPIIIa was detected. The glycosylation of GPIIb-IIIa was examined in megakaryocytes by metabolic labeling in the presence of tunicamycin, monensin, or treatment with endoglycosidase H. The polypeptide backbones of the GPIIb and the GPIIIa have molecular masses of 120 and 90 kD, respectively. High-mannose oligosaccharides are added to these polypeptide backbones co-translationally. The GPIIb precursor is then processed with conversion of high-mannose to complex type carbohydrates yielding the mature subunits GPIIb alpha (Mr 116,000) and GPIIb beta (Mr 25,000). No posttranslational processing of GPIIIa was detected.

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.

21 CFR 864.6675 - Platelet aggregometer.

Code of Federal Regulations, 2010 CFR

2010-04-01

... shape and platelet aggregation following the addition of an aggregating reagent to a platelet rich... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Platelet aggregometer. 864.6675 Section 864.6675...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Manual Hematology Devices § 864.6675 Platelet...

21 CFR 864.6675 - Platelet aggregometer.

Code of Federal Regulations, 2014 CFR

2014-04-01

... shape and platelet aggregation following the addition of an aggregating reagent to a platelet rich... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Platelet aggregometer. 864.6675 Section 864.6675...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Manual Hematology Devices § 864.6675 Platelet...

21 CFR 864.6675 - Platelet aggregometer.

Code of Federal Regulations, 2011 CFR

2011-04-01

... shape and platelet aggregation following the addition of an aggregating reagent to a platelet rich... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Platelet aggregometer. 864.6675 Section 864.6675...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Manual Hematology Devices § 864.6675 Platelet...

21 CFR 864.6675 - Platelet aggregometer.

Code of Federal Regulations, 2012 CFR

2012-04-01

... shape and platelet aggregation following the addition of an aggregating reagent to a platelet rich... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Platelet aggregometer. 864.6675 Section 864.6675...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Manual Hematology Devices § 864.6675 Platelet...

21 CFR 864.6675 - Platelet aggregometer.

Code of Federal Regulations, 2013 CFR

2013-04-01

... shape and platelet aggregation following the addition of an aggregating reagent to a platelet rich... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Platelet aggregometer. 864.6675 Section 864.6675...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Manual Hematology Devices § 864.6675 Platelet...

Three-dimensional architecture and cell composition of a Choukroun's platelet-rich fibrin clot and membrane.

PubMed

Dohan Ehrenfest, David M; Del Corso, Marco; Diss, Antoine; Mouhyi, Jaafar; Charrier, Jean-Baptiste

2010-04-01

Platelet-rich fibrin (PRF; Choukroun's technique) is a second-generation platelet concentrate for surgical use. This easy protocol allows the production of leukocyte and platelet-rich fibrin clots and membranes starting from 10-ml blood samples. The purposes of this study were to determine the cell composition and three-dimensional organization of this autologous biomaterial and to evaluate the influence of different collection tubes (dry glass or glass-coated plastic tubes) and compression procedures (forcible or soft) on the final PRF-membrane architecture. After centrifugation, blood analyses were performed on the residual waste plasmatic layers after collecting PRF clots. The PRF clots and membranes were processed for examination by light microscopy and scanning electron microscopy. Approximately 97% of the platelets and >50% of the leukocytes were concentrated in the PRF clot and showed a specific three-dimensional distribution, depending on the centrifugation forces. Platelets and fibrin formed large clusters of coagulation in the first millimeters of the membrane beyond the red blood cell base. The fibrin network was very mature and dense. Moreover, there was no significant difference in the PRF architecture between groups using the different tested collection tubes and compression techniques, even if these two parameters could have influenced the growth factor content and biologic matrix properties. The PRF protocol concentrated most platelets and leukocytes from a blood harvest into a single autologous fibrin biomaterial. This protocol offers reproducible results as long as the main production principles are respected.

Platelet Activation by Streptococcus pyogenes Leads to Entrapment in Platelet Aggregates, from Which Bacteria Subsequently Escape

PubMed Central

Svensson, Lisbeth; Baumgarten, Maria; Mörgelin, Matthias

2014-01-01

Platelet activation and aggregation have been reported to occur in response to a number of Gram-positive pathogens. Here, we show that platelet aggregates induced by Streptococcus pyogenes were unstable and that viable bacteria escaped from the aggregates over time. This was not due to differential activation in response to the bacteria compared with physiological activators. All the bacterial isolates induced significant platelet activation, including integrin activation and alpha and dense-granule release, at levels equivalent to those induced by potent physiological platelet activators that induced stable aggregates. The ability to escape the aggregates and to resist the antibacterial effects of platelets was dependent on active protein synthesis by the bacteria within the aggregate. We conclude that S. pyogenes bacteria can temporarily cover themselves with activated platelets, and we propose that this may facilitate survival of the bacteria in the presence of platelets. PMID:25069984

Platelet activation in pregnancy-induced hypertension.

PubMed

Karalis, Ioannis; Nadar, Sunil K; Al Yemeni, Eman; Blann, Andrew D; Lip, Gregory Y H

2005-01-01

Although excess platelet activation, as indicated by increased plasma beta thromboglobulin (beta-TG), has been shown in pregnancy-induced hypertension (PIH), platelet adhesion, platelet morphology and a comparison of platelet and soluble (plasma) levels of the adhesion molecules P-selectin (pPsel and sPsel, respectively) have not been studied. We conducted a cross-sectional study of 35 consecutive women with PIH (age 31+/-6 years), 31 consecutive women with normotensive pregnancies (age 29+/-5 years) and 30 normotensive non pregnant women (age 30+/-5 years). Platelet adhesion was studied in vitro by binding to fibrinogen-coated microwells, platelet morphology [mass and volume by flow cytometry], whole-platelet P-selectin (pPsel) by ELISA of the lysate of 2 x 10(8) cells, and the plasma markers soluble P-selectin (sP-sel) and beta-TG, by ELISA. The women with PIH had significantly raised sPsel, pPsel and (as expected) beta-TG (all p<0.05), when compared to the normotensive pregnant women and controls. However, in PIH platelet adhesion was similar to that in the normotensive pregnancy, but still higher than the normal controls (p<0.001). There was no difference among the three groups with respect to platelet mass and volume. pPsel and platelet adhesion correlated with gestational age and with systolic and diastolic blood pressure (all p<0.05). Increased platelet activation and adhesion develop during normal pregnancy, with some indices being further altered in PIH.

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

Platelet storage lesion in interim platelet unit concentrates: A comparison with buffy-coat and apheresis concentrates.

PubMed

Singh, Sukhi; Shams Hakimi, Caroline; Jeppsson, Anders; Hesse, Camilla

2017-12-01

Platelet storage lesion is characterized by morphological changes and impaired platelet function. The collection method and storage medium may influence the magnitude of the storage lesion. The aim of this study was to compare the newly introduced interim platelet unit (IPU) platelet concentrates (PCs) (additive solution SSP+, 40% residual plasma content) with the more established buffy-coat PCs (SSP, 20% residual plasma content) and apheresis PCs (autologous plasma) in terms of platelet storage lesions. Thirty PCs (n=10 for each type) were assessed by measuring metabolic parameters (lactate, glucose, and pH), platelet activation markers, and in vitro platelet aggregability on days 1, 4, and 7 after donation. The expression of platelet activation markers CD62p (P-selectin), CD63 (LAMP-3), and phosphatidylserine was measured using flow cytometry and in vitro aggregability was measured with multiple electrode aggregometry. Higher platelet activation and lower in vitro aggregability was observed in IPU than in buffy-coat PCs on day 1 after donation. In contrast, metabolic parameters, expression of platelet activation markers, and in vitro aggregability were better maintained in IPU than in buffy-coat PCs at the end of the storage period. Compared to apheresis PCs, IPU PCs had higher expression of activation markers and lower in vitro aggregability throughout storage. In conclusion, the results indicate that there are significant differences in platelet storage lesions between IPU, buffy-coat, and apheresis PCs. The quality of IPU PCs appears to be at least comparable to buffy-coat preparations. Further studies are required to distinguish the effect of the preparation methods from storage conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tocotrienols-induced inhibition of platelet thrombus formation and platelet aggregation in stenosed canine coronary arteries.

PubMed

Qureshi, Asaf A; Karpen, Charles W; Qureshi, Nilofer; Papasian, Christopher J; Morrison, David C; Folts, John D

2011-04-14

Dietary supplementation with tocotrienols has been shown to decrease the risk of coronary artery disease. Tocotrienols are plant-derived forms of vitamin E, which have potent anti-inflammatory, antioxidant, anticancer, hypocholesterolemic, and neuroprotective properties. Our objective in this study was to determine the extent to which tocotrienols inhibit platelet aggregation and reduce coronary thrombosis, a major risk factor for stroke in humans. The present study was carried out to determine the comparative effects of α-tocopherol, α-tocotrienol, or tocotrienol rich fraction (TRF; a mixture of α-+γ-+δ-tocotrienols) on in vivo platelet thrombosis and ex vivo platelet aggregation (PA) after intravenous injection in anesthetized dogs, by using a mechanically stenosed circumflex coronary artery model (Folts' cyclic flow model). Collagen-induced platelet aggregation (PA) in platelet rich plasma (PRP) was decreased markedly after treatment with α-tocotrienol (59%; P<0.001) and TRF (92%; P<0.001). α-Tocopherol treatment was less effective, producing only a 22% (P<0.05) decrease in PA. Adenosine diphosphate-induced (ADP) PA was also decreased after treatment with α-tocotrienol (34%; P<0.05) and TRF (42%; P<0.025). These results also indicate that intravenously administered tocotrienols were significantly better than tocopherols in inhibiting cyclic flow reductions (CFRs), a measure of the acute platelet-mediated thrombus formation. Tocotrienols (TRF) given intravenously (10 mg/kg), abolished CFRs after a mean of 68 min (range 22 -130 min), and this abolition of CFRs was sustained throughout the monitoring period (50-160 min).Next, pharmacokinetic studies were carried out and tocol levels in canine plasma and platelets were measured. As expected, α-Tocopherol treatment increased levels of total tocopherols in post- vs pre-treatment specimens (57 vs 18 μg/mL in plasma, and 42 vs 10 μg/mL in platelets). However, treatment with α-tocopherol resulted in slightly

Tocotrienols-induced inhibition of platelet thrombus formation and platelet aggregation in stenosed canine coronary arteries

PubMed Central

2011-01-01

Background Dietary supplementation with tocotrienols has been shown to decrease the risk of coronary artery disease. Tocotrienols are plant-derived forms of vitamin E, which have potent anti-inflammatory, antioxidant, anticancer, hypocholesterolemic, and neuroprotective properties. Our objective in this study was to determine the extent to which tocotrienols inhibit platelet aggregation and reduce coronary thrombosis, a major risk factor for stroke in humans. The present study was carried out to determine the comparative effects of α-tocopherol, α-tocotrienol, or tocotrienol rich fraction (TRF; a mixture of α- + γ- + δ-tocotrienols) on in vivo platelet thrombosis and ex vivo platelet aggregation (PA) after intravenous injection in anesthetized dogs, by using a mechanically stenosed circumflex coronary artery model (Folts' cyclic flow model). Results Collagen-induced platelet aggregation (PA) in platelet rich plasma (PRP) was decreased markedly after treatment with α-tocotrienol (59%; P < 0.001) and TRF (92%; P < 0.001). α-Tocopherol treatment was less effective, producing only a 22% (P < 0.05) decrease in PA. Adenosine diphosphate-induced (ADP) PA was also decreased after treatment with α-tocotrienol (34%; P < 0.05) and TRF (42%; P < 0.025). These results also indicate that intravenously administered tocotrienols were significantly better than tocopherols in inhibiting cyclic flow reductions (CFRs), a measure of the acute platelet-mediated thrombus formation. Tocotrienols (TRF) given intravenously (10 mg/kg), abolished CFRs after a mean of 68 min (range 22 -130 min), and this abolition of CFRs was sustained throughout the monitoring period (50 - 160 min). Next, pharmacokinetic studies were carried out and tocol levels in canine plasma and platelets were measured. As expected, α-Tocopherol treatment increased levels of total tocopherols in post- vs pre-treatment specimens (57 vs 18 μg/mL in plasma, and 42 vs 10 μg/mL in platelets). However, treatment with

Platelet aggregation caused by a partially purified jellyfish toxin from Carybdea rastonii.

PubMed

Azuma, H; Sekizaki, S; Satoh, A; Nakajima, T; Ishikawa, M

1986-01-01

A partially purified toxin (pCrTX) was obtained from the tentacles of the jellyfish, Carybdea rastonii. When pCrTX (3 X 10(-8) - 3 X 10(-7) g/ml) was added to citrated platelet-rich plasma, aggregation was produced in a concentration-dependent manner. Scanning electron microscopic examination revealed that both pCrTX and collagen produced aggregates of platelets possessing many pseudopods. The concentration which produced 50% aggregation for pCrTX was 1.8 X 10(-7) g/ml, as compared to 2.3 X 10(-6) g/ml for collagen. The pCrTX-induced aggregation was only slightly inhibited by indomethacin and quinacrine in concentrations sufficient to inhibit arachidonic acid- and collagen-induced aggregation. pCrTX was less active in washed platelets suspended in Ca2+ free medium, whereas the pCrTX-induced aggregation was significantly augmented in the presence of Ca2+. The augmentation of aggregation by Ca2+ was only slightly attenuated by pretreatment with 100 microM verapamil. pCrTX significantly increased the concentration of cytoplasmic free Ca2+ ([Ca2+]i) and depolarized the platelet membrane in concentrations that produced aggregation. The increase in [Ca2+]i caused by pCrTX was little affected by verapamil. The depolarization by pCrTX was unchanged in the presence or absence of Ca2+, or by sodium or potassium transport inhibitors. The movement of 22Na+ into platelets was significantly increased by pCrTX. This increase in the movement of 22N+ into platelets was unaffected by tetrodotoxin. On the other hand, pCrTX-induced aggregation, depolarization and the increase in [Ca2+]i were all significantly attenuated in low Na+ medium. These results suggest that pCrTX causes a massive depolarization by increasing cation permeability indiscriminately and this generalized depolarization permits an inward movement of calcium down an electrochemical gradient which, in turn triggers platelet aggregation.

Use of indium-111 oxine to study the effects of terbutaline on pulmonary and hepatic platelet sequestration in endotoxin shock

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

Christenson, J.T.; Sigurdsson, G.H.; Mousawi, M.

This study describes the effects of the beta 2 receptor agonist terbutaline on platelet sequestration in sheep exposed to endotoxin shock. The in vivo behavior of Indium-111-labeled platelets was followed simultaneously in the lungs, liver, spleen, and kidneys. The effects on the respiratory function and the central hemodynamics were also followed. Twelve adult sheep were given endotoxin (10 micrograms/kg bw), and six of those received a continuous intravenous infusion of terbutaline (20 micrograms/kg/hr) during 4 hr, starting 30 min after injection of endotoxin. The other six acted as controls. It was found that a marked pulmonary and hepatic platelet sequestrationmore » occurred during and just after the endotoxin infusion and was followed by a marked platelet disaggregation within 30 min in both groups. Three hours after the endotoxin a second wave of platelet trapping occurred in the control animals in both the lungs and the liver, while no such increase was seen in the terbutaline-treated animals. In the spleen, however, there was a decrease in platelet sequestration after endotoxin in both groups, and in the kidneys only minor changes occurred. Furthermore, less marked hemodynamic and respiratory alterations occurred in the terbutaline group compared with the controls. It was concluded that terbutaline decreased sequestration of platelets in the lungs and in the liver of sheep in endotoxin shock, which may be of importance in the development of multiple organ failure.« less

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

Relationship between platelet phospholipid FA and mean platelet volume in healthy men.

PubMed

Li, Duo; Turner, Alan; Sinclair, Andrew J

2002-09-01

Increased mean platelet volume (MPV) has been suggested as an independent risk factor for acute myocardial infarction and the increased reactivity of large platelets. The aim of this study was to investigate the correlation between platelet phospholipid (PL) PUFA composition and MPV in 139 free-living healthy men ages 20-55 yr (vegans, n = 18; ovolacto vegetarians, n = 43; moderate meat-eaters, n = 60; and high meateaters, n = 18). Each subject completed a semiquantitative Food Frequency Questionnaire and gave a blood sample. Platelet PL FA composition and MPV were determined by standard methods. MPV was significantly greater in the vegans than in the ovolacto vegetarian, moderate, or high meat-eater groups (P < 0.01). Both vegan and ovolacto vegetarian groups had significantly higher platelet PL 18:2n-6 and 22:4n-6, and lower 20:5n-3 and 22:6n-3 compared with the moderate and high meat-eater groups. The vegans demonstrated a significant reduction in 20:4n-6 and 22:5n-3 compared with the ovolacto vegetarian, high meat-eater, and moderate meat-eater groups. Bivariate analysis results showed that MPV was significantly positively correlated with platelet PL 18:2n-6 (P = 0.048) and negatively correlated with 20:3n-6 (P = 0.02), 20:5n-3 (P = 0.005), and 22:5n-3 (P< 0.0001), respectively. In a multiple linear regression analysis, after controlling for potential confounding factors such as dietary group, age, exercise, body mass index, and dietary polyunsaturated and saturated fat, cholesterol, carbohydrate, and fiber intake, the MPV was still strongly negatively correlated with platelet PL 20:3n-6 (P = 0.003) and 22:5n-3 (P = 0.001). The present data suggest that 22:5n-3 and 20:3n-6 may play a role in the structural function of the platelet membrane.

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

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

Aggregation and disaggregation kinetics of human blood platelets: Part II. Shear-induced platelet aggregation.

PubMed Central

Huang, P Y; Hellums, J D

1993-01-01

A population balance equation (PBE) mathematical model for analyzing platelet aggregation kinetics was developed in Part I (Huang, P. Y., and J. D. Hellums. 1993. Biophys. J. 65: 334-343) of a set of three papers. In this paper, Part II, platelet aggregation and related reactions are studied in the uniform, known shear stress field of a rotational viscometer, and interpreted by means of the model. Experimental determinations are made of the platelet-aggregate particle size distributions as they evolve in time under the aggregating influence of shear stress. The PBE model is shown to give good agreement with experimental determinations when either a reversible (aggregation and disaggregation) or an irreversible (no disaggregation) form of the model is used. This finding suggests that for the experimental conditions studied disaggregation processes are of only secondary importance. During shear-induced platelet aggregation, only a small fraction of platelet collisions result in the binding together of the involved platelets. The modified collision efficiency is approximately zero for shear rates below 3000 s-1. It increases with shear rates above 3000 s-1 to about 0.01 for a shear rate of 8000 s-1. Addition of platelet chemical agonists yields order of magnitude increases in collision efficiency. The collision efficiency for shear-induced platelet aggregation is about an order of magnitude less at 37 degrees C than at 24 degrees C. The PBE model gives a much more accurate representation of aggregation kinetics than an earlier model based on a monodispersed particle size distribution. PMID:8369442

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 effect of sibutramine, a serotonin-norepinephrine reuptake inhibitor, on platelets and fibrin networks of male Sprague-Dawley rats: a descriptive study.

PubMed

van der Schoor, Ciska; Oberholzer, Hester Magdalena; Bester, Megan Jean; van Rooy, Mia-Jeanne

2014-12-01

Sibutramine is used in the treatment of obesity due to its ability to influence feelings of hunger and satiety by inhibiting the re-uptake of serotonin and noradrenalin in the central nervous system (CNS). Sibutramine use has been associated with numerous adverse events in particular cardiovascular complications possibly due to the formation of thrombi. This ultrastructural descriptive study investigated the effect of sibutramine on blood coagulation, specifically the effect on morphology of platelets and fibrin networks using scanning electron microscopy. Male Sprague-Dawley rats treated with either a recommended therapeutic dose [low dosage 1.32 mg/kg] or a toxicological higher dose [high dosage 13.2 mg/kg] of sibutramine for 28 days were used and compared to control animals. Blood samples were collected and plasma smears were prepared for platelet evaluation. Following the addition of thrombin to the plasma samples, the morphology of the fibrin clots was evaluated. Platelet evaluation by scanning electron microscopy revealed morphology typical of a prothrombotic state with a characteristic excessive platelet activation in both low-dose (LD) and high-dose (HD) rats. The fibrin clots of sibutramine-treated rats, LD and HD revealed fused thick fibers with thin fibers forming a net-like structure over the thick fibers which differ considerably from the organized structure of the control animals. It can be concluded that sibutramine alters the ultrastructure of platelets and fibrin networks creating a prothrombotic state.

Carica papaya Leaves Juice Significantly Accelerates the Rate of Increase in Platelet Count among Patients with Dengue Fever and Dengue Haemorrhagic Fever

PubMed Central

Subenthiran, Soobitha; Choon, Tan Chwee; Cheong, Kee Chee; Thayan, Ravindran; Teck, Mok Boon; Muniandy, Prem Kumar; Afzan, Adlin; Abdullah, Noor Rain; Ismail, Zakiah

2013-01-01

The study was conducted to investigate the platelet increasing property of Carica papaya leaves juice (CPLJ) in patients with dengue fever (DF). An open labeled randomized controlled trial was carried out on 228 patients with DF and dengue haemorrhagic fever (DHF). Approximately half the patients received the juice, for 3 consecutive days while the others remained as controls and received the standard management. Their full blood count was monitored 8 hours for 48 hours. Gene expression studies were conducted on the ALOX 12 and PTAFR genes. The mean increase in platelet counts were compared in both groups using repeated measure ANCOVA. There was a significant increase in mean platelet count observed in the intervention group (P < 0.001) but not in the control group 40 hours since the first dose of CPLJ. Comparison of mean platelet count between intervention and control group showed that mean platelet count in intervention group was significantly higher than control group after 40 and 48 hours of admission (P < 0.01). The ALOX 12 (FC  =  15.00) and PTAFR (FC  =  13.42) genes were highly expressed among those on the juice. It was concluded that CPLJ does significantly increase the platelet count in patients with DF and DHF. PMID:23662145

Platelet count and platelet indices in women with preeclampsia.

PubMed

AlSheeha, Muneera A; Alaboudi, Rafi S; Alghasham, Mohammad A; Iqbal, Javed; Adam, Ishag

2016-01-01

Although the exact pathophysiology of preeclampsia is not completely understood, the utility of different platelets indices can be utilized to predict preeclampsia. To compare platelet indices, namely platelet count (PC), mean platelet volume (MPV), platelet distribution width (PDW), and PC to MPV ratio in women with preeclampsia compared with healthy controls. Qassim Hospital, Kingdom of Saudi Arabia. A case-control study. Sixty preeclamptic women were the cases and an equal number of healthy pregnant women were the controls. There was no significant difference in age, parity, and body mass index between the study groups. Sixteen and 44 of the cases were severe and mild preeclampsia, respectively. There was no significant difference in PDW and MPV between the preeclamptic and control women. Both PC and PC to MPV ratios were significantly lower in the women with preeclampsia compared with the controls. There was no significant difference in the PC, PDW, MPV, and PC to MPV ratio when women with mild and severe preeclampsia were compared. Using receiver operating characteristic (ROC) curves, the PC cutoff was 248.0×10 3 /µL for diagnosis of pre-eclampsia ( P =0.019; the area under the ROC curve was 62.4%). Binary regression suggests that women with PC <248.010×10 3 /µL were at higher risk of preeclampsia (odds ratio =2.2, 95% confidence interval =1.08-4.6, P =0.03). The PC/MPV cutoff was 31.2 for diagnosis of preeclampsia ( P =0.035, the area under the ROC curve was 62.2%). PC <248.010×10 3 /µL and PC to MPV ratio 31.2 are valid predictors of preeclampsia.

Platelet Glycoprotein Ib-IX and Malignancy

DTIC Science & Technology

2010-09-01

provide a unique microenvironment supporting the accumulation of more platelets and the elaboration of a fibrin - rich network produced by coagulation...process and can initiate the formation of a platelet - rich thrombus by tethering the platelet to a thrombogenic surface. Several ligands binding to GP Ib... Platelet Glycoprotein Ib-IX and Malignancy PRINCIPAL INVESTIGATOR: Jerry Ware, Ph.D

Antiplatelet Agents Can Promote Two-Peaked Thrombin Generation in Platelet Rich Plasma: Mechanism and Possible Applications

PubMed Central

Tarandovskiy, Ivan D.; Artemenko, Elena O.; Panteleev, Mikhail A.; Sinauridze, Elena I.; Ataullakhanov, Fazoil I.

2013-01-01

Background Thrombin generation assay is a convenient and widely used method for analysis of the blood coagulation system status. Thrombin generation curve (TGC) is usually bell-shaped with a single peak, but there are exceptions. In particular, TGC in platelet-rich plasma (PRP) can sometimes have two peaks. Objective We sought to understand the mechanism underlying the occurrence of two peaks in the PRP thrombin generation curve. Methods Tissue factor-induced thrombin generation in PRP and platelet-poor plasma (PPP) was monitored using continuous measurement of the hydrolysis rate of the thrombin-specific fluorogenic substrate Z-Gly-Gly-Arg-AMC. Expression of phosphatidylserine (PS) and CD62P on the surface of activated platelets was measured by flow cytometry using corresponding fluorescently labeled markers. Results The addition of the P2Y12 receptor antagonist MeS-AMP (160 µM), 83 nM prostaglandin E1 (PGE1), or 1.6% DMSO to PRP caused the appearance of two peaks in the TGC. The PS exposure after thrombin activation on washed platelets in a suspension supplemented with DMSO, PGE1 or MeS-AMP was delayed, which could indicate mechanism of the second peak formation. Supplementation of PRP with 1.6% DMSO plus 830 nM PGE1 mediated the disappearance of the second peak and decreased the amplitude of the first peak. Increasing the platelet concentration in the PRP promoted the consolidation of the two peaks into one. Conclusions Procoagulant tenase and prothrombinase complexes in PRP assemble on phospholipid surfaces containing PS of two types - plasma lipoproteins and the surface of activated platelets. Thrombin generation in the PRP can be two-peaked. The second peak appears in the presence of platelet antagonists as a result of delayed PS expression on platelets, which leads to delayed assembly of the membrane-dependent procoagulant complexes and a second wave of thrombin generation. PMID:23405196

Novel platelet substitutes: disk-shaped biodegradable nanosheets and their enhanced effects on platelet aggregation.

PubMed

Okamura, Yosuke; Fukui, Yoshihito; Kabata, Koki; Suzuki, Hidenori; Handa, Makoto; Ikeda, Yasuo; Takeoka, Shinji

2009-10-21

We have studied biocompatible spherical carriers carrying a dodecapeptide, HHLGGAKQAGDV (H12), on their surface as platelet substitutes. This peptide is a fibrinogen γ-chain carboxy-terminal sequence (γ400-411) and specifically recognizes the active form of glycoprotein IIb/IIIa on activated platelets. Our purpose is to assess the possibility of making a novel platelet substitute consisting of disk-shaped nanosheets having a large contact area for the targeting site, rather than conventional small contact area spherical carriers. The H12 peptide was conjugated to the surface of the free-standing nanosheets made of biodegradable poly(d,l-lactide-co-glycolide) (PLGA). These H12-PLGA nanosheets were fabricated onto 3 μm disk-shaped patterned hydrophobic octadecyl regions on a SiO(2) substrate. By way of comparison, spherical H12-PLGA microparticles with the same surface area and conjugation number of H12 were also prepared. The resulting H12-PLGA nanosheets specifically interacted with the activated platelets adhered on the collagen surface at twice the rate of the H12-PLGA microparticles under flow conditions, and showed platelet thrombus formation in a two-dimensional spreading manner. Thus, H12-PLGA nanosheets might be a suitable candidate novel platelet alternative substitute for infused human platelet concentrates for the treatment of bleeding in patients with severe thrombocytopenia.

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.

Maximising platelet availability by delaying cold storage.

PubMed

Wood, B; Johnson, L; Hyland, R A; Marks, D C

2018-04-06

Cold-stored platelets may be an alternative to conventional room temperature (RT) storage. However, cold-stored platelets are cleared more rapidly from circulation, reducing their suitability for prophylactic transfusion. To minimise wastage, it may be beneficial to store platelets conventionally until near expiry (4 days) for prophylactic use, transferring them to refrigerated storage to facilitate an extended shelf life, reserving the platelets for the treatment of acute bleeding. Two ABO-matched buffy-coat-derived platelets (30% plasma/70% SSP+) were pooled and split to produce matched pairs (n = 8 pairs). One unit was stored at 2-6°C without agitation (day 1 postcollection; cold); the second unit was stored at 20-24°C with constant agitation until day 4 then stored at 2-6°C thereafter (delayed-cold). All units were tested for in vitro quality periodically over 21 days. During storage, cold and delayed-cold platelets maintained a similar platelet count. While pH and HSR were significantly higher in delayed-cold platelets, other metabolic markers, including lactate production and glucose consumption, did not differ significantly. Furthermore, surface expression of phosphatidylserine and CD62P, release of soluble CD62P and microparticles were not significantly different, suggesting similar activation profiles. Aggregation responses of delayed-cold platelets followed the same trend as cold platelets once transferred to cold storage, gradually declining over the storage period. The metabolic and activation profile of delayed-cold platelets was similar to cold-stored platelets. These data suggest that transferring platelets to refrigerated storage when near expiry may be a viable option for maximising platelet inventories. © 2018 International Society of Blood Transfusion.

Effects of different shear rates on the attachment and detachment of platelet thrombi.

PubMed

Shi, Xiaofeng; Yang, Jichun; Huang, Jiansong; Long, Zhangbiao; Ruan, Zheng; Xiao, Bing; Xi, Xiaodong

2016-03-01

Thrombosis and hemostasis take place in flowing blood, which generates shear forces. The effect of different shear rates, particularly pathological forces, on platelet thrombus formation remains to be fully elucidated. The present study observed the morphological characteristics and hierarchical structure of thrombi on the collagen surface at a wide range of wall shear rates (WSRs) and examined the underlying mechanisms. Calcein AM‑labeled whole blood was perfused over a collagen‑coated surface at different shear rates set by a Bioflux 200 microfluidic device and the thrombi formed were assessed for area coverage, the height and the hierarchical structure defined by the extent of platelet activation and packing density. The factors that affect thrombus formation were also investigated. Platelet thrombus formation varied under different WSRs, for example, dispersed platelet adhesion mixed with erythrocytes was observed at 125‑250 s(‑1), extensive and thin platelet thrombi were observed at 500‑1,500 s(‑1), and sporadic, thick thrombi were observed at pathological WSRs of 2,500‑5,000 s(‑1), which showed a tendency to be shed. With increasing WSRs, the height of the thrombi showed an increasing linear trend, whereas the total fluorescence intensity and area of the thrombi exhibited a parabolic curve‑like change, with a turning point at a WSR of 2,500 s(‑1). The number of thrombi, the average fluorescence intensity and the area per thrombus showed similar trends, with an initial upwards incline followed by a decline. The thrombi formed at higher WSRs had a thicker shell, which led to a more densely packed core. Platelet thrombus formation under shear‑flow was regulated by the adhesive strength, which was mediated by receptor‑ligand interaction, the platelet deposition induced by shear rates and the detachment by the dynamic force of flow. This resulted in a balance between thrombus attachment, including adhesion and aggregation, and detachment

Platelet-rich preparations to improve healing. Part II: platelet activation and enrichment, leukocyte inclusion, and other selection criteria.

PubMed

Davis, Vicki L; Abukabda, Alaeddin B; Radio, Nicholas M; Witt-Enderby, Paula A; Clafshenkel, William P; Cairone, J Vito; Rutkowski, James L

2014-08-01

Multiple platelet-rich preparations have been reported to improve wound and bone healing, such as platelet-rich plasma (PRP) and platelet rich fibrin (PRF). The different methods employed during their preparation are important, as they influence the quality of the product applied to a wound or surgical site. Besides the general protocol for preparing the platelet-rich product (discussed in Part 1 of this review), multiple choices need to be considered during its preparation. For example, activation of the platelets is required for the release and enmeshment of growth factors, but the method of activation may influence the resulting matrix, growth factor availability, and healing. Additionally, some methods enrich leukocytes as well as platelets, but others are designed to be leukocyte-poor. Leukocytes have many important roles in healing and their inclusion in PRP results in increased platelet concentrations. Platelet and growth factor enrichment reported for the different types of platelet-rich preparations are also compared. Generally, TGF-β1 and PDGF levels were higher in preparations that contain leukocytes compared to leukocyte-poor PRP. However, platelet concentration may be the most reliable criterion for comparing different preparations. These and other criteria are described to help guide dental and medical professionals, in large and small practices, in selecting the best procedures for their patients. The healing benefits of platelet-rich preparations along with the low risk and availability of simple preparation procedures should encourage more clinicians to incorporate platelet-rich products in their practice to accelerate healing, reduce adverse events, and improve patient outcomes.

Thrombospondin-1 controls vascular platelet recruitment and thrombus adherence in mice by protecting (sub)endothelial VWF from cleavage by ADAMTS13

PubMed Central

Bonnefoy, Arnaud; Daenens, Kim; Feys, Hendrik B.; De Vos, Rita; Vandervoort, Petra; Vermylen, Jos; Lawler, Jack; Hoylaerts, Marc F.

2006-01-01

The function of thrombospondin-1 (TSP-1) in hemostasis was investigated in wild-type (WT) and Tsp1-/- mice, via dynamic platelet interaction studies with A23187-stimulated mesenteric endothelium and with photochemically injured cecum subendothelium. Injected calcein-labeled WT platelets tethered or firmly adhered to almost all A23187-stimulated blood vessels of WT mice, but Tsp1-/- platelets tethered to 45% and adhered to 25.8% of stimulated Tsp1-/- vessels only. Stimulation generated temporary endothelium-associated ultralarge von Willebrand factor (VWF) multimers, triggering platelet string formation in 48% of WT versus 20% of Tsp1-/- vessels. Injection of human TSP-1 or thrombotic thrombocytopenic purpura (TTP) patient-derived neutralizing anti-ADAMTS13 antibodies corrected the defective platelet recruitment in Tsp1-/- mice, while having a moderate effect in WT mice. Photochemical injury of intestinal blood vessels induced thrombotic occlusions with longer occlusion times in Tsp1-/- venules (1027 ± 377 seconds) and arterioles (858 ± 289 seconds) than in WT vessels (559 ± 241 seconds, P < .001; 443 ± 413 seconds, P < .003) due to defective thrombus adherence, resulting in embolization of complete thrombi, a defect restored by both human TSP-1 and anti-ADAMTS13 antibodies. We conclude that in a shear field, soluble or local platelet-released TSP-1 can protect unfolded endothelium-bound and subendothelial VWF from degradation by plasma ADAMTS13, thus securing platelet tethering and thrombus adherence to inflamed and injured endothelium, respectively. PMID:16204318

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

Platelet bioreactor-on-a-chip

PubMed Central

Mazutis, Linas; Wu, Stephen; Sylman, Joanna L.; Ehrlicher, Allen; Machlus, Kellie R.; Feng, Qiang; Lu, Shijiang; Lanza, Robert; Neeves, Keith B.; Weitz, David A.; Italiano, Joseph E.

2014-01-01

Platelet transfusions total >2.17 million apheresis-equivalent units per year in the United States and are derived entirely from human donors, despite clinically significant immunogenicity, associated risk of sepsis, and inventory shortages due to high demand and 5-day shelf life. To take advantage of known physiological drivers of thrombopoiesis, we have developed a microfluidic human platelet bioreactor that recapitulates bone marrow stiffness, extracellular matrix composition, micro-channel size, hemodynamic vascular shear stress, and endothelial cell contacts, and it supports high-resolution live-cell microscopy and quantification of platelet production. Physiological shear stresses triggered proplatelet initiation, reproduced ex vivo bone marrow proplatelet production, and generated functional platelets. Modeling human bone marrow composition and hemodynamics in vitro obviates risks associated with platelet procurement and storage to help meet growing transfusion needs. PMID:25606631

Biomaterial Property Effects on Platelets and Macrophages: An in Vitro Study.

PubMed

Fernandes, Kelly R; Zhang, Yang; Magri, Angela M P; Renno, Ana C M; van den Beucken, Jeroen J J P

2017-12-11

The purpose of this study was to evaluate the effects of surface properties of bone implants coated with hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) on platelets and macrophages upon implant installation and compare them to grit-blasted Ti and Thermanox used as a control. Surface properties were characterized using scanning electron microscopy, profilometry, crystallography, Fourier transform infrared spectroscopy, and coating stability. For platelets, platelet adherence and morphology were assessed. For macrophages, morphology, proliferation, and polarization were evaluated. Surface characterization showed similar roughness of ∼2.5 μm for grit-blasted Ti discs, both with and without coating. Coating stability assessment showed substantial dissolution of HA and β-TCP coatings. Platelet adherence was significantly higher for grit-blasted Ti, Ti-HA, and Ti-β-TCP coatings compared to that of cell culture control Thermanox. Macrophage cultures revealed a decreased proliferation on both HA and β-TCP coated discs compared to both Thermanox and grit-blasted Ti. In contrast, secretion of pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine TGF-β were marginal for grit-blasted Ti and Thermanox, while a coating-dependent increased secretion of pro- and anti-inflammatory cytokines was observed for HA and β-TCP coatings. The results demonstrated a significantly upregulated pro-inflammatory and anti-inflammatory cytokine secretion and marker gene expression of macrophages on HA and β-TCP coatings. Furthermore, HA induced an earlier M1 macrophage polarization but more M2 phenotype potency than β-TCP. In conclusion, our data showed that material surface affects the behaviors of first cell types attached to implants. Due to the demonstrated crucial roles of platelets and macrophages in bone healing and implant integration, this information will greatly aid the design of metallic implants for a higher rate of success in patients.

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.

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.

Comparison of bacterial attachment to platelet bags with and without preconditioning with plasma.

PubMed

Loza-Correa, M; Kalab, M; Yi, Q-L; Eltringham-Smith, L J; Sheffield, W P; Ramirez-Arcos, S

2017-07-01

Canadian Blood Services produces apheresis and buffy coat pooled platelet concentrates (PCs) stored in bags produced by two different manufacturers (A and B, respectively), both made of polyvinyl chloride-butyryl trihexyl citrate. This study was aimed at comparing Staphylococcus epidermidis adhesion to the inner surface of both bag types in the presence or absence of plasma factors. Sets (N = 2-6) of bags type A and B were left non-coated (control) or preconditioned with platelet-rich, platelet-poor or defibrinated plasma (PRP, PPP and DefibPPP, respectively). Each bag was inoculated with a 200-ml S. epidermidis culture adjusted to 0·5 colony-forming units/ml. Bags were incubated under platelet storage conditions for 7 days. After culture removal, bacteria attached to the plastic surface were either dislodged by sonication for bacterial quantification or examined in situ by scanning electron microscopy (SEM). Higher bacterial adhesion was observed to preconditioned PC bags than control containers for both bag types (P < 0·0001). Bacterial attachment to preconditioned bags was confirmed by SEM. Bacteria adhered equally to both types of containers in the presence of PRP, PPP and DefibPPP residues (P > 0·05). By contrast, a significant increase in bacterial adherence was observed to type A bags compared with type B bags in the absence of plasma (P < 0·05) [Correction added on 16 June 2017, after first online publication: this sentence has been corrected]. The ability of S. epidermidis to adhere to preconditioned platelet collection bags depends on the presence of plasma factors. Future efforts should be focused on reducing plasma proteins' attachment to platelet storage containers to decrease subsequent bacterial adhesion. © 2017 International Society of Blood Transfusion.

Pathogen-Reduced, Platelet Additive Solution, Extended Stored Platelets (PREPS)

DTIC Science & Technology

2015-10-01

Sherrill J. Slichter, MD CONTRACTING ORGANIZATION: Bloodworks Northwest Seattle, WA 98104 REPORT DATE : October 2015 TYPE OF REPORT: Annual...NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE October 2015 2. REPORT TYPE Annual 3. DATES COVERED 15-SEP-14 to 14-SEP-15 4. TITLE AND...accomplishments to date . Brrr Study Our study of platelets stored as whole blood at 4°C demonstrated that end-over-end rotation is required to reduce platelet

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

Characteristics of platelet gels combined with silk

PubMed Central

Pallotta, Isabella; Kluge, Jonathan A.; Moreau, Jodie; Calabrese, Rossella

2014-01-01

Platelet gel, a fibrin network containing activated platelets, is widely used in regenerative medicine due the capacity of platelet-derived growth factors to accelerate and direct healing processes. However, limitations to this approach include poor mechanical properties, relatively rapid degradation, and the lack of control of release of growth factors at the site of injection. These issues compromise the ability of platelet gels for sustained function in regenerative medicine. In the present study, a combination of platelet gels with silk fibroin gel was studied to address the above limitations. Mixing sonicated silk gels with platelet gels extended the release of growth factors without inhibiting gel forming ability. The released growth factors were biologically active and their delivery was modified further by manipulation of the charge of the silk protein. Moreover, the silk gel augmented both the rheological properties and compressive stiffness of the platelet gel, tuned by the silk concentration and/or silk/platelet gel ratio. Silk-platelet gel injections in nude rats supported enhanced cell infiltration and blood vessel formation representing a step towards new platelet gel formulations with enhanced therapeutic impact. PMID:24480538

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 ...

Platelet concentrates for transfusion-metabolic and storage aspects.

PubMed

Farrugia, A

1994-01-01

Transfusion of platelets concentrated from donated blood is an established therapeutic modality in clinical medicine. Over the past 25 years much effort has gone into optimising the conditions for the collection, preparation and storage of platelets for transfusion. Despite significant advances, platelet production is still a costly process requiring a dedicated environment and the use of specially formulated plastic storage containers. A progressive lesion over storage limits the shelf life and the availability of donated platelets, while the need to store platelets in the donor's autologous plasma also results in a loss of valuable fresh plasma for fractionation. Recent studies have addressed the issues of platelet quality and plasma economy by examining the possibility of storing platelets in a synthetic medium. Platelets stored in a variety of crystalloid solutions have been shown to retain in vitro and in vivo properties equivalent or superior to platelets stored in autologous donor plasma. Some additional insight has been gained on the metabolic patterns of stored platelets. In particular, studies have shown that, under these conditions, platelets are unable to oxidise dextrose to any significant extent, and that dextrose is invariably broken down to lactate, irrespective of the oxygen tensions in the platelet's environment. This in turn leads to the metabolic lesion of platelet storage, whereby low pH results in loss of platelet viability. Platelets stored in synthetic dextrose-free media are capable of maintaining aerobic ATP generation, and acetate-a component of many media studied-has been shown to be metabolised by platelets. Similarly, platelets prepared from blood collected into a dextrose-free anticoagulant have satisfactory properties both when suspended in autologous plasma or in a dextrose-free synthetic medium. The requirements for storage in special, high gas-permeable, containers, and for constant agitation during storage, were both found to be

PET scanning in head and neck oncology: a review.

PubMed

McGuirt, W F; Greven, K; Williams, D; Keyes, J W; Watson, N; Cappellari, J O; Geisinger, K R

1998-05-01

The objective of this study was to review and describe the usage of fluorine-labeled deoxyglucose (FDG) and positron emission tomography (PET) in the diagnosis and management of head and neck cancer. Several prospective series,-including 159 newly diagnosed and previously untreated and 23 previously irradiated head and neck squamous cell carcinoma patients initially seen at the Wake Forest University Medical Center and evaluated by clinical examination, conventional computed tomography/ magnetic resonance imaging (CT/MRI) scans, PET scans, and histopathologic studies,-were reviewed and the findings summarized for comparison of the correct differentiation of primary and metastatic cancers and for postirradiation tumor clearance in a subsegment of those cases. Positron emission tomography scanning using a fluorine-labeled deoxyglucose (FDG) radiotracer proved as reliable as conventional scanning for primary and metastatic tumor identification. Compared with clinical examination, PET was better for identification of nodal metastatic tumors but poorer for small primary tumors. For previously irradiated patients treated at least 4 months before the test, PET scanning was clearly superior to clinical examination and conventional imaging in differentiating tumor recurrence from soft-tissue irradiation effects. Fluorine-labeled deoxyglucose-PET scanning is comparable to conventional imaging of head and neck cancers in detecting primary and metastatic carcinoma. Lack of anatomic detail remains its major drawback. Currently, its greatest role is in the evaluation of the postradiotherapy patient.

Gallic Acid Attenuates Platelet Activation and Platelet-Leukocyte Aggregation: Involving Pathways of Akt and GSK3β

PubMed Central

Chang, Shih-Sheng; Lee, Viola S. Y.; Tseng, Yu-Lun; Chang, Kuan-Cheng; Chen, Kuen-Bao; Chen, Yuh-Lien; Li, Chi-Yuan

2012-01-01

Platelet activation and its interaction with leukocytes play an important role in atherothrombosis. Cardiovascular diseases resulted from atherothrombosis remain the major causes of death worldwide. Gallic acid, a major constituent of red wine and tea, has been believed to have properties of cardiovascular protection, which is likely to be related to its antioxidant effects. Nonetheless, there were few and inconsistent data regarding the effects of gallic acid on platelet function. Therefore, we designed this in vitro study to determine whether gallic acid could inhibit platelet activation and the possible mechanisms. From our results, gallic acid could concentration-dependently inhibit platelet aggregation, P-selectin expression, and platelet-leukocyte aggregation. Gallic acid prevented the elevation of intracellular calcium and attenuated phosphorylation of PKCα/p38 MAPK and Akt/GSK3β on platelets stimulated by the stimulants ADP or U46619. This is the first mechanistic explanation for the inhibitory effects on platelets from gallic acid. PMID:22811749

Assessment of platelet function in healthy sedated cats using three whole blood platelet function tests.

PubMed

Ho, Kimberly K; Abrams-Ogg, Anthony C G; Wood, R Darren; O'Sullivan, M Lynne; Kirby, Gordon M; Blois, Shauna L

2015-05-01

The objectives of this study were to establish feline references intervals for 3 commercial whole blood platelet function test analyzer systems: Multiplate analyzer (MP; Roche Diagnostics International Ltd., Rotkreuz, Switzerland), Platelet Function Analyzer-100 (PF: Siemens Canada, Mississauga, Ontario, Canada), and Plateletworks Combo-25 kit (PW; Helena Laboratories, Beaumont, TX). Venipuncture was performed on 55 healthy sedated cats, and platelet aggregation in response to adenosine diphosphate (ADP), collagen (COL), and arachidonic acid (AA; MP only) was assessed using citrated blood. For the MP analyzer, median (95% confidence intervals [CIs]) area under curve (Units) for ADP, COL, and AA agonists were 87 (11-176), 81 (32-129), and 91 (59-129), respectively. For the PF analyzer, median (95% CIs) closure time, using COL-ADP cartridges, was 69 (46-89) sec. For the PW assay, median (95% CIs) percent aggregations for ADP and COL agonists were 71 (18-92) and 49 (9-96), respectively, using impedance hematology analyzer platelet counts, and 94 (25-98) and 68 (14-119), respectively, using flow cytometry hematology analyzer platelet counts. There were low correlations between the PF analyzer (COL-ADP cartridge) and MP analyzer (COL agonist; ρ = 0.11), and between the PF analyzer (COL-ADP cartridge) and PW assay (COL agonist using impedance platelet counts; ρ = 0.14). The PW assay percent aggregations using impedance and flow cytometric platelet counts were correlated for both ADP (ρ = 0.64) and COL (ρ = 0.64) agonists. Platelet function testing using these tests are feasible in cats, but 95% CIs are wide, so single results may be difficult to interpret. Platelet counting by impedance or flow cytometry may be used for the PW assay but are not interchangeable. © 2015 The Author(s).

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

Tyrosine phosphorylation of P-selectin in intact platelets and in a disulphide-linked complex with immunoprecipitated pp60c-src.

PubMed Central

Modderman, P W; von dem Borne, A E; Sonnenberg, A

1994-01-01

P-selectin is a 140 kDa membrane glycoprotein found in secretory granules of platelets and endothelial cells where it is rapidly translocated to the plasma membrane upon cell activation. It then functions as a receptor for various types of leucocytes. Metabolic labelling of resting platelets with 32Pi showed that P-selectin is primarily phosphorylated on serine residues, although some tyrosine phosphorylation was observed as well. However, tyrosine phosphorylation of P-selectin was greatly stimulated by treatment with the permeating phosphatase inhibitor, pervanadate. When P-selectin immunoprecipitates were incubated with [gamma-32P]ATP (in vitro kinase assay), a fraction of P-selectin was phosphorylated on its tyrosine residues by a co-precipitated kinase. P-selectin phosphorylated in vitro co-migrated with 140 kDa surface-labelled 125I-P-selectin during SDS/PAGE under reducing conditions. Under non-reducing conditions, however, phosphorylated P-selectin was disulphide-linked to unknown protein(s) in a 205 kDa complex. In vitro kinase assays of the most abundant platelet tyrosine kinase, pp60c-src, demonstrated the presence of similar 140 and 205 kDa phosphorylated proteins in SDS/PAGE under reducing and non-reducing conditions respectively. Extraction and reprecipitation studies with proteins phosphorylated in vitro indicated that P-selectin and pp60c-src form a 205 kDa 1:1 disulphide-linked complex. In the complex, pp60c-src autophosphorylation is inhibited and P-selectin is phosphorylated on tyrosine residues. As protein disulphides in the cytoplasm of intact cells are extremely rare, our results suggest that P-selectin and pp60c-src, which co-localize in platelet dense granules, may be non-covalently associated and spontaneously form disulphide bridges during lysis. In addition, the observed tyrosine phosphorylation of P-selectin in intact platelets suggests that its function might be regulated by phosphorylation by pp60c-src. Images Figure 1 Figure 2 Figure 3

Clinical significance of residual platelet reactivity in patients treated with platelet P2Y12 inhibitors.

PubMed

Thomas, Mark R; Storey, Robert F

2016-09-01

Platelet P2Y12 inhibitors have become an essential component of the treatment strategy for patients with acute coronary syndromes and patients undergoing percutaneous coronary intervention. It is now well-established that approximately 30% of patients treated with the P2Y12 inhibitor clopidogrel display high residual platelet reactivity despite treatment. Patients with high on-treatment platelet reactivity have approximately 2-3-fold greater risk of adverse cardiovascular events and stent thrombosis than those without high platelet reactivity. Conversely, clopidogrel-treated patients with low platelet reactivity display approximately 1.7-fold increased risk of major bleeding. High platelet reactivity is uncommon during treatment with prasugrel and ticagrelor, which achieve a greater reduction in adverse cardiovascular events compared to clopidogrel in ACS patients treated with PCI. This is at the expense of an increase in spontaneous bleeding, however. Minor bleeding events, such as skin haematomas, are more common in prasugrel- and ticagrelor-treated patients that have particularly low platelet reactivity values. These minor bleeding events may occasionally prompt discontinuation of therapy, but their overall prognostic impact is uncertain. However, risk factors for bleeding tend to overlap with risk factors for adverse cardiovascular events. Therefore, patients with these minor bleeding events may also be at higher risk of adverse cardiovascular events, conferring a benefit from low platelet reactivity. Further work is needed to determine the optimal level of platelet reactivity in individuals by taking into account their risk of subsequent adverse cardiovascular events and bleeding. Copyright © 2016 Elsevier Inc. All rights reserved.

Platelet chemokines in vascular disease

PubMed Central

Gleissner, Christian A.; von Hundelshausen, Philipp; Ley, Klaus

2009-01-01

Platelets are a rich source of different chemokines and express chemokine receptors. CXCL4 is highly abundant in platelets and involved in promoting monocyte arrest from rolling and monocyte differentiation to macrophages. CXCL4 can also associate with CCL5 and amplify its effect on monocytes. The megakaryocyte CXCL7 gene product is proteolytically cleaved into the strong neutrophil chemoattractant, NAP-2, which has also been implicated in repair cell homing to vascular lesions. Platelet adhesion can induce release of CCL2 and CXCL8 from endothelial cells. Conversely, the chemokines CCL17, CCL22 and CXCL12 made by other cells amplify platelet activation. Platelet chemokines enhance recruitment of various hematopoietic cells to the vascular wall, fostering processes such as neointima formation, atherosclerosis, and thrombosis but also vessel repair and regeneration after vascular injury. PMID:18723831

Platelet inhibition with ticagrelor versus clopidogrel in Hispanic patients with stable coronary artery disease with or without diabetes mellitus.

PubMed

Clavijo, Leonardo C; Maya, Juan; Carlson, Glenn; Angiolillo, Dominick J; Teng, Renli; Caplan, Richard; Price, Matthew J

2015-12-01

Diabetes mellitus (DM) disproportionately affects Hispanic patients. DM patients have enhanced platelet reactivity and reduced sensitivity to clopidogrel. Ticagrelor demonstrated a more rapid onset and greater magnitude of platelet inhibition than clopidogrel in Hispanic patients with stable coronary artery disease (CAD). This subgroup analysis examined the onset and level of platelet inhibition of ticagrelor and clopidogrel in Hispanic patients with DM. This was a subgroup analysis of a randomized, open-label, crossover study in which 40 Hispanic patients with stable CAD received ticagrelor 180 mg loading dose (LD)/90 mg twice-daily maintenance dose (MD) then clopidogrel 600 mg LD/75 mg once-daily MD, or vice versa. The primary end point was on-treatment platelet reactivity at 2 hours post-LD using the VerifyNow™ P2Y12 test. 21 patients had DM and 19 were non-diabetic. At 2 hours post-LD, mean platelet reactivity in the diabetic group was 34.5 PRU with ticagrelor versus 219.3 PRU with clopidogrel (P<0.001), and in the non-diabetic group was 33.7 PRU with ticagrelor versus 181.0 PRU with clopidogrel (P<0.001). In both diabetic and non-diabetic subgroups, mean platelet reactivity declined to a significantly greater extent with ticagrelor than clopidogrel at all time points evaluated (0.5, 2, and 8 hours post LD and after 7-9 days of MD). Patients were significantly more likely to have high on-treatment platelet reactivity (≥208 PRU) during treatment with clopidogrel compared with ticagrelor, regardless of diabetic status. Among Hispanic patients with stable CAD, ticagrelor achieves a faster onset and greater magnitude of platelet inhibition compared with clopidogrel, irrespective of diabetic status. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

The superiority of indium ratio over blood pool subtraction in analysis of indium-111 platelet deposition on thoraco-abdominal prosthetic grafts

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

Ripley, S.; Wakefield, T.; Spaulding, S.

1985-05-01

In this investigation platelet deposition in polytetrafluroethylene (PTFE) thoracoabdominal grafts (TAC's) was evaluated using two different semi-quantitative techniques. Ten PTFE TAG's 6 mm in diameter and 30 cm in length were inserted into 10 mongrel dogs. One, 4 and 6 weeks after graft implantation the animals were injected with autologous In-111 platelets labelled by a modified Thakur technique. Platelet imaging in grafts was performed 48 hrs after injection. Blood pool was determined by Tc99m labelled RBC's (in vivo/in vitro technique). Semi-quantitative analysis was performed by subdividing the imaged graft into three major regions and selecting a reference region from eithermore » the native aorta or common iliac artery. Excess platelet deposition was determined by two methods: 1) the ratio of In-111 counts in the graft ROI''s to the reference region and 2) the percent In-111 excess using the Tc99m blood pool subtraction technique (TBPST). Animals were sacrificed 7 weeks after implantation and radioactivity in the excised grafts was determined using a well counter. A positive correlation was found to exist between the In-111 ratio percent analysis (IRPA) and the direct gamma counting (DCC) for all three segments of the prosthetic graft. Correlation coefficients for the thorax, midsegment and abdominal segments were 0.80, 0.73 and 0.48 respectivly. There was no correlation between TBPST and DGC. Using the IRPA technique the thrombogenicity of TAG's can be routinely assessed and is clinically applicable for patient use. TBPST should probably be limited to the extremities to avoid error due to free Tc99m counts from kidneys and ureters.« less

Platelet cyclooxygenase expression in normal dogs.

PubMed

Thomason, J; Lunsford, K; Mullins, K; Stokes, J; Pinchuk, L; Wills, R; McLaughlin, R; Langston, C; Pruett, S; Mackin, A

2011-01-01

Human platelets express both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). Variation in COX-2 expression could be a mechanism for variable response to aspirin. The hypotheses were that circulating canine platelets express COX-1 and COX-2, and that aspirin alters COX expression. The objective was to identify changes in platelet COX expression and in platelet function caused by aspirin administration to dogs. Eight female, intact hounds. A single population, repeated measures design was used to evaluate platelet COX-1 and COX-2 expression by flow cytometry before and after aspirin (10 mg/kg Q12h for 10 days). Platelet function was analyzed via PFA-100(®) (collagen/epinephrine), and urine 11-dehydro-thromboxane B(2) (11-dTXB(2)) was measured and normalized to urinary creatinine. Differences in COX expression, PFA-100(®) closure times, and urine 11-dTXB(2 ): creatinine ratio were analyzed before and after aspirin administration. Both COX-1 and COX-2 were expressed in canine platelets. COX-1 mean fluorescent intensity (MFI) increased in all dogs, by 250% (range 63-476%), while COX-2 expression did not change significantly (P = 0.124) after aspirin exposure, with large interindividual variation. PFA-100(®) closure times were prolonged and urine 11-dTXB(2) concentration decreased in all dogs after aspirin administration. Canine platelets express both COX isoforms. After aspirin exposure, COX-1 expression increased despite impairment of platelet function, while COX-2 expression varied markedly among dogs. Variability in platelet COX-2 expression should be explored as a potential mechanism for, or marker of, variable aspirin responsiveness. Copyright © 2011 by the American College of Veterinary Internal Medicine.

13C-TRIPLY Labeled Ethyl Cyanide Submillimeterwave Study with Lille's Fast Scan Dds-Based Spectrometer

NASA Astrophysics Data System (ADS)

Pienkina, A.; Motiyenko, R. A.; Margulès, L.; Müller, Holger S. P.; Guillemin, J.-C.

2016-06-01

This study of the 13C-triply labeled species of ethyl cyanide (CH_3CH_2CN) follows our recent work on the three 13C-doubly-labeled that allowed their detection in the line survey recently obtained with ALMA (EMoCA). The detection of isotopologues could improve the knowledge of the astrochemistry. The other goal is to clean the surveys from the lines of known molecules in order to detect new ones, this is especially important for the abundant complex organic molecules like ethyl cyanide. As in the case of the doubly substitued species, no spectroscopic studies exist up to now for 13CH_313CH_213CN, the first predictions were thus obtained from scaled ab initio calculations. The spectra were recorded and analyzed up to 1 THz. More than 5500 lines were fitted with quantum numbers J and K_a up to 95 and 25 respectively. The spectra were obtained with the new version of the Lille's solid state spectrometers. This new version used Direct Digital Synthesizer in order to speed up acquisition time. We constructed a spectrometer covering a decade, from 150 to 1500 GHz, it scans the full range in 24 hours with high sensitivity and accuracy. This work was supported by the CNES and the Action sur Projets de l'INSU, PCMI. This work was also done under ANR-13-BS05-0008-02 IMOLABS Margules, L.; et al. 2015, 69th International Symposium on Molecular Spectroscopy, RI06 Belloche, A.; et al. 2014, Science, 345, 1584

Peri-Procedural Platelet Reactivity in Percutaneous Coronary Intervention.

PubMed

Alexopoulos, Dimitrios; Xenogiannis, Iosif; Vlachakis, Panagiotis; Tantry, Udaya; Gurbel, Paul A

2018-06-04

Platelet activation and aggregation play a pivotal role in thrombotic complications occurring during percutaneous coronary intervention (PCI), and peri-PCI anti-platelet therapy represents a standard of care. High platelet reactivity prior to PCI has been correlated with an increased incidence of peri-procedural myonecrosis. Pre-PCI platelet reactivity predicts post-PCI platelet reactivity and has a prognostic impact on subsequent ischaemic and bleeding events, so as the platelet inhibition measured post-PCI. Many anti-platelet treatment strategies, including aspirin, glycoprotein IIb/IIIa inhibitors, P2Y 12 receptor blockers and vorapaxar, are being used in the routine clinical practice to modify platelet reactivity at each stage, e.g. pre-, during and post-PCI. Anti-platelet strategies with a 'stronger and faster' pharmacodynamic effect than clopidogrel have been mostly adopted in patients with acute coronary syndromes. However, several issues regarding the anti-platelet treatment such as benefits/risks of anti-platelet therapy pre-treatment and duration, and definite association between speed and potency of various anti-platelet agents and clinical outcomes remain controversial. We believe that a better understanding of peri-PCI platelet reactivity and its relations to outcomes may lead to the development of more effective and safe treatment strategies. Schattauer GmbH Stuttgart.

Peptide-Mediated Platelet Capture at Gold Micropore Arrays.

PubMed

Adamson, Kellie; Spain, Elaine; Prendergast, Una; Moran, Niamh; Forster, Robert J; Keyes, Tia E

2016-11-30

Ordered spherical cap gold cavity arrays with 5.4, 1.6, and 0.98 μm diameter apertures were explored as capture surfaces for human blood platelets to investigate the impact of surface geometry and chemical modification on platelet capture efficiency and their potential as platforms for surface enhanced Raman spectroscopy of single platelets. The substrates were chemically modified with single-constituent self-assembled monolayers (SAM) or mixed SAMs comprised of thiol-functionalized arginine-glycine-aspartic acid (RGD, a platelet integrin target) with or without 1-octanethiol (adhesion inhibitor). As expected, platelet adhesion was promoted and inhibited at RGD and alkanethiol modified surfaces, respectively. Platelet adhesion was reversible, and binding efficiency at the peptide modified substrates correlated inversely with pore diameter. Captured platelets underwent morphological change on capture, the extent of which depended on the topology of the underlying substrate. Regioselective capture of the platelets enabled study for the first time of the surface enhanced Raman spectroscopy of single blood platelets, yielding high quality Raman spectroscopy of individual platelets at 1.6 μm diameter pore arrays. Given the medical importance of blood platelets across a range of diseases from cancer to psychiatric illness, such approaches to platelet capture may provide a useful route to Raman spectroscopy for platelet related diagnostics.

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

Endothelial Cell and Platelet Bioenergetics: Effect of Glucose and Nutrient Composition

PubMed Central

Fink, Brian D.; Herlein, Judy A.; O’Malley, Yunxia; Sivitz, William I.

2012-01-01

It has been suggested that cells that are independent of insulin for glucose uptake, when exposed to high glucose or other nutrient concentrations, manifest enhanced mitochondrial substrate oxidation with consequent enhanced potential and generation of reactive oxygen species (ROS); a paradigm that could predispose to vascular complications of diabetes. Here we exposed bovine aortic endothelial (BAE) cells and human platelets to variable glucose and fatty acid concentrations. We then examined oxygen consumption and acidification rates using recently available technology in the form of an extracellular oxygen and proton flux analyzer. Acute or overnight exposure of confluent BAE cells to glucose concentrations from 5.5 to 25 mM did not enhance or change the rate of oxygen consumption (OCR) under basal conditions, during ATP synthesis, or under uncoupled conditions. Glucose also did not alter OCR in sub-confluent cells, in cells exposed to low serum, or in cells treated with added pyruvate. Likewise, overnight exposure to fatty acids of varying saturation had no such effects. Overnight exposure of BAE cells to low glucose concentration decreased maximal uncoupled respiration, but not basal or ATP related oxygen consumption. Labeled glucose oxidation to CO2 increased, but only marginally after high glucose exposure while oleate oxidation to CO2 decreased. Overnight exposure to linolenic acid, but not oleic or linoleic acid increased extracellular acidification consistent with enhanced glycolytic metabolism. We were unable to detect an increase in production of reactive oxygen species (ROS) from BAE cells exposed to high medium glucose. Like BAE cells, exposure of human platelets to glucose did not increase oxygen consumption. As opposed to BAE cells, platelet mitochondria demonstrate less respiratory reserve capacity (beyond that needed for basal metabolism). Our data do not support the concept that exposure to high glucose or fatty acids accelerates mitochondrial

Platelet morphology, soluble P selectin and platelet P-selectin in acute ischaemic stroke. The West Birmingham Stroke Project.

PubMed

Nadar, Sunil K; Lip, Gregory Y H; Blann, Andrew D

2004-12-01

The pathophysiology of ischaemic stroke involves the platelet. In this study, we hypothesised that abnormalities in platelet morphology, as well as soluble (sPsel) and total platelet P-selectin (pPsel) levels would be present in patients presenting with an acute ischaemic stroke, and that these changes would improve at > or = 3 months' follow-up. We studied 59 hypertensive patients (34 male; mean age 68 +/- 12 years) who presented with an acute ischaemic stroke (ictus < 24 hours), and compared them with 2 groups: (i) age-, sex- and ethnic- origin matched normotensive healthy controls; and (ii) uncomplicated 'high risk' hypertensive patients as 'risk factor control' subjects. Platelet morphology (volume and mass) was quantified, and sPsel (plasma marker of platelet activation) was measured (ELISA) in citrated plasma. The mass of P-selectin in each platelet (pPsel) was determined by lysing a fixed number of platelets and then determining the levels of P-selectin in the lysate. Results show that patients who presented with a stroke had significantly higher levels of sPsel and pPsel (both p < 0.001), compared to the normal controls and the hypertensive patients. Patients with an acute stroke had lower mean platelet mass (MPM) and mean platelet volume (MPV) as compared to the uncomplicated hypertensive patients, who had significantly higher mean MPM and MPV values, as compared to normal controls. On follow-up, the levels of both sPsel (p = 0.011), pPsel (< 0.001) and MPV (p = 0.03) were significantly lower. Mean MPM levels remained unchanged. We conclude that patients presenting with an acute ischaemic stroke have activated platelets, as evident by the increased levels of soluble and platelet P-selectin. Further study of platelet activation and the role of P-selectin is warranted.

Blood platelets: computerized morphometry applied on optical images

NASA Astrophysics Data System (ADS)

Korobova, Farida V.; Ivanova, Tatyana V.; Gusev, Alexander A.; Shmarov, Dmitry A.; Kozinets, Gennady I.

2000-11-01

The new technology of computerized morphometric image analysis of platelets on blood smears was developed. In a basis of the device is included analysis of cytophotometric and morphometric parameters of platelets. Geometrical and optical parameters of platelets on 35 donors, platelet concentrates and 15 patients with haemorrhagic thrombocythaemia were investigated, average meanings for the area, diameter, its logarithms and optical density of platelets in norm were received. Distribution of the areas, diameters and optical densities of platelets of patients with haemorrhagic thrombocythaemia differed from those at the healthy people. After a course of treatment these meanings came nearer to normal. The important characteristics of platelets in platelet concentrates after three days of storage were in limits of normal meanings, but differed from those in whole blood platelets. Obtained data allow to enter the quantitative standards into investigation of platelets of the healthy people and at various alteration of thrombocytopoieses.

Regulating billions of blood platelets: glycans and beyond

PubMed Central

Grozovsky, Renata; Giannini, Silvia; Falet, Hervé

2015-01-01

The human body produces and removes 1011 platelets daily to maintain a normal steady state platelet count. Platelet production must be regulated to avoid spontaneous bleeding or arterial occlusion and organ damage. Multifaceted and complex mechanisms control platelet production and removal in physiological and pathological conditions. This review will focus on different mechanisms of platelet senescence and clearance with specific emphasis on the role of posttranslational modifications. It will also briefly address platelet transfusion and the role of glycans in the clearance of stored platelets. PMID:26330242

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

1993-06-01

Normal mice received hematoporphyrin derivative (HpD) i.v. prior to red light irradiation and the platelet-rich plasma was prepared and irradiated by red light. The platelets were processed for EM examination and stereological analysis. It was shown the 16 hrs after irradiation almost all platelets were necrotized; 8 hours after irradiation about 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 deformated, showing significantly increased mean area, perimeter and short axis, and mean cell volume and cell surface area. The findings indicate that platelets are highly sensitive to PDT action and can be directly and rapidly damaged by PDT even in the absence of vascular endothelial cells. The early platelet photoactivation may play an important role in the initiation of early vascular damage and microcirculatory alterations induced by PDT in vivo.

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.

Investigations of human platelet-type 12-lipoxygenase: role of lipoxygenase products in platelet activation1[S

PubMed Central

Ikei, Kenneth N.; Yeung, Jennifer; Apopa, Patrick L.; Ceja, Jesús; Vesci, Joanne; Holinstat, Michael

2012-01-01

Human platelet-type 12-lipoxygenase (12-LOX) has recently been shown to play an important role in regulation of human platelet function by reacting with arachidonic acid (AA). However, a number of other fatty acids are present on the platelet surface that, when cleaved from the phospholipid, can be oxidized by 12-LOX. We sought to characterize the substrate specificity of 12-LOX against six essential fatty acids: AA, dihomo-γ-linolenic acid (DGLA), eicosapentaenoic acid (EPA), α-linolenic acid (ALA), eicosadienoic acid (EDA), and linoleic acid (LA). Three fatty acids were comparable substrates (AA, DGLA, and EPA), one was 5-fold slower (ALA), and two showed no reactivity with 12-LOX (EDA and LA). The bioactive lipid products resulting from 12-LOX oxidation of DGLA, 12-(S)-hydroperoxy-8Z,10E,14Z-eicosatrienoic acid [12(S)-HPETrE], and its reduced product, 12(S)-HETrE, resulted in significant attenuation of agonist-mediated platelet aggregation, granule secretion, αIIbβ3 activation, Rap1 activation, and clot retraction. Treatment with DGLA similarly inhibited PAR1-mediated platelet activation as well as platelet clot retraction. These observations are in surprising contrast to our recent work showing 12(S)-HETE is a prothrombotic bioactive lipid and support our hypothesis that the overall effect of 12-LOX oxidation of fatty acids in the platelet is dependent on the fatty acid substrates available at the platelet membrane. PMID:22984144

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

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.

21 CFR 864.6650 - Platelet adhesion test.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Platelet adhesion test. 864.6650 Section 864.6650...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Manual Hematology Devices § 864.6650 Platelet adhesion test. (a) Identification. A platelet adhesion test is a device used to determine in vitro platelet...

21 CFR 864.6650 - Platelet adhesion test.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Platelet adhesion test. 864.6650 Section 864.6650...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Manual Hematology Devices § 864.6650 Platelet adhesion test. (a) Identification. A platelet adhesion test is a device used to determine in vitro platelet...

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 morphology and plasma indices of platelet activation in essential hypertension: effects of amlodipine-based antihypertensive therapy.

PubMed

Nadar, Sunil; Blann, Andrew D; Lip, Gregory Y H

2004-01-01

Platelet abnormalities have been described in hypertension, especially in the presence of target organ damage. Our aim was to study the differences in morphology and indices of platelet activation in treatment-naive patients with essential hypertension as compared to normotensive controls and secondly, to study the effects of amlodipine-based antihypertensive therapy on these indices. We recruited 42 previously untreated, newly diagnosed hypertensive patients (25 men; mean age 53 years) for the cross-sectional study, where data were compared with those from 30 normotensive controls (20 men; mean age 57 years). Of the 42 untreated hypertensive patients who were recruited, 27 patients successfully completed, the six-month treatment phase with amlodipine-based antihypertensive therapy. Platelet morphology (volume and mass) was quantified, and plasma markers of platelet activation (betaTG and sPsel) measured in citrated plasma. The mass of P-selectin in each platelet (pPsel) was determined by lysing a fixed number of platelets and then determining the levels of P-selectin in the lysate. Hypertensive patients had significantly higher platelet volume (P = 0.01) and mass (P = 0.003), plasma betaTG and sPsel, and pPsel levels (all P < 0.001) compared to the controls. After a mean treatment time of 6 months, there was a decrease in platelet volume (P < 0.001) and mass (P = 0.02), with lower pPsel, sPsel and BTG levels (all P < 0.001) compared to the untreated state. Treatment of uncomplicated essential hypertension using amlodipine-based anti-hypertensive therapy results in a reversal of the platelet morphology abnormalities and indices of platelet activation. This may contribute to a reduction in thrombosis-related complications seen in those whose blood pressure lowering is effective.

A Novel Variant in the Platelet Endothelial Aggregation Receptor-1 Gene is Associated with Increased Platelet Aggregabili

PubMed Central

Herrera-Galeano, J. Enrique; Becker, Diane M.; Wilson, Alexander F.; Yanek, Lisa R.; Bray, Paul; Vaidya, Dhananjay; Faraday, Nauder; Becker, Lewis C

2009-01-01

Objective: Platelet endothelial aggregation receptor-1 (PEAR1) is a recently identified platelet transmembrane protein that becomes activated by platelet contact. We looked for novel genetic variants in PEAR1 and studied their association with agonist-induced native platelet aggregation and with aspirin's inhibitory effect on platelets. Methods and Results: We genotyped PEAR1 for 10 single nucleotide polymorphisms (SNPs), selected for optimal gene coverage at a density of 4kb, in 1486 apparently healthy individuals from two generations of families with premature CAD. Subjects had a mean age of 45 years; 62% were white and 38% African American. Platelet aggregation to collagen, epinephrine, and ADP was measured in platelet rich plasma, at baseline and after 2 weeks of aspirin (ASA, 81 mg/day), and genotype-phenotype associations were examined separately by ethnicity using multivariable generalized linear models adjusted for covariates. The C allele of SNP rs2768759 [A/C], located in the promoter region of the gene, was common in whites and uncommon in African Americans (allele frequency 70.2% vs 17.7%). The C allele was generally associated in both ethnic groups with increased aggregation of native platelets to each agonist. Following ASA, the associations were stronger and more consistent, and remained significant when post ASA aggregation was adjusted for baseline aggregation, consistent with a relationship between the C allele and reduced platelet responsiveness to ASA. The PEAR1 SNP explained up to 6.9% of the locus specific genetic variance in African Americans and up to 2.5% of the genetic variance in whites following ASA. Conclusion: PEAR1 appears to play an important role in agonist-induced platelet aggregation and in the response to ASA in both whites and African Americans. PMID:18511696

Platelet concentration in platelet-rich plasma affects tenocyte behavior in vitro.

PubMed

Giusti, Ilaria; D'Ascenzo, Sandra; Mancò, Annalisa; Di Stefano, Gabriella; Di Francesco, Marianna; Rughetti, Anna; Dal Mas, Antonella; Properzi, Gianfranco; Calvisi, Vittorio; Dolo, Vincenza

2014-01-01

Since tendon injuries and tendinopathy are a growing problem, sometimes requiring surgery, new strategies that improve conservative therapies are needed. Platelet-rich plasma (PRP) seems to be a good candidate by virtue of its high content of growth factors, most of which are involved in tendon healing. This study aimed to evaluate if different concentrations of platelets in PRP have different effects on the biological features of normal human tenocytes that are usually required during tendon healing. The different platelet concentrations tested (up to 5 × 10(6) plt/µL) stimulated differently tenocytes behavior; intermediate concentrations (0.5 × 10(6), 1 × 10(6) plt/µL) strongly induced all tested processes (proliferation, migration, collagen, and MMPs production) if compared to untreated cells; on the contrary, the highest concentration had inhibitory effects on proliferation and strongly reduced migration abilities and overall collagen production but, at the same time, induced increasing MMP production, which could be counterproductive because excessive proteolysis could impair tendon mechanical stability. Thus, these in vitro data strongly suggest the need for a compromise between extremely high and low platelet concentrations to obtain an optimal global effect when inducing in vivo tendon healing.

Platelet Concentration in Platelet-Rich Plasma Affects Tenocyte Behavior In Vitro

PubMed Central

Rughetti, Anna; Dal Mas, Antonella; Properzi, Gianfranco; Calvisi, Vittorio

2014-01-01

Since tendon injuries and tendinopathy are a growing problem, sometimes requiring surgery, new strategies that improve conservative therapies are needed. Platelet-rich plasma (PRP) seems to be a good candidate by virtue of its high content of growth factors, most of which are involved in tendon healing. This study aimed to evaluate if different concentrations of platelets in PRP have different effects on the biological features of normal human tenocytes that are usually required during tendon healing. The different platelet concentrations tested (up to 5 × 106 plt/µL) stimulated differently tenocytes behavior; intermediate concentrations (0.5 × 106, 1 × 106 plt/µL) strongly induced all tested processes (proliferation, migration, collagen, and MMPs production) if compared to untreated cells; on the contrary, the highest concentration had inhibitory effects on proliferation and strongly reduced migration abilities and overall collagen production but, at the same time, induced increasing MMP production, which could be counterproductive because excessive proteolysis could impair tendon mechanical stability. Thus, these in vitro data strongly suggest the need for a compromise between extremely high and low platelet concentrations to obtain an optimal global effect when inducing in vivo tendon healing. PMID:25147809

Do nutrition labels influence healthier food choices? Analysis of label viewing behaviour and subsequent food purchases in a labelling intervention trial.

PubMed

Ni Mhurchu, Cliona; Eyles, Helen; Jiang, Yannan; Blakely, Tony

2018-02-01

There are few objective data on how nutrition labels are used in real-world shopping situations, or how they affect dietary choices and patterns. The Starlight study was a four-week randomised, controlled trial of the effects of three different types of nutrition labels on consumer food purchases: Traffic Light Labels, Health Star Rating labels, or Nutrition Information Panels (control). Smartphone technology allowed participants to scan barcodes of packaged foods and receive randomly allocated labels on their phone screen, and to record their food purchases. The study app therefore provided objectively recorded data on label viewing behaviour and food purchases over a four-week period. A post-hoc analysis of trial data was undertaken to assess frequency of label use, label use by food group, and association between label use and the healthiness of packaged food products purchased. Over the four-week intervention, study participants (n = 1255) viewed nutrition labels for and/or purchased 66,915 barcoded packaged products. Labels were viewed for 23% of all purchased products, with decreasing frequency over time. Shoppers were most likely to view labels for convenience foods, cereals, snack foods, bread and bakery products, and oils. They were least likely to view labels for sugar and honey products, eggs, fish, fruit and vegetables, and meat. Products for which participants viewed the label and subsequently purchased the product during the same shopping episode were significantly healthier than products where labels were viewed but the product was not subsequently purchased: mean difference in nutrient profile score -0.90 (95% CI -1.54 to -0.26). In a secondary analysis of a nutrition labelling intervention trial, there was a significant association between label use and the healthiness of products purchased. Nutrition label use may therefore lead to healthier food purchases. Copyright © 2017 The Authors. Published by Elsevier Ltd.. 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.

Preclinical Investigation of Lyophilized Platelet Preparations

DTIC Science & Technology

1994-10-31

Western blots of rehydrated platelet preparations. The AMAC antibody reacted strongly with a high molecular weight protein in the fresh platelet lysate , and...to a lesser degree with a protein of identical molecular weight in the rehydrated platelet lysate . The antibody to fibrinogen reacted strongly with

Effects of protopine on blood platelet aggregation. II. Effect on metabolic system of adenosine 3',5'-cyclic monophosphate in platelets.

PubMed

Shiomoto, H; Matsuda, H; Kubo, M

1990-08-01

The mode of action of protopine on rabbit platelet aggregation was investigated in the metabolic system of adenosine 3',5'-cyclic monophosphate (cyclic AMP) in vitro experimental models. The inhibitory activity of protopine on adenosine 5'-diphosphate induced platelet aggregation was increased in the presence of prostaglandin I2 or papaverine in platelets. Protopine elevated content of the basal cyclic AMP accumulation in platelets and enhanced activity of crude adenylate cyclase prepared from platelets, but was ineffective on cyclic AMP phosphodiesterase. It is concluded that protopine has an inhibitory activity on platelet aggregation, activates adenylate cyclase and increases cyclic AMP content in platelets, in addition to other inhibitory actions in the metabolic system of cyclic AMP.

An effector role for platelets in systemic and local lipopolysaccharide-induced toxicity in mice, mediated by a CD11a- and CD54-dependent interaction with endothelium.

PubMed Central

Piguet, P F; Vesin, C; Ryser, J E; Senaldi, G; Grau, G E; Tacchini-Cottier, F

1993-01-01

The role of platelets was investigated in two models of lipopolysaccharide (LPS)-induced toxicity in mice: the systemic reaction, provoked by intravenous LPS injection in D-galactosamine-sensitized recipients, which results in host death, and the local reaction, elicited in the skin by sequential injections of LPS and tumor necrosis factor alpha at 24-h intervals, which results in hemorrhagic necrosis. In both models, the depletion of platelets with a rabbit polyclonal or a mouse monoclonal antiplatelet immunoglobulin G afforded significant protection. In the local reaction, studies of the distribution of 111In-labelled platelets as well as optical and electron microscopy showed that platelets are localized in the dermal venules before hemorrhage occurs. Anti-CD11a (LFA-1) and anti-CD54 (ICAM-1) monoclonal antibodies prevented both platelet localization and hemorrhagic necrosis, and these determinants were detected on mouse platelets by immunofluorescence. The antiplatelet monoclonal antibody did not reduce the localization of polymorphonuclear leukocytes in the dermal venules, as shown by histological sections. Thus, in the local reaction, the stimulation with LPS and tumor necrosis factor alpha leads to a binding of platelets to the endothelium of venules by their beta 2 integrins, which seems necessary for the development of the hemorrhagic necrosis. Images PMID:8104895

Human plasma platelet-derived exosomes: effects of aspirin.

PubMed

Goetzl, Edward J; Goetzl, Laura; Karliner, Joel S; Tang, Norina; Pulliam, Lynn

2016-05-01

Platelet-derived exosomes mediate platelet atherogenic interactions with endothelial cells and monocytes. A new method for isolation of plasma platelet-derived exosomes is described and used to examine effects of aging and aspirin on exosome cargo proteins. Exosome secretion by purified platelets in vitro did not increase after exposure to thrombin or collagen, as assessed by exosome counts and quantification of the CD81 exosome marker. Thrombin and collagen increased exosome content of α-granule chemokines CXCL4 and CXCL7 and cytoplasmic high-mobility group box 1 (HMGB1) protein, but not membrane platelet glycoprotein VI (GPVI), with dependence on extracellular calcium. Aspirin consumption significantly blocked thrombin- and collagen-induced increases in exosome cargo levels of chemokines and HMGB1, without altering total exosome secretion or GPVI cargo. Plasma platelet-derived exosomes, enriched by absorption with mouse antihuman CD42b [platelet glycoprotein Ib (GPIb)] mAb, had sizes and cargo protein contents similar to those of exosomes from purified platelets. The plasma platelet-derived exosome number is lower and its chemokine and HMGB1 levels higher after age 65 yr. Aspirin consumption significantly suppressed cargo protein levels of plasma platelet-derived exosomes without altering total levels of exosomes. Cargo proteins of human plasma platelet-derived exosomes may biomark platelet abnormalities and in vivo effects of drugs.- Goetzl, E. J., Goetzl, L., Karliner, J. S., Tang, N., Pulliam, L. Human plasma platelet-derived exosomes: effects of aspirin. © FASEB.

Do Labels Matter When Implementing Change? Implications of Labelling an Academic as a Champion--Results from a Case Study

ERIC Educational Resources Information Center

Cordiner, Moira; Thomas, Sharon; Green, Wendy

2018-01-01

Organisational change literature is littered with labels for those who instigate, support, resist, or implement change. Absent is research into the perspectives of those who are given these labels. This paper reports findings from a literature search, journal scan and a case study of an Australian university where change agents were labelled…

Dabigatran and rivaroxaban do not affect AA- and ADP-induced platelet aggregation in patients receiving concomitant platelet inhibitors.

PubMed

Olivier, Christoph B; Weik, Patrick; Meyer, Melanie; Weber, Susanne; Diehl, Philipp; Bode, Christoph; Moser, Martin; Zhou, Qian

2016-08-01

Dabigatran and rivaroxaban are novel, vitamin K-independent oral anticoagulants (NOACs) and act via antagonism of the coagulation factor (F) IIa (dabigatran) or FXa (rivaroxaban), respectively. Compared to vitamin-K-antagonists, NOACs have shown non-inferiority of risk and benefit in patients with non valvular atrial fibrillation (AF). In clinical practice there is increasing use of NOACs combined with platelet inhibitors in patients with AF and coronary artery disease. However, whether NOACs affect the function of platelet inhibitors remains incompletely known. This observational study aimed to assess the platelet function in patients receiving dabigatran or rivaroxaban and concomitant platelet inhibitors. A single centre observational study was performed analysing the platelet aggregation of patients treated with dabigatran or rivaroxaban with or without concomitant platelet inhibitors. Measurements before the initiation of NOAC therapy served as the respective control group. Platelet aggregation was measured by multiple electrode aggregometry and was induced with adenosine diphosphate (ADP, 6.5 µM) and arachidonic acid (AA, 0.5 mM), respectively. In order to evaluate whether NOACs interact with platelet inhibition by ASA or the P2Y12-antagonist clopidogrel, 87 patients were grouped according to their concomitant antiplatelet medication. Comparing the ADP- and AA-induced platelet aggregation in patients without concomitant platelet inhibitors (n = 45) no significant differences under therapy with dabigatran (d) or rivaroxaban (r) compared to the control group (c) were observed. In patients taking clopidogrel as a concomitant platelet inhibitor (n = 21), neither dabigatran nor rivaroxaban affected the ADP-induced platelet aggregation (c 20 ± 11, d 21 ± 14, r 18 ± 8 AU*min, p = 0.200). Patients receiving dabigatran or rivaroxaban in combination with ASA (n = 42; 21 ASA only, 21 ASA + clopidogrel) showed no significant differences of the AA

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

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.

Platelet activation by Histophilus somni and its lipooligosaccharide induces endothelial cell proinflammatory responses and platelet internalization.

PubMed

Kuckleburg, Christopher J; McClenahan, Dave J; Czuprynski, Charles J

2008-02-01

Histophilus somni is a gram-negative coccobacillus that causes respiratory and reproductive disease in cattle. The hallmark of systemic H. somni infection is diffuse vascular inflammation that can lead to an acute central nervous system disease known as thrombotic meningoencephalitis. Previously, we demonstrated that H. somni and its lipooligosaccharide (LOS) activate bovine platelets, leading to expression of P selectin, CD40L, and FasL. Because activated platelets have been reported to induce endothelial cell cytokine production and adhesion molecule expression, we sought to determine if bovine platelets induce proinflammatory and procoagulative changes in bovine pulmonary artery endothelial cells. Endothelial cells were incubated with platelets activated with adenosine diphosphate, H. somni, or H. somni LOS. Incubation with activated bovine platelets significantly increased expression of in adhesion molecules (intercellular adhesion molecule 1, E selectin) and tissue factor, as measured by flow cytometry, real-time polymerase chain reaction, and Western blot analysis. Activated platelets also up-regulated expression of endothelial cell IL-1beta, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1alpha as determined by real-time polymerase chain reaction and an IL-1beta enzyme-linked immunosorbent assay. An interesting and surprising finding was that bovine platelets activated by H. somni or its LOS were internalized by bovine endothelial cells as visualized by transmission electron microscopy. This internalization seemed to correlate with endothelial cell activation and morphological changes indicative of cell stress. These findings suggest that activated platelets might play a role in promoting vascular inflammation during H. somni infection.

Evaluation of four methods for platelet compatibility testing

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

McFarland, J.G.; Aster, R.H.

1987-05-01

Four platelet compatibility assays were performed on serum and platelet or lymphocyte samples from 38 closely HLA-matched donor/recipient pairs involved in 55 single-donor platelet transfusions. The 22 patients studied were refractory to transfusions of pooled random-donor platelets. Of the four assays (platelet suspension immunofluorescence, PSIFT; /sup 51/Cr release; microlymphocytotoxicity; and a monoclonal anti-IgG assay, MAIA), the MAIA was most predictive of platelet transfusion outcome (predictability, 74% for one-hour posttransfusion platelet recovery and 76% for 24-hour recovery). The only other assay to reach statistical significance was the PSIFT (63% predictability for one-hour posttransfusion recovery). The degree of HLA compatibility between donormore » and recipient (exact matches v those utilizing cross-reactive associations) was unrelated to the ability of the MAIA to predict transfusion results. The MAIA may be capable of differentiating HLA antibodies, ABO antibodies, and platelet-specific antibodies responsible for failure of HLA-matched and selectively mismatched single-donor platelet transfusions.« less

The effect of molar pregnancies on platelet parameters.

PubMed

Soylu Karapınar, Oya; Benk Şilfeler, Dilek; Dolapçıoğlu, Kenan; Keskin Kurt, Raziye; Beyazıt, Ahmet

2016-10-01

The aim of this study was to compare platelet parameters between abortus groups with gestational trophoblastic disease (GTD) (molar pregnancy, invasive mole, choriocarcinoma, etc) and without disease according to pathological result. The study population consisted of patients with GTD (n = 53) and aborted patients without disease as a control group (n = 53) who were seen in our clinic between January 2010 and December 2013. In this retrospective study, age, gravidity, levels of haemoglobin, white blood cell count, platelets, platelet parameters (mean platelet volume (MPV), platelet distrubition width (PDW), platelet crit (PCT), which shows platelet functions were recorded. The pathological diagnosis of GTD was recorded. The mean platelet count, MPV, PDW and PCT levels were similar between the groups. There is no statistically significiant difference between types of GTN in these parameters according to pathological diagnosis. According to our study results, platelet count and levels of MPV, PDW ve PCT in GTD patients were similar to aborted patients without disease.

The life cycle of platelet granules.

PubMed

Sharda, Anish; Flaumenhaft, Robert

2018-01-01

Platelet granules are unique among secretory vesicles in both their content and their life cycle. Platelets contain three major granule types-dense granules, α-granules, and lysosomes-although other granule types have been reported. Dense granules and α-granules are the most well-studied and the most physiologically important. Platelet granules are formed in large, multilobulated cells, termed megakaryocytes, prior to transport into platelets. The biogenesis of dense granules and α-granules involves common but also distinct pathways. Both are formed from the trans -Golgi network and early endosomes and mature in multivesicular bodies, but the formation of dense granules requires trafficking machinery different from that of α-granules. Following formation in the megakaryocyte body, both granule types are transported through and mature in long proplatelet extensions prior to the release of nascent platelets into the bloodstream. Granules remain stored in circulating platelets until platelet activation triggers the exocytosis of their contents. Soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, located on both the granules and target membranes, provide the mechanical energy that enables membrane fusion during both granulogenesis and exocytosis. The function of these core fusion engines is controlled by SNARE regulators, which direct the site, timing, and extent to which these SNAREs interact and consequently the resulting membrane fusion. In this review, we assess new developments in the study of platelet granules, from their generation to their exocytosis.

Glutamate mediates platelet activation through the AMPA receptor

PubMed Central

Morrell, Craig N.; Sun, Henry; Ikeda, Masahiro; Beique, Jean-Claude; Swaim, Anne Marie; Mason, Emily; Martin, Tanika V.; Thompson, Laura E.; Gozen, Oguz; Ampagoomian, David; Sprengel, Rolf; Rothstein, Jeffrey; Faraday, Nauder; Huganir, Richard; Lowenstein, Charles J.

2008-01-01

Glutamate is an excitatory neurotransmitter that binds to the kainate receptor, the N-methyl-D-aspartate (NMDA) receptor, and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR). Each receptor was first characterized and cloned in the central nervous system (CNS). Glutamate is also present in the periphery, and glutamate receptors have been identified in nonneuronal tissues, including bone, heart, kidney, pancreas, and platelets. Platelets play a central role in normal thrombosis and hemostasis, as well as contributing greatly to diseases such as stroke and myocardial infarction. Despite the presence of glutamate in platelet granules, the role of glutamate during hemostasis is unknown. We now show that activated platelets release glutamate, that platelets express AMPAR subunits, and that glutamate increases agonist-induced platelet activation. Furthermore, we demonstrate that glutamate binding to the AMPAR increases intracellular sodium concentration and depolarizes platelets, which are important steps in platelet activation. In contrast, platelets treated with the AMPAR antagonist CNQX or platelets derived from GluR1 knockout mice are resistant to AMPA effects. Importantly, mice lacking GluR1 have a prolonged time to thrombosis in vivo. Our data identify glutamate as a regulator of platelet activation, and suggest that the AMPA receptor is a novel antithrombotic target. PMID:18283118

Platelet growth factors from allogeneic platelet-rich plasma for clinical improvement in split-thickness skin graft.

PubMed

Sonker, Atul; Dubey, Anju; Bhatnagar, Ankur; Chaudhary, Rajendra

2015-01-01

Platelets are a source of numerous growth factors which facilitate repair and healing. Thus platelet rich plasma has been increasingly used as a treatment modality in the field of reconstructive surgeries for wound healing. This preliminary study was carried out to explore whether platelet growth factors from platelet rich plasma could be used for enhancement of split thickness skin graft survival. Twenty patients (13 males and 7 females) requiring split thickness skin graft for various clinical reasons were enrolled in the study. Platelet rich plasma was collected by apheresis and frozen at -80° C. It was thawed at room temperature immediately before its intended application. PRP was applied only on one half of the wound, while another half served as control. Patient was followed for 6 weeks. The effect was assessed at first dressing in terms of graft uptake and subsequently as time taken for complete healing. There was 100% uptake of the graft in the area where platelet rich plasma was applied. In the control area, there was complete graft loss in 4 cases, partial loss in 7 cases and complete uptake in 9 cases. This study demonstrated promising results on application of PRP to split thickness skin grafts. Further randomized studies with greater sample size may be undertaken to establish platelet rich plasma as a validated treatment modality.

Chitosan inhibits platelet-mediated clot retraction, increases platelet-derived growth factor release, and increases residence time and bioactivity of platelet-rich plasma in vivo.

PubMed

Deprés-Tremblay, Gabrielle; Chevrier, Anik; Tran-Khanh, Nicolas; Nelea, Monica; Buschmann, Michael D

2017-11-10

Platelet-rich plasma (PRP) has been used to treat different orthopedic conditions, however, the clinical benefits of using PRP remain uncertain. Chitosan (CS)-PRP implants have been shown to improve meniscus, rotator cuff and cartilage repair in pre-clinical models. The purpose of this current study was to investigate in vitro and in vivo mechanisms of action of CS-PRP implants. Freeze-dried formulations containing 1% (w/v) CS (80% degree of deacetylation and number average molar mass 38 kDa), 1% (w/v) trehalose as a lyoprotectant and 42.2 mM calcium chloride as a clot activator were solubilized in PRP. Gravimetric measurements and molecular/cellular imaging studies revealed that clot retraction is inhibited in CS-PRP hybrid clots through physical coating of platelets, blood cells and fibrin strands by chitosan, which interferes with platelet aggregation and platelet-mediated clot retraction. Flow cytometry and ELISA assays revealed that platelets are activated and granules secreted in CS-PRP hybrid clots and that cumulative release of platelet-derived growth factor (PDGF-AB) and epidermal growth factor is higher from CS-PRP hybrid clots compared to PRP clots in vitro. Finally, CS-PRP implants resided for up to 6 weeks in a subcutaneous implantation model and induced cell recruitment and granulation tissue synthesis, confirming greater residency and bioactivity compared to PRP in vivo.

Platelet Glycoprotein lb-1X and Malignancy

DTIC Science & Technology

2010-09-01

supporting the accumulation of more platelets and the elaboration of a fibrin - rich network produced by coagulation factors. This paradigm has been...a platelet - rich thrombus by tethering the platelet to a thrombogenic surface. Several ligands binding to GP Ib-IX have been identified, including...08-1-0576 TITLE: Platelet Glycoprotein lb-1X and Malignancy PRINCIPAL INVESTIGATOR: Dr. Jerry Ware

Is platelet function as measured by Thrombelastograph monitoring in whole blood affected by platelet inhibitors?

PubMed

Bailey, Lori A; Sistino, Joseph J; Uber, Walter E

2005-03-01

Platelet inhibitors, especially the glycoprotein (GP) IIb/IIIa receptor antagonists, have demonstrated their effectiveness in reducing the acute ischemic complications of percutaneous coronary intervention (PCI) and in improving clinical outcomes in patients with acute coronary crisis. Three common platelet inhibitors observed in emergent cardiopulmonary bypass (CPB) for failed PCI are abciximab, eptifibatide, and tirofiban. An in vitro model was constructed in two parts to determine whether platelet aggregation inhibition induced by platelet inhibitors would be demonstrated by the Thrombelastograph (TEG) monitor when compared with baseline samples with no platelet inhibitor. In part A, 20 mL of fresh whole blood was divided into four groups: group I = baseline, group A = abcix-imab microg/mL, group E = eptifibatide ng/mL, and group T = tirofiban ng/mL. Platelet inhibitor concentrations in whole blood were derived starting with reported serum concentrations with escalation to achieve 80% platelet inhibition using the Medtronic hemoSTATUS and/or Lumi-aggregometer. A concentration range determined by our in vitro tests were chosen for each drug using concentrations achieving less than, equal to, or greater than 80% platelet inhibition. In part B, TEG analysis was then performed using baseline and concentrations for each drug derived in part A. Parameters measured were clot formation reaction time (R), coagulation time (K), maximum amplitude (MA) and alpha angle (A). Groups E1000 and E2000 extended R over control by 37% and 23%, respectively (p = 0.01 and 0.03). Groups E1000 and E2000 increased K times by 45% and 58% (p = .02 and .04). T160 samples prolonged K by 20% (p = 0.01). The angle or clot strength (A) was decreased in groups T160 and E1000 by 23% (+ 7.06 SD) and 18% (+ 11.23 SD), respectively (p = 0.001 and 0.01). The MA decrease was statistically significant in the T160, E1000 and E2000 by 9%, 6% and 13% respectively (p = 0.01). Samples treated with abciximab

Nicergoline inhibits human platelet Ca(2+) signalling through triggering a microtubule-dependent reorganization of the platelet ultrastructure.

PubMed

Walford, T; Musa, F I; Harper, A G S

2016-01-01

Recently, we demonstrated that a pericellular Ca(2+) recycling system potentiates agonist-evoked Ca(2+) signalling and granule secretion in human platelets and hypothesized a role for the membrane complex (MC) in orchestrating the accumulation of Ca(2+) in the pericellular region. Previous work has demonstrated that treatment with high concentrations of nicergoline may disrupt the MC through an ability to trigger a re-organization of the dense tubular system. Experiments were therefore performed to assess whether nicergoline-induced changes in platelet ultrastructure affects thrombin-evoked Ca(2+) fluxes and dense granule secretion. Thrombin-evoked Ca(2+) fluxes were monitored in Fura-2- or Fluo-5N-loaded human platelets, or using platelet suspensions containing Fluo-4 or Rhod-5N K(+) salts. Fluorescence microscopy was utilized to monitor microtubule structure and intracellular Ca(2+) store distribution in TubulinTracker- and Fluo-5N-loaded platelets respectively. Dense granule secretion was monitored using luciferin-luciferase. Nicergoline treatment inhibited thrombin-evoked Ca(2+) signalling and induced alterations in the microtubule structure and the distribution of intracellular Ca(2+) stores in platelets. Nicergoline altered the generation and spreading of thrombin-induced pericellular Ca(2+) signals and almost completely prevented dense granule secretion. Stabilization of microtubules using taxol reversed most effects of nicergoline on platelet Ca(2+) signalling and partially reversed its effects on dense granule secretion. Nicergoline-induced alterations to platelet ultrastructure disrupt platelet Ca(2+) signalling in a manner that would be predicted if the MC had been disrupted. These data suggest that nicergoline may be a useful prototype for the discovery of novel MC-disrupting anti-thrombotics. © 2015 The British Pharmacological Society.

Platelets, neutrophils, and vasoconstriction after arterial injury by angioplasty in pigs: effects of MK-886, a leukotriene biosynthesis inhibitor

PubMed Central

Provost, Patrick; Borgeat, Pierre; Merhi, Yahye

1998-01-01

Leukotrienes constitute a class of potent bioactive mediators known to play a pivotal role in inflammation. Since their biosynthesis has been shown to be enhanced by platelet-neutrophil interactions, leukotrienes may be involved in these interactions and the arterial response to injury. Therefore, we investigated the effects of the selective leukotriene biosynthesis inhibitor 3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethylpropanoic acid (MK-886) on the acute thrombotic and vasomotor responses after arterial injury by angioplasty.Carotid arterial injury was produced by balloon dilatation in control (molecusol vehicle; n=10) and treated (MK-886, 10 mg kg−1, i.v.; n=9) pigs. The acute thrombotic reaction following deep arterial wall injury was quantified with 51Cr labelled platelets and 111In labelled neutrophils, and the vasomotor response was determined angiographically.Platelet deposition at the site of deep arterial wall injury averaged 56.4±11.0×106 platelets cm−2 in the control group, and was significantly reduced to 18.2±3.8×106 platelets cm−2 (P<0.005) by treatment with MK-886. Neutrophil deposition was also decreased by MK-886, from 242.8±36.8 to 120.9±20.3×103 neutrophils cm−2 (P<0.01). MK-886-treated animals had a significant decrease in the postangioplasty vasoconstrictive response at the site of endothelial injury distally, from 37.5±3.1% in the control group to 13.5±2.5% (P<0.001).The effects of MK-886 were associated with a profound inhibition of ex vivo leukotriene B4 (LTB4) synthesis in blood stimulated by the calcium ionophore A23187 and a significant reduction of neutrophil aggregation in whole blood (P<0.01), whereas neutrophil superoxide anion production, serum thromboxane B2 and platelet aggregation in whole blood were not influenced.The relevant effects of MK-886 are primarily related to inhibition of neutrophil function and suggest an important modulatory role for leukotrienes in the

[Effect of protopine on rabbit platelet function].

PubMed

Ma, G Y; Zhang, Z Z; Chen, Z H

1994-07-01

Protopine (Pro) inhibited dose-dependently rabbit platelet aggregation induced by ADP, arachidonic acid (AA), collagen, or aggregoserpentin of Trimeresurus mucrosquamatus venom (TMVA) in vitro. Their IC50 were 25.3, 30.5, 46.9, 33.4 mumol.L-1, respectively. Pro 10, 20 mg.kg-1 iv also inhibited the platelet aggregation induced by these inducers. The effects (maximal at 5 min) lasted 1 h. By using fluorophotometry and RIA, it was seen that Pro suppressed the release of 5-HT from platelets during aggregation induced by collagen, AA, or TMVM in vitro. Pro did not block the formation of thromboxane A2 during aggregation induced by AA and did not increase the content of cAMP in rabbit platelet, but increased the content of cGMP in rabbit platelets. The antiplatelet effect of Pro may be related to an increase cGMP in rabbit platelets and the suppression of the release of the active substances from platelets.

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.

The Non-Hemostatic Aspects of Transfused Platelets

PubMed Central

Sut, Caroline; Tariket, Sofiane; Aubron, Cécile; Aloui, Chaker; Hamzeh-Cognasse, Hind; Berthelot, Philippe; Laradi, Sandrine; Greinacher, Andreas; Garraud, Olivier; Cognasse, Fabrice

2018-01-01

Platelets transfusion is a safe process, but during or after the process, the recipient may experience an adverse reaction and occasionally a serious adverse reaction (SAR). In this review, we focus on the inflammatory potential of platelet components (PCs) and their involvement in SARs. Recent evidence has highlighted a central role for platelets in the host inflammatory and immune responses. Blood platelets are involved in inflammation and various other aspects of innate immunity through the release of a plethora of immunomodulatory cytokines, chemokines, and associated molecules, collectively termed biological response modifiers that behave like ligands for endothelial and leukocyte receptors and for platelets themselves. The involvement of PCs in SARs—particularly on a critically ill patient’s context—could be related, at least in part, to the inflammatory functions of platelets, acquired during storage lesions. Moreover, we focus on causal link between platelet activation and immune-mediated disorders (transfusion-associated immunomodulation, platelets, polyanions, and bacterial defense and alloimmunization). This is linked to the platelets’ propensity to be activated even in the absence of deliberate stimuli and to the occurrence of time-dependent storage lesions. PMID:29536007

Immature platelet fraction in bacterial sepsis severity assessment

NASA Astrophysics Data System (ADS)

Djuang, M. H.; Ginting, F.; Hariman, H.

2018-03-01

Sepsis is an infection-induced syndrome, mostly caused by bacteria, of organ dysfunctions that caused by host response dysregulations. One of the simplest sepsis-indicator is platelet and its indexes. A new platelet parameter called immature platelet count (IPF) became theinterest in this study. The study aims to see whether IPF could assess sepsis severity by procalcitonin (PCT).Sixty-four of seventy-one patients with increased PCT were included in this cross-sectional study and separated into three groups based on their PCT levels. IPF showed no significance among the three groups (p-value>0.05) while platelet count was significant (p-value<0.05). Mean Platelet Volume (MPV) and Platelet Distribution Width (PDW) showed a strongpositive correlation with IPF. Higher sepsis severity based on PCT showed larger platelet count, as the result of platelet destructions caused by pro-inflammatory cytokines and endotoxins.

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

Differential Dynamics of Platelet Contact and Spreading

PubMed Central

Lee, Dooyoung; Fong, Karen P.; King, Michael R.; Brass, Lawrence F.; Hammer, Daniel A.

2012-01-01

Platelet spreading is critical for hemostatic plug formation and thrombosis. However, the detailed dynamics of platelet spreading as a function of receptor-ligand adhesive interactions has not been thoroughly investigated. Using reflection interference contrast microscopy, we found that both adhesive interactions and PAR4 activation affect the dynamics of platelet membrane contact formation during spreading. The initial growth of close contact area during spreading was controlled by the combination of different immobilized ligands or PAR4 activation on fibrinogen, whereas the growth of the total area of spreading was independent of adhesion type and PAR4 signaling. We found that filopodia extend to their maximal length and then contract over time; and that filopodial protrusion and expansion were affected by PAR4 signaling. Upon PAR4 activation, the integrin αIIbβ3 mediated close contact to fibrinogen substrata and led to the formation of ringlike patterns in the platelet contact zone. A systematic study of platelet spreading of GPVI-, α2-, or β3-deficient platelets on collagen or fibrinogen suggests the integrin α2 is indispensable for spreading on collagen. The platelet collagen receptors GPVI and α2 regulate integrin αIIbβ3-mediated platelet spreading on fibrinogen. This work elucidates quantitatively how receptor-ligand adhesion and biochemical signals synergistically control platelet spreading. PMID:22325269

Blood Platelets in the Progression of Alzheimer’s Disease

PubMed Central

Gowert, Nina S.; Donner, Lili; Chatterjee, Madhumita; Eisele, Yvonne S.; Towhid, Seyda T.; Münzer, Patrick; Walker, Britta; Ogorek, Isabella; Borst, Oliver; Grandoch, Maria; Schaller, Martin; Fischer, Jens W.; Gawaz, Meinrad; Weggen, Sascha; Lang, Florian; Jucker, Mathias; Elvers, Margitta

2014-01-01

Alzheimer’s disease (AD) is characterized by neurotoxic amyloid-ß plaque formation in brain parenchyma and cerebral blood vessels known as cerebral amyloid angiopathy (CAA). Besides CAA, AD is strongly related to vascular diseases such as stroke and atherosclerosis. Cerebrovascular dysfunction occurs in AD patients leading to alterations in blood flow that might play an important role in AD pathology with neuronal loss and memory deficits. Platelets are the major players in hemostasis and thrombosis, but are also involved in neuroinflammatory diseases like AD. For many years, platelets were accepted as peripheral model to study the pathophysiology of AD because platelets display the enzymatic activities to generate amyloid-ß (Aß) peptides. In addition, platelets are considered to be a biomarker for early diagnosis of AD. Effects of Aß peptides on platelets and the impact of platelets in the progression of AD remained, however, ill-defined. The present study explored the cellular mechanisms triggered by Aß in platelets. Treatment of platelets with Aß led to platelet activation and enhanced generation of reactive oxygen species (ROS) and membrane scrambling, suggesting enhanced platelet apoptosis. More important, platelets modulate soluble Aß into fibrillar structures that were absorbed by apoptotic but not vital platelets. This together with enhanced platelet adhesion under flow ex vivo and in vivo and platelet accumulation at amyloid deposits of cerebral vessels of AD transgenic mice suggested that platelets are major contributors of CAA inducing platelet thrombus formation at vascular amyloid plaques leading to vessel occlusion critical for cerebrovascular events like stroke. PMID:24587388

Platelet Transfusion Practices in Critically Ill Children.

PubMed

Nellis, Marianne E; Karam, Oliver; Mauer, Elizabeth; Cushing, Melissa M; Davis, Peter J; Steiner, Marie E; Tucci, Marisa; Stanworth, Simon J; Spinella, Philip C

2018-05-04

Little is known about platelet transfusions in pediatric critical illness. We sought to describe the epidemiology, indications, and outcomes of platelet transfusions among critically ill children. Prospective cohort study. Multicenter (82 PICUs), international (16 countries) from September 2016 to April 2017. Children ages 3 days to 16 years prescribed a platelet transfusion in the ICU during screening days. None. Over 6 weeks, 16,934 patients were eligible, and 559 received at least one platelet transfusion (prevalence, 3.3%). The indications for transfusion included prophylaxis (67%), minor bleeding (21%), and major bleeding (12%). Thirty-four percent of prophylactic platelet transfusions were prescribed when the platelet count was greater than or equal to 50 × 10 cells/L. The median (interquartile range) change in platelet count post transfusion was 48 × 10 cells/L (17-82 × 10 cells/L) for major bleeding, 42 × 10 cells/L (16-80 × 10 cells/L) for prophylactic transfusions to meet a defined threshold, 38 × 10 cells/L (17-72 × 10 cells/L) for minor bleeding, and 25 × 10 cells/L (10-47 × 10 cells/L) for prophylaxis in patients at risk of bleeding from a device. Overall ICU mortality was 25% but varied from 18% to 35% based on indication for transfusion. Upon adjusted analysis, total administered platelet dose was independently associated with increased ICU mortality (odds ratio for each additional 1 mL/kg platelets transfused, 1.002; 95% CI, 1.001-1.003; p = 0.005). The majority of platelet transfusions are given as prophylaxis to nonbleeding children, and significant variation in platelet thresholds exists. Studies are needed to clarify appropriate indications, with focus on prophylactic transfusions.

Metabolic response of granulocytes and platelets to synthetic vascular grafts: preliminary results with an in vitro technique.

PubMed

Pärsson, H; Nässberger, L; Thörne, J; Norgren, L

1995-04-01

The metabolism of granulocytes as well as platelets evoked by incubation with different synthetic vascular grafts was monitored during 6-h batch experiments using microcalorimetry. Standard knitted Dacron grafts, ePTFE-grafts, knitted Dacron grafts with collagen impregnation, and knitted Dacron grafts with external collagen-coating were used. The heat production per cell was calculated. A rapid increase of metabolic activity followed by a gradual decrease was demonstrated with both granulocyte suspension and platelet concentrate. Significant differences were obtained between the materials with a maximum response of Dacron grafts with collagen impregnation for both granulocyte and platelet response. The materials had different surface morphologies regarding cell adhesion after incubation as demonstrated with scanning electron microscopy with more pronounced adhesion on the collagen-impregnated grafts. The results suggest that microcalorimetry may be useful for the evaluation of cellular reactions on different biomaterials. However, further studies have to reveal the specificity of the reactions.

Concentration of platelets and growth factors in platelet-rich plasma from Goettingen minipigs.

PubMed

Jungbluth, Pascal; Grassmann, Jan-Peter; Thelen, Simon; Wild, Michael; Sager, Martin; Windolf, Joachim; Hakimi, Mohssen

2014-01-01

In minipigs little is known about the concentration of growth factors in plasma, despite their major role in several patho-physiological processes such as healing of fractures. This prompted us to study the concentration of platelets and selected growth factors in plasma and platelet-rich plasma (PRP) preparation of sixteen Goettingen minipigs. Platelet concentrations increased significantly in PRP in comparison to native blood plasma. Generally, significant increase in the concentration of all growth factors tested was observed in the PRP in comparison to the corresponding plasma or serum. Five of the plasma samples examined contained detectable levels of bone morphogenic protein 2 (BMP-2) whereas eleven of the plasma or serum samples contained minimal amounts of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF-bb) respectively. On the other hand variable concentrations of bone morphogenic protein 7 (BMP-7) and transforming growth factor β1 (TGF-β1) were measured in all plasma samples. In contrast, all PRP samples contained significantly increased amounts of growth factors. The level of BMP-2, BMP-7, TGF-β1, VEGF and PDGF-bb increased by 17.6, 1.5, 7.1, 7.2 and 103.3 fold, in comparison to the corresponding non-enriched preparations. Moreover significant positive correlations were found between platelet count and the concentrations of BMP-2 (r=0.62, p<0.001), TGF-β1 (r=0.85, p<0.001), VEGF (r=0.46, p<0.01) and PDGF-bb (r=0.9, p<0.001). Our results demonstrate that selected growth factors are present in the platelet-rich plasma of minipigs which might thus serve as a source of autologous growth factors.

Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation, platelet aggregation, and P-selectin expression

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

Nygaard, Gyrid; Department of Biomedicine, University of Bergen, Bergen; Herfindal, Lars

Highlights: • We investigated the impact of cyclic nucleotide analogues on platelet activation. • Different time dependence were found for inhibition of platelet activation. • Additive effect was found using PKA- and PKG-activating analogues. • Our results may explain some of the discrepancies reported for cNMP signalling. - Abstract: In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigatedmore » whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation.« less

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.

Pathogen-reduced platelets for the prevention of bleeding

PubMed Central

Estcourt, Lise J; Malouf, Reem; Hopewell, Sally; Trivella, Marialena; Doree, Carolyn; Stanworth, Simon J; Murphy, Michael F

2017-01-01

Background Platelet transfusions are used to prevent and treat bleeding in people who are thrombocytopenic. Despite improvements in donor screening and laboratory testing, a small risk of viral, bacterial, or protozoal contamination of platelets remains. There is also an ongoing risk from newly emerging blood transfusion-transmitted infections for which laboratory tests may not be available at the time of initial outbreak. One solution to reduce the risk of blood transfusion-transmitted infections from platelet transfusion is photochemical pathogen reduction, in which pathogens are either inactivated or significantly depleted in number, thereby reducing the chance of transmission. This process might offer additional benefits, including platelet shelf-life extension, and negate the requirement for gamma-irradiation of platelets. Although current pathogen-reduction technologies have been proven to reduce pathogen load in platelet concentrates, a number of published clinical studies have raised concerns about the effectiveness of pathogen-reduced platelets for post-transfusion platelet count recovery and the prevention of bleeding when compared with standard platelets. This is an update of a Cochrane review first published in 2013. Objectives To assess the effectiveness of pathogen-reduced platelets for the prevention of bleeding in people of any age requiring platelet transfusions. Search methods We searched for randomised controlled trials (RCTs) in the Cochrane Central Register of Controlled Trials (CENTRAL) (the Cochrane Library 2016, Issue 9), MEDLINE (from 1946), Embase (from 1974), CINAHL (from 1937), the Transfusion Evidence Library (from 1950), and ongoing trial databases to 24 October 2016. Selection criteria We included RCTs comparing the transfusion of pathogen-reduced platelets with standard platelets, or comparing different types of pathogen-reduced platelets. Data collection and analysis We used the standard methodological procedures expected by

Platelet bioreactor: accelerated evolution of design and manufacture.

PubMed

Thon, Jonathan N; Dykstra, Brad J; Beaulieu, Lea M

2017-07-01

Platelets, responsible for clot formation and blood vessel repair, are produced by megakaryocytes in the bone marrow. Platelets are critical for hemostasis and wound healing, and are often provided following surgery, chemotherapy, and major trauma. Despite their importance, platelets today are derived exclusively from human volunteer donors. They have a shelf life of just five days, making platelet shortages common during long weekends, civic holidays, bad weather, and during major emergencies when platelets are needed most. Megakaryocytes in the bone marrow generate platelets by extruding long cytoplasmic extensions called proplatelets through gaps/fenestrations in blood vessels. Proplatelets serve as assembly lines for platelet production by sequentially releasing platelets and large discoid-shaped platelet intermediates called preplatelets into the circulation. Recent advances in platelet bioreactor development have aimed to mimic the key physiological characteristics of bone marrow, including extracellular matrix composition/stiffness, blood vessel architecture comprising tissue-specific microvascular endothelium, and shear stress. Nevertheless, how complex interactions within three-dimensional (3D) microenvironments regulate thrombopoiesis remains poorly understood, and the technical challenges associated with designing and manufacturing biomimetic microfluidic devices are often under-appreciated and under-reported. We have previously reviewed the major cell culture, platelet quality assessment, and regulatory roadblocks that must be overcome to make human platelet production possible for clinical use [1]. This review builds on our previous manuscript by: (1) detailing the historical evolution of platelet bioreactor design to recapitulate native platelet production ex vivo, and (2) identifying the associated challenges that still need to be addressed to further scale and validate these devices for commercial application. While platelets are among the first

Opposing Effects of Platelet-Activating Factor and Lyso-Platelet-Activating Factor on Neutrophil and Platelet ActivationS⃞

PubMed Central

Welch, Emily J.; Naikawadi, Ram P.; Li, Zhenyu; Lin, Phoebe; Ishii, Satoshi; Shimizu, Takao; Tiruppathi, Chinnaswamy; Du, Xiaoping; Subbaiah, Papasani V.; Ye, Richard D.

2009-01-01

Platelet-activating factor (PAF) is a potent, bioactive phospholipid that acts on multiple cells and tissues through its G protein-coupled receptor (GPCR). PAF is not stored but is rapidly generated via enzymatic acetylation of the precursor 1-O-hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine (lysoPAF). The bioactivity of PAF is effectively and tightly regulated by PAF acetylhydrolases, which convert PAF back to lysoPAF. Previous studies report that lysoPAF is an inactive precursor and metabolite of PAF. However, lysoPAF has not been carefully studied in its own context. Here we report that lysoPAF has an opposing effect of PAF in the activation of neutrophils and platelets. Whereas PAF potentiates neutrophil NADPH oxidase activation, lysoPAF dose-dependently inhibits this function. Inhibition by lysoPAF is not affected by the use of a PAF receptor antagonist or genetic deletion of the PAF receptor gene. The mechanism of lysoPAF-mediated inhibition of neutrophils involves an elevation in the intracellular cAMP level, and pharmacological blockade of adenylyl cyclase completely reverses the inhibitory effect of lysoPAF. In addition, lysoPAF increases intracellular cAMP levels in platelets and inhibits thrombin-induced platelet aggregation, which can be reversed by inhibition of protein kinase A. These findings identify lysoPAF as a bioactive lipid with opposing functions of PAF and suggest a novel and intrinsic regulatory mechanism for balance of the potent activity of PAF. PMID:18931035

Radioisotope scanning in osseous sarcoidosis

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

Rohatgi, P.K.

1980-01-01

Technetium-99m (/sup 99m/Tc)-labeled pyrophosphate or diphosphonate compounds and gallium-67 citrate (/sup 67/Ga) are two radionuclide scanning agents that are in widespread use in clinical practice. Technetium-99m pyrophosphate is used extensively for bone scanning to detect metastatic bone disease, benign bone tumors, osteomyelitis, benign hypertrophic osteoarthropathy, and Paget's disease. Only two reports describe abnormal /sup 99m/Tc/ pyrophosphate bone scans in four patients with osseous sarcoidosis. Gallium-67 scans are used primarily to localize neoplastic or inflammatory lesions anywhere in the body. In recent years /sup 67/Ga scans have also been used to detect the presence of both pulmonary and extrapulmonary sarcoidosis, butmore » there are no reports describing abnormal uptake of gallium in patients with osseous sarcoidosis. This report describes experience with radioisotope scanning in two patients with osseous sarcoidosis.« less

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 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.

Platelet reactivity and mean platelet volume as risk markers of thrombogenesis in atrial fibrillation.

PubMed

Makowski, Marcin; Smorag, Ireneusz; Makowska, Joanna; Bissinger, Andrzej; Grycewicz, Tomasz; Paśnik, Jarek; Kidawa, Michal; Lubiński, Andrzej; Zielińska, Marzenna; Baj, Zbigniew

2017-05-15

Atrial fibrillation (AF) is associated with increased risk of thromboembolic complications. One of the markers of the increased risk of hypercoagulable state is platelet hyperreactivity. The aim of the study was to assess impact of arrhythmia on platelet reactivity. The study included 36 (mean age 48,3; range 21-60) male patients with lone atrial fibrillation, with exclusion of concomitant diseases known to trigger hypercoagulable state. The AF patients underwent cardioversion to restore sinus rhythm and were subsequently under observation for 1month. Echocardiography, ECG and blood collection was performed before cardioversion (T0) and 4weeks after successful cardioversion (T1). During the study period patients have been contacted and examined every week and 24h ECG monitoring was performed. Platelet reactivity was assessed based on changes of CD62 and CD42b expression on platelet surface after stimulation with thrombin. Also changes in MPV were assessed. In all patients sinus rhythm was maintained at the end of the study period, however in 14 patients recurrences of AF were observed, confirmed by 24h ECG monitoring (atrial fibrillation recurrence group - AFR) and 22 patients maintained sinus rhythm throughout the whole study period (SR group). Mean fluorescence intensity (MFI) of CD62 on thrombin stimulated platelets decreased significantly 4weeks after electrical cardioversion as compared to T0 (48.04±22.42 vs 41.47±16.03; p<0.01). Also MFI of CD42b on thrombin stimulated platelets decreased significantly 4weeks after electrical cardioversion as compared to T0 (22.16±10.82 vs 12.06±5.99; p<0.0001). Platelets reactivity estimated by CD 62 expression in SR group decreased significantly after 4weeks observation (58.01±15.26 vs 46.57±13.44; p<0.001) opposite to AFR group 35.66±21.87 vs 34.54±16.4; p-ns). Moreover there were significant differences between basal reactivity during AF between SR and AFR groups (58.01±15.26 vs 35.66±21.87; p-0.01). MFI of CD42

Nicergoline inhibits human platelet Ca2+ signalling through triggering a microtubule‐dependent reorganization of the platelet ultrastructure

PubMed Central

Walford, T; Musa, F I

2015-01-01

Background and Purpose Recently, we demonstrated that a pericellular Ca2+ recycling system potentiates agonist‐evoked Ca2+ signalling and granule secretion in human platelets and hypothesized a role for the membrane complex (MC) in orchestrating the accumulation of Ca2+ in the pericellular region. Previous work has demonstrated that treatment with high concentrations of nicergoline may disrupt the MC through an ability to trigger a re‐organization of the dense tubular system. Experiments were therefore performed to assess whether nicergoline‐induced changes in platelet ultrastructure affects thrombin‐evoked Ca2+ fluxes and dense granule secretion. Experimental Approach Thrombin‐evoked Ca2+ fluxes were monitored in Fura‐2‐ or Fluo‐5N‐loaded human platelets, or using platelet suspensions containing Fluo‐4 or Rhod‐5N K+ salts. Fluorescence microscopy was utilized to monitor microtubule structure and intracellular Ca2+ store distribution in TubulinTracker‐ and Fluo‐5N‐loaded platelets respectively. Dense granule secretion was monitored using luciferin–luciferase. Key Results Nicergoline treatment inhibited thrombin‐evoked Ca2+ signalling and induced alterations in the microtubule structure and the distribution of intracellular Ca2+ stores in platelets. Nicergoline altered the generation and spreading of thrombin‐induced pericellular Ca2+ signals and almost completely prevented dense granule secretion. Stabilization of microtubules using taxol reversed most effects of nicergoline on platelet Ca2+ signalling and partially reversed its effects on dense granule secretion. Conclusions and Implications Nicergoline‐induced alterations to platelet ultrastructure disrupt platelet Ca2+ signalling in a manner that would be predicted if the MC had been disrupted. These data suggest that nicergoline may be a useful prototype for the discovery of novel MC‐disrupting anti‐thrombotics. PMID:26450366

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.

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.

Effect of ticagrelor with clopidogrel on high on-treatment platelet reactivity in acute stroke or transient ischemic attack (PRINCE) trial: Rationale and design.

PubMed

Wang, Yilong; Lin, Yi; Meng, Xia; Chen, Weiqi; Chen, Guohua; Wang, Zhimin; Wu, Jialing; Wang, Dali; Li, Jianhua; Cao, Yibin; Xu, Yuming; Zhang, Guohua; Li, Xiaobo; Pan, Yuesong; Li, Hao; Liu, Liping; Zhao, Xingquan; Wang, Yongjun

2017-04-01

Rationale and aim Little is known about the safety and efficacy of the combination of ticagrelor and aspirin in acute ischemic stroke. This study aimed to evaluate whether the combination of ticagrelor and aspirin was superior to that of clopidogrel and aspirin in reducing the 90-day high on-treatment platelet reactivity for acute minor stroke or transient ischemic attack, especially for carriers of cytochrome P450 2C19 loss-of-function allele. Sample size and design This study was designed as a prospective, multicenter, randomized, open-label, active-controlled, and blind-endpoint, phase II b trial. The required sample size was 952 patients. It was registered with ClinicalTrials.gov (NCT02506140). Study outcomes The primary outcome was the proportion of patients with high on-treatment platelet reactivity at 90 days. High on-treatment platelet reactivity is defined as the P2Y12 reaction unit >208 measured using the VerifyNow P2Y12 assay. Conclusion The Platelet Reactivity in Acute Non-disabling Cerebrovascular Events study explored whether ticagrelor combined with aspirin could reduce further the proportion of patients with high on-treatment platelet reactivity at 90 days after acute minor stroke or transient ischemic attack compared with clopidogrel and aspirin.

Transformation and motility of human platelets: details of the shape change and release reaction observed by optical and electron microscopy

PubMed Central

1979-01-01

Blood platelets from 10 normal human subjects have been examined with a sensitive differential interference contrast (DIC) microscope. The entire transformation process during adhesion to glass is clearly visible and has been recorded cinematographically, including the disk to sphere change of shape, the formation of sessile protuberances, the extension and retraction of pseudopodia, and the spreading, ruffling, and occasional regression of the hyalomere. The exocytosis of intact dense bodies can be observed either by DIC microscopy, or by epifluorescence microscopy in platelets stained with mepacrine. Details of fluorescent flashes indicate that the dense bodies usually release their contents extracellularly, may do so intracytoplasmically under the influence of strong, short wavelength light on some preparations of mepacrine-stained platelets. The release of one or more dense bodies leaves a crater of variable size on the upper surface of the granulomere. Such craters represent the surface component of the open canalicular system and their formation and disappearance can be directly observed. Because these techniques permit quantitation of several parameters of motility which are not readily observable by other techniques, it is suggested that high extinction DIC microscope examination may become a rapid and useful method of studying congenital and acquired platelet disorders. Many features of platelet transformation have been confirmed and extended by scanning electron micrographs. These can in turn be interpreted by reference to time- lapse films of living platelets. PMID:511936

Determinants of ABH expression on human blood platelets.

PubMed

Cooling, Laura L W; Kelly, Kathleen; Barton, James; Hwang, Debbie; Koerner, Theodore A W; Olson, John D

2005-04-15

Platelets express ABH antigens, which can adversely effect platelet transfusion recovery and survival in ABH-incompatible recipients. To date, there has been no large, comprehensive study comparing specific donor factors with ABH expression on platelet membranes and glycoconjugates. We studied ABH expression in 166 group A apheresis platelet donors by flow cytometry, Western blotting, and thin layer chromatography relative to donor age, sex, A1/A2 subgroup, and Lewis phenotype. Overall, A antigen on platelet membranes, glycoproteins, and glycosphingolipids was linked to an A1 red blood cell (RBC) phenotype. Among A1 donors, platelet ABH varied significantly between donors (0%-87%). Intradonor variability, however, was minimal, suggesting that platelet ABH expression is a stable, donor-specific characteristic, with 5% of A1 donors typing as either ABH high- or low-expressers. Group A2 donors, in contrast, possessed a Bombay-like phenotype, lacking both A and H antigens. Unlike RBCs, ABH expression on platelets may be determined primarily by H-glycosyltransferase (FUT1) activity. Identification of A2 and A1 low expressers may increase the availability and selection of crossmatched and HLA-matched platelets. Platelets from group A2 may also be a superior product for patients undergoing A/O major mismatch allogeneic progenitor cell transplantation.

Antioxidants change platelet responses to various stimulating events

PubMed Central

Sobotková, Alžběta; Mášová-Chrastinová, Leona; Suttnar, Jiří; Štikarová, Jana; Májek, Pavel; Reicheltová, Zuzana; Kotlín, Roman; Weisel, John W.; Malý, Martin; Dyr, Jan E.

2010-01-01

The role of platelets in hemostasis may be influenced by alteration of the platelet redox state—the presence of antioxidants and the formation of reactive oxygen and nitrogen species. We investigated the effects of two antioxidants, resveratrol and trolox, on platelet activation. Trolox and resveratrol inhibited aggregation of washed platelets and platelet-rich plasma activated by ADP, collagen, and thrombin receptor-activating peptide. Resveratrol was a more effective agent in reducing platelet static and dynamic adhesion in comparison with trolox. The antioxidant capacity of resveratrol was, however, the same as that of trolox. After incubation of platelets with antioxidants, the resveratrol intraplatelet concentration was about five times lower than the intracellular concentration of trolox. Although both antioxidants comparably lowered hydroxyl radical and malondialdehyde production in platelets stimulated with collagen, TxB2 levels were decreased by resveratrol much more effectively than by trolox. Cyclooxygenase 1 was inhibited by resveratrol and not by trolox. Our data indicate that antioxidants, apart from nonspecific redox or radical-quenching mechanisms, inhibit platelet activation also by specific interaction with target proteins. The results also show the importance of studying platelet activation under conditions of real blood flow in contact with reactive surfaces, e.g., using dynamic adhesion experiments. PMID:19766712

Antioxidants change platelet responses to various stimulating events.

PubMed

Sobotková, Alzbeta; Másová-Chrastinová, Leona; Suttnar, Jirí; Stikarová, Jana; Májek, Pavel; Reicheltová, Zuzana; Kotlín, Roman; Weisel, John W; Malý, Martin; Dyr, Jan E

2009-12-15

The role of platelets in hemostasis may be influenced by alteration of the platelet redox state-the presence of antioxidants and the formation of reactive oxygen and nitrogen species. We investigated the effects of two antioxidants, resveratrol and trolox, on platelet activation. Trolox and resveratrol inhibited aggregation of washed platelets and platelet-rich plasma activated by ADP, collagen, and thrombin receptor-activating peptide. Resveratrol was a more effective agent in reducing platelet static and dynamic adhesion in comparison with trolox. The antioxidant capacity of resveratrol was, however, the same as that of trolox. After incubation of platelets with antioxidants, the resveratrol intraplatelet concentration was about five times lower than the intracellular concentration of trolox. Although both antioxidants comparably lowered hydroxyl radical and malondialdehyde production in platelets stimulated with collagen, TxB(2) levels were decreased by resveratrol much more effectively than by trolox. Cyclooxygenase 1 was inhibited by resveratrol and not by trolox. Our data indicate that antioxidants, apart from nonspecific redox or radical-quenching mechanisms, inhibit platelet activation also by specific interaction with target proteins. The results also show the importance of studying platelet activation under conditions of real blood flow in contact with reactive surfaces, e.g., using dynamic adhesion experiments.

Effect of BN 52021, a specific antagonist of platelet activating factor (PAF-acether), on calcium movements and phosphatidic acid production induced by PAF-acether in human platelets

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

Simon, M.F.; Chap, H.; Braquet, P.

1987-02-15

/sup 32/P-labelled human platelets loaded with quin 2 and pretreated with aspirin were stimulated with 1-100 nM platelet activating factor (PAF-acether or 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in a medium containing the ADP-scavenging system creatine phosphate/creatine phosphokinase. Under these conditions, PAF-acether evoked a characteristic fluorescence change allowing to quantify elevations in cytoplasmic free Ca/sup 2 +/ from internal stores (Ca/sup 2 +/ mobilization) or from external medium (Ca/sup 2 +/ influx), as well as an increased production of phosphatidic acid, reflecting phospholipase C activation. These effects, which can be attributed to PAF-acether only and not to released products such as ADP or thromboxane A2,more » were strongly inhibited in a dose-dependent manner by BN 52021, a specific antagonist of PAF-acether isolated from Ginkgo biloba. As the drug remained inactive against the same effects elicited by thrombin, it is concluded that BN 52021 does not interfere directly with the mechanism of transmembrane signalling involving inositol-phospholipids or (and) some putative receptor-operated channels, but rather acts on the binding of PAF-acether to its presumed membrane receptor.« less

Rapid-scan EPR of immobilized nitroxides

NASA Astrophysics Data System (ADS)

Yu, Zhelin; Quine, Richard W.; Rinard, George A.; Tseitlin, Mark; Elajaili, Hanan; Kathirvelu, Velavan; Clouston, Laura J.; Boratyński, Przemysław J.; Rajca, Andrzej; Stein, Richard; Mchaourab, Hassane; Eaton, Sandra S.; Eaton, Gareth R.

2014-10-01

X-band electron paramagnetic resonance spectra of immobilized nitroxides were obtained by rapid scan at 293 K. Scan widths were 155 G with 13.4 kHz scan frequency for 14N-perdeuterated tempone and for T4 lysozyme doubly spin labeled with an iodoacetamide spirocyclohexyl nitroxide and 100 G with 20.9 kHz scan frequency for 15N-perdeuterated tempone. These wide scans were made possible by modifications to our rapid-scan driver, scan coils made of Litz wire, and the placement of highly conducting aluminum plates on the poles of a Bruker 10″ magnet to reduce resistive losses in the magnet pole faces. For the same data acquisition time, the signal-to-noise for the rapid-scan absorption spectra was about an order of magnitude higher than for continuous wave first-derivative spectra recorded with modulation amplitudes that do not broaden the lineshapes.

In vitro platelet activation, aggregation and platelet-granulocyte complex formation induced by surface modified single-walled carbon nanotubes.

PubMed

Fent, János; Bihari, Péter; Vippola, Minnamari; Sarlin, Essi; Lakatos, Susan

2015-08-01

Surface modification of single-walled carbon nanotubes (SWCNTs) such as carboxylation, amidation, hydroxylation and pegylation is used to reduce the nanotube toxicity and render them more suitable for biomedical applications than their pristine counterparts. Toxicity can be manifested in platelet activation as it has been shown for SWCNTs. However, the effect of various surface modifications on the platelet activating potential of SWCNTs has not been tested yet. In vitro platelet activation (CD62P) as well as the platelet-granulocyte complex formation (CD15/CD41 double positivity) in human whole blood were measured by flow cytometry in the presence of 0.1mg/ml of pristine or various surface modified SWCNTs. The effect of various SWCNTs was tested by whole blood impedance aggregometry, too. All tested SWCNTs but the hydroxylated ones activate platelets and promote platelet-granulocyte complex formation in vitro. Carboxylated, pegylated and pristine SWCNTs induce whole blood aggregation as well. Although pegylation is preferred from biomedical point of view, among the samples tested by us pegylated SWCNTs induced far the most prominent activation and a well detectable aggregation of platelets in whole blood. Copyright © 2015 Elsevier Ltd. All rights reserved.

Platelet interactions in thrombosis.

PubMed

Andrews, Robert K; Gardiner, Elizabeth E; Shen, Yang; Berndt, Michael C

2004-01-01

Patho/physiological platelet aggregate (thrombus) formation is initiated by engagement of platelet surface receptors, glycoprotein (GP)Ib-IX-V and GPVI that bind von Willebrand factor or collagen. Although beneficial in response to vascular injury by preventing blood loss (haemostasis), platelet aggregation in a sclerotic coronary artery or other diseased blood vessel (thrombosis) can cause thrombotic diseases like heart attack and stroke. At the molecular level, ligand interactions with GPIb-IX-V or GPVI trigger signalling responses, including elevation of cytosolic Ca2+, dissociation of calmodulin from their cytoplasmic domains, cytoskeletal actin-filament rearrangements, activation of src-family kinases or PI 3-kinase, and 'inside-out' activation of the integrin, alphaIIbbeta3 (GPIIb-llla), that binds von Willebrand factor or fibrinogen and mediates platelet aggregation. Furthermore, emerging evidence supports a topographical co-association of these receptors of the leucine-rich repeat family (GPIb-IX-V) and immunoglobulin superfamily (GPVI) in an adhesive cluster or 'adhesosome'. This arrangement may underlie common mechanisms of initiating thrombus formation in haemostasis or thrombotic disease.

Identification of platelet refractoriness in oncohematologic patients

PubMed Central

Ferreira, Aline Aparecida; Zulli, Roberto; Soares, Sheila; de Castro, Vagner; Moraes-Souza, Helio

2011-01-01

OBJECTIVES: To identify the occurrence and the causes of platelet refractoriness in oncohematologic patients. INTRODUCTION: Platelet refractoriness (unsatisfactory post-transfusion platelet increment) is a severe problem that impairs the treatment of oncohematologic patients and is not routinely investigated in most Brazilian services. METHODS: Forty-four episodes of platelet concentrate transfusion were evaluated in 16 patients according to the following parameters: corrected count increment, clinical conditions and detection of anti-platelet antibodies by the platelet immunofluorescence test (PIFT) and panel reactive antibodies against human leukocyte antigen class I (PRA-HLA). RESULTS: Of the 16 patients evaluated (median age: 53 years), nine (56%) were women, seven of them with a history of pregnancy. An unsatisfactory increment was observed in 43% of the transfusion events, being more frequent in transfusions of random platelet concentrates (54%). Platelet refractoriness was confirmed in three patients (19%), who presented immunologic and non-immunologic causes. Alloantibodies were identified in eight patients (50%) by the PIFT and in three (19%) by the PRA-HLA. Among alloimmunized patients, nine (64%) had a history of transfusion, and three as a result of pregnancy (43%). Of the former, two were refractory (29%). No significant differences were observed, probably as a result of the small sample size. CONCLUSION: The high rate of unsatisfactory platelet increment, refractoriness and alloimmunization observed support the need to set up protocols for the investigation of this complication in all chronically transfused patients, a fundamental requirement for the guarantee of adequate management. PMID:21437433

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.

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.

21 CFR 864.5700 - Automated platelet aggregation system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... addition of an aggregating reagent to a platelet-rich plasma. (b) Classification. Class II (performance... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Automated platelet aggregation system. 864.5700... § 864.5700 Automated platelet aggregation system. (a) Identification. An automated platelet aggregation...

21 CFR 864.5700 - Automated platelet aggregation system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... addition of an aggregating reagent to a platelet-rich plasma. (b) Classification. Class II (performance... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Automated platelet aggregation system. 864.5700... § 864.5700 Automated platelet aggregation system. (a) Identification. An automated platelet aggregation...

21 CFR 864.5700 - Automated platelet aggregation system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... addition of an aggregating reagent to a platelet-rich plasma. (b) Classification. Class II (performance... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Automated platelet aggregation system. 864.5700... § 864.5700 Automated platelet aggregation system. (a) Identification. An automated platelet aggregation...

21 CFR 864.5700 - Automated platelet aggregation system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... addition of an aggregating reagent to a platelet-rich plasma. (b) Classification. Class II (performance... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Automated platelet aggregation system. 864.5700... § 864.5700 Automated platelet aggregation system. (a) Identification. An automated platelet aggregation...

21 CFR 864.5700 - Automated platelet aggregation system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... addition of an aggregating reagent to a platelet-rich plasma. (b) Classification. Class II (performance... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Automated platelet aggregation system. 864.5700... § 864.5700 Automated platelet aggregation system. (a) Identification. An automated platelet aggregation...

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.

Improving platelet transfusion safety: biomedical and technical considerations

PubMed Central

Garraud, Olivier; Cognasse, Fabrice; Tissot, Jean-Daniel; Chavarin, Patricia; Laperche, Syria; Morel, Pascal; Lefrère, Jean-Jacques; Pozzetto, Bruno; Lozano, Miguel; Blumberg, Neil; Osselaer, Jean-Claude

2016-01-01

Platelet concentrates account for near 10% of all labile blood components but are responsible for more than 25% of the reported adverse events. Besides factors related to patients themselves, who may be particularly at risk of side effects because of their underlying illness, there are aspects of platelet collection and storage that predispose to adverse events. Platelets for transfusion are strongly activated by collection through disposal equipment, which can stress the cells, and by preservation at 22 °C with rotation or rocking, which likewise leads to platelet activation, perhaps more so than storage at 4 °C. Lastly, platelets constitutively possess a very large number of bioactive components that may elicit pro-inflammatory reactions when infused into a patient. This review aims to describe approaches that may be crucial to minimising side effects while optimising safety and quality. We suggest that platelet transfusion is complex, in part because of the complexity of the “material” itself: platelets are highly versatile cells and the transfusion process adds a myriad of variables that present many challenges for preserving basal platelet function and preventing dysfunctional activation of the platelets. The review also presents information showing - after years of exhaustive haemovigilance - that whole blood buffy coat pooled platelet components are extremely safe compared to the gold standard (i.e. apheresis platelet components), both in terms of acquired infections and of immunological/inflammatory hazards. PMID:26674828

Thrombin-activated human platelets acutely generate oxidized docosahexaenoic-acid-containing phospholipids via 12-lipoxygenase.

PubMed

Morgan, Lloyd T; Thomas, Christopher P; Kühn, Hartmut; O'Donnell, Valerie B

2010-10-01

Arachidonate-containing oxidized phospholipids are acutely generated by 12-LOX (12-lipoxygenase) in agonist-activated platelets. In the present study, formation of structurally related lipids by oxidation of DHA (docosahexaenoic acid)-containing phospholipids is demonstrated using lipidomic approaches. Precursor scanning reverse-phase LC (liquid chromatography)-MS/MS (tandem MS) identified a new family of lipids that comprise phospholipid-esterified HDOHE (hydroxydocosahexaenoic acid). Two diacyl and two plasmalogen PEs (phosphatidylethanolamines) containing predominantly the 14-HDOHE positional isomer (18:0p/14-HDOHE-PE, 18:0a/14-HDOHE-PE, 16:0a/14-HDOHE-PE and 16:0p/14-HDOHE-PE) were structurally characterized using MS/MS and by comparison with biogenic standards. An involvement of 12-LOX was indicated as purified recombinant human 12-LOX also generated the 14-HDOHE isomer from DHA. Pharmacological studies using inhibitors and recombinant platelet 12-LOX indicate that they form via esterification of newly formed non-esterified HDOHE. HDOHE-PEs formed at significant rates (2-4 ng/4×10(7) cells) within 2-180 min of thrombin stimulation, and their formation was blocked by calcium chelation. In summary, a new family of oxidized phospholipid was identified in thrombin-activated human platelets.

Heparin-associated thrombocytopenia: antibody binding specificity to platelet antigens.

PubMed

Lynch, D M; Howe, S E

1985-11-01

Sera from four patients with heparin-associated thrombocytopenia (HAT) were evaluated by a quantitative enzyme-linked immunosorbent assay (ELISA) to detect heparin-dependent serum platelet-bindable immunoglobulin (S-PBIg) and by Western blotting and immunoprecipitation to investigate the specificity of the antibody binding. All HAT sera showed mildly increased S-PBIg (mean, 7.8 fg per platelet; normal, less than 6.0 fg per platelet) to intact target platelets in the ELISA, which was markedly increased in the presence of heparin (mean, 20.9 fg per platelet). This increase was 20-fold greater than normal control sera, which showed a mean differential increase of only 0.5 fg per platelet. Immunoglobulin binding specificity to platelet antigens was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of platelet lysate with transfer of the platelet fractions onto nitrocellulose strips (Western blotting) and subsequent immunoassay using HAT and normal sera. In the presence of heparin, the four HAT patients demonstrated increased binding of immunoglobulin to platelet antigens of apparent molecular weights of 180, 124, and 82 kd. Radiolabeled heparin when incubated with HAT sera, normal sera, or albumin blanks bound to platelet proteins of the same apparent molecular weights. These observations are consistent with current hypotheses suggesting that HAT antibody is directed to heparin-platelet complexes or, alternatively, that heparin induces conformational change of antigenic sites on the platelet membrane.

Evaluating platelet aggregation dynamics from laser speckle fluctuations.

PubMed

Hajjarian, Zeinab; Tshikudi, Diane M; Nadkarni, Seemantini K

2017-07-01

Platelets are key to maintaining hemostasis and impaired platelet aggregation could lead to hemorrhage or thrombosis. We report a new approach that exploits laser speckle intensity fluctuations, emanated from a drop of platelet-rich-plasma (PRP), to profile aggregation. Speckle fluctuation rate is quantified by the speckle intensity autocorrelation, g 2 (t) , from which the aggregate size is deduced. We first apply this approach to evaluate polystyrene bead aggregation, triggered by salt. Next, we assess dose-dependent platelet aggregation and inhibition in human PRP spiked with adenosine diphosphate and clopidogrel. Additional spatio-temporal speckle analyses yield 2-dimensional maps of particle displacements to visualize platelet aggregate foci within minutes and quantify aggregation dynamics. These findings demonstrate the unique opportunity for assessing platelet health within minutes for diagnosing bleeding disorders and monitoring anti-platelet therapies.

A System for Anesthesia Drug Administration Using Barcode Technology: The Codonics Safe Label System and Smart Anesthesia Manager.

PubMed

Jelacic, Srdjan; Bowdle, Andrew; Nair, Bala G; Kusulos, Dolly; Bower, Lynnette; Togashi, Kei

2015-08-01

Many anesthetic drug errors result from vial or syringe swaps. Scanning the barcodes on vials before drug preparation, creating syringe labels that include barcodes, and scanning the syringe label barcodes before drug administration may help to prevent errors. In contrast, making syringe labels by hand that comply with the recommendations of regulatory agencies and standards-setting bodies is tedious and time consuming. A computerized system that uses vial barcodes and generates barcoded syringe labels could address both safety issues and labeling recommendations. We measured compliance of syringe labels in multiple operating rooms (ORs) with the recommendations of regulatory agencies and standards-setting bodies before and after the introduction of the Codonics Safe Label System (SLS). The Codonics SLS was then combined with Smart Anesthesia Manager software to create an anesthesia barcode drug administration system, which allowed us to measure the rate of scanning syringe label barcodes at the time of drug administration in 2 cardiothoracic ORs before and after introducing a coffee card incentive. Twelve attending cardiothoracic anesthesiologists and the OR satellite pharmacy participated. The use of the Codonics SLS drug labeling system resulted in >75% compliant syringe labels (95% confidence interval, 75%-98%). All syringe labels made using the Codonics SLS system were compliant. The average rate of scanning barcodes on syringe labels using Smart Anesthesia Manager was 25% (730 of 2976) over 13 weeks but increased to 58% (956 of 1645) over 8 weeks after introduction of a simple (coffee card) incentive (P < 0.001). An anesthesia barcode drug administration system resulted in a moderate rate of scanning syringe label barcodes at the time of drug administration. Further, adaptation of the system will be required to achieve a higher utilization rate.

Platelet activation suppresses HIV-1 infection of T cells

PubMed Central

2013-01-01

Background Platelets, anucleate cell fragments abundant in human blood, can capture HIV-1 and platelet counts have been associated with viral load and disease progression. However, the impact of platelets on HIV-1 infection of T cells is unclear. Results We found that platelets suppress HIV-1 spread in co-cultured T cells in a concentration-dependent manner. Platelets containing granules inhibited HIV-1 spread in T cells more efficiently than degranulated platelets, indicating that the granule content might exert antiviral activity. Indeed, supernatants from activated and thus degranulated platelets suppressed HIV-1 infection. Infection was inhibited at the stage of host cell entry and inhibition was independent of the viral strain or coreceptor tropism. In contrast, blockade of HIV-2 and SIV entry was less efficient. The chemokine CXCL4, a major component of platelet granules, blocked HIV-1 entry and neutralization of CXCL4 in platelet supernatants largely abrogated their anti-HIV-1 activity. Conclusions Release of CXCL4 by activated platelets inhibits HIV-1 infection of adjacent T cells at the stage of virus entry. The inhibitory activity of platelet-derived CXCL4 suggests a role of platelets in the defense against infection by HIV-1 and potentially other pathogens. PMID:23634812

Platelet activation suppresses HIV-1 infection of T cells.

PubMed

Solomon Tsegaye, Theodros; Gnirß, Kerstin; Rahe-Meyer, Niels; Kiene, Miriam; Krämer-Kühl, Annika; Behrens, Georg; Münch, Jan; Pöhlmann, Stefan

2013-05-01

Platelets, anucleate cell fragments abundant in human blood, can capture HIV-1 and platelet counts have been associated with viral load and disease progression. However, the impact of platelets on HIV-1 infection of T cells is unclear. We found that platelets suppress HIV-1 spread in co-cultured T cells in a concentration-dependent manner. Platelets containing granules inhibited HIV-1 spread in T cells more efficiently than degranulated platelets, indicating that the granule content might exert antiviral activity. Indeed, supernatants from activated and thus degranulated platelets suppressed HIV-1 infection. Infection was inhibited at the stage of host cell entry and inhibition was independent of the viral strain or coreceptor tropism. In contrast, blockade of HIV-2 and SIV entry was less efficient. The chemokine CXCL4, a major component of platelet granules, blocked HIV-1 entry and neutralization of CXCL4 in platelet supernatants largely abrogated their anti-HIV-1 activity. Release of CXCL4 by activated platelets inhibits HIV-1 infection of adjacent T cells at the stage of virus entry. The inhibitory activity of platelet-derived CXCL4 suggests a role of platelets in the defense against infection by HIV-1 and potentially other pathogens.

Measurement of brain perfusion in newborns: Pulsed arterial spin labeling (PASL) versus pseudo-continuous arterial spin labeling (pCASL)

PubMed Central

Boudes, Elodie; Gilbert, Guillaume; Leppert, Ilana Ruth; Tan, Xianming; Pike, G. Bruce; Saint-Martin, Christine; Wintermark, Pia

2014-01-01

Background Arterial spin labeling (ASL) perfusion-weighted imaging (PWI) by magnetic resonance imaging (MRI) has been shown to be useful for identifying asphyxiated newborns at risk of developing brain injury, whether or not therapeutic hypothermia was administered. However, this technique has been only rarely used in newborns until now, because of the challenges to obtain sufficient signal-to-noise ratio (SNR) and spatial resolution in newborns. Objective To compare two methods of ASL-PWI (i.e., single inversion-time pulsed arterial spin labeling [single TI PASL], and pseudo-continuous arterial spin labeling [pCASL]) to assess brain perfusion in asphyxiated newborns treated with therapeutic hypothermia and in healthy newborns. Design/methods We conducted a prospective cohort study of term asphyxiated newborns meeting the criteria for therapeutic hypothermia; four additional healthy term newborns were also included as controls. Each of the enrolled newborns was scanned at least once during the first month of life. Each MRI scan included conventional anatomical imaging, as well as PASL and pCASL PWI-MRI. Control and labeled images were registered separately to reduce the effect of motion artifacts. For each scan, the axial slice at the level of the basal ganglia was used for comparisons. Each scan was scored for its image quality. Quantification of whole-slice cerebral blood flow (CBF) was done afterwards using previously described formulas. Results A total number of 61 concomitant PASL and pCASL scans were obtained in nineteen asphyxiated newborns treated with therapeutic hypothermia and four healthy newborns. After discarding the scans with very poor image quality, 75% (46/61) remained for comparison between the two ASL methods. pCASL images presented a significantly superior image quality score compared to PASL images (p < 0.0001). Strong correlation was found between the CBF measured by PASL and pCASL (r = 0.61, p < 0.0001). Conclusion This study

Cystamine immobilization on TiO 2 film surfaces and the influence on inhibition of collagen-induced platelet activation

NASA Astrophysics Data System (ADS)

Zhou, Yujuan; Weng, Yajun; Zhang, Liping; Jing, Fengjuan; Huang, Nan; Chen, Junying

2011-12-01

Poor haemocompatibility is a main issue of artificial cardiovascular materials in clinical application. Nitric oxide (NO), produced by vascular endothelial cells, is a well known inhibitor of platelet adhesion and activation. Thus, NO-releasing biomaterials are beneficial for improving haemocompatibility of blood-contacting biomedical devices. In this paper, a novel method was developed for enhancement of haemocompatibility by exploiting endogenous NO donors. TiO 2 films were firstly synthesized on Si (1 0 0) wafers via unbalanced magnetron sputtering technology, and then polydopamine was grafted on TiO 2 films and used as a linker for further immobilization of cystamine. The obtained surfaces were characterized by scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) analysis. NO generation is evaluated by saville-griess reagents, and it shows that cystamine immobilized samples are able to catalytically generate NO by decomposing endogenous S-nitrosothiols (RSNO). In vitro platelet adhesion results reveal that cystamine modified surfaces can inhibit collagen-induced platelet activation. ELISA analysis reveals that cGMP in platelets obviously increases on cystamine immobilized surface, which suggests the reducing of platelet activation is through NO/cGMP signal channel. It can be concluded that cystamine immobilized surface shows better blood compatibility by catalyzing NO release from the endogenous NO donor. It may be a promising method for improvement of haemocompatibility of blood-contacting implants.

Scott Murphy, MD: platelet storage pioneer.

PubMed

Vassallo, Ralph

2011-04-01

Dr Scott Murphy was a leading authority in the science of platelet storage for transfusion. Among the first to advocate room-temperature storage, his work enabled the extension of platelet shelf life, which facilitated the development of aggressive medical and surgical therapies requiring intensive platelet support. During a 38-year career, he characterized the metabolism of stored platelets, worked to optimize storage conditions, and developed a landmark standard for the assessment of platelet quality. As a clinical hematologist, he was devoted to his patients, providing compassionate, comforting care. He was also a cherished mentor, a sage advisor, a fast friend, and a dedicated family man. Dr Murphy was one of those rare, great, humble men who inspire and enrich everyone around him. Truly one of transfusion medicine's leaders, Scott is remembered by those whose lives he touched for his warmth, wit, and wisdom. Copyright © 2011 Elsevier Inc. All rights reserved.

Ristocetin induces phosphorylated-HSP27 (HSPB1) release from the platelets of type 2 DM patients: Anti-platelet agent-effect on the release.

PubMed

Tokuda, Haruhiko; Kuroyanagi, Gen; Onuma, Takashi; Enomoto, Yukiko; Doi, Tomoaki; Iida, Hiroki; Otsuka, Takanobu; Ogura, Shinji; Iwama, Toru; Kojima, Kumi; Kozawa, Osamu

2018-04-01

It has been previously reported that HSP27 is released from platelets in type 2 diabetes mellitus (DM) patients according to phosphorylation. In the present study, we investigated the effect of ristocetin, a glycoprotein (GP)Ib/IX/V activator, on the release of HSP27 and the effect of anti-platelet agents, such as acetylsalicylic acid (ASA), on this release. Forty-six patients with type 2 DM were recruited, and classified into two groups depending on administration of anti-platelet agents, resulting in 31 patients without these agents (control group) and 15 patients with them (anti-platelet group). Ristocetin potently induced the aggregation of platelets in the two groups. Ristocetin-induced changes of the area under the curve of light transmittance and the ratio of the size of platelet aggregates in the anti-platelet group were slightly different from those in the control group. On the other hand, the levels of phosphorylated-HSP27 induced by ristocetin in the platelets from a patient of the anti-platelet group taking ASA were significantly lower than those from a patient of the control group. The levels of HSP27 release from the ristocetin-stimulated platelets were significantly correlated with the levels of phosphorylated-HSP27 in the platelets from patients in the two groups. The levels of HSP27 release and those of platelet-derived growth factor-AB (PDGF-AB) secretion stimulated by ristocetin in the anti-platelet group were lower than those in the control group. In addition, the levels of HSP27 release and those of PDGF-AB secretion stimulated by ADP in the anti-platelet group were lower than those in the control group. These results strongly suggest that anti-platelet agents inhibit the HSP27 release from platelets stimulated by ristocetin but not the aggregation in type 2 DM patients.

Thrombopoietin contributes to enhanced platelet activation in cigarette smokers.

PubMed

Lupia, Enrico; Bosco, Ornella; Goffi, Alberto; Poletto, Cesare; Locatelli, Stefania; Spatola, Tiziana; Cuccurullo, Alessandra; Montrucchio, Giuseppe

2010-05-01

Thrombopoietin (TPO) is a humoral growth factor that primes platelet activation in response to several agonists. We recently showed that TPO enhances platelet activation in unstable angina and sepsis. Aim of this study was to investigate the role of TPO in platelet function abnormalities described in cigarette smokers. In a case-control study we enrolled 20 healthy cigarette smokers and 20 nonsmokers, and measured TPO and C-reactive protein (CRP), as well as platelet-leukocyte binding and P-selectin expression. In vitro we evaluated the priming activity of smoker or control plasma on platelet activation, and the role of TPO in this effect. We then studied the effects of acute smoking and smoking cessation on TPO levels and platelet activation indices. Chronic cigarette smokers had higher circulating TPO levels than nonsmoking controls, as well as increased platelet-leukocyte binding, P-selectin expression, and CRP levels. Serum cotinine concentrations correlated with TPO concentrations, platelet-monocyte aggregates and P-selectin expression. In addition, TPO levels significantly correlated with ex vivo platelet-monocyte aggregation and P-selectin expression. In vitro, the plasma from cigarette smokers, but not from nonsmoking controls, primed platelet-monocyte binding, which was reduced when an inhibitor of TPO was used. We also found that acute smoking slightly increased TPO levels, but did not affect platelet-leukocyte binding, whereas smoking cessation induced a significant decrease in both circulating TPO and platelet-leukocyte aggregation. Elevated TPO contributes to enhance platelet activation and platelet-monocyte cross-talk in cigarette smokers. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

Equid Herpesvirus Type 1 Activates Platelets

PubMed Central

Stokol, Tracy; Yeo, Wee Ming; Burnett, Deborah; DeAngelis, Nicole; Huang, Teng; Osterrieder, Nikolaus; Catalfamo, James

2015-01-01

Equid herpesvirus type 1 (EHV-1) causes outbreaks of abortion and neurological disease in horses. One of the main causes of these clinical syndromes is thrombosis in placental and spinal cord vessels, however the mechanism for thrombus formation is unknown. Platelets form part of the thrombus and amplify and propagate thrombin generation. Here, we tested the hypothesis that EHV-1 activates platelets. We found that two EHV-1 strains, RacL11 and Ab4 at 0.5 or higher plaque forming unit/cell, activate platelets within 10 minutes, causing α-granule secretion (surface P-selectin expression) and platelet microvesiculation (increased small events double positive for CD41 and Annexin V). Microvesiculation was more pronounced with the RacL11 strain. Virus-induced P-selectin expression required plasma and 1.0 mM exogenous calcium. P-selectin expression was abolished and microvesiculation was significantly reduced in factor VII- or X-deficient human plasma. Both P-selectin expression and microvesiculation were re-established in factor VII-deficient human plasma with added purified human factor VIIa (1 nM). A glycoprotein C-deficient mutant of the Ab4 strain activated platelets as effectively as non-mutated Ab4. P-selectin expression was abolished and microvesiculation was significantly reduced by preincubation of virus with a goat polyclonal anti-rabbit tissue factor antibody. Infectious virus could be retrieved from washed EHV-1-exposed platelets, suggesting a direct platelet-virus interaction. Our results indicate that EHV-1 activates equine platelets and that α-granule secretion is a consequence of virus-associated tissue factor triggering factor X activation and thrombin generation. Microvesiculation was only partly tissue factor and thrombin-dependent, suggesting the virus causes microvesiculation through other mechanisms, potentially through direct binding. These findings suggest that EHV-1-induced platelet activation could contribute to the thrombosis that occurs in

Platelets in liver disease, cancer and regeneration.

PubMed

Kurokawa, Tomohiro; Ohkohchi, Nobuhiro

2017-05-14

Although viral hepatitis treatments have evolved over the years, the resultant liver cirrhosis still does not completely heal. Platelets contain proteins required for hemostasis, as well as many growth factors required for organ development, tissue regeneration and repair. Thrombocytopenia, which is frequently observed in patients with chronic liver disease (CLD) and cirrhosis, can manifest from decreased thrombopoietin production and accelerated platelet destruction caused by hypersplenism; however, the relationship between thrombocytopenia and hepatic pathogenesis, as well as the role of platelets in CLD, is poorly understood. In this paper, experimental evidence of platelets improving liver fibrosis and accelerating liver regeneration is summarized and addressed based on studies conducted in our laboratory and current progress reports from other investigators. In addition, we describe our current perspective based on the results of these studies. Platelets improve liver fibrosis by inactivating hepatic stellate cells, which decreases collagen production. The regenerative effect of platelets in the liver involves a direct effect on hepatocytes, a cooperative effect with liver sinusoidal endothelial cells, and a collaborative effect with Kupffer cells. Based on these observations, we ascertained the direct effect of platelet transfusion on improving several indicators of liver function in patients with CLD and liver cirrhosis. However, unlike the results of our previous clinical study, the smaller incremental changes in liver function in patients with CLD who received eltrombopag for 6 mo were due to patient selection from a heterogeneous population. We highlight the current knowledge concerning the role of platelets in CLD and cancer and anticipate a novel application of platelet-based clinical therapies to treat liver disease.

Influence of mental stress on platelet bioactivity

PubMed Central

Koudouovoh-Tripp, Pia; Sperner-Unterweger, Barbara

2012-01-01

It is well established that various mental stress conditions contribute, or at least influence, underlying pathophysiological mechanisms in somatic, as well as in psychiatric disorders; blood platelets are supposed to represent a possible link in this respect. The anculeated platelets are the smallest corpuscular elements circulating in the human blood. They display different serotonergic markers which seem to reflect the central nervous serotonin metabolism. They are known as main effectors in haematological processes but recent research highlights their role in the innate and adaptive immune system. Platelets are containing a multitude of pro-inflammatory and immune-modulatory bioactive compounds in their granules and are expressing immune-competent surface markers. Research gives hint that platelets activation and reactivity is increased by mental stress. This leads to enhanced cross talk with the immune system via paracrine secretion, receptor interaction and formation of platelet leucocyte-aggregates. Recently it has been demonstrated that the immune system can have a remarkable impact in the development of psychiatric disorders. Therefore platelets represent an interesting research area in psychiatry and their role as a possible biomarker has been investigated. We review the influence of mental stress on what is termed platelet bioactivity in this article, which subsumes the mainly immune-modulatory activity of platelets in healthy volunteers, elderly persons with chronic care-giving strain, patients with cardiovascular diseases who are prone to psychosocial stress, as well as in patients with posttraumatic stress disorder. Research data suggest that stress enhances platelet activity, reactivity and immune-modulatory capacities. PMID:24175179