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Sample records for platelet function assay

  1. Normalization methods in time series of platelet function assays

    PubMed Central

    Van Poucke, Sven; Zhang, Zhongheng; Roest, Mark; Vukicevic, Milan; Beran, Maud; Lauwereins, Bart; Zheng, Ming-Hua; Henskens, Yvonne; Lancé, Marcus; Marcus, Abraham

    2016-01-01

    Abstract Platelet function can be quantitatively assessed by specific assays such as light-transmission aggregometry, multiple-electrode aggregometry measuring the response to adenosine diphosphate (ADP), arachidonic acid, collagen, and thrombin-receptor activating peptide and viscoelastic tests such as rotational thromboelastometry (ROTEM). The task of extracting meaningful statistical and clinical information from high-dimensional data spaces in temporal multivariate clinical data represented in multivariate time series is complex. Building insightful visualizations for multivariate time series demands adequate usage of normalization techniques. In this article, various methods for data normalization (z-transformation, range transformation, proportion transformation, and interquartile range) are presented and visualized discussing the most suited approach for platelet function data series. Normalization was calculated per assay (test) for all time points and per time point for all tests. Interquartile range, range transformation, and z-transformation demonstrated the correlation as calculated by the Spearman correlation test, when normalized per assay (test) for all time points. When normalizing per time point for all tests, no correlation could be abstracted from the charts as was the case when using all data as 1 dataset for normalization. PMID:27428217

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

    PubMed

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

    2002-01-01

    Platelets have a pivotal role in the initial defense against insult to the vasculature and are also recognized of critical importance in the acute care settings of percutaneous coronary intervention and cardiopulmonary bypass. In these environments both platelet count and function may be markedly compromised. Unfortunately, current assays to evaluate the parameters of platelet count and function are of limited utility for bed-side testing. Moreover, it is suggested that there may be significant inter patient variation in response to antiplatelet therapy that may be exacerbated by other agents (e.g. heparin) that are routinely administered during cardiac intervention. Here we describe a practical, rapid and user-friendly whole blood platelet function assay that has been developed for use in bed-side settings. Platelet agonists were formulated with an anticoagulant and lyophilized in blood collection tubes standardised to receive a l mL fresh whole blood sample. In the presence of an agonist, platelets are activated and interact (aggregate). Using traditional cell counting principles, non-aggregated platelets are counted whereas aggregated platelets are not. The percentage (%) of functional platelets in reference to a baseline tube may then be determined. Results are available within four minutes. Platelet aggregation in whole blood demonstrated good correlation with turbidometric aggregometry for both ADP (r=0.91) and collagen (r=0.88). Moreover, in clinical settings where antiplatelet agents were administered, this rapid, bed-side, platelet function assay demonstrated utility in monitoring patient response to these therapies. This novel bed-side assay of platelet function is extremely suitable for the clinical environment with a rapid turn-around time. In addition, it provides a full haematology profile, including platelet count, and should permit enhancement of transfusion and interventional decisions. PMID:17890800

  3. Functional assay of antiplatelet drugs based on margination of platelets in flowing blood.

    PubMed

    Eichinger, Colin D; Fogelson, Aaron L; Hlady, Vladimir

    2016-06-01

    A novel functional assay of antiplatelet drug efficacy was designed by utilizing the phenomena of platelet margination in flowing blood and transient platelet contacts with surface-immobilized platelet agonists. Flow margination enhances transient contacts of platelets with the walls of flow chambers covered with surface-immobilized proteins. Depending on the type and the surface density of the immobilized agonists, such transient interactions could "prime" the marginated platelet subpopulation for enhanced activation and adhesion downstream. By creating an upstream surface patch with an immobilized platelet agonist, platelet flow margination was used to test how effective antiplatelet drugs are in suppressing downstream platelet activation and adhesion. The platelet adhesion downstream was measured by a so-called "capture" patch region close to the distal end of the flow chamber. Platelet adhesion downstream was found to be dose-dependent on the upstream surface coverage of the "priming" patch, with immobilized fibrinogen acting as a platelet agonist. Several antiplatelet agents (acetylsalicylic acid, eptifibatide, and tirofiban) were evaluated for their efficacy in attenuating downstream adhesion after upstream platelet priming. The activation of the platelet population was found to be dependent on both the extent of the upstream agonist stimulus and the antiplatelet drug concentration. Such a relationship provides an opportunity to measure the efficacy of specific antiplatelet agents against the type and concentration of upstream platelet agonists. PMID:27030476

  4. Radioimmune assay of human platelet prostaglandin synthetase

    SciTech Connect

    Roth, G.J.; Machuga, E.T.

    1982-02-01

    Normal platelet function depends, in part, on platelet PG synthesis. PG synthetase (cyclo-oxygenase) catalyzes the first step in PG synthesis, the formation of PGH/sub 2/ from arachidonic acid. Inhibition of the enzyme by ASA results in an abnormality in the platelet release reaction. Patients with pparent congenital abnormalities in the enzyme have been described, and the effects have been referred to as ''aspirin-like'' defects of the platelet function. These patients lack platelet PG synthetase activity, but the actual content of PG synthetase protein in these individuals' platelets is unknown. Therefore an RIA for human platelet PG synthetase would provide new information, useful in assessing the aspirin-like defects of platelet function. An RIA for human platelet PG synthetase is described. The assay utilizes a rabbit antibody directed against the enzyme and (/sup 125/I)-labelled sheep PG synthetase as antigen. The human platelet enzyme is assayed by its ability to inhibit precipitation of the (/sup 125/I)antigen. The assay is sensitive to 1 ng of enzyme. By the immune assay, human platelets contain approximately 1200 ng of PG synethetase protein per 1.5 mg of platelet protein (approximately 10/sup 9/ platelets). This content corresponds to 10,000 enzyme molecules per platelet. The assay provides a rapid and convenient assay for the human platelet enzyme, and it can be applied to the assessment of patients with apparent platelet PG synthetase (cyclo-oxygenase) deficiency.

  5. Normalization methods in time series of platelet function assays: A SQUIRE compliant study.

    PubMed

    Van Poucke, Sven; Zhang, Zhongheng; Roest, Mark; Vukicevic, Milan; Beran, Maud; Lauwereins, Bart; Zheng, Ming-Hua; Henskens, Yvonne; Lancé, Marcus; Marcus, Abraham

    2016-07-01

    Platelet function can be quantitatively assessed by specific assays such as light-transmission aggregometry, multiple-electrode aggregometry measuring the response to adenosine diphosphate (ADP), arachidonic acid, collagen, and thrombin-receptor activating peptide and viscoelastic tests such as rotational thromboelastometry (ROTEM).The task of extracting meaningful statistical and clinical information from high-dimensional data spaces in temporal multivariate clinical data represented in multivariate time series is complex. Building insightful visualizations for multivariate time series demands adequate usage of normalization techniques.In this article, various methods for data normalization (z-transformation, range transformation, proportion transformation, and interquartile range) are presented and visualized discussing the most suited approach for platelet function data series.Normalization was calculated per assay (test) for all time points and per time point for all tests.Interquartile range, range transformation, and z-transformation demonstrated the correlation as calculated by the Spearman correlation test, when normalized per assay (test) for all time points. When normalizing per time point for all tests, no correlation could be abstracted from the charts as was the case when using all data as 1 dataset for normalization. PMID:27428217

  6. Different Influences of Hematocrit on the Results of Two Point-Of-Care Platelet Function Tests, the VerifyNow Assay and Multiple Electrode Platelet Aggregometry

    PubMed Central

    Kim, Yun Gi; Suh, Jung-Won; Park, Jin Joo; Oh, Il-Young; Yoon, Chang-Hwan; Cho, Young-Seok; Youn, Tae-Jin; Chae, In-Ho; Choi, Dong-Ju

    2014-01-01

    Objective Previous studies have reported a considerable association between the VerifyNow (Accumetrics, San Diego, CA, USA) P2Y12 assay results and hematocrit. No reports, however, have described an association between the multiple electrode platelet aggregometry (MEA; Dynabyte, Munich, Germany) adenosine diphosphate (ADP) assay results and hematocrit. This study was conducted to evaluate the influence of hematocrit on the results of 2 different point-of-care platelet function tests. Methods A total of 462 consecutive patients who were undergoing percutaneous coronary intervention were enrolled. Platelet function was evaluated with both the VerifyNow P2Y12 and MEA ADP assays. Results Anemic patients (n = 152, 32.9%) demonstrated a significantly higher rate of cardiac death, myocardial infarction, and stroke (5.3% vs. 2.3%, p = 0.046) during the follow-up (median: 18.8 months). Although the VerifyNow P2Y12 assay results demonstrated a significant inverse correlation with hematocrit (r = −0.409, p<0.001), there was no such correlation between the MEA ADP assay results and hematocrit (r = 0.039, p = 0.401). In the multivariate analysis, anemia was an independent predictor of high on-treatment platelet reactivity, defined as a VerifyNow P2Y12 reaction unit level of ≥252.5 (odds ratio = 2.21, 95% confidence interval = 1.39–3.52; p = 0.001). Importantly, this association was independent of an intrinsic change in platelet reactivity as measured by the MEA ADP assay. Adjusting for the influence of hematocrit improved the strength of the correlation between the VerifyNow P2Y12 and MEA ADP assay results. Conclusions Hematocrit significantly influenced the VerifyNow P2Y12 assay results, a phenomenon that was presumably in-vitro. Hematocrit level should therefore be considered when interpreting results of the VerifyNow P2Y12 assay. PMID:25427105

  7. Platelet function and ageing.

    PubMed

    Jones, Chris I

    2016-08-01

    There are clear age-related changes in platelet count and function, driven by changes in hematopoietic tissue, the composition of the blood and vascular health. Platelet count remains relatively stable during middle age (25-60 years old) but falls in older people. The effect of age on platelet function is slightly less clear. The longstanding view is that platelet reactivity increases with age in an almost linear fashion. There are, however, serious limitations to the data supporting this dogma. We can conclude that platelet function increases during middle age, but little evidence exists on the changes in platelet responsiveness in old age (>75 years old). This change in platelet function is driven by differential mRNA and microRNA expression, an increase in oxidative stress and changes in platelet receptors. These age-related changes in platelets are particularly pertinent given that thrombotic disease and use of anti-platelet drugs is much more prevalent in the elderly population, yet the majority of platelet research is carried out in young to middle-aged (20-50 years old) human volunteers and young mice (2-6 months old). We know relatively little about exactly how platelets from people over 75 years old differ from those of middle-aged subjects, and we know even less about the mechanisms that drive these changes. Addressing these gaps in our knowledge will provide substantial understanding in how cell signalling changes during ageing and will enable the development of more precise anti-platelet therapies. PMID:27068925

  8. Platelet Function Tests

    MedlinePlus

    ... of the clotting process in the body ( in vivo ). A person with normal platelet function test results may still experience excessive bleeding or inappropriate clotting during and after a surgery. Most samples for platelet function testing are only stable for a very short period ...

  9. Platelet function tests: a comparative review

    PubMed Central

    Paniccia, Rita; Priora, Raffaella; Alessandrello Liotta, Agatina; Abbate, Rosanna

    2015-01-01

    In physiological hemostasis a prompt recruitment of platelets on the vessel damage prevents the bleeding by the rapid formation of a platelet plug. Qualitative and/or quantitative platelet defects promote bleeding, whereas the high residual reactivity of platelets in patients on antiplatelet therapies moves forward thromboembolic complications. The biochemical mechanisms of the different phases of platelet activation – adhesion, shape change, release reaction, and aggregation – have been well delineated, whereas their complete translation into laboratory assays has not been so fulfilled. Laboratory tests of platelet function, such as bleeding time, light transmission platelet aggregation, lumiaggregometry, impedance aggregometry on whole blood, and platelet activation investigated by flow cytometry, are traditionally utilized for diagnosing hemostatic disorders and managing patients with platelet and hemostatic defects, but their use is still limited to specialized laboratories. To date, a point-of-care testing (POCT) dedicated to platelet function, using pertinent devices much simpler to use, has now become available (ie, PFA-100, VerifyNow System, Multiplate Electrode Aggregometry [MEA]). POCT includes new methodologies which may be used in critical clinical settings and also in general laboratories because they are rapid and easy to use, employing whole blood without the necessity of sample processing. Actually, these different platelet methodologies for the evaluation of inherited and acquired bleeding disorders and/or for monitoring antiplatelet therapies are spreading and the study of platelet function is strengthening. In this review, well-tried and innovative platelet function tests and their methodological features and clinical applications are considered. PMID:25733843

  10. Platelet Function Tests in Bleeding Disorders.

    PubMed

    Lassila, Riitta

    2016-04-01

    Functional disorders of platelets can involve any aspect of platelet physiology, with many different effects or outcomes. These include platelet numbers (thrombocytosis or thrombocytopenia); changes in platelet production or destruction, or capture to the liver (Ashwell receptor); altered adhesion to vascular injury sites and/or influence on hemostasis and wound healing; and altered activation or receptor functions, shape change, spreading and release reactions, procoagulant and antifibrinolytic activity. Procoagulant membrane alterations, and generation of thrombin and fibrin, also affect platelet aggregation. The above parameters can all be studied, but standardization and quality control of assay methods have been limited despite several efforts. Only after a comprehensive clinical bleeding assessment, including family history, information on drug use affecting platelets, and exclusion of coagulation factor, and tissue deficits, should platelet function testing be undertaken to confirm an abnormality. Current diagnostic tools include blood cell counts, platelet characteristics according to the cell counter parameters, peripheral blood smear, exclusion of pseudothrombocytopenia, whole blood aggregometry (WBA) or light transmission aggregometry (LTA) in platelet-rich plasma, luminescence, platelet function analysis (PFA-100) for platelet adhesion and deposition to collagen cartridges under blood flow, and finally transmission electron microscopy to exclude rare structural defects leading to functional deficits. The most validated test panels are included in WBA, LTA, and PFA. Because platelets are isolated from their natural environment, many simplifications occur, as circulating blood and interaction with vascular wall are omitted in these assays. The target to reach a highly specific platelet disorder diagnosis in routine clinical management can be exhaustive, unless needed for genetic counseling. The elective overall assessment of platelet function disorder

  11. 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

  12. The platelet serotonin-release assay.

    PubMed

    Warkentin, Theodore E; Arnold, Donald M; Nazi, Ishac; Kelton, John G

    2015-06-01

    Few laboratory tests are as clinically useful as The platelet serotonin-release assay (SRA): a positive SRA in the appropriate clinical context is virtually diagnostic of heparin-induced thrombocytopenia (HIT), a life- and limb-threatening prothrombotic disorder caused by anti-platelet factor 4 (PF4)/heparin antibodies that activate platelets, thereby triggering serotonin-release. The SRA's performance characteristics include high sensitivity and specificity, although caveats include indeterminate reaction profiles (observed in ∼4% of test sera) and potential for false-positive reactions. As only a subset of anti-PF4/heparin antibodies detectable by enzyme-immunoassay (EIA) are additionally platelet-activating, the SRA has far greater diagnostic specificity than the EIA. However, requiring a positive EIA, either as an initial screening test or as an SRA adjunct, will reduce risk of a false-positive SRA (since a negative EIA in a patient with a "positive" SRA should prompt critical evaluation of the SRA reaction profile). The SRA also provides useful information on whether a HIT serum produces strong platelet activation even in the absence of heparin: such heparin-"independent" platelet activation is a marker of unusually severe HIT, including delayed-onset HIT and severe HIT complicated by consumptive coagulopathy with risk for microvascular thrombosis. PMID:25775976

  13. Dietary manipulation of platelet function.

    PubMed

    Bachmair, E M; Ostertag, L M; Zhang, X; de Roos, B

    2014-11-01

    Activated platelets contribute to plaque formation within blood vessels in the early and late stages of atherogenesis, and therefore they have been proposed as risk factor for cardiovascular disease. Anti-platelet drugs, such as aspirin, are now the most prescribed pharmacological treatment in Europe. Certain dietary bioactives also beneficially affect platelet function, and with less side effects, albeit that effects are generally more subtle. Therefore, consumption of dietary bioactives could play a role in the prevention of atherothrombotic vascular disease. Here we review the efficacy of dietary treatment strategies, especially those involving certain dietary fatty acids and polyphenols, to modulate platelet function in healthy subjects or in patients with cardiovascular disease. Variation in study populations, small study sizes and lack of comparability between methods to assess platelet function currently limit robust evidence on the efficacy of dietary bioactives in healthy subjects or specific patient groups. Also, limited knowledge of the metabolism of dietary bioactives, and therefore of the bioavailability of bioactive ingredients, restricts our ability to identify the most effective dietary regimes to improve platelet function. Implementation of uniform point-of-care tests to assess platelet function, and enhanced knowledge of the efficacy by which specific dietary compounds and their metabolites affect platelet function, may enable the identification of functional anti-platelet ingredients that are eligible for a health claim, or combined treatment strategies, including both pharmacological anti-platelet treatment as well as dietary intervention, to tackle atherothrombotic vascular disease. PMID:24858060

  14. Acquired platelet function defect

    MedlinePlus

    ... dark black, or tarry bowel movements ; or vomiting blood or material that looks like coffee grounds Nosebleeds ... Tests that may done include: Bleeding time Platelet aggregation test Platelet count PT and PTT

  15. Congenital platelet function defects

    MedlinePlus

    Kottke-Marchant K. Platelet disorders. In: Hsi ED, ed. Hematopathology . 2nd ed. Philadelphia, PA: Elsevier Saunders; 2012:chap 2. Nichols WL. Von Willebrand disease and hemorrhagic abnormalities of platelet ...

  16. Hormonal contraception and platelet function.

    PubMed

    Saleh, A A; Ginsburg, K A; Duchon, T A; Dorey, L G; Hirata, J; Alshameeri, R S; Dombrowski, M P; Mammen, E F

    1995-05-15

    73 healthy women (29 controls, 25 using OCs, and 19 using Norplant) were selected from the clinic population at North Oakland Medical Center for inclusion in this study after obtaining informed consent. Age, race, height, weight, blood pressure, and cigarette smoking were recorded for each subject. 12 patients were on monophasic OCs while 13 were on triphasic preparations. Both hormonal contraceptive groups had used their particular contraceptive for at least 3 months prior to blood drawing. Platelet tests were performed within 2 hours of sample collection: platelet counts (PLC) and mean platelet volume (MPV) were determined on an Automated Platelet Counter (Baker 810 Platelet Analyzer). Whole blood aggregation was performed on a platelet aggregometer (Chrono-Log, Model 550) using both ADP (ADP, 5 mM) and collagen (COLL, 2 mcg/ml) as inducing agents. Demographic differences were not significant (p 0.05) among the 3 treatment groups, whose average age was 25.3-25.8 years old. Furthermore, no significant differences (p 0.05) in platelet function were detected among controls or subjects receiving either oral contraceptives or Norplant, compared to control patients. The mean platelet counts (X 10/9/L) were 223 for OC users, 231 for Norplant users, and 232 for controls. The respective platelet aggregation (ADP, ohms) values were 12.5, 18.0, and 19.2 as well as (COLL, ohms) 35.6, 40.7, and 39.0. These results demonstrated that there is no evidence for altered platelet function, with the testing methods employed, in women using either Norplant or combination low dose oral contraceptives. To date, several studies have examined this issue, with contradictory reports about the effects of hormonal contraceptives in platelet function. After controlling for differences between various steroid preparations and other such confounding variables, some of these conflicting conclusions could be the result of a lack of uniformity among the methods used to evaluate platelet aggregation

  17. Sources of variability in platelet accumulation on type 1 fibrillar collagen in microfluidic flow assays.

    PubMed

    Neeves, Keith B; Onasoga, Abimbola A; Hansen, Ryan R; Lilly, Jessica J; Venckunaite, Diana; Sumner, Meghan B; Irish, Andrew T; Brodsky, Gary; Manco-Johnson, Marilyn J; Di Paola, Jorge A

    2013-01-01

    Microfluidic flow assays (MFA) that measure shear dependent platelet function have potential clinical applications in the diagnosis and treatment of bleeding and thrombotic disorders. As a step towards clinical application, the objective of this study was to measure how phenotypic and genetic factors, as well as experimental conditions, affect the variability of platelet accumulation on type 1 collagen within a MFA. Whole blood was perfused over type 1 fibrillar collagen at wall shear rates of 150, 300, 750 and 1500 s⁻¹ through four independent channels with a height of 50 µm and a width of 500 µm. The accumulation of platelets was characterized by the lag time to 1% platelet surface coverage (Lag(T)), the rate of platelet accumulation (V(PLT)), and platelet surface coverage (SC). A cohort of normal donors was tested and the results were correlated to plasma von Willebrand factor (VWF) levels, platelet count, hematocrit, sex, and collagen receptors genotypes. VWF levels were the strongest determinant of platelet accumulation. VWF levels were positively correlated to V(PLT) and SC at all wall shear rates. A longer Lag(T) for platelet accumulation at arterial shear rates compared to venous shear rates was attributed to the time required for plasma proteins to adsorb to collagen. There was no association between platelet accumulation and hematocrit or platelet count. Individuals with the AG genotype of the GP6 gene had lower platelet accumulation than individuals with the AA genotype at 150 s⁻¹ and 300 s⁻¹. Recalcified blood collected into sodium citrate and corn trypsin inhibitor (CTI) resulted in diminished platelet accumulation compared to CTI alone, suggesting that citrate irreversibly diminishes platelet function. This study the largest association study of MFA in healthy donors (n = 104) and will likely set up the basis for the determination of the normal range of platelet responses in this type of assay. PMID:23355889

  18. Monoclonal platelet antigen capture assays (MAIPA) and reagents: a statement.

    PubMed

    Kaplan, C; Freedman, J; Foxcroft, Z; Husebekk, A; Metcalfe, P; Muniz-Diaz, E; Ouwehand, W; Panzer, S; Rozman, P; Skogen, B

    2007-11-01

    This statement concerning the monoclonal-specific immobilization of platelet antigens (MAIPA) has been written on behalf of the International Society of Blood Transfusion--Working Party on Platelet Immunology. The MAIPA technique is considered as the gold standard reference technique in platelet immunology. The assay performed with reagents labelled for 'research only' is acceptable as long as it is regularly evaluated by participation of laboratories in national or international workshops held with reference laboratories. PMID:18070272

  19. [Assessment of platelet function in man].

    PubMed

    Gaussem, Pascale

    2006-01-01

    Assessment of platelet function was primarily designed to explore patients with hemostatic disorders, but is becoming important for the monitoring of anti platelet agents, mostly aspirin and clopidogrel. Beside platelet counting, morphological analysis and bleeding time, a number of dedicated platelet function instruments are now available, generally allowing a rapid evaluation of platelet function in whole blood. The other tests including aggregometry and ELISA measurement of activation markers are generally restricted to specialized laboratories. Although aggregometry is still considered as the "gold standard", the recently developed flow cytometric-based platelet function analysis provides a wide choice of tests that assess the number of surface receptors, the measure of secretion and aggregation, the quantification of microparticules and leukocyte-platelet aggregates. It also allows the measure of the function of the ADP receptor P2Y12 by the phosphorylation level of the VASP protein, method currently under evaluation to monitor the platelet response to clopidogrel treatment. PMID:17243268

  20. Platelet function defects in chronic alcoholism.

    PubMed Central

    Mikhailidis, D P; Jenkins, W J; Barradas, M A; Jeremy, J Y; Dandona, P

    1986-01-01

    Platelet function in alcoholic patients was assessed on admission and during abstinence in hospital. On admission platelets from these patients were significantly less responsive (percentage aggregation and thromboxane A2 release) to conventional in vitro aggregating agents (adrenaline, adenosine diphosphate, and collagen) than platelets from healthy, moderate drinkers. Initially, platelet counts in platelet rich plasma tended to be low and the Simplate II bleeding times frequently prolonged. Platelet aggregation and thromboxane A2 release, however, were inhibited even in patients with normal platelet counts on admission. Platelet aggregation and thromboxane A2 release returned to normal or became hyper-responsive during two to three weeks of abstinence. Platelet counts rose during this period, the largest responses occurring in those patients with the lowest counts on admission. Bleeding times reverted to normal during abstinence and correlated significantly with changes in platelet aggregation, thromboxane A2 release, and platelet count and with the estimated ethanol consumption during the week before admission. Chronic, heavy alcohol ingestion evidently exerts an inhibitory effect on platelet function even in the absence of alcohol in the blood, and this phenomenon is reversible on abstaining. The impaired platelet function, together with the reduced platelet count, may contribute to the bleeding diathesis associated with chronic alcoholism and to the increased incidence and recurrence of gastrointestinal haemorrhage associated with excessive alcohol intake. PMID:3094624

  1. Dynamic light scattering can determine platelet function

    NASA Astrophysics Data System (ADS)

    Lee, Nathan

    2011-10-01

    Platelet transfusions are life-saving procedures for patients who are bleeding or undergoing chemotherapy. The effectiveness of transfusions depends on the number of platelets transfused and the platelet function. Platelet function correlates with proportion of discoid to activated platelets, morphology response to temperature stress, and inversely correlates with microparticle content. ThromboLUX is a novel device that determines platelet function by measuring all of these characteristics using dynamic light scattering (DLS). During periods of stress, such as decreased temperature, cytoskeletal rearrangements will cause normal, discoid platelets to activate and become spiny spheres. The formation of pseudopods of various lengths facilitates the clotting cascade and also increases the apparent size of platelets. ThromboLUX uses a 37-20-37 C temperature cycle that mimics the bleeding, storage, and transfusion process. As the temperature fluctuates, DLS will measure the changing platelet hydrodynamic radius and the size of any microparticles present. ThromboLUX analysis of platelet concentrates in vitro would allow determination of high platelet function units before transfusion and would therefore improve transfusion outcomes and patient safety. This study examined how DLS is able to distinguish between discoid and activated platelets as well as measure the parameters that contribute to high platelet function.

  2. [Research Progress on Cytometric Bead Assay for Platelet Antibody Detection].

    PubMed

    Ling, Yun; Kong, Xin; Chen, Bao-An

    2015-08-01

    Anti-platelet specific antibody is one of the most important reasons leading to thrombocytopenia and megakaryocyte dysmaturity. The detection of platelet autoantibodies is an important step in the diagnosis of ITP because of the absence of specific clinic feature. The monoclonal antibody-specific immobilization of platelet antigens (MAIPA) has become a "gold standard" for determination of PLT specific antibody, which has high specificity and low sensitivity. However, this assay is time-consuming and tedious work. Routine use of this assay in hospital is difficult. Recently, some researches reporded the cytometric bead assay that has higher sensitivity than MAIPA, and so probably solves the problem of time-consuming partly, that also can use different beads for simultaneous detection. This review focuses on recent progress of the cytometric bead assay. PMID:26314475

  3. P2 receptors and platelet function.

    PubMed

    Hechler, Béatrice; Gachet, Christian

    2011-09-01

    Following vessel wall injury, platelets adhere to the exposed subendothelium, become activated and release mediators such as TXA(2) and nucleotides stored at very high concentration in the so-called dense granules. Released nucleotides and other soluble agents act in a positive feedback mechanism to cause further platelet activation and amplify platelet responses induced by agents such as thrombin or collagen. Adenine nucleotides act on platelets through three distinct P2 receptors: two are G protein-coupled ADP receptors, namely the P2Y(1) and P2Y(12) receptor subtypes, while the P2X(1) receptor ligand-gated cation channel is activated by ATP. The P2Y(1) receptor initiates platelet aggregation but is not sufficient for a full platelet aggregation in response to ADP, while the P2Y(12) receptor is responsible for completion of the aggregation to ADP. The latter receptor, the molecular target of the antithrombotic drugs clopidogrel, prasugrel and ticagrelor, is responsible for most of the potentiating effects of ADP when platelets are stimulated by agents such as thrombin, collagen or immune complexes. The P2X(1) receptor is involved in platelet shape change and in activation by collagen under shear conditions. Each of these receptors is coupled to specific signal transduction pathways in response to ADP or ATP and is differentially involved in all the sequential events involved in platelet function and haemostasis. As such, they represent potential targets for antithrombotic drugs. PMID:21792575

  4. Altered Platelet Function in Intrauterine Growth Restriction: A Cause or a Consequence of Uteroplacental Disease?

    PubMed

    Müllers, Sieglinde M; Burke, Naomi; Flood, Karen; Cowman, Jonathan; O'connor, Hugh; Cotter, Brian; Kearney, Morgan; Dempsey, Mark; Dicker, Patrick; Tully, Elizabeth; Geary, Michael P; Kenny, Dermot; Malone, Fergal D

    2016-07-01

    Objective A limited number of platelet function studies in intrauterine growth restriction (IUGR) have yielded conflicting results. We sought to evaluate platelet reactivity in IUGR using a novel platelet aggregation assay. Study Design Pregnancies with IUGR were recruited from 24 weeks' gestation (estimated fetal weight < 10th centile) and had platelet function testing performed after diagnosis. A modification of light transmission aggregometry created dose-response curves of platelet reactivity in response to multiple agonists at differing concentrations. Findings were compared with healthy third trimester controls. IUGR cases with a subsequent normal birth weight were analyzed separately. Results In this study, 33 pregnancies retained their IUGR diagnosis at birth, demonstrating significantly reduced platelet reactivity in response to all agonists (arachidonic acid, adenosine diphosphate, collagen, thrombin receptor-activating peptide, and epinephrine) when compared with 36 healthy pregnancy controls (p < 0.0001). Similar results were obtained for cases demonstrating an increasing in utero growth trajectory. When IUGR preceded preeclampsia or gestational hypertension, platelet function was significantly reduced compared with normotensive IUGR. Conclusion Using this comprehensive platelet assay, we have demonstrated a functional impairment of platelets in IUGR. This may reflect platelet-derived placental growth factor release. Further evaluation of platelet function may aid in the development of future platelet-targeted therapies for uteroplacental disease. PMID:26906182

  5. Sodium-hydrogen exchange and platelet function.

    PubMed

    Rosskopf, D

    1999-07-01

    On stimulation of platelets with agonists, for example, thrombin, a rapid rise in intracellular pH is observed. This alkalinization is mediated by an increase in transport activity of the Na(+)/H(+) exchanger isoform NHE1. In addition to this Na(+)/H(+) exchange mechanism, platelets express bicarbonate/chloride exchangers, which also contribute to pH(i) homeostasis. The main functions of NHE1 in platelets include pH(i) control, volume regulation, and participation in cell signaling. The isoform NHE1 is highly sensitive toward inhibition by EIPA, Hoe694, and Hoe642. The regulation of NHE1 activity is complex and is not completely understood. It includes the MAP kinase cascade, the Ca/calmodulin system, several heterotrimeric G proteins (Galpha12, Galpha13, Galphaq, and Galphai), small G proteins (ras, cdc42, rhoA), and downstream kinases (e.g., p160ROCK). Volume challenges stimulate tyrosine phosphorylation of cytoplasmic proteins, which ultimately activate NHE1. Thrombin, thromboxane, platelet-activating factor, angiotensin II, endothelin, phorbol ester, and Ca(2+) ionophors stimulate NHE1 activity in platelets. Blockade of platelet NHE1 can inhibit platelet activation. With the development of highly specific NHE1 inhibitors, detailed investigation of the relationships between NHE1 activity and platelet activation now becomes feasible. PMID:10481210

  6. Inherited Platelet Function Disorders: Algorithms for Phenotypic and Genetic Investigation.

    PubMed

    Gresele, Paolo; Bury, Loredana; Falcinelli, Emanuela

    2016-04-01

    Inherited platelet function disorders (IPFDs) manifest with mucocutaneous bleeding and are frequently difficult to diagnose due to their heterogeneity, the complexity of the platelet activation pathways and a lack of standardization of the platelet function laboratory assays and of their use for this purpose. A rational diagnostic approach to IPFDs should follow an algorithm where clinical examination and a stepwise laboratory evaluation play a crucial role. A streamlined panel of laboratory tests, with consecutive steps of increasing level of complexity, allows the phenotypic characterization of most IPFDs. A first-line diagnosis of a significant fraction of the IPFD may be made also at nonspecialized centers by using relatively simple tests, including platelet count, peripheral blood smear, light transmission aggregometry, measurement of platelet granule content and release, and the expression of glycoproteins by flow cytometry. Some of the most complex, second- and third-step tests may be performed only in highly specialized laboratories. Genotyping, including the widespread application of next-generation sequencing, has enabled discovery in the last few years of several novel genes associated with platelet disorders and this method may eventually become a first-line diagnostic approach; however, a preliminary clinical and laboratory phenotypic characterization nowadays still remains crucial for diagnosis of IPFDs. PMID:26962877

  7. Viability and functional integrity of washed platelets.

    PubMed

    Pineda, A A; Zylstra, V W; Clare, D E; Dewanjee, M K; Forstrom, L A

    1989-01-01

    The viability and functional integrity of saline- and ACD-saline-washed platelets were compared with those of unwashed platelets. After template bleeding time (TBT) was measured, 15 healthy volunteers underwent plateletpheresis and ingested 600 mg of aspirin. Autologous 111In-labeled platelets were transfused: unwashed (n = 5), washed with 0.9 percent saline solution (SS) (n = 5), and washed with a buffered 12.6 percent solution of ACD-A in 0.9 percent saline solution (n = 5). After transfusion, we measured TBT at 1, 4, and 24 hours; platelet survival at 10 minutes and 1, 4, and 24 hours and daily for 6 days; and the percentage of uptake in liver and spleen by quantitative whole-body radionuclide scintigraphy at 24 and 190 hours. We found that saline washing affected platelet recovery, 23.47 +/- 12 percent (p less than 0.001) as compared to 52.43 +/- 17 percent (p less than 0.002) for ACD-saline and 73.17 +/- 8 percent for control; that saline washing resulted in a greater liver uptake than control and ACD-saline-washed platelets (31.9 +/- 8% [p less than 0.001] vs 17.7 +/- 4.1 and 19.3 +/- 2.1% [p greater than 0.1], respectively); that, unlike control and ACD-saline-washed platelets, saline-washed platelets did not shorten bleeding time; and that neither type of washing affected survival. Although ACD-saline washing affects recovery, it also results in intact function, normal survival, higher recovery than SS platelets, and no significant liver uptake. PMID:2749876

  8. Viability and functional integrity of washed platelets

    SciTech Connect

    Pineda, A.A.; Zylstra, V.W.; Clare, D.E.; Dewanjee, M.K.; Forstrom, L.A.

    1989-07-01

    The viability and functional integrity of saline- and ACD-saline-washed platelets were compared with those of unwashed platelets. After template bleeding time (TBT) was measured, 15 healthy volunteers underwent plateletpheresis and ingested 600 mg of aspirin. Autologous /sup 111/In-labeled platelets were transfused: unwashed (n = 5), washed with 0.9 percent saline solution (SS) (n = 5), and washed with a buffered 12.6 percent solution of ACD-A in 0.9 percent saline solution (n = 5). After transfusion, we measured TBT at 1, 4, and 24 hours; platelet survival at 10 minutes and 1, 4, and 24 hours and daily for 6 days; and the percentage of uptake in liver and spleen by quantitative whole-body radionuclide scintigraphy at 24 and 190 hours. We found that saline washing affected platelet recovery, 23.47 +/- 12 percent (p less than 0.001) as compared to 52.43 +/- 17 percent (p less than 0.002) for ACD-saline and 73.17 +/- 8 percent for control; that saline washing resulted in a greater liver uptake than control and ACD-saline-washed platelets (31.9 +/- 8% (p less than 0.001) vs 17.7 +/- 4.1 and 19.3 +/- 2.1% (p greater than 0.1), respectively); that, unlike control and ACD-saline-washed platelets, saline-washed platelets did not shorten bleeding time; and that neither type of washing affected survival. Although ACD-saline washing affects recovery, it also results in intact function, normal survival, higher recovery than SS platelets, and no significant liver uptake.

  9. Detection and effects on platelet function of anti-platelet antibody in mule foals with experimentally induced neonatal alloimmune thrombocytopenia.

    PubMed

    Ramirez, S; Gaunt, S D; McClure, J J; Oliver, J

    1999-01-01

    Horse mares carrying mule foals were immunized during the last trimester of pregnancy with whole acid-citrate-dextrose-anticoagulated donkey blood to experimentally induce neonatal alloimmune thrombocytopenia. Thrombocytopenia occurred in the neonatal mule foals born to immunized horse mares within 24 hours after ingestion of their dams' colostrum. Mule foals born to mares not immunized with donkey blood did not develop thrombocytopenia. These findings suggest that antibodies may have been directed against a donkey platelet antigen present in the mule foals but not present in their dams. The objectives of this study were to determine whether anti-platelet antibody could be detected in mule foals with experimentally induced neonatal alloimmune thrombocytopenia, to identify any platelet proteins recognized by serum antibody in these foals, and to determine if platelet function was altered by sera from these mule foals. An indirect enzyme-linked immunosorbent assay demonstrated significantly higher absorption at 1:200 of platelet-bindable immunoglobulin G in serum from thrombocytopenic mule foals, compared with nonthrombocytopenic mule foals. Sera from thrombocytopenic and nonthrombocytopenic mule foals produced similar binding patterns in western immunoblots with donkey platelet proteins separated on sodium dodecyl sulfate polyacrylamide gels. Maximal platelet aggregation and relative slope of aggregation in response to collagen were significantly inhibited after incubation with sera from thrombocytopenic mule foals. These results suggest that mule foals with induced alloimmune thrombocytopenia have serum antibodies that bind to platelets and may compete with collagen binding sites to impair platelet aggregation. PMID:10587252

  10. 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

  11. Effects of irradiation on platelet function

    SciTech Connect

    Rock, G.; Adams, G.A.; Labow, R.S.

    1988-09-01

    Current medical practice involves the irradiation of blood components, including platelet concentrates, before their administration to patients with severe immunosuppression. The authors studied the effect of irradiation on in vitro platelet function and the leaching of plasticizers from the bag, both immediately and after 5 days of storage. The platelet count, white cell count, pH, glucose, lactate, platelet aggregation and release reaction, and serotonin uptake were not altered by the irradiation of random-donor or apheresis units with 2000 rads carried out at 0 and 24 hours and 5 days after collection. The leaching of di(2-ethylhexyl)phthalate from the plastic bags followed by the conversion to mono(2-ethylhexyl)phthalate was not increased by irradiation. Therefore, it is possible to irradiate platelet concentrates on the day of collection and subsequently store them for at least 5 days while maintaining in vitro function. This procedure could have considerable benefit for blood banks involved in the provision of many platelet products.

  12. Rapid in vitro biocompatibility assay of endovascular stents by flow cytometry using platelet activation and platelet-leukocyte aggregation.

    PubMed

    Tárnok, A; Mahnke, A; Müller, M; Zotz, R J

    1999-02-15

    Clinical studies suggest that stent design and surface texture are responsible for differences in biocompatibility of metallic endovascular stents. A simple in vitro experimental setup was established to test stent-induced degree of platelet and leukocyte activation and platelet-leukocyte aggregation by flow cytometry. Heparin-coated tantalum stents and gold-coated and uncoated stainless steel stents were tested. Stents were implanted into silicone tubes and exposed to blood from healthy volunteers. Platelet and leukocyte activation and percentage of leukocyte-platelet aggregates were determined in a whole-blood assay by subsequent staining for activation-associated antigens (CD41a, CD42b, CD62p, and fibrinogen binding) and leukocyte antigens (CD14 and CD45) and flow cytometric analysis. Blood taken directly after venous puncture or exposed to the silicone tube alone was used as negative controls. Positive control was in vitro stimulation with thrombin receptor activating peptide (TRAP-6). Low degree of platelet activation and significant increase in monocyte- and neutrophil-platelet aggregation were observed in blood exposed to stents (P < 0.05). In addition, leukocyte activation was induced as measured by increased CD45 and CD14 expression. Heparin coated stents continuously induced less platelet activation and leukocyte-platelet aggregation than uncoated stainless steel stents of the same length and shorter stents of the same structure. Stent surface coating and texture plays a role in platelet and leukocyte activation and leukocyte-platelet aggregation. Using this simple in vitro assay and whole blood and flow cytometry, it seems possible to differentiate stents by their potency to activate platelets and/or leukocytes. This assay could be applied for improving the biocompatibility of coronary stents. PMID:10088974

  13. STIM and Orai in platelet function.

    PubMed

    Varga-Szabo, David; Braun, Attila; Nieswandt, Bernhard

    2011-09-01

    Physiological platelet activation and thrombus formation are essential to stop bleeding in case of vascular injury, whereas inadequate triggering of the same process in diseased vessels can lead to fatal thromboembolism and tissue ischemia of vital organs. A central step in platelet activation is agonist-induced elevation of the intracellular Ca(2+) concentration. This happens on the one hand through the release of Ca(2+) from intracellular stores and on the other hand through Ca(2+) influx from the extracellular space. In platelets, the major Ca(2+) influx pathway is the so-called store operated Ca(2+) entry (SOCE), induced by store depletion. Studies in the last five years discovered the molecular background of platelet SOCE. Stromal interaction molecule 1 (STIM1) and Orai1, two so far unknown molecules, got in the focus of research. STIM1 was found to be the Ca(2+) sensor in the endoplasmic reticulum (ER) membrane, whereas Orai1 was identified as the major store operated Ca(2+) (SOC) channel in the plasma membrane. These two molecules and their role in platelet function and thrombus formation are the topic of the present review with a special focus on apoptosis and apoptosis-like processes in platelet physiology. PMID:21616531

  14. An Assay of Measuring Platelet Reactivity Using Monoclonal Antibody against Activated Platelet Glycoprotein IIb/IIIa in Patients Taking Clopidogrel

    PubMed Central

    Choi, Joon-Hyouk; Kim, Song-Yi; Kim, Ki-Seok; Kim, Young Ree; Kang, Sung Ha

    2015-01-01

    Background and Objectives Residual platelet reactivity in patients who are taking clopidogrel is commonly measured with VerifyNow assay, which is based on the principle of light transmission aggregometry. However, to evaluate the residual platelet reactivity, it would be more accurate if the reactivity of platelet glycoprotein (GP) IIb/IIIa is directly monitored. In this study, PAC1, a monoclonal antibody against activated platelet GP IIb/IIIa, was used to measure the residual platelet reactivity. Subjects and Methods Twenty seven patients with coronary artery disease taking clopidogrel were enrolled. Platelets in whole blood were stained with fluorescein isothiocyanate (FITC)-conjugated PAC1. Mean fluorescence intensity (MFI) and % positive platelets (PP) were measured with flow cytometry, and the binding index (BI; MFI × %PP/100) was calculated. P2Y12 reaction unit (PRU) and % inhibition of VerifyNow assay were also measured in the usual manner. Results PRU of VerifyNow assay correlated significantly with MFI, %PP, and BI at 10 µM (r=0.59, 0.73, and 0.60, respectively, all p<0.005) and 20 µM of adenosine diphosphate (ADP; r=0.61, 0.75, and 0.63, respectively, all p<0.005). The % inhibition also correlated significantly with MFI, %PP, and BI at 10 µM (r=-0.60, -0.69, and -0.59, respectively, all p<0.005) and 20 µM of ADP (r=-0.63, -0.71, and -0.62, respectively, all p<0.005). Conclusion Direct measurements of the reactivity of platelet GP IIb/IIIa were feasible using PAC1 and flow cytometry in patients taking clopidogrel. Further clinical studies are required to determine the cut-off values which would define high residual platelet reactivity in patients on this treatment protocol. PMID:26413105

  15. Platelet function tests, independent of platelet count, are associated with bleeding severity in ITP

    PubMed Central

    Grace, Rachael F.; Gerrits, Anja J.; Berny-Lang, Michelle A.; Brown, Travis; Carmichael, Sabrina L.; Neufeld, Ellis J.; Michelson, Alan D.

    2015-01-01

    Immune thrombocytopenia (ITP) patients with similarly low platelet counts differ in their tendency to bleed. To determine if differences in platelet function in ITP patients account for this variation in bleeding tendency, we conducted a single-center, cross-sectional study of pediatric patients with ITP. Bleeding severity (assessed by standardized bleeding score) and platelet function (assessed by whole blood flow cytometry) with and without agonist stimulation was evaluated in 57 ITP patients (median age, 9.9 years). After adjustment for platelet count, higher levels of thrombin receptor activating peptide (TRAP)-stimulated percent P-selectin- and activated glycoprotein (GP)IIb-IIIa–positive platelets were significantly associated with a lower bleeding score, whereas higher levels of immature platelet fraction (IPF), TRAP-stimulated platelet surface CD42b, unstimulated platelet surface P-selectin, and platelet forward light scatter (FSC) were associated with a higher bleeding score. Thus, platelet function tests related to platelet age (IPF, FSC) and activation through the protease activated receptor 1 (PAR1) thrombin receptor (TRAP-stimulated P-selectin, activated GPIIb-IIIa, and CD42b), independent of platelet count, are associated with concurrent bleeding severity in ITP. These tests may be useful markers of future bleeding risk in ITP. PMID:26138687

  16. Resveratrol preserves the function of human platelets stored for transfusion.

    PubMed

    Lannan, Katie L; Refaai, Majed A; Ture, Sara K; Morrell, Craig N; Blumberg, Neil; Phipps, Richard P; Spinelli, Sherry L

    2016-03-01

    Stored platelets undergo biochemical, structural and functional changes that lead to decreased efficacy and safety of platelet transfusions. Not only do platelets acquire markers of activation during storage, but they also fail to respond normally to agonists post-storage. We hypothesized that resveratrol, a cardioprotective antioxidant, could act as a novel platelet storage additive to safely prevent unwanted platelet activation during storage, while simultaneously preserving normal haemostatic function. Human platelets treated with resveratrol and stored for 5 d released less thromboxane B2 and prostaglandin E2 compared to control platelets. Resveratrol preserved the ability of platelets to aggregate, spread and respond to thrombin, suggesting an improved ability to activate post-storage. Utilizing an in vitro model of transfusion and thromboelastography, clot strength was improved with resveratrol treatment compared to conventionally stored platelets. The mechanism of resveratrol's beneficial actions on stored platelets was partly mediated through decreased platelet apoptosis in storage, resulting in a longer half-life following transfusion. Lastly, an in vivo mouse model of transfusion demonstrated that stored platelets are prothrombotic and that resveratrol delayed vessel occlusion time to a level similar to transfusion with fresh platelets. We show resveratrol has a dual ability to reduce unwanted platelet activation during storage, while preserving critical haemostatic function. PMID:26683619

  17. [Analysis of platelet-derived factors that modulate functions of polymorphonuclear leukocytes].

    PubMed

    Sasada, M; Asagoe, K

    1994-04-01

    Interactions between platelets and polymorphonuclear leukocytes (PMN) modulate their functions and play a role in the development of pathogenesis of some disease. Platelets secret various kinds of factors that affect PMN functions. They seemed to have important role in vivo, but little has been elucidated on exact mechanism of action and physiological meaning of each factor in relation to PMN functions. We studied the effects of platelets and released substances from activated platelets on the functions of PMN. Results were as follows. 1) Platelets enhanced bactericidal activities of PMN against E.coli. 2) Platelets had effects on the generation of superoxide anion (O2-) of PMN. Their effects were quite different according to the assay condition of PMN, that is, platelets inhibited O2- generation when PMN were at rest or stimulated slightly and they enhanced O2-generation of PMN that were stimulated with optimal condition. 3) Thrombin-activated platelets and their supernatant elicited a transient elevation of [Ca2] of PMN. The activity of the supernatant decreased by treating with hexokinase that decomposed ATP. Further treatment with trypsin abolished its activity almost completely. Considering with our additional experiments, factors that induced [Ca2+] elevation of PMN were ATP, beta-thromboglobulin and some trypsin-sensitive factor(s). 4) Supernatant of thrombin-activated platelets decreased random migration and chemokinesis of PMN. PMID:8028184

  18. In vivo effects of eltrombopag on platelet function in immune thrombocytopenia: no evidence of platelet activation

    PubMed Central

    Psaila, Bethan; Bussel, James B.; Linden, Matthew D.; Babula, Bracken; Li, Youfu; Barnard, Marc R.; Tate, Chinara; Mathur, Kanika; Frelinger, Andrew L.

    2012-01-01

    The effects of eltrombopag, a thrombopoietin-receptor agonist, on platelet function in immune thrombocytopenia (ITP) are not fully characterized. This study used whole blood flow cytometry to examine platelet function in 20 patients receiving eltrombopag treatment at days 0, 7, and 28. Platelet surface expression of activated GPIIb/IIIa, P-selectin, and GPIb was measured with and without low and high adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP) concentrations. Before eltrombopag treatment with no ex vivo agonist, platelet activation was higher in ITP patients than controls. Platelet GPIb and activated GPIIb/IIIa expression without added agonist was unchanged following eltrombopag treatment, whereas a slight increase in P-selectin was observed. Expression of P-selectin and activated GPIIb/IIIa in response to high-dose ADP was lower during eltrombopag treatment than at baseline. Eltrombopag led to a slight increase in platelet reactivity to TRAP only in responders to eltrombopag but not to levels above those in controls; whole blood experiments demonstrated that this increase was probably because of higher platelet counts rather than higher platelet reactivity. In conclusion, although thrombocytopenic ITP patients have higher baseline platelet activation than controls, eltrombopag did not cause platelet activation or hyper-reactivity, irrespective of whether the platelet count increased. PMID:22294727

  19. Platelets

    MedlinePlus

    ... are related to immunity and fighting infection. Platelet Production Platelets are produced in the bone marrow, the ... platelet destruction and also decreased bone marrow platelet production. These problems are caused by autoantibodies. Antibodies are ...

  20. Bidirectional effects of dexmedetomidine on human platelet functions in vitro.

    PubMed

    Kawamoto, Shuji; Hirakata, Hideo; Sugita, Naoko; Fukuda, Kazuhiko

    2015-11-01

    Platelets express the imidazoline (I)-receptor, I1 and I2, as well as the α2-adrenoceptor. Although dexmedetomidine, a selective α2-adrenoceptor agonist with some affinity for the I-receptor is expected to affect platelet function, the effects of dexmedetomidine on platelet functions remain unclear. In the present study, we investigated the effects of dexmedetomidine on human platelet functions in vitro. The effects of dexmedetomidine on platelet aggregation were examined using aggregometers. The formation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in platelets was measured by an enzyme immunoassay. In addition, P-selectin expression in platelets was estimated by flow cytometry. We showed that dexmedetomidine enhances platelet aggregation. But in the presence of yohimbine, an α2-antagonist, dexmedetomidine suppressed platelet aggregation. Efaroxan, an I1-antagonist, and methylene blue, a soluble guanylate cyclase inhibitor, abolished the suppressive effect of dexmedetomidine, whereas idazoxan, an I2-antagonist, showed no effect. Dexmedetomidine suppressed cAMP formation and enhanced P-selectin expression in platelets, and these effects were inhibited by yohimbine. Dexmedetomidine increased cGMP formation in platelets in the presence of yohimbine, and this increase was suppressed by efaroxan. These results demonstrated that dexmedetomidine has both enhancing and suppressive effects on human platelet functions through its action on the α2-adrenoceptor and on the I1-imidazoline receptor, respectively. PMID:26435028

  1. Impact of Aspirin and Clopidogrel Interruption on Platelet Function in Patients Undergoing Major Vascular Surgery

    PubMed Central

    Le Manach, Yannick; Kahn, David; Bachelot-Loza, Christilla; Le Sache, Frederic; Smadja, David M.; Remones, Veronique; Loriot, Marie-Anne; Coriat, Pierre; Gaussem, Pascale

    2014-01-01

    Aims To investigate functional platelet recovery after preoperative withdrawal of aspirin and clopidogrel and platelet function 5 days after treatment resumption. Methods/Results We conducted an observational study, which prospectively included consecutive patients taking aspirin, taking clopidogrel, and untreated controls (15 patients in each group). The antiplatelet drugs were withdrawn five days before surgery (baseline) and were reintroduced two days after surgery. Platelet function was evaluated by optical aggregation in the presence of collagen, arachidonic acid (aspirin) and ADP (clopidogrel) and by VASP assay (clopidogrel). Platelet-leukocyte complex (PLC) level was quantified at each time-point. At baseline, platelet function was efficiently inhibited by aspirin and had recovered fully in most patients 5 days after drug withdrawal. PLC levels five days after aspirin reintroduction were similar to baseline (+4±10%; p = 0.16), in line with an effective platelet inhibition. Chronic clopidogrel treatment was associated with variable platelet inhibition and its withdrawal led to variable functional recovery. PLC levels were significantly increased five days after clopidogrel reintroduction (+10±15%; p = 0.02), compared to baseline. Conclusions Aspirin withdrawal 5 days before high-bleeding-risk procedures was associated with functional platelet recovery, and its reintroduction two days after surgery restored antiplaletet efficacy five days later. This was not the case of clopidogrel, and further work is therefore needed to define its optimal perioperative management. PMID:25141121

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

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

  3. Heparin inhibition of von Willebrand factor-dependent platelet function in vitro and in vivo.

    PubMed Central

    Sobel, M; McNeill, P M; Carlson, P L; Kermode, J C; Adelman, B; Conroy, R; Marques, D

    1991-01-01

    The intravenous administration of heparin to patients before open heart surgery reduced ristocetin cofactor activity by 58% (P less than 0.01, t test), and this impairment of von Willebrand factor-dependent platelet function was closely related to plasma heparin levels (r2 = 0.9), but not to plasma von Willebrand factor (vWF) levels. We hypothesized that heparin may inhibit vWF-dependent platelet hemostatic functions by directly binding vWF in solution and interfering with vWF-GpIb binding. Using the in vitro techniques of ristocetin-induced platelet agglutination, fluorescent flow cytometric measurement of vWF-platelet binding, and conventional radioligand binding assays we observed that heparin inhibited both vWF-dependent platelet function and vWF-platelet binding in a parallel and dose-dependent manner. Heparin also inhibited platelet agglutination induced by bovine vWF and inhibited the binding of human asialo-vWF to platelets in ristocetin-free systems. The inhibitory potency of heparin was not dependent upon its affinity for antithrombin III, but was molecular weight dependent: homogeneous preparations of lower molecular weight were less inhibitory. Heparin impairment of vWF function may explain why some hemorrhagic complications of heparin therapy are not predictable based on techniques for monitoring the conventional anticoagulant effects of heparin. PMID:2022745

  4. Biochemical and functional abnormalities in hypercholesterolemic rabbit platelets

    SciTech Connect

    Dalal, K.B.; Ebbe, S.; Mazoyer, E.; Carpenter, D.; Yee, T. )

    1990-02-01

    This study was designed to elucidate changes in rabbit platelet lipids induced by a cholesterol rich diet and to explore the possible correlation of these lipid changes with platelet abnormalities. Pronounced biochemical alterations were observed when serum cholesterol levels of 700-1000 mg% were reached. Hypercholesterolemic (HC) platelets contained 37% more neutral lipids and 16% less phospholipids than the controls. Lysolecithin, cholesterol esters and phosphatidylinositol (PI) levels were increased in HC platelets, and the levels of phosphatidylcholine (PC) were decreased. The cholesterol/phospholipid molar ratio of lipidemic platelets increased from 0.55 +/- 0.011 to 0.89 +/- 0.016 (P less than 0.01) in eight weeks. HC platelets had 90% more arachidonic acid (AA) in the PI than normal platelets. No significant changes in AA of PC were observed. Platelet function was monitored by the uptake and release of (14C)serotonin in platelet rich plasma (PRP), using varying concentrations of collagen as an aggregating agent. The uptake of (14C)serotonin in HC and normal platelets ranged from 78-94%. The percent of (14C)serotonin released from normal and HC platelets was proportional to the concentration of collagen. However, lipidemic platelets were hyperreactive to low concentrations of collagen. Incorporation of 50 microM acetylsalicylic acid into the aggregating medium suppressed the release of (14C)serotonin in normal PRP by more than 90%, but had only a partial effect on lipidemic PRP.

  5. Misshapen/NIK-related kinase (MINK1) is involved in platelet function, hemostasis, and thrombus formation.

    PubMed

    Yue, Ming; Luo, Dongjiao; Yu, Shanshan; Liu, Pu; Zhou, Qi; Hu, Mengjiao; Liu, Yangyang; Wang, Shuai; Huang, Qian; Niu, Yuxi; Lu, Linrong; Hu, Hu

    2016-02-18

    The sterile-20 kinase misshapen/Nck-interacting kinase (NIK)-related kinase 1 (MINK1) is involved in many important cellular processes such as growth, cytoskeletal rearrangement, and motility. Here, with MINK1-deficient (MINK1(-/-)) mice, we showed that MINK1 plays an important role in hemostasis and thrombosis via the regulation of platelet functions. In the tail-bleeding assay, MINK1(-/-) mice exhibited a longer bleeding time than wild-type (WT) mice (575.2 ± 59.7 seconds vs 419.6 ± 66.9 seconds). In a model of ferric chloride-induced mesenteric arteriolar thrombosis, vessel occlusion times were twice as long in MINK1(-/-) mice as in WT mice. In an in vitro microfluidic whole-blood perfusion assay, thrombus formation on a collagen matrix under arterial shear conditions was significantly reduced in MINK1(-/-) platelets. Moreover, MINK1(-/-) platelets demonstrated impaired aggregation and secretion in response to low doses of thrombin and collagen. Furthermore, platelet spreading on fibrinogen was largely hampered in MINK1(-/-) platelets. The functional differences of MINK1(-/-) platelets could be attributed to impaired adenosine 5'-diphosphate secretion. Signaling events associated with MINK1 appeared to involve extracellular signal-regulated kinase, p38, and Akt. Hence, MINK1 may be an important signaling molecule that mediates mitogen-activated protein kinase signaling and participates in platelet activation and thrombus formation. PMID:26598717

  6. Platelet Function Tests: A Review of Progresses in Clinical Application

    PubMed Central

    Choi, Jae-Lim; Li, Shuhua

    2014-01-01

    The major goal of traditional platelet function tests has been to screen and diagnose patients who present with bleeding problems. However, as the central role of platelets implicated in the etiology of arterial thrombotic diseases such as myocardial infarction and stroke became widely known, platelet function tests are now being promoted to monitor the efficacy of antiplatelet drugs and also to potentially identify patients at increased risk of thrombosis. Beyond hemostasis and thrombosis, an increasing number of studies indicate that platelets play an integral role in intercellular communication, are mediators of inflammation, and have immunomodulatory activity. As new potential biomarkers and technologies arrive at the horizon, platelet functions testing appears to take on a new aspect. This review article discusses currently available clinical application of platelet function tests, placing emphasis on essential characteristics. PMID:24895576

  7. Effects of Physical (In)activity on Platelet Function

    PubMed Central

    Heber, Stefan; Volf, Ivo

    2015-01-01

    As platelet activation is closely related to the liberation of growth factors and inflammatory mediators, platelets play a central role in the development of CVD. Virtually all cardiovascular risk factors favor platelet hyperreactivity and, accordingly, also physical (in)activity affects platelet function. Within this paper, we will summarize and discuss the current knowledge on the impact of acute and habitual exercise on platelet function. Although there are apparent discrepancies regarding the reported effects of acute, strenuous exercise on platelet activation, a deeper analysis of the available literature reveals that the applied exercise intensity and the subjects' cardiorespiratory fitness represent critical determinants for the observed effects. Consideration of these factors leads to the summary that (i) acute, strenuous exercise can lead to platelet activation, (ii) regular physical activity and/or physical fitness diminish or prevent platelet activation in response to acute exercise, and (iii) habitual physical activity and/or physical fitness also favorably modulate platelet function at physical rest. Notably, these effects of exercise on platelet function show obvious similarities to the well-recognized relation between exercise and the risk for cardiovascular events where vigorous exercise transiently increases the risk for myocardial infarction and a physically active lifestyle dramatically reduces cardiovascular mortality. PMID:26557653

  8. Characterization of multiple platelet activation pathways in patients with bleeding as a high-throughput screening option: use of 96-well Optimul assay

    PubMed Central

    Lordkipanidzé, Marie; Lowe, Gillian C.; Kirkby, Nicholas S.; Chan, Melissa V.; Lundberg, Martina H.; Morgan, Neil V.; Bem, Danai; Nisar, Shaista P.; Leo, Vincenzo C.; Jones, Matthew L.; Mundell, Stuart J.; Daly, Martina E.; Mumford, Andrew D.; Warner, Timothy D.; Watson, Steve P.; Watson, Steve P.; Mumford, Andrew D.; Mundell, Stuart J.; Gissen, Paul; Daly, Martina E.; Lester, Will; Clark, Justin; Williams, Mike; Motwani, Jayashree; Marshall, Dianne; Nyatanga, Priscilla; Mann, Pat; Kirwan, Julie; Wilde, Jonathan; Dunkley, Tracey; Greenway, April; Makris, Michael; Pavord, Sue; Dattani, Rashesh; Grimley, Gerry Dolan Charlotte; Stokley, Simone; Astwood, Emma; Chang, Cherry; Foros, Merri; Trower, Linda; Thachil, Jecko; Hay, Charlie; Pike, Gill; Will, Andrew; Grainger, John; Foulkes, Matt; Fareh, Mona; Talks, Kate; Biss, Tina; Kesteven, Patrick; Hanley, John; Vowles, Julie; Basey, Lesley; Barnes, Michelle; Collins, Peter; Rayment, Rachel; Alikhan, Raza; Morris, Ana Guerrero Rebecca; Mansell, Dianne; Toh, Cheng Hock; Martlew, Vanessa; Murphy, Elaine; Lachmann, Robin; Rose, Peter; Chapman, Oliver; Lokare, Anand; Marshall, Kathryn; Khan, Naseem; Keeling, David; Giangrande, Paul; Austin, Steve; Bevan, David; Alamelu, Jayanthi

    2014-01-01

    Up to 1% of the population have mild bleeding disorders, but these remain poorly characterized, particularly with regard to the roles of platelets. We have compared the usefulness of Optimul, a 96-well plate-based assay of 7 distinct pathways of platelet activation to characterize inherited platelet defects in comparison with light transmission aggregometry (LTA). Using Optimul and LTA, concentration-response curves were generated for arachidonic acid, ADP, collagen, epinephrine, Thrombin receptor activating-peptide, U46619, and ristocetin in samples from (1) healthy volunteers (n = 50), (2) healthy volunteers treated with antiplatelet agents in vitro (n = 10), and (3) patients with bleeding of unknown origin (n = 65). The assays gave concordant results in 82% of cases (κ = 0.62, P < .0001). Normal platelet function results were particularly predictive (sensitivity, 94%; negative predictive value, 91%), whereas a positive result was not always substantiated by LTA (specificity, 67%; positive predictive value, 77%). The Optimul assay was significantly more sensitive at characterizing defects in the thromboxane pathway, which presented with normal responses with LTA. The Optimul assay is sensitive to mild platelet defects, could be used as a rapid screening assay in patients presenting with bleeding symptoms, and detects changes in platelet function more readily than LTA. This trial was registered at www.isrctn.org as #ISRCTN 77951167. PMID:24408324

  9. Characterization of multiple platelet activation pathways in patients with bleeding as a high-throughput screening option: use of 96-well Optimul assay.

    PubMed

    Lordkipanidzé, Marie; Lowe, Gillian C; Kirkby, Nicholas S; Chan, Melissa V; Lundberg, Martina H; Morgan, Neil V; Bem, Danai; Nisar, Shaista P; Leo, Vincenzo C; Jones, Matthew L; Mundell, Stuart J; Daly, Martina E; Mumford, Andrew D; Warner, Timothy D; Watson, Steve P

    2014-02-20

    Up to 1% of the population have mild bleeding disorders, but these remain poorly characterized, particularly with regard to the roles of platelets. We have compared the usefulness of Optimul, a 96-well plate-based assay of 7 distinct pathways of platelet activation to characterize inherited platelet defects in comparison with light transmission aggregometry (LTA). Using Optimul and LTA, concentration-response curves were generated for arachidonic acid, ADP, collagen, epinephrine, Thrombin receptor activating-peptide, U46619, and ristocetin in samples from (1) healthy volunteers (n = 50), (2) healthy volunteers treated with antiplatelet agents in vitro (n = 10), and (3) patients with bleeding of unknown origin (n = 65). The assays gave concordant results in 82% of cases (κ = 0.62, P < .0001). Normal platelet function results were particularly predictive (sensitivity, 94%; negative predictive value, 91%), whereas a positive result was not always substantiated by LTA (specificity, 67%; positive predictive value, 77%). The Optimul assay was significantly more sensitive at characterizing defects in the thromboxane pathway, which presented with normal responses with LTA. The Optimul assay is sensitive to mild platelet defects, could be used as a rapid screening assay in patients presenting with bleeding symptoms, and detects changes in platelet function more readily than LTA. This trial was registered at www.isrctn.org as #ISRCTN 77951167. PMID:24408324

  10. The caspase-3 inhibitor (peptide Z-DEVD-FMK) affects the survival and function of platelets in platelet concentrate during storage

    PubMed Central

    Shiri, Reza; Ahmadinejad, Minoo; Vaeli, Shahram; Tabatabaei, Mohammad Reza

    2014-01-01

    Background Although apoptosis occurs in nucleated cells, studies show that this event also occurs in some anucleated cells such as platelets. During storage of platelets, the viability of platelets decreased, storage lesions were observed, and cells underwent apoptosis. We investigated the effects of caspase-3 inhibitor on the survival and function of platelets after different periods of storage. Methods Platelet concentrates were obtained from the Iranian Blood Transfusion Organization in plastic blood bags. Caspase-3 inhibitor (Z-DEVD-FMK) was added to the bags. These bags along with control bags to which no inhibitor was added were stored in a shaking incubator at 22℃ for 7 days. The effects of Z-DEVD-FMK on the functionality of platelets were analyzed by assessing their ability to bind to von Willebrand factor (vWF) and to aggregate in the presence of arachidonic acid and ristocetin. Cell survival was surveyed by MTT assay. Results At day 4 of storage, ristocetin-induced platelet aggregation was significantly higher in the inhibitor-treated (test) than in control samples; the difference was not significant at day 7. There was no significant difference in arachidonic acid-induced platelet aggregation between test and control samples. However, at day 7 of storage, the binding of platelets to vWF was significantly higher in test than in control samples. The MTT assay revealed significantly higher viability in test than in control samples at both days of study. Conclusion Treatment of platelets with caspase-3 inhibitor could increase their functionality and survival. PMID:24724067

  11. Function of human platelets during extracorporeal circulation.

    PubMed

    Hennessy, V L; Hicks, R E; Niewiarowski, S; Edmunds, L H; Colman, R W

    1977-06-01

    The interaction between human platelets and nonbiologic surfaces was studied during in vitro recirculation of 500 ml of fresh, heparinized human blood in four different perfusion circuits. Circuits differed in surface area (0.1 m2 or 0.9 m2) and in surface composition. No important differences were observed between standard silicone-rubber and filler-free, silicone-rubber surfaces. Platelet counts decreased to 85% of control in 0.1- m2 circuits, but retained normal sensitivity to aggregating agents and released only small amounts of platelet factor 4 (PF4). In contrast, platelet counts in 0.9-m2 circuits decreased to 20% of control within 2 min and platelet sensitivity was depressed out of proportion to the fall in platelet count. Plasma PF4 progressively increased and platelet PF4 content progressively decreased during 6 h of recirculation. The results indicate that human platelets may exist in three conditions during extracorporeal circulation. Some platelets are unaltered, some are less sensitive to aggregating agents, and others have undergone extensive release. The ratio of blood volume to surface area appears to be an important determinant of platelet-surface interaction. PMID:18017

  12. Changes in platelet function following cold storage of RBC suspensions

    PubMed Central

    Dang, Qian-Li; Li, Jian-Gang; Sun, Yang; Jin, Zhan-Kui; Gao, Ying; Xu, Cui-Xiang; Chen, Ping; Ma, Ting; Yang, Jiang-Cun

    2015-01-01

    Objective: To provide a basis for the cold-storage of human platelets as a way to assess changes in platelet function. Methods: Red blood cell suspensions (11 U and 50 U) were randomly selected at different storage times (3-28 days) and evidence of platelet activation (CD62P) and thromboelastography (TEG) reaction times were investigated. Results: After 21 days of storage at 4°C, a large number of activated platelets (PAC1+62P+, PAC1-62P+) within the red blood cell suspension (RBCs) retained their function and had TEG-maximum amplitude (TEG-MA) indices in the normal range. Conclusion: We report that platelets in RBC suspensions retain high activity when stored at 4°C for 21 days. The results provide important information for studies that involve storing platelets under cold conditions. PMID:26770402

  13. Platelet Consumption by Arterial Prostheses: The Effects of Endothelialization and Pharmacologic Inhibition of Platelet Function

    PubMed Central

    Harker, Laurence A.; Slichter, Sherrill J.; Sauvage, Lester R.

    1977-01-01

    The thrombogenic mechanism of arterial grafts has been studied by determining the relative utilization of platelets, fibrinogen and plasminogen by human arterial prostheses, and by direct examination of arterial grafts in a baboon model. Forty-one survival and turnover measurements of 51Crplatelets, 131I-fibrinogen and 125I-plasminogen in ten patients with aortofemoral knitted Dacron prostheses demonstrated platelet consumption after graft placement (platelet survival 4.2 days ± 0.5 and turnover 68,000 plat/ul/day ±10,000 compared with 8.2 days ± 0.3 and 35,000 plat/ul/day ± 5,000 respectively for control subjects with stable vascular disease, p < 0.01). In vitro platelet function test results were normal. Platelet consumption was interrupted by dipyridamole or a combination of dipyridamole and acetylsalicylic acid, and platelet survival normalized spontaneously during nine months postoperatively. No significantly increased consumption of fibrinogen or plasminogen was found in these patients with arterial grafts. Placement of impervious knitted Dacron velour aortic grafts in baboons reproduced platelet consumption that progressively normalized over six weeks postoperatively. Platelet survival measurements correlated directly with endothelial cell coverage of the graft luminal surface in these animals implying that endothelialization of the graft surface was also occurring postoperatively in patients. ImagesFig. 4.Fig. 5. PMID:411428

  14. Role of platelet function and platelet membrane glycoproteins in children with primary immune thrombocytopenia

    PubMed Central

    Liu, Wen-Jun; Bai, Jing; Guo, Qu-Lian; Huang, Zhe; Yang, Hong; Bai, Yong-Qi

    2016-01-01

    The aim of the present study was to examine and understand changes in platelet functions prior to and after the treatment of primary immune thrombocytopenia (ITP) in children. An automatic hematology analyzer and whole blood flow cytometry were used to detect immature platelet fraction (IPF), IPC and membrane glycoproteins (CD62p, PAC-1 and CD42b) in ITP children (ITP group), children with complete response after ITP treatment (ITP-CR group) and children with elective surgery (normal control group). The results showed that, levels of platelet count (PLT) and plateletcrit in the ITP group were lower alhtough the levels of mean platelet volume, platelet distribution width and platelet-large cell ratio (P-LCR) were higher than those in the normal control and ITP-CR groups. PLT in the ITP-CR group was lower than that in the normal controls. Additionally, IPF% was higher in the normal control and ITP-CR groups, IPC was lower in the ITP group compared to the normal control and ITP-CR groups. Furthermore, prior to ADP activation, the expression levels of CD62p, PAC-1 and CD42b in the ITP group were lower in ITP group than those in the normal control and ITP-CR groups. The expression level of PAC-1 was lower in the ITP-CR and normal control groups. No differences were identified in CD62p and CD42b expression levels. Following ATP activation, CD62p, PAC-1 and CD42b expression in the ITP group was lower than that in the normal control and ITP-CR groups. PAC-1 expression was lower while CD62p expression was higher in the ITP-CR group compared to the normal control group. In conclusion, the activation of platelets in ITP children was low. Decreased platelet function, platelet parameters and platelet glycoproteins may be used as markers for monitoring the treatment efficacy in ITP children. PMID:27431926

  15. Metastasis: new functional implications of platelets and megakaryocytes.

    PubMed

    Leblanc, Raphael; Peyruchaud, Olivier

    2016-07-01

    Platelets are essential components of hemostasis. Due to a plethora of factors released on activation, platelet functions are also connected to tumor growth, notably by acting on angiogenesis. It is now well recognized that major roles of platelets in the poor outcome of cancer patients occurs during hematogenous dissemination of cancer cells. In this review, we describe recent insights into the molecular mechanisms supporting the prometastatic activity of platelets. Platelets have been shown to promote survival of circulating tumor cells (CTCs) in the bloodstream by conferring resistance to the shear stress and attack from natural killer cells. Recently, platelets were found to promote and/or maintain the state of epithelial to mesenchymal transition on CTCs through platelet secretion of transforming growth factor β in response to CTC activation. At a later stage in the metastatic process, platelets promote extravasation and establishment of metastatic cells in distant organs as observed in bone. This particular environment is also the site of hematopoiesis, megakaryocytopoiesis, and platelet production. Increasing the number of megakaryocytes (MKs) in the bone marrow results in a high bone mass phenotype and inhibits skeletal metastasis formation of prostate cancer cells. As a result of their specific location in vascular niches in the bone marrow, MK activity might contribute to the "seed and soil" suitability between CTCs and bone. In conclusion, recent findings have made a great advance in our knowledge on how platelets contribute to the metastatic dissemination of cancer cells and that may support the development of new antimetastasis therapies. PMID:27154188

  16. Methodological issues in the preparation and assay of platelet 3H-imipramine binding.

    PubMed

    Severson, J A; Schneider, L S; Fredrickson, E R

    1990-07-01

    Several methodological factors in the preparation of platelets and the determination of platelet 3H-imipramine (3H-IMI) binding were examined. The ionic composition of the assay significantly affected platelet 3H-IMI binding. Approximately 25% of the specific binding of 3H-IMI to intact platelet preparations was retained in the absence of sodium and chloride ions. The addition of sodium ions enhanced the specific binding of 3H-IMI, but the addition of chloride in the presence of sodium had a more pronounced effect, enhancing binding approximately five-fold over that observed with the addition of sodium. Sodium was the only cation tested that enhanced binding. Only halides enhanced binding in the presence of sodium with the following order of potency: Cl- greater than Br- greater than I- = F-. Ions increased the density of binding sites (Bmax) and did not affect the affinity of the binding sites for 3H-IMI. In the presence of sodium and chloride, the use of serotonin (5HT) to define nonspecific binding in saturation experiments resulted in lower binding densities (Bmax) than when desipramine was used to define nonspecific binding. The component of binding that was insensitive to 5HT was roughly equal to the Bmax of 3H-IMI binding obtained in the absence of sodium and chloride using desipramine to define nonspecific binding. Overall, these data suggest that not all 3H-IMI binding that is displaced by desipramine is related to serotonergic mechanisms, and suggest that 5HT is a better choice than desipramine for the determination of the nonspecific binding of 3H-IMI. In addition, the binding of 3H-IMI to different platelet preparations was compared. The binding of 3H-IMI to intact platelets was less than that obtained using lysed platelet membranes when data were expressed per mg protein. The Coomassie Blue dye-binding method to determine platelet protein resulted in greater Bmax values than were obtained with the Folin phenol reagent method. The method of platelet

  17. The effect of time of day and exercise on platelet functions and platelet-neutrophil aggregates in healthy male subjects.

    PubMed

    Aldemir, Hatice; Kiliç, Nedret

    2005-12-01

    Platelet activation state changes by exercise. The effect of exercise time on platelet activation state and formation of platelet-neutrophil aggregates are not known yet. In this study the effect of exercise and time of day were examined on platelet activity with platelet-neutrophil aggregates. Ten moderately active males aged 27+/- 1.63 (mean+/-S.D.) years completed sub-maximal (70% VO(2max)) exercise trials for 30 min. Blood pressure (BP) was recorded. Venous blood samples were obtained at rest, immediately post-exercise and after 30 min of recovery. Whole blood was analysed for haematocrit (Hct), haemoglobin (Hb), platelet count (PC), mean platelet count (MPV) and platelet aggregation (PA). Platelet-neutrophil aggregates and beta-thromboglobulin (beta-TG) levels were assayed. Platelet count showed significant increase after morning exercise ((236+/- 32)x10(9) l(-1) versus (202+/- 34)x10(9) l(-1) baseline, p < 0.05). Exercise resulted in significantly lower MPV after the evening exercise (9.16+/- 0.5 fl versus 9.65+/- 0.36 fl, p < 0.05). Platelet aggregation by adenosine diphosphate (ADP) decreased after morning exercise and the recovery aggregation levels were significantly different at two different times of the day (68+/- 20% a.m. versus 80+/- 12% p.m., p < 0.05). It was also showed that platelet-neutrophil aggregates increased significantly from baseline after both exercises. Exercise-induced platelet-neutrophil aggregates were higher in the evening (10.7+/- 1.3% p.m. versus 6.4+/- 1.8% a.m., p < 0.0001). It is therefore concluded that besides platelet-platelet aggregation, exercise can cause platelet- neutrophil aggregates. In addition, time of day has an effect on platelet activation related events. Circadian variations of physiological parameters may have an effect on thrombus formation by platelet activation. PMID:16311912

  18. Effect of circulating epinephrine on platelet function and hematocrit.

    PubMed

    Kjeldsen, S E; Weder, A B; Egan, B; Neubig, R; Zweifler, A J; Julius, S

    1995-05-01

    We investigated the effect of raising arterial plasma epinephrine within the lower pathophysiological concentration range on various indicators of blood platelet function and hematocrit. Epinephrine was raised over 60 minutes by a stepwise increasing intravenous infusion in 40 healthy men aged 20 to 40 years. Platelet count increased progressively with increasing arterial epinephrine to a maximal change of 69 +/- 6 x 10(9)/L in EDTA-anticoagulated blood and a maximal change of 42 +/- 6 x 10(9)/L in acid-citrate-dextrose (ACD)-anticoagulated blood, and the weight of circulating platelets increased by 29% (P < .001). Platelet size increased significantly in EDTA and decreased in ACD, and the difference between EDTA and ACD was significant (P < .0001) for both count and size, suggesting that epinephrine not only recruits platelets into the circulation but also induces some microaggregation in vivo or adhesion ex vivo. Aggregation of platelets in vitro induced by epinephrine decreased (P < .003 for delta optical density and P = .038 for maximal optical density) after epinephrine infusion compared with saline but did not change when stimulated with ADP or collagen. These findings suggest a selective downregulation of the epinephrine-activating mechanisms concomitant with a rise in the platelet content of epinephrine by 81% (P < .001) and no change in the platelet sodium-proton membrane exchange. The release of granular content (beta-thromboglobulin and platelet factor 4) to the circulation in response to epinephrine was not significant. Thus, under acute conditions it seems that the platelets may protect themselves against inappropriate overstimulation by epinephrine. The importance of platelet epinephrine uptake is still unknown, but sodium-proton exchange does not seem to be involved in regulating the effects of circulating epinephrine on platelet function. Epinephrine has a pronounced effect on raising hematocrit (maximal change of 1.74 +/- 0.13 x 10(-2), P < .0001

  19. Ultraviolet irradiation of platelet concentrate abrogates lymphocyte activation without affecting platelet function in vitro

    SciTech Connect

    Kahn, R.A.; Duffy, B.F.; Rodey, G.G.

    1985-11-01

    We studied the effect of ultraviolet (UV) radiation on platelet concentrates. Samples irradiated at 310 mm for 30 minutes at a dose of 1782 J per m2 showed no loss of platelet function in vitro as determined by adenosine diphosphate, collagen, or ristocetin-induced aggregation. Lymphocytes isolated from irradiated units were unable to act as responders or stimulators in a mixed-lymphocyte reaction. These data suggest that UV radiation of platelet concentrates may result in a cell suspension that is unable to evoke an immunological response.

  20. Decreased platelet function in aortic valve stenosis: high shear platelet activation then inactivation.

    PubMed Central

    O'Brien, J. R.; Etherington, M. D.; Brant, J.; Watkins, J.

    1995-01-01

    OBJECTIVE--To elucidate the mechanism of the bleeding tendency observed in patients with aortic valve stenosis. DESIGN--A prospective study of high and low shear platelet function tests in vitro in normal controls compared with that in patients with severe aortic valve stenosis with a mean (SD) systolic gradient by Doppler of 75 (18) mm Hg before and at least 4 months after aortic valve replacement. SETTING--District general hospital. RESULTS--The patients showed reduced retention in the high shear platelet function tests. (a) Platelet retention in the filter test was 53.6 (12.6)% in patients with aortic valve stenosis and 84.8 (9.6)% in the controls (P < 0.001). (b) Retention in the glass bead column test was 49.8 (19.2) in the patients and 87.4 (8.7) in the controls (P < 0.001). (c) The standard bleeding time was longer in the patients (P < 0.06). Results of the high shear tests (a, b, and c) after aortic valve replacement were within the normal range. The platelet count was low but within the normal range before surgery and increased postoperatively (P < 0.01). There were no differences in the results of standard clotting tests, plasma and intraplatelet von Willebrand's factor, or in 15 platelet aggregation tests using five agonists between patients with aortic valve stenosis and controls. CONCLUSIONS--The high shear haemodynamics of aortic valve stenosis modify platelet function in vivo predisposing to a bleeding tendency. This abnormality of platelet function is detectable only in vitro using high shear tests. The abnormal function is reversed by aortic valve replacement. High shear forces in vitro activate and then inactivate platelets. By the same mechanisms aortic valve stenosis seems to lead to high shear damage in vivo, resulting in a clinically important bleeding tendency in some patients. PMID:8541170

  1. Morphological and functional platelet abnormalities in Berkeley sickle cell mice.

    PubMed

    Shet, Arun S; Hoffmann, Thomas J; Jirouskova, Marketa; Janczak, Christin A; Stevens, Jacqueline R M; Adamson, Adewole; Mohandas, Narla; Manci, Elizabeth A; Cynober, Therese; Coller, Barry S

    2008-01-01

    Berkeley sickle cell mice are used as animal models of human sickle cell disease but there are no reports of platelet studies in this model. Since humans with sickle cell disease have platelet abnormalities, we studied platelet morphology and function in Berkeley mice (SS). We observed elevated mean platelet forward angle light scatter (FSC) values (an indirect measure of platelet volume) in SS compared to wild type (WT) (37+/-3.2 vs. 27+/-1.4, mean+/-SD; p<0.001), in association with moderate thrombocytopenia (505+/-49 x 10(3)/microl vs. 1151+/-162 x 10(3)/microl; p<0.001). Despite having marked splenomegaly, SS mice had elevated levels of Howell-Jolly bodies and "pocked" erythrocytes (p<0.001 for both) suggesting splenic dysfunction. SS mice also had elevated numbers of thiazole orange positive platelets (5+/-1% vs. 1+/-1%; p<0.001), normal to low plasma thrombopoietin levels, normal plasma glycocalicin levels, normal levels of platelet recovery, and near normal platelet life spans. Platelets from SS mice bound more fibrinogen and antibody to P-selectin following activation with a threshold concentration of a protease activated receptor (PAR)-4 peptide compared to WT mice. Enlarged platelets are associated with a predisposition to arterial thrombosis in humans and some humans with SCD have been reported to have large platelets. Thus, additional studies are needed to assess whether large platelets contribute either to pulmonary hypertension or the large vessel arterial occlusion that produces stroke in some children with sickle cell disease. PMID:18374611

  2. Effects of the new glycopeptide antibiotic teicoplanin on platelet function and blood coagulation.

    PubMed Central

    Agnelli, G; Longetti, M; Guerciolini, R; Menichetti, F; Grasselli, S; Boldrini, F; Bucaneve, G; Nenci, G G; Del Favero, A

    1987-01-01

    Teicoplanin, a new glycopeptide antibiotic, is structurally related to ristocetin, an antibiotic known to induce human platelet agglutination and, thus, thrombocytopenia and thromboembolic side effects. The aim of this study was to evaluate the effects of teicoplanin on platelet function in vitro and ex vivo and on blood coagulation ex vivo. In the in vitro studies, spontaneous platelet aggregation; platelet aggregation induced by ADP, collagen, and ristocetin; and the release of beta-thromboglobulin from platelets were assessed. Platelets from healthy subjects were incubated with teicoplanin at final concentrations of 100, 1,500, 5,000, and 10,000 micrograms/ml. The maximal achievable concentration with therapeutic doses is 100 micrograms/ml. When compared with saline, teicoplanin at concentrations of 100 and 1,500 micrograms/ml had no effect on platelet function, but at concentrations of 5,000 and 10,000 micrograms/ml, it induced greater spontaneous platelet aggregation (P less than 0.01) and inhibited platelet aggregation induced by ADP, collagen, and ristocetin (P less than 0.01). Teicoplanin at concentrations of 100, 1,500, and 5,000 micrograms/ml did not induce the release of beta-thromboglobulin, in contrast to teicoplanin at a concentration of 10,000 micrograms/ml and ristocetin at a concentration of 1.5 mg/ml (P less than 0.01). In the ex vivo studies, platelet count, bleeding time, plasma beta-thromboglobulin, platelet aggregation induced by ADP, ristocetin, and epinephrine, activated partial thromboplastin time, prothrombin time, thrombin clotting time, and serum fibrinogen degradation products were evaluated at days 0, 3, and 6 and at 72 h after the end of therapy. All subjects completed the study without evidence of side effects. When compared with the pretreatment values, none of the values from these assays showed a significant change at any time during and after treatment. We concluded that platelet function and blood coagulation are not affected by

  3. Point-of-care platelet function tests: detection of platelet inhibition induced by nonopioid analgesic drugs.

    PubMed

    Scharbert, Gisela; Gebhardt, Kristina; Sow, Zacharia; Duris, Monika; Deusch, Engelbert; Kozek-Langenecker, Sibylle

    2007-12-01

    Detection of platelet inhibition is of clinical relevance in the preinterventional risk-benefit assessment in chronic low-back-pain patients scheduled for invasive pain therapy. We evaluated the sensitivity of various point-of-care platelet function tests for the detection of platelet inhibition induced by nonopioid analgesic drugs. After Institutional Review Board approval and informed consent, citrated whole blood from 40 patients with chronic unspecific low back pain was investigated before and 30 min after intravenous infusion of the study medication consisting of diclofenac 75 mg (plus orphenadrin 30 mg; Neodolpasse; Fresenius Kabi Austria GmbH, Austria), parecoxib 40 mg (Dynastat; Pharmacia Europe EEIG, UK), paracetamol 1 g (Perfalgan; Bieffe Medital S.P.A., Italy), or normal saline in a randomized, cross-over, double-blinded, placebo-controlled study. Platelet function was assessed using the PFA-100 platelet function analyzer and thromboelastometry, as well as impedance aggregometry (in the last 17 patients recruited after it became commercially available). Sensitivity for detecting diclofenac-induced platelet inhibition was 85% for the PFA-100 using epinephrine as agonist and 94% for arachidonic acid-induced impedance aggregometry. ADP-induced platelet function tests, as well as cytochalasin D-modified thromboelastometry were unreliable. All tests had a low incidence of false-positive test results after normal saline. Paracetamol and parecoxib had no significant platelet inhibiting effect. The PFA-100 using epinephrine as agonist and arachidonic acid-induced impedance aggregometry are recommended for the detection of cyclooxygenase-I-inhibiting effects of nonsteroidal anti-inflammatory drugs such as diclofenac. Our findings confirm that a single rescue dose of paracetamol and parecoxib has no antiplatelet effect. PMID:17982319

  4. Platelet Serotonin Transporter Function Predicts Default-Mode Network Activity

    PubMed Central

    Kasess, Christian H.; Meyer, Bernhard M.; Hofmaier, Tina; Diers, Kersten; Bartova, Lucie; Pail, Gerald; Huf, Wolfgang; Uzelac, Zeljko; Hartinger, Beate; Kalcher, Klaudius; Perkmann, Thomas; Haslacher, Helmuth; Meyer-Lindenberg, Andreas; Kasper, Siegfried; Freissmuth, Michael; Windischberger, Christian; Willeit, Matthäus; Lanzenberger, Rupert; Esterbauer, Harald; Brocke, Burkhard; Moser, Ewald; Sitte, Harald H.; Pezawas, Lukas

    2014-01-01

    Background The serotonin transporter (5-HTT) is abundantly expressed in humans by the serotonin transporter gene SLC6A4 and removes serotonin (5-HT) from extracellular space. A blood-brain relationship between platelet and synaptosomal 5-HT reuptake has been suggested, but it is unknown today, if platelet 5-HT uptake can predict neural activation of human brain networks that are known to be under serotonergic influence. Methods A functional magnetic resonance study was performed in 48 healthy subjects and maximal 5-HT uptake velocity (Vmax) was assessed in blood platelets. We used a mixed-effects multilevel analysis technique (MEMA) to test for linear relationships between whole-brain, blood-oxygen-level dependent (BOLD) activity and platelet Vmax. Results The present study demonstrates that increases in platelet Vmax significantly predict default-mode network (DMN) suppression in healthy subjects independent of genetic variation within SLC6A4. Furthermore, functional connectivity analyses indicate that platelet Vmax is related to global DMN activation and not intrinsic DMN connectivity. Conclusion This study provides evidence that platelet Vmax predicts global DMN activation changes in healthy subjects. Given previous reports on platelet-synaptosomal Vmax coupling, results further suggest an important role of neuronal 5-HT reuptake in DMN regulation. PMID:24667541

  5. Effects of microgravity and hypergravity on platelet functions.

    PubMed

    Dai, Kesheng; Wang, Yuedan; Yan, Rong; Shi, Quanwei; Wang, Zhicheng; Yuan, Yanhong; Cheng, Hong; Li, Suping; Fan, Yubo; Zhuang, Fengyuan

    2009-05-01

    Many serious thrombotic and haemorrhagic diseases or fatalities have been documented in human being exposed to microgravity or hypergravity environments, such as crewmen in space, roller coaster riders, and aircrew subjected to high-G training. Some possible related organs have been examined to explore the mechanisms underlying these gravity change-related diseases. However, the role of platelets which are the primary players in both thrombosis and haemostasis is unknown. Here we show that platelet aggregation induced by ristocetin or collagen and platelet adhesion to von Willebrand factor (VWF) were significantly decreased after platelets were exposed to simulated microgravity. Conversely, these platelet functions were increased after platelets were exposed to hypergravity. The tail bleeding time in vivo was significantly shortened in mice exposed to high-G force, whereas, was prolonged in hindlimb unloaded mice. Furthermore, three of 23 mice died after 15 minutes of -8 Gx stress. Platelet thrombi disseminated in the heart ventricle and blood vessels in the brain, lung, and heart from the dead mice. Finally, glycoprotein (GP) Ibalpha surface expression and its association with the cytoskeleton were significantly decreased in platelets exposed to simulated microgravity, and obviously increased in hypergravity-exposed platelets. These data indicate that the platelet functions are inhibited in microgravity environments, and activated under high-G conditions, suggesting a novel mechanism for gravity change-related haemorrhagic and thrombotic diseases. This mechanism has important implications for preventing and treating gravity change-related diseases, and also suggests that special attentions should be paid to human actions under different gravity conditions. PMID:19404544

  6. Hemostatic Function, Survival, and Membrane Glycoprotein Changes in Young versus Old Rabbit Platelets

    PubMed Central

    Blajchman, Morris A.; Senyi, Andrew F.; Hirsh, Jack; Genton, Edward; George, James N.

    1981-01-01

    Although in vitro studies have demonstrated functional differences between young and old platelets, in vivo differences have not been precisely established. Therefore the in vivo hemostatic function of young and old platelets and the survival time have been examined in rabbits. The hemostatic function was measured by performing serial ear bleeding times in irradiation-induced thrombocytopenic rabbits. After irradiation with 930 rad the platelet count gradually diminished reaching a nadir (∼20 × 103/μl) at 10 d. The platelets present in the circulation, 7-10 d after irradiation, were considered old platelets, and the platelets present after recovery, 11-14 d postirradiation, young platelets. The measurement of platelet size was consistent with the hypothesis that platelets become smaller with age: the mean size was 3.84 μm3 for old platelets and 5.86 μm3 for young platelets. Regression analysis of the relationship between the bleeding time and the platelet count in 18 rabbits showed a significantly different slope for rabbits with predominantly old platelets compared with rabbits with predominantly young platelets (P < 0.001). Young platelets were more effective giving much shorter bleeding times than old platelets at comparable platelet counts. Survival times of young and old platelets were measured using platelets harvested on day 8 postirradiation (old platelets) and day 12 postirradiation (young platelets) that were labeled and then reinjected into normal recipient animals. The mean platelet survival time, calculated by gamma function, of old platelets was 28.8 h; of young platelets, 87.4 h; and of normally circulating heterogeneous platelets, (normal platelets) 53.0 h. Notably, the survival of old platelets was found to be exponential, and of young platelets, linear. Analysis of the membrane glycoproteins in young, old and normal platelets indicated that there was no qualitative difference amongst the young, normal, and old platelets. The relative

  7. Role of platelet function and platelet membrane glycoproteins in children with primary immune thrombocytopenia.

    PubMed

    Liu, Wen-Jun; Bai, Jing; Guo, Qu-Lian; Huang, Zhe; Yang, Hong; Bai, Yong-Qi

    2016-09-01

    The aim of the present study was to examine and understand changes in platelet functions prior to and after the treatment of primary immune thrombocytopenia (ITP) in children. An automatic hematology analyzer and whole blood flow cytometry were used to detect immature platelet fraction (IPF), IPC and membrane glycoproteins (CD62p, PAC-1 and CD42b) in ITP children (ITP group), children with complete response after ITP treatment (ITP-CR group) and children with elective surgery (normal control group). The results showed that, levels of platelet count (PLT) and plateletcrit in the ITP group were lower alhtough the levels of mean platelet volume, platelet distribution width and platelet-large cell ratio (P-LCR) were higher than those in the normal control and ITP-CR groups. PLT in the ITP-CR group was lower than that in the normal controls. Additionally, IPF% was higher in the normal control and ITP-CR groups, IPC was lower in the ITP group compared to the normal control and ITP-CR groups. Furthermore, prior to ADP activation, the expression levels of CD62p, PAC-1 and CD42b in the ITP group were lower in ITP group than those in the normal control and ITP-CR groups. The expression level of PAC-1 was lower in the ITP-CR and normal control groups. No differences were identified in CD62p and CD42b expression levels. Following ATP activation, CD62p, PAC-1 and CD42b expression in the ITP group was lower than that in the normal control and ITP-CR groups. PAC-1 expression was lower while CD62p expression was higher in the ITP-CR group compared to the normal control group. In conclusion, the activation of platelets in ITP children was low. Decreased platelet function, platelet parameters and platelet glycoproteins may be used as markers for monitoring the treatment efficacy in ITP children. PMID:27431926

  8. Modification of Solid Phase Red Cell Adherence Assay for the Detection of Platelet Antibodies in Patients With Thrombocytopenia

    PubMed Central

    Vongchan, Preeyanat; Nawarawong, Weerasak; Linhardt, Robert J.

    2009-01-01

    Platelet refractoriness is caused by HLA antibodies and platelet-specific antibodies. Current methods used to detect antiplatelet antibodies have limitations. Solid phase red cell adherence (SPRCA) lacks sensitivity and requires a second assay using chloroquine-treated intact platelets to specify the response due to anti-HLA. We modified SPRCA by using 2 types of antihuman platelet antibodies with different specificities toward platelet lysate and tested samples from 361 patients (69 with unexplained thrombocytopenia and 292 with poor response to platelet transfusions not explicable by alloimmunization or the clinical situation) and 50 from healthy volunteers. Our method compared favorably with platelet suspension direct immunofluorescence. All samples from healthy volunteers were negative; of the samples from the patient population, 240 were positive (147 samples had only antiplatelet and 3 samples had only anti-HLA antibodies). This modified technique had a sensitivity of 98% and a specificity of 91%. PMID:18701420

  9. Platelet functional and transcriptional changes induced by intralipid infusion.

    PubMed

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

    2016-06-01

    Multiple studies have shown the effects of long-term exposure to high-fat or western diets on the vascular system. There is limited knowledge on the acute effects of high circulating fat levels, specifically on platelets, which have a role in many processes, including thrombosis and inflammation. This study investigated the effects of acute, high-fat exposure on platelet function and transcript profile. Twenty healthy participants were given an intravenous infusion of 20% Intralipid emulsion and heparin over 6 hours. Blood samples were taken prior to and the day after infusion to measure platelet function and transcript expression levels. Platelet aggregation was not significantly affected by Intralipid infusion, but, when mitochondria function was inhibited by carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or oligomycin, platelet aggregation was higher in the post-infusion state compared to baseline. Through RNA sequencing, and verified by RT-qPCR, 902 miRNAs and 617 mRNAs were affected by Intralipid infusion. MicroRNAs increased include miR-4259 and miR-346, while miR-517b and miR-517c are both decreased. Pathway analysis identified two clusters significantly enriched, including cell motility. In conclusion, acute exposure to high fat affects mitochondrial-dependent platelet function, as well as the transcript profile. PMID:26940969

  10. Platelet hexosaminidase a enzyme assay effectively detects carriers missed by targeted DNA mutation analysis.

    PubMed

    Nakagawa, Sachiko; Zhan, Jie; Sun, Wei; Ferreira, Jose Carlos; Keiles, Steven; Hambuch, Tina; Kammesheidt, Anja; Mark, Brian L; Schneider, Adele; Gross, Susan; Schreiber-Agus, Nicole

    2012-01-01

    Biochemical testing of hexosaminidase A (HexA) enzyme activity has been available for decades and has the ability to detect almost all Tay-Sachs disease (TSD) carriers, irrespective of ethnic background. This is increasingly important, as the gene pool of those who identify as Ashkenazi Jewish is diversifying. Here we describe the analysis of a cohort of 4,325 individuals arising from large carrier screening programs and tested by the serum and/or platelet HexA enzyme assays and by targeted DNA mutation analysis. Our results continue to support the platelet assay as a highly effective method for TSD carrier screening, with a low inconclusive rate and the ability to detect possible disease-causing mutation carriers that would have been missed by targeted DNA mutation analysis. Sequence analysis performed on one such platelet assay carrier, who had one non-Ashkenazi Jewish parent, identified the amino acid change Thr259Ala (A775G). Based on crystallographic modeling, this change is predicted to be deleterious, as threonine 259 is positioned proximal to the HexA alpha subunit active site and helps to stabilize key residues therein. Accordingly, if individuals are screened for TSD in broad-based programs by targeted molecular testing alone, they must be made aware that there is a more sensitive and inexpensive test available that can identify additional carriers. Alternatively, the enzyme assays can be offered as a first tier test, especially when screening individuals of mixed or non-Jewish ancestry. PMID:23430931

  11. Utility of the ISTH bleeding assessment tool in predicting platelet defects in participants with suspected inherited platelet function disorders

    PubMed Central

    Lowe, G C; Lordkipanidzé, M; Watson on behalf of the uk gapp study group, S P

    2013-01-01

    Background The ISTH bleeding assessment tool (ISTH-BAT) was developed to record bleeding symptoms and to aid diagnosis in patients with a possible bleeding disorder. Objectives To investigate the utility of the ISTH-BAT in predicting functional defects in platelet activation in participants with suspected inherited platelet function disorders. Patients/Methods Participants with clinical evidence of excessive bleeding and suspected inherited platelet function disorders and healthy volunteers were recruited to the Genotyping and Phenotyping of Platelets study (GAPP; ISRCTN 77951167). The ISTH-BAT questionnaire was applied by a trained investigator prior to lumiaggregometry. Results One hundred participants were included (79 with suspected inherited platelet function disorders, and 21 healthy volunteers). The ISTH-BAT score in participants with suspected inherited platelet function disorders (median 12; interquartile range [IQR] 8–16) was significantly higher than in healthy volunteers (median 0; IQR 0–0). There was no difference between participants with suspected inherited platelet function disorders with a platelet defect detected by lumiaggregometry (median 11; IQR 8–16) and those with normal platelet function (median 12; IQR 8–14) (P > 0.05). The ISTH-BAT score was not associated with a demonstrable platelet defect on platelet function testing (area under the receiver operating characteristic curve = 0.501 [95% confidence interval 0.372–0.630, P = 0.98] and odds ratio 1.01 [95% confidence interval 0.93–1.09, P = 0.91]). Conclusions The ISTH-BAT is a powerful tool for documenting lifelong bleeding history. However, the score obtained is not predictive of the presence of a platelet defect on lumiaggregometry in patients with suspected inherited platelet function disorders. PMID:23809206

  12. Identification of platelet function defects by multi-parameter assessment of thrombus formation

    PubMed Central

    de Witt, Susanne M.; Swieringa, Frauke; Cavill, Rachel; Lamers, Moniek M. E.; van Kruchten, Roger; Mastenbroek, Tom; Baaten, Constance; Coort, Susan; Pugh, Nicholas; Schulz, Ansgar; Scharrer, Inge; Jurk, Kerstin; Zieger, Barbara; Clemetson, Kenneth J.; Farndale, Richard W.; Heemskerk, Johan W. M.; Cosemans, Judith M.E.M.

    2014-01-01

    Assays measuring platelet aggregation (thrombus formation) at arterial shear rate mostly use collagen as only platelet-adhesive surface. Here we report a multi-surface and multi-parameter flow assay to characterize thrombus formation in whole blood from healthy subjects and patients with platelet function deficiencies. A systematic comparison is made of 52 adhesive surfaces with components activating the main platelet-adhesive receptors, and of eight output parameters reflecting distinct stages of thrombus formation. Three types of thrombus formation can be identified with a predicted hierarchy of the following receptors: glycoprotein (GP)VI, C-type lectin-like receptor-2 (CLEC-2)>GPIb>α6β1, αIIbβ3>α2β1>CD36, α5β1, αvβ3. Application with patient blood reveals distinct abnormalities in thrombus formation in patients with severe combined immune deficiency, Glanzmann’s thrombasthenia, Hermansky–Pudlak syndrome, May–Hegglin anomaly or grey platelet syndrome. We suggest this test may be useful for the diagnosis of patients with suspected bleeding disorders or a pro-thrombotic tendency. PMID:25027852

  13. Influence of gold nanoparticles on platelets functional activity in vitro

    NASA Astrophysics Data System (ADS)

    Akchurin, Garif G.; Akchurin, George G.; Ivanov, Alexey N.; Kirichuk, Vyacheslav F.; Terentyuk, George S.; Khlebtsov, Boris N.; Khlebtsov, Nikolay G.

    2008-02-01

    Now in the leading biomedical centers of the world approved new technology of laser photothermal destruction of cancer cells using plasmon gold nanoparticles. Investigations of influence of gold nanoparticles on white rat platelets aggregative activity in vitro have been made. Platelet aggregation was investigated in platelet rich plasma (PRP) with help of laser analyzer 230 LA <>, Russia). Aggregation inductor was ADP solution in terminal concentration 2.5 micromole (<>, Russia). Gold nanoshells soluted in salt solution were used for experiments. Samples of PRP were incubated with 50 or 100 μl gold nanoshells solution in 5 minute, after that we made definition ADP induced platelet aggregation. We found out increase platelet function activity after incubation with nanoparticles solution which shown in maximum ADP-induced aggregation degree increase. Increase platelet function activity during intravenous nanoshells injection can be cause of thrombosis on patients. That's why before clinical application of cancer cell destruction based on laser photothermal used with plasmon gold nanoparticles careful investigations of thrombosis process and detail analyze of physiological blood parameters are very necessary.

  14. Platelet Function During Hypothermia in Experimental Mock Circulation.

    PubMed

    Van Poucke, Sven; Stevens, Kris; Kicken, Cécile; Simons, Antoine; Marcus, Abraham; Lancé, Marcus

    2016-03-01

    Alterations in platelet function are a common finding in surgical procedures involving cardiopulmonary bypass and hypothermia. Although the combined impact of hypothermia and artificial circulation on platelets has been studied before, the ultimate strategy to safely minimize the risk for bleeding and thrombosis is yet unknown. The aim of this study was to evaluate the use of a mock circulation loop to study the impact of hypothermia for platelet-related hemostatic changes. Venous blood was collected from healthy adult humans (n = 3). Closed mock circulation loops were assembled, each consisting of a centrifugal pump, an oxygenator with integrated heat exchanger, and a hardshell venous reservoir. The experiment started with the mock circulation temperature set at 37°C (T0 [0 h]). Cooling was then initiated at T1 (+2 h), where temperature was adjusted from 37°C to 32°C. Hypothermia was maintained from T2 (+4 h) to T3 (+28 h). From that point in time, rewarming from 32°C to 37°C was initiated with similar speed as cooling. From time point T4 (+30 h), normothermia (37°C) was maintained until the experiment ended at T5 (+32 h). Blood samples were analyzed in standard hematological tests: light transmission aggregometry (LTA) (arachidonic acid [AA], adenosine diphosphate [ADP], collagen [COL], thrombin-receptor-activating-peptide-14 [TRAP]), multiple electrode aggregometry (MEA) (AA, ADP, COL, TRAP), and rotational thromboelastometry (ROTEM) (EXTEM, FIBTEM, PLTEM). Hemoglobin, hematocrit, and platelet count decrease more substantially during temperature drop (37-32°C) than during hypothermia maintenance. Hb and Hct continue to follow this trend during active rewarming (32-37°C). PC increase from the moment active rewarming was initiated. None of the values return to the initial values. LTA values demonstrate a similar decrease in aggregation after stimulation with the platelet agonists between the start of the mock circulation and the start of cooling. Except

  15. Optimized flow cytometric assay for the measurement of platelet microparticles in plasma: pre-analytic and analytic considerations.

    PubMed

    Kim, H K; Song, K S; Lee, E S; Lee, Y J; Park, Y S; Lee, K R; Lee, S N

    2002-07-01

    Platelet microparticles (PMP) are submicroscopic membrane vesicles released by platelets during activation. Flow cytometry is the most widely used method for quantifying PMP, but the optimization of the technical method has not yet been fully evaluated. This study was designed to assess the pre-analytical variables including blood sampling conditions, and to evaluate the analytical variations including effect of the platelet-specific antibodies and quantitative beads, precision, linearity and accuracy in comparison with beta-thromboglobulin, which is one of the platelet activation markers. Numbers of PMP collected into citrate-theophylline-adenosine-dipyridamole (CTAD) tubes were increased with time, but to a lesser extent than when collected into sodium citrate tubes. The precision of the PMP assay was relatively high. Excellent linear correlation was observed for dilution linearity. Regarding the platelet-specific antibodies used, anti-CD41a-labeled samples resulted in higher PMP levels than those labeled with anti-CD61 and anti-CD42a. There was no significant difference of PMP counts according to the quantitative beads. The PMP assay is well correlated with beta-thromboglobulin levels. Our findings suggest that blood samples for the PMP assay should be collected in a CTAD tube and delayed measurement is not allowed to avoid artefactual platelet activation. The PMP assay can be used successfully as a useful marker of the detection of in vivo platelet activation, provided that pre-analytical and technical points are optimally taken into consideration. PMID:12138366

  16. Microfluidic assessment of functional culture-derived platelets in human thrombi under flow.

    PubMed

    Kamat, Viraj; Muthard, Ryan W; Li, Ruizhi; Diamond, Scott L

    2015-10-01

    Despite their clinical significance, human platelets are not amenable to genetic manipulation, thus forcing a reliance on mouse models. Culture-derived platelets (CDPs) from human peripheral blood CD34(+) cells can be genetically altered and may eventually be used for transfusions. By use of microfluidics, the time-dependent incorporation of CD41(+)CD42(+) CDPs into clots was measured using only 54,000 CDPs doped into 27 μL of human whole blood perfused over collagen at a wall shear rate of 100 sec(-1). With the use of fluorescence-labeled human platelets (instead of CDPs) doped between 0.25% and 2% of total platelets, incorporation was highly quantitative and allowed monitoring of the anti-αIIbβ3 antagonism that occurred after collagen adhesion. CDPs were only 15% as efficient as human platelets in their incorporation into human thrombi under flow, although both cell types were equally antagonized by αIIbβ3 inhibition. Transient transfection allowed the monitoring of GFP(+) human CDP incorporation into clots. This assay quantifies genetically altered CDP function under flow. PMID:26145051

  17. Platelet function in dogs with bacterial infections and leishmaniasis.

    PubMed

    Abid, Monia; Kalbantner, Kerstin; Mischke, Reinhard

    2015-01-01

    The objective of this study was to examine the influence of bacterial infections or leishmaniasis on primary haemostasis in dogs. Capillary bleeding time, automatic platelet function analysis (PFA-100), turbidimetric platelet aggregation, impedance aggregometry, platelet count and, in addition, the haematocrit were investigated in 25 dogs with bacterial infections or leishmaniasis . Results of these diseased dogs were compared to the control group and additionally classified into two subgroups based on criteria of systemic inflammatory response syndrome (SIRS) (groups "SIRS" and "Non-SIRS"). Dogs with infections had a significantly prolonged closure time of the PFA-100 using both cartridges (e. g., collagen/ADP: 83 [55-301] vs. 65 [47-99 s; median [minimum-maximum]; p < 0.0001), a significant decrease in maximal aggregation of the turbidimetric aggregometry (e. g., ADP-induced: 45.2 ± 26.8 vs. 67.3 ± 21.8%; mean ± SD; P = 0.003), a significant increase of collagen-induced impedance aggregometry and a significant suppression of arachidonic acid-induced impedance aggregometry. An enhanced collagen-induced impedance aggregation was the only significant difference between subgroups "SIRS"and "Non-SIRS". In conclusion, although individual tests indicate enhanced platelet aggregation, most of the in vitro tests revealed a normal to moderately reduced functionality. The reduced aggregabiity may partly indicate preactivation of platelets. PMID:26281441

  18. p38 mitogen-activated protein kinase activation during platelet storage: consequences for platelet recovery and hemostatic function in vivo

    PubMed Central

    Canault, Matthias; Duerschmied, Daniel; Brill, Alexander; Stefanini, Lucia; Schatzberg, Daphne; Cifuni, Stephen M.

    2010-01-01

    Platelets undergo several modifications during storage that reduce their posttransfusion survival and functionality. One important feature of these changes, which are known as platelet storage lesion, is the shedding of the surface glycoproteins GPIb-α and GPV. We recently demonstrated that tumor necrosis factor-α converting enzyme (TACE/ADAM17) mediates mitochondrial injury-induced shedding of adhesion receptors and that TACE activity correlates with reduced posttransfusion survival of these cells. We now confirm that TACE mediates receptor shedding and clearance of platelets stored for 16 hours at 37°C or 22°C. We further demonstrate that both storage and mitochondrial injury lead to the phosphorylation of p38 mitogen-activated kinase (MAPK) in platelets and that TACE-mediated receptor shedding from mouse and human platelets requires p38 MAP kinase signaling. Protein kinase C, extracellular regulated-signal kinase MAPK, and caspases were not involved in TACE activation. Both inhibition of p38 MAPK and inactivation of TACE during platelet storage led to a markedly improved posttransfusion recovery and hemostatic function of platelets in mice. p38 MAPK inhibitors had only minor effects on the aggregation of fresh platelets under static or flow conditions in vitro. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets. PMID:19965619

  19. Platelet aggregation inhibitors from Philippine marine invertebrate samples screened in a new microplate assay.

    PubMed

    Pimentel, Sheila Marie V; Bojo, Zenaida P; Roberto, Amy V D; Lazaro, Jose Enrico H; Mangalindan, Gina C; Florentino, Leila M; Lim-Navarro, Pilar; Tasdemir, Deniz; Ireland, Chris M; Concepcion, Gisela P

    2003-01-01

    A new microplate assay for Ca(2+)-induced platelet aggregation as detected by Giemsa dye was used to screen marine invertebrate samples from the Philippines for inhibitors of human platelet aggregation. Out of 261 crude methanol extracts of marine sponges and tunicates, 25 inhibited aggregation at 2 mg/ml. Inhibition of agonist-induced aggregation in an aggregometer was used to confirm results of the microplate assay and to determine the specific mode of inhibition of 2 samples. The marine sponge Xestospongia sp. yielded a xestospongin/araguspongine-type molecule that inhibited collagen-induced aggregation by 87% at 2 micro g/ml, and epinephrine-induced aggregation by 78% at 20 micro g/ml, while the marine sponge Aplysina sp. yielded 5,6-dibromotryptamine, which inhibited epinephrine-induced aggregation by 51% at 20 micro g/ml. In this study we have found that the microplate assay is a simple, inexpensive, yet useful preliminary tool to qualitatively screen a large number of marine samples for antiplatelet aggregation activity. PMID:14719168

  20. New gene functions in megakaryopoiesis and platelet formation

    PubMed Central

    Gieger, Christian; Radhakrishnan, Aparna; Cvejic, Ana; Tang, Weihong; Porcu, Eleonora; Pistis, Giorgio; Serbanovic-Canic, Jovana; Elling, Ulrich; Goodall, Alison H.; Labrune, Yann; Lopez, Lorna M.; Mägi, Reedik; Meacham, Stuart; Okada, Yukinori; Pirastu, Nicola; Sorice, Rossella; Teumer, Alexander; Voss, Katrin; Zhang, Weihua; Ramirez-Solis, Ramiro; Bis, Joshua C.; Ellinghaus, David; Gögele, Martin; Hottenga, Jouke-Jan; Langenberg, Claudia; Kovacs, Peter; O’Reilly, Paul F.; Shin, So-Youn; Esko, Tõnu; Hartiala, Jaana; Kanoni, Stavroula; Murgia, Federico; Parsa, Afshin; Stephens, Jonathan; van der Harst, Pim; van der Schoot, C. Ellen; Allayee, Hooman; Attwood, Antony; Balkau, Beverley; Bastardot, François; Basu, Saonli; Baumeister, Sebastian E.; Biino, Ginevra; Bomba, Lorenzo; Bonnefond, Amélie; Cambien, François; Chambers, John C.; Cucca, Francesco; D’Adamo, Pio; Davies, Gail; de Boer, Rudolf A.; de Geus, Eco J. C.; Döring, Angela; Elliott, Paul; Erdmann, Jeanette; Evans, David M.; Falchi, Mario; Feng, Wei; Folsom, Aaron R.; Frazer, Ian H.; Gibson, Quince D.; Glazer, Nicole L.; Hammond, Chris; Hartikainen, Anna-Liisa; Heckbert, Susan R.; Hengstenberg, Christian; Hersch, Micha; Illig, Thomas; Loos, Ruth J. F.; Jolley, Jennifer; Khaw, Kay Tee; Kühnel, Brigitte; Kyrtsonis, Marie-Christine; Lagou, Vasiliki; Lloyd-Jones, Heather; Lumley, Thomas; Mangino, Massimo; Maschio, Andrea; Leach, Irene Mateo; McKnight, Barbara; Memari, Yasin; Mitchell, Braxton D.; Montgomery, Grant W.; Nakamura, Yusuke; Nauck, Matthias; Navis, Gerjan; Nöthlings, Ute; Nolte, Ilja M.; Porteous, David J.; Pouta, Anneli; Pramstaller, Peter P.; Pullat, Janne; Ring, Susan M.; Rotter, Jerome I.; Ruggiero, Daniela; Ruokonen, Aimo; Sala, Cinzia; Samani, Nilesh J.; Sambrook, Jennifer; Schlessinger, David; Schreiber, Stefan; Schunkert, Heribert; Scott, James; Smith, Nicholas L.; Snieder, Harold; Starr, John M.; Stumvoll, Michael; Takahashi, Atsushi; Tang, W. H. Wilson; Taylor, Kent; Tenesa, Albert; Thein, Swee Lay; Tönjes, Anke; Uda, Manuela; Ulivi, Sheila; van Veldhuisen, Dirk J.; Visscher, Peter M.; Völker, Uwe; Wichmann, H.-Erich; Wiggins, Kerri L.; Willemsen, Gonneke; Yang, Tsun-Po; Zhao, Jing Hua; Zitting, Paavo; Bradley, John R.; Dedoussis, George V.; Gasparini, Paolo; Hazen, Stanley L.; Metspalu, Andres; Pirastu, Mario; Shuldiner, Alan R.; van Pelt, L. Joost; Zwaginga, Jaap-Jan; Boomsma, Dorret I.; Deary, Ian J.; Franke, Andre; Froguel, Philippe; Ganesh, Santhi K.; Jarvelin, Marjo-Riitta; Martin, Nicholas G.; Meisinger, Christa; Psaty, Bruce M.; Spector, Timothy D.; Wareham, Nicholas J.; Akkerman, Jan-Willem N.; Ciullo, Marina; Deloukas, Panos; Greinacher, Andreas; Jupe, Steve; Kamatani, Naoyuki; Khadake, Jyoti; Kooner, Jaspal S.; Penninger, Josef; Prokopenko, Inga; Stemple, Derek; Toniolo, Daniela; Wernisch, Lorenz; Sanna, Serena; Hicks, Andrew A.; Rendon, Augusto; Ferreira, Manuel A.; Ouwehand, Willem H.; Soranzo, Nicole

    2012-01-01

    Platelets are the second most abundant cell type in blood and are essential for maintaining haemostasis. Their count and volume are tightly controlled within narrow physiological ranges, but there is only limited understanding of the molecular processes controlling both traits. Here we carried out a high-powered meta-analysis of genome-wide association studies (GWAS) in up to 66,867 individuals of European ancestry, followed by extensive biological and functional assessment. We identified 68 genomic loci reliably associated with platelet count and volume mapping to established and putative novel regulators of megakaryopoiesis and platelet formation. These genes show megakaryocyte-specific gene expression patterns and extensive network connectivity. Using gene silencing in Danio rerio and Drosophila melanogaster, we identified 11 of the genes as novel regulators of blood cell formation. Taken together, our findings advance understanding of novel gene functions controlling fate-determining events during megakaryopoiesis and platelet formation, providing a new example of successful translation of GWAS to function. PMID:22139419

  1. Antiplatelet drugs in patients with enhanced platelet turnover: biomarkers versus platelet function testing.

    PubMed

    Freynhofer, Matthias K; Gruber, Susanne C; Grove, Erik L; Weiss, Thomas W; Wojta, Johann; Huber, Kurt

    2015-08-31

    Platelets are key players in atherothrombosis. Antiplatelet therapy comprising aspirin alone or with P2Y12-inhibitors are effective for prevention of atherothrombotic complications. However, there is interindividual variability in the response to antiplatelet drugs, leaving some patients at increased risk of recurrent atherothrombotic events. Several risk factors associated with high on-treatment platelet reactivity (HTPR), including elevated platelet turnover, have been identified. Platelet turnover is adequately estimated from the fraction of reticulated platelets. Reticulated platelets are young platelets, characterised by residual messenger RNA. They are larger, haemostatically more active and there is evidence that platelet turnover is a causal and prognostic factor in atherothrombotic disease. Whether platelet turnover per se represents a key factor in pathogenesis, progression and prognosis of atherothrombotic diseases (with focus on acute coronary syndromes) or whether it merely facilitates insufficient platelet inhibition will be discussed in this state-of-the art review. PMID:26272640

  2. Common variants in the human platelet PAR4 thrombin receptor alter platelet function and differ by race

    PubMed Central

    Edelstein, Leonard C.; Simon, Lukas M.; Lindsay, Cory R.; Kong, Xianguo; Teruel-Montoya, Raúl; Tourdot, Benjamin E.; Chen, Edward S.; Ma, Lin; Coughlin, Shaun; Nieman, Marvin; Holinstat, Michael; Shaw, Chad A.

    2014-01-01

    Human platelets express 2 thrombin receptors: protease-activated receptor (PAR)-1 and PAR4. Recently, we reported 3.7-fold increased PAR4-mediated aggregation kinetics in platelets from black subjects compared with white subjects. We now show that platelets from blacks (n = 70) express 14% more PAR4 protein than those from whites (n = 84), but this difference is not associated with platelet PAR4 function. Quantitative trait locus analysis identified 3 common single nucleotide polymorphisms in the PAR4 gene (F2RL3) associated with PAR4-induced platelet aggregation. Among these single nucleotide polymorphisms, rs773902 determines whether residue 120 in transmembrane domain 2 is an alanine (Ala) or threonine (Thr). Compared with the Ala120 variant, Thr120 was more common in black subjects than in white subjects (63% vs 19%), was associated with higher PAR4-induced human platelet aggregation and Ca2+ flux, and generated greater inositol 1,4,5-triphosphate in transfected cells. A second, less frequent F2RL3 variant, Phe296Val, was only observed in blacks and abolished the enhanced PAR4-induced platelet aggregation and 1,4,5-triphosphate generation associated with PAR4-Thr120. PAR4 genotype did not affect vorapaxar inhibition of platelet PAR1 function, but a strong pharmacogenetic effect was observed with the PAR4-specific antagonist YD-3 [1-benzyl-3(ethoxycarbonylphenyl)-indazole]. These findings may have an important pharmacogenetic effect on the development of new PAR antagonists. PMID:25293779

  3. Methodological considerations for the human platelet 5-HT2A receptor binding kinetic assay.

    PubMed

    Khait, V D; Huang, Y Y; Mann, J J

    1999-01-01

    Analysis of an extensive database of human platelet 5-HT2A receptor binding assays has been conducted in order to identify factors that may affect the assay results. Despite anecdotal reports that storage of frozen platelet pellets may affect 5-HT2A binding affinity and capacity, no quantitative study has been reported in the literature. Analysis of binding data for 373 frozen samples with a storage time up to three years is presented in this paper. It is shown that prolonged storage significantly decreases binding. The loss of binding capacity begins in the first six month of storage. Bmax declines by half after 17 month. The impact of storage time on the binding affinity is much smaller. There is only about 20% increase in the value of affinity K(D) during the half-life of Bmax. Differences in sample storage time may partly explain discrepancies in results between different research groups. Nonspecific binding due to binding to filter material diminishes accuracy and reliability of the binding assays as a result of a decrease in the ratio of specific to nonspecific ratio. A data analysis based on our suggested mathematical model shows that this effect depends on tissue concentration in test tube and becomes pronounced when the concentration is below 0.1 mg protein/ml (at 0.2 nM of ligand). Above 0.1 mg protein/ml, percentage of specific to total binding exceeds 65%, which is an acceptable level for the ratio. The majority of the binding studies reported in the literature employed a tissue concentration more than 0.5 mg/ml, well above the minimal limit sufficient for a reliable assay. However, development of microassays to conserve precious tissue must take the limit into consideration. PMID:10619369

  4. A study on the impact of CYP2C19 genotype and platelet reactivity assay on patients undergoing PCI

    PubMed Central

    Rath, P.C.; Chidambaram, Sundar; Rath, Pallavi; Dikshit, Byomakesh; Naik, Sudhir; Sahoo, Prashant K.; Das, Brajraj; Mahalingam, Mohanshankar; Khandrika, Lakshmipathi; Jain, Jugnu

    2015-01-01

    Background A thorough understanding of the patient's genotype and their functional response to a medication is necessary for improving event free survival. Several outcome studies support this view particularly if the patient is to be started on clopidogrel due to the prevalence of clopidogrel resistance. Such guided therapy has reduced the incidence of Major Adverse Cardiac Events (MACE) after stent implantation. Methods Between August 2013 and August 2014, 200 patients with coronary artery disease undergoing percutaneous coronary intervention (PCI) were prescribed any one of the anti-platelet medications such as clopidogrel, prasugrel or ticagrelor and offered testing to detect CYP2C19 gene mutations along with a platelet reactivity assay (PRA). Intended outcome was modification of anti-platelet therapy defined as either dose escalation of clopidogrel or replacement of clopidogrel with prasugrel or ticagrelor for the patients in clopidogrel arm, and replacement of ticagrelor or prasugrel with clopidogrel if those patients were non-carrier of mutant genes and also if they demonstrated bleeding tendencies in the ticagrelor and prasugrel arms. Conclusion Clopidogrel resistance was observed to be 16.5% in our study population. PRA was useful in monitoring the efficacy of thienopyridines. By having this test, one can be safely maintained on clopidogrel in non-carriers, or with increased dose of clopidogrel in intermediate metabolizers or with newer drugs such as ticagrelor or prasugrel in poor metabolizers. Patients on ticagrelor and prasugrel identified as non-carriers of gene mutations for clopidogrel metabolism could be offered clopidogrel resulting in economic benefits to the patients. Patients at high risk of bleeding were also identified by the PRA. PMID:26071289

  5. The interaction of sodium nitroprusside with human endothelial cells and platelets: nitroprusside and prostacyclin synergistically inhibit platelet function

    SciTech Connect

    Levin, R.I.; Weksler, B.B.; Jaffe, E.A.

    1982-12-01

    Sodium nitroprusside (NP) is a potent vasodilator that also inhibits platelet aggregation. To test the hypothesis that NP causes both of these effects by altering the balance between prostacyclin (PGI2) produced by endothelial cells and thromboxane A2 (TXA2) produced by platelets, we incubated each of these cell types with NP for 5 minutes and assayed the PGI2 and TXA2 produced. NP at pharmacologically achieved doses (0.01--30 micrograms/ml) inhibited platelet aggregation and resultant TXA2 synthesis in a dose- and time-dependent manner (p less than 0.001). The inhibition was not dependent on cAMP production, external calcium concentration, or suppression of TXA2 synthesis. NP did not alter the production of PGI2 by cultured human endothelial cells as measured by radioimmunoassay for 6-Keto-PGF1 alpha, the stable hydrolysis product of PGI2. However, supernates of NP-treated endothelial cells containing low, noninhibitory concentrations of NP unexpectedly inhibited platelet aggregation. This inhibition of platelet aggregation was due to synergy between PGI2 (0.1--3 nM) and NP (p interaction less than 0.03). The synergistic inhibition by NP and PGI2 of platelet aggregation and TXA2 synthesis in vivo may explain some of the beneficial actions of NP in the treatment of hypertension and congestive heart failure.

  6. Assays for B lymphocyte function.

    PubMed

    Bondada, Subbarao; Robertson, Darrell A

    2003-11-01

    This unit describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. The first basic protocol is a generalized system for inducing in vitro antibody production and can accommodate various types of antigens under study. Secreted antibodies can then be measured with an enzyme-linked immunosorbent assay (ELISA) or other soluble-antibody detection systems. Alternatively, the number of antibody-producing cells can be quantified by plaque-forming cell (PFC) assays presented in this unit: the Cunningham-Szenberg and the Jerne-Nordin techniques. Both methods employ specially prepared slide chambers, described here, in which the antibody-producing B cells are mixed with complement and indicator sheep red blood cells (SRBC), or with trinitrophenol-modified SRBC (TNP-SRBC), with subsequent lysis and counting of plaques. Because IgM antibodies fix complement efficiently, whereas IgG and IgA antibodies do not, unmodified PFC assays measure only IgM antibodies. The assay can be modified, however, to measure all classes of antibodies or to enumerate total immunoglobulin-secreting B cells, as described in alternate protocols. Yet another method of measuring the number of antibody-producing B cells (in a class-specific fashion) is to use the ELISPOT technique described in UNIT 7.14. The resting B cells used in these procedures are prepared as described in the final support protocols for Percoll gradient centrifugation. PMID:18432909

  7. Dicer1-mediated miRNA processing shapes the mRNA profile and function of murine platelets.

    PubMed

    Rowley, Jesse W; Chappaz, Stéphane; Corduan, Aurélie; Chong, Mark M W; Campbell, Robert; Khoury, Amanda; Manne, Bhanu Kanth; Wurtzel, Jeremy G T; Michael, James V; Goldfinger, Lawrence E; Mumaw, Michele M; Nieman, Marvin T; Kile, Benjamin T; Provost, Patrick; Weyrich, Andrew S

    2016-04-01

    Human platelets contain microRNAs (miRNAs) and miRNA processing machinery, but their contribution to platelet function remains incompletely understood. Here, we show that murine megakaryocyte (MK)-specific knockdown of Dicer1, the ribonuclease that cleaves miRNA precursors into mature miRNAs, reduces the level of the majority of miRNAs in platelets. This leads to altered platelet messenger RNA (mRNA) expression profiles and mild thrombocytopenia. Fibrinogen receptor subunits Itga2b (αIIb) and Itgb3 (β3) mRNAs were among the differentially expressed transcripts that are increased in platelets lacking Dicer1. Argonaute 2 (Ago2), a member of the miRNA silencing complex, co-immunoprecipitated with αIIband β3mRNAs in wild-type platelets. Furthermore, co-immunoprecipitation experiments suggested reduced αIIb/β3/Ago2 complexes in miRNA-deficient platelets. These results suggested that miRNAs regulate both integrin subunits. Subsequent 3' untranslated region luciferase reporter assays confirmed that the translation of both αIIband β3mRNAs can be regulated by miRNAs miR-326, miR-128, miR-331, and miR-500. Consistent with these molecular changes, the deletion ofDicer1resulted in increased surface expression of integrins αIIband β3, and enhanced platelet binding to fibrinogen in vivo and in vitro. Heightened platelet reactivity, shortened tail-bleeding time, and reduced survival following collagen/epinephrine-induced pulmonary embolism were also observed in Dicer1-deficient animals. CombinedPf4-cre-mediated deletion of Drosha and Dicer1 did not significantly exacerbate phenotypes observed in single Dicer1 knockout mice. In summary, these findings indicate that Dicer1-dependent generation of mature miRNAs in late-stage MKs and platelets modulates the expression of target mRNAs important for the hemostatic and thrombotic function of platelets. PMID:26773046

  8. Variability in platelet responses to collagen--comparison between whole blood perfusions, traditional platelet function tests and PFA-100.

    PubMed

    Lepäntalo, A; Beer, J H; Siljander, P; Syrjälä, M; Lassila, R

    2001-07-15

    The purpose of this study was to determine if the results obtained in platelet function tests and whole blood perfusions are associated with those in platelet function analyser (PFA)-100. We used collagen type I monomers and fibrils to analyse the distinct roles of glycoprotein (GP) Ia/IIa and other collagen receptors in flowing blood under a high shear rate (1600/s) and in aggregation studies. Also, anticoagulation [citrate vs. D-phenylalanyl-1-prolyl-1 arginine chloromethyl ketone (PPACK)] was varied to enhance the functions of GP Ia/IIa, since it has been shown that the cation-poor environment of citrated blood impairs GP Ia/IIa-dependent platelet recruitment. Large interindividual variability (45-fold) was detected in deposition of platelets in whole blood perfusions over collagen monomers, whereas this variation was only fourfold in fibrils. In PFA, this variation was reduced to 2.5-fold. However, platelet deposition on monomers is associated with epinephrine-enhanced PFA (r=-.49, P<.03), whereas platelet deposition on fibrils is correlated with adenosine diphosphate (ADP)-enhanced PFA (r=-.47, P<.05), suggesting a distinct synergism between epinephrine and monomers (GP Ia/IIa) as well as ADP with fibrils (other collagen receptors). Donors with 807 C/C polymorphism of GP Ia (n=14) had longer lag phase in aggregation experiments compared with C/T (n=7) both by monomers and fibrils (P<.04), but these polymorphisms with their mild impact on GP Ia/IIa activity did not markedly differ in other tests. In conclusion, the results obtained in perfusion studies and PFA experiments correlated, but PFA fails to reveal the large-scale variability related to collagen-induced platelet responses. PMID:11457470

  9. Usefulness of platelet function tests to predict bleeding with antithrombotic medications.

    PubMed

    Gorog, Diana A; Otsui, Kazunori; Inoue, Nobutaka

    2015-01-01

    The pharmacological inhibition of platelets has always been regarded as a double-edged sword: the challenge of balancing the antithrombotic effect against the bleeding risk. Potent antiplatelet agents and novel oral anticoagulants, sometimes in combination, are increasingly used in the treatment of cardiovascular disease and for thromboprophylaxis in atrial fibrillation. Although such treatment has reduced the risk of thrombotic events, the potential for major bleeding has increased, and a technique to identify those at increased bleeding risk is greatly needed. Platelet function tests (PFTs), most frequently VerifyNow and also the vasodilator-stimulated phosphoprotein -phosphorylation assay, have been used to identify low on-treatment platelet reactivity, to identify individuals who may be at increased bleeding risk. Such results predict nuisance bleeding, but many individuals have low on-treatment platelet reactivity and yet do not exhibit major or even minor bleeding. Although PFTs may be useful in assessing populations, they do not allow identification of individual patients at risk of bleeding on either antiplatelet or novel oral anticoagulant therapy, nor do they allow the tailoring of such therapy to optimize the risk:benefit ratio. Thrombin plays a cardinal role in both arterial thrombus formation and hemostasis, yet most PFTs fail to assess the contribution of thrombin, because they employ anticoagulated blood. Techniques such as the calibrated automated thrombogram and the point-of-care global thrombosis test, performed on native blood, which measure endogenous thrombin potential, seem to show the most promise for profiling bleeding risk, as tests that most physiologically assess the effects of medications on thrombin. PMID:25839991

  10. Thrombocytopenia model with minimal manipulation of blood cells allowing whole blood assessment of platelet function.

    PubMed

    Tiedemann Skipper, Mette; Rubak, Peter; Halfdan Larsen, Ole; Hvas, Anne-Mette

    2016-06-01

    In vitro models of thrombocytopenia are useful research tools. Previously published models have shortcomings altering properties of platelets and other blood components. The aim of the present study was to develop a whole blood method to induce thrombocytopenia with minimal manipulation, and to describe platelet function in induced thrombocytopenia in individuals with healthy platelets. Hirudin anticoagulated blood was obtained from 20 healthy volunteers. One part of the blood was gently centrifuged at 130g for 15 minutes. The platelet-rich plasma was replaced with phosphate-buffered saline to establish thrombocytopenia. Various levels of thrombocytopenia were achieved by combining different volumes of baseline whole blood and thrombocytopenic blood. Platelet counts were measured by flow cytometry (Navios, Beckman Coulter) and routine haematological analyser (Sysmex XE-5000). Platelet function was analysed by impedance aggregometry (Multiplate® Analyzer, Roche) and by flow cytometry (Navios, Beckman Coulter) using collagen, adenosine diphosphate, thrombin receptor activating peptide-6 and ristocetin as agonists. Median baseline platelet count was 227×10(9)/l. The in vitro model yielded median platelet counts at 51×10(9)/l (range 26-93×10(9)/l). We observed minor, yet significant, changes in platelet size and maturity from baseline to modelled thrombocytopenia. In the thrombocytopenic samples, significant and positive linear associations were found between platelet count and platelet aggregation across all agonists (all p-values<0.001). Platelet function assessed by flow cytometry showed minimal alterations in the thrombocytopenic samples. A new whole blood-based model of thrombocytopenia was established and validated. This new model serves as a useful future tool, particularly to explore platelet function in patients with thrombocytopenia. PMID:26555800

  11. Microfluidic assay of hemophilic blood clotting: Distinct deficits in platelet and fibrin deposition at low factor levels

    PubMed Central

    Colace, T.; Fogarty, Patrick F.; Panckeri, Karen A.; Li, Ruizhi; Diamond, S.L.

    2014-01-01

    Background Coagulation factor deficiencies create a range of bleeding phenotypes. Microfluidic devices offer controlled hemodynamics and defined procoagulant triggers for measurement of clotting under flow. Objectives We tested a flow assay of contact pathway-triggered clotting to quantify platelet and fibrin deposition distal of dysfunctional thrombin production. Microfluidic metrics were then compared with PTT or % factor activity assays. Methods Whole blood (WB) treated with low level corn trypsin inhibitor (4 µg/ml) from 9 healthy donors and 27 patients [deficient in: Factor VIII (19 patients); IX (1); XI (1); VWF (6)] was perfused over fibrillar collagen at wall shear rate=100 s−1. Results Using healthy WB, platelets deposited within 30 sec, while fibrin appeared within 6 min. Compared to healthy controls, WB from patients displayed a 50% reduction in platelet deposition only at <1 % factor activity. In contrast, striking defects in fibrin deposition occurred for patients with <13% factor activity (or PTT >40 sec). Full occlusion of the 60-micron high channel was completely absent over the 15 min test in patients with <1% factor activity, while an intermediate defect was present in patients with >1% factor. Conclusion Spontaneous bleeding in patients with < 1% factor activity may be linked to deficits in both platelet and fibrin deposition, a risk known to be mitigated when factor levels are raised to >1 % activity (PTT of ~40–60 sec), a level that does not necessarily rescue fibrin formation under flow. PMID:24261634

  12. Platelet C1- inhibitor. A secreted alpha-granule protein.

    PubMed Central

    Schmaier, A H; Smith, P M; Colman, R W

    1985-01-01

    In order to characterize which proteins of the contact phase of coagulation interact with platelets, human platelets were studied immunochemically and functionally to determine if they contain C1- inhibitor. By means of monospecific antibody to C1- inhibitor, a competitive enzyme-linked immunosorbent assay (CELISA) was developed to measure directly platelet C1- inhibitor. With the CELISA, from 33 to 115 ng of C1- inhibitor antigen per 10(8) platelets from 15 normal donors was quantified in lysates of washed human platelets solubilized in nonionic detergent. The mean concentration in 10(8) platelets was 62 +/- 33 ng (SD). Plasma C1- inhibitor either in the platelet suspension medium or on the surface of the platelets could account for only from 6.5 to 16% of the total antigen measured in the solubilized platelets. Upon functional studies, platelets contained 84 +/- 36 ng (SD) of C1- inhibitor activity in 10(8) platelets. As assessed by the CELISA, platelet C1- inhibitor antigen was immunochemically identical to plasma and purified C1- inhibitor. In contrast, the mean concentration of platelet C1- inhibitor antigen in platelets from four patients with classical hereditary angioedema was 8.3 ng/10(8) platelets (range, 5.3 to 11.3 ng/10(8) platelets). 25 and 31% of the total platelet C1- inhibitor was secreted without cell lysis from normal platelets after exposure to collagen (20 micrograms/ml) and thrombin (1 U/ml), respectively, and this secretion was blocked by metabolic inhibitors. Platelet subcellular fractionation showed that platelet C1- inhibitor resided mostly in alpha-granules, similar to the location of platelet fibrinogen. Thus, human platelets contained C1- inhibitor, which became available by platelet secretion. The identification of platelet C1- inhibitor suggests that platelets may modulate the activation of the proteins of early blood coagulation and the classical complement pathways. Images PMID:3965505

  13. Circulating primers enhance platelet function and induce resistance to antiplatelet therapy

    PubMed Central

    Blair, T A; Moore, S F; Hers, I

    2015-01-01

    Background Aspirin and P2Y12 antagonists are antiplatelet compounds that are used clinically in patients with thrombosis. However, some patients are ‘resistant’ to antiplatelet therapy, which increases their risk of developing acute coronary syndromes. These patients often present with an underlying condition that is associated with altered levels of circulating platelet primers and platelet hyperactivity. Platelet primers cannot stimulate platelet activation, but, in combination with physiologic stimuli, significantly enhance platelet function. Objectives To explore the role of platelet primers in resistance to antiplatelet therapy, and to evaluate whether phosphoinositide 3-kinase (PI3K) contributes to this process. Methods and Results We used platelet aggregation, thromboxane A2 production and ex vivo thrombus formation as functional readouts of platelet activity. Platelets were treated with the potent P2Y12 inhibitor AR-C66096, aspirin, or a combination of both, in the presence or absence of the platelet primers insulin-like growth factor-1 (IGF-1) and thrombopoietin (TPO), or the Gz-coupled receptor ligand epinephrine. We found that platelet primers largely overcame the inhibitory effects of antiplatelet compounds on platelet functional responses. IGF-1-mediated and TPO-mediated, but not epinephrine-mediated, enhancements in the presence of antiplatelet drugs were blocked by the PI3K inhibitors wortmannin and LY294002. Conclusions These results demonstrate that platelet primers can contribute to antiplatelet resistance. Furthermore, our data demonstrate that there are PI3K-dependent and PI3K-independent mechanisms driving primer-mediated resistance to antiplatelet therapy. PMID:26039631

  14. Complement Activation in Trauma Patients Alters Platelet Function.

    PubMed

    Atefi, Gelareh; Aisiku, Omozuanvbo; Shapiro, Nathan; Hauser, Carl; Dalle Lucca, Jurandir; Flaumenhaft, Robert; Tsokos, George C

    2016-09-01

    Trauma remains the main cause of death for both civilians and those in uniform. Trauma-associated coagulopathy is a complex process involving inflammation, coagulation, and platelet dysfunction. It is unknown whether activation of complement, which occurs invariably in trauma patients, is involved in the expression of trauma-associated coagulopathy. We designed a prospective study in which we enrolled 40 trauma patients and 30 healthy donors upon arrival to the emergency department of BIDMC. Platelets from healthy individuals were incubated with sera from trauma patients and their responsiveness to a thrombin receptor-activating peptide was measured using aggregometry. Complement deposition on platelets from trauma patients was measured by flow cytometry. Normal platelets displayed hypoactivity after incubation with trauma sera even though exposure to trauma sera resulted in increased agonist-induced calcium flux. Depletion of complement from sera further blocked activation of hypoactive platelets. Conversely, complement activation increased aggregation of platelets. Platelets from trauma patients were found to have significantly higher amounts of C3a and C4d on their surface compared with platelets from controls. Depletion of complement (C4d, C3a) reversed the ability of trauma sera to augment agonist-induced calcium flux in donor platelets. Our data indicate that complement enhances platelet aggregation. Despite its complement content, trauma sera render platelets hypoactive and complement depletion further blocks activation of hypoactive platelets. The defect in platelet activation induced by trauma sera is distal to receptor activation since agonist-induced Ca2+ flux is elevated in the presence of trauma sera owing to complement deposition. PMID:27355402

  15. Platelet aggregation test

    MedlinePlus

    The platelet aggregation blood test checks how well platelets , a part of blood, clump together and cause blood to clot. ... Decreased platelet aggregation may be due to: Autoimmune ... Fibrin degradation products Inherited platelet function defects ...

  16. Alterations in bovine platelet function and acute phase proteins induced by Pasteurella haemolytica A1.

    PubMed Central

    Cheryk, L A; Hooper-McGrevy, K E; Gentry, P A

    1998-01-01

    Platelet function was assessed by aggregometry in 10 Holstein calves before and after exposure to Pasteurella haemolytica (biotype A, serotype 1) by intrabronchial challenge. At 24 h after exposure the platelets had become more reactive to stimulation with known platelet agonists such as adenosine diphosphate (ADP) and platelet-activating factor (PAF) and the platelet aggregates that formed were more resistant to disaggregation. The activation of platelets was an early response in the challenged calves as platelet function had returned to pretreatment levels 72 h after exposure to the bacteria while the acute phase reactant proteins, haptoglobin and fibrinogen, were approaching their peak values and alpha 2-macroglobulin levels had also risen significantly (P < 0.05) at this time. The plasma levels of these proteins were still elevated and albumin levels were depressed 6 d post-treatment. At post-mortem all calves exhibited pneumonic tissue damage. When P. haemolytica leukotoxin was added directly to bovine platelet suspensions both spontaneous aggregation and an increase in the aggregation response to ADP and PAF stimulation were observed. The morphological appearance of the platelet aggregates exhibited the typical pattern for bovine platelets with 2 distinct zones of cells being visible within each aggregate. One zone contained platelets in which the cytoplasmic granules were still evident and the other zone contained irregularly shaped platelets devoid of granular content. In the latter zone, discrete gaps, or pores, were evident in the plasma membrane of numerous platelets. This pore formation is characteristic of leukotoxin action and is not observed in ADP or PAF induced aggregates. Images Figure 2. PMID:9442932

  17. Platelet function and activation in Cavalier King Charles Spaniels with subclinical chronic valvular heart disease.

    PubMed

    Tong, Linda J; Hosgood, Giselle L; French, Anne T; Irwin, Peter J; Shiel, Robert E

    2016-08-01

    OBJECTIVE To assess platelet closure time (CT), mean platelet component (MPC) concentration, and platelet component distribution width (PCDW) in dogs with subclinical chronic valvular heart disease. ANIMALS 89 Cavalier King Charles Spaniels (CKCSs) and 39 control dogs (not CKCSs). PROCEDURES Platelet count, MPC concentration, PCDW, and Hct were measured by use of a hematology analyzer, and CT was measured by use of a platelet function analyzer. Murmur grade and echocardiographic variables (mitral valve regurgitant jet size relative to left atrial area, left atrial-to-aortic diameter ratio, and left ventricular internal dimensions) were recorded. Associations between explanatory variables (sex, age, murmur grade, echocardiographic variables, platelet count, and Hct) and outcomes (CT, MPC concentration, and PCDW) were examined by use of multivariate regression models. RESULTS A model with 5 variables best explained variation in CT (R(2), 0.74), with > 60% of the variance of CT explained by mitral valve regurgitant jet size. The model of best fit to explain variation in MPC concentration included only platelet count (R(2), 0.24). The model of best fit to explain variation in PCDW included platelet count and sex (R(2), 0.25). CONCLUSIONS AND CLINICAL RELEVANCE In this study, a significant effect of mitral valve regurgitant jet size on CT was consistent with platelet dysfunction. However, platelet activation, as assessed on the basis of the MPC concentration and PCDW, was not a feature of subclinical chronic valvular heart disease in CKCSs. PMID:27463549

  18. Differences in Platelet Function In Patients with Acute Myeloid Leukaemia and Myelodysplasia Compared to Equally Thrombocytopenic Patients with Immune Thrombocytopenia

    PubMed Central

    Psaila, Bethan; Bussel, James B.; Frelinger, Andrew L.; Babula, Bracken; Linden, Matthew D.; Li, Youfu; Barnard, Marc R.; Tate, Chinara; Feldman, Eric J.; Michelson, Alan D.

    2011-01-01

    Background Severe thrombocytopenia is a major risk factor for haemorrhage, and yet platelet function and bleeding risk at low platelet counts are poorly understood because of limitations of platelet function testing at very low platelet counts. Objectives To examine and compare platelet function in severely thrombocytopenic patients with acute myeloid leukaemia (AML) or myelodysplasia (MDS) to patients with immune thrombocytopenia (ITP). Methods Whole blood flow cytometric measurement of platelet activation and platelet reactivity to agonists was correlated with the immature platelet fraction (IPF) and bleeding symptoms. Results Compared with patients with ITP, patients with AML/MDS had smaller platelets, lower IPF, and substantially lower platelet surface expression of activated GPIIb/IIIa and GPIb both with and without addition of ex vivo ADP or TRAP. In both ITP and AML/MDS, increased platelet surface GPIb on circulating platelets and expression of activated GPIIb/IIIa and GPIb on ex vivo activated platelets correlated with a higher IPF. Whereas platelet reactivity was higher for AML/MDS patients with bleeding than those with no bleeding, platelet reactivity was lower for ITP patients with bleeding than those with no bleeding. Conclusions AML/MDS patients have lower in vivo platelet activation and ex vivo platelet reactivity than patients with ITP. The proportion of newly-produced platelets correlates with the expression of platelet surface markers of activation. These differences might contribute to differences in bleeding tendency between AML/MDS and ITP. This study is the first to define differences in platelet function between AML/MDS patients and ITP patients with equivalent degrees of thrombocytopenia. PMID:21920014

  19. Effect of Fibrinogen on Platelet Reactivity Measured by the VerifyNow P2Y12 Assay.

    PubMed

    Dobrovolsky, A B; Laguta, P S; Guskova, E V; Yarovaya, E B; Titaeva, E V; Storozhilova, A N; Panchenko, E P

    2016-05-01

    The VerifyNow assay is based upon the ability of activated platelets to cross-link beads coated with fibrinogen. However, fibrinogen is an abundant protein of blood, and therefore it may affect test results by competing with fibrinogen of beads for binding to platelets. To test this assumption, we assessed the influence of artificial alteration of fibrinogen level in blood samples obtained from donors (n = 9) and patients on clopidogrel therapy (n = 8) on the results of the VerifyNow P2Y12 assay. Fibrinogen level was altered by adding to blood samples 1/10 volume of fibrinogen solution (10.56 g/liter) or corresponding buffer. Relative to baseline, addition of buffer significantly increased platelet reactivity, whereas addition of fibrinogen decreased it. Analysis of the relationship between change in platelet reactivity values (dBase and dPRU) and change in fibrinogen concentration (dFg) revealed strong negative correlations: dBase = -63.3 × dFg - 27.1 (r = -0.924, p < 0.0005) and dPRU = -54.4 × dFg - 21.8 (r = -0.764, p < 0.0005). Thus, the results of our experiments suggest that: (i) blood fibrinogen strongly influences results of the VerifyNow P2Y12 assay, and (ii) correcting for fibrinogen effect may be needed to improve the accuracy of the test in the measuring of antiplatelet effect of clopidogrel therapy. PMID:27297894

  20. Evaluation of store lesion in platelet obtained by apheresis compared to platelet derived from whole blood and its impact on the in vitro functionality.

    PubMed

    Quintero, M; Núñez, M; Mellado, S; Maldonado, M; Wehinger, S

    2015-12-01

    Platelet units for transfusion purposes are obtained manually from whole blood or by apheresis, in an automated process. In both methods, platelets during storage present a characteristics grouped under the name "storage lesion" that are associated with adverse effects on platelet units. Oxidative stress has been claimed to be one of major causes, leading to activation and apoptosis processes affecting their post transfusion functionality. In this work, we observed an association between apheresis and a reduced presence of oxidative stress and better results in functional markers in stored platelets, compared to manually obtained platelets. Then, apheresis which would ensure a greater number of functional platelets during the 5 days of storage, compared to concentrates obtained from whole blood. PMID:26043812

  1. Using a Filtration Technique to Isolate Platelet Free Plasma for Assaying Pyrophosphate

    PubMed Central

    TOLOUIAN, RAMIN; CONNERY, SEAN M.; O’NEILL, W. CHARLES; GUPTA, AJAY

    2015-01-01

    SUMMARY Background Vascular calcification (VC) is a strong prognostic marker of mortality from cardiovascular disease. Extracellular inorganic pyrophosphate (PPi) is a critical inhibitor of vascular calcification and it has been reported that hemodialysis patients have reduced plasma PPi levels, suggesting that altered PPi metabolism could contribute to VC in hemodialysis patients. Platelets are rich in PPi and release of PPi from platelets during storage or processing of plasma can lead to falsely elevated plasma PPi levels. To prepare plasma samples that are suitable for measuring PPi levels, ultracentrifugation has been used to remove platelets. Consequently, plasma PPi measurements have been limited to research laboratories since the majority of clinical laboratories do not have access to an ultracentrifuge. The purpose of the present study was to test the validity of an improved method of preparing platelet free plasma that uses filtration with a 300,000 Dalton molecular weight cut-off filter to exclude platelets, while minimizing their release of PPi. Methods In 20 maintenance hemodialysis patients, PPi levels were measured in plasma samples prepared by the conventional technique of low-speed centrifugation to remove red and white blood cells versus a novel filtration technique. Results Plasma prepared by filtration had significantly lower platelet counts (0 vs. 3 – 7 103/μL) and PPi levels (1.39 ± 0.30 μM vs. 2.74 ± 1.19 μM; mean ± SD, p < 0.01). Conclusions The filtration method appears effective in excluding platelets without causing trauma to platelets and can be used by clinical laboratories to prepare platelet-depleted plasma for PPi measurement. PMID:23289181

  2. An overview of platelet indices for evaluating platelet function in children with scorpion envenomation

    PubMed Central

    Konca, Capan; Tekin, Mehmet; Colak, Pinar; Uckardes, Fatih; Turgut, Mehmet

    2014-01-01

    The aim of this study was to assess the correlation between platelet indices and scorpion envenomations (SE). Medical records of 76 children who were hospitalised for scorpion stings in the paediatric intensive care unit (PICU) between February 2013 and November 2013, and 55 healthy children who were similar to the patient group in terms of age and sex, were analysed retrospectively. The leucocyte (WBC), thrombocyte (PLT), plateletcrit (PCT), platelet distribution width (PDW) and mean platelet volume (MPV) values of the 76 children with SE were recorded. These values were compared with the healthy control group. Significantly higher WBC and PDW values were noted in patients with SE in comparison to the controls. Patients with SE had significantly lower mean MPV values compared to the healthy controls (9.03 ± 1.26 compared to 10.43 ± 1.44 fL, respectively; p < 0.001). Although the mean platelet count was slightly elevated in the SE group, no statistically significant difference existed between the two groups (p = 0.097). Furthermore, the mean PCT values in the SE group compared to the control group were slightly decreased, but this decrease was not statistically significant (p = 0.141). A significant inverse correlation existed between the MPV values and the WBC (r = -0.450, p < 0.01) and PLT counts (r = -0.420, p < 0.01). The PLT values were significantly correlated with the PCT values (r = 0.687, p < 0.01). This study demonstrated that SE may lead to several alterations in platelet indices. Significantly lower values of MPV and higher values of PDW were detected in SE patients. However, the increase in the platelet counts and the decrease in the PCT values were not significant. PMID:26417303

  3. An overview of platelet indices for evaluating platelet function in children with scorpion envenomation.

    PubMed

    Konca, Capan; Tekin, Mehmet; Colak, Pinar; Uckardes, Fatih; Turgut, Mehmet

    2014-01-01

    The aim of this study was to assess the correlation between platelet indices and scorpion envenomations (SE). Medical records of 76 children who were hospitalised for scorpion stings in the paediatric intensive care unit (PICU) between February 2013 and November 2013, and 55 healthy children who were similar to the patient group in terms of age and sex, were analysed retrospectively. The leucocyte (WBC), thrombocyte (PLT), plateletcrit (PCT), platelet distribution width (PDW) and mean platelet volume (MPV) values of the 76 children with SE were recorded. These values were compared with the healthy control group. Significantly higher WBC and PDW values were noted in patients with SE in comparison to the controls. Patients with SE had significantly lower mean MPV values compared to the healthy controls (9.03 ± 1.26 compared to 10.43 ± 1.44 fL, respectively; p < 0.001). Although the mean platelet count was slightly elevated in the SE group, no statistically significant difference existed between the two groups (p = 0.097). Furthermore, the mean PCT values in the SE group compared to the control group were slightly decreased, but this decrease was not statistically significant (p = 0.141). A significant inverse correlation existed between the MPV values and the WBC (r = -0.450, p < 0.01) and PLT counts (r = -0.420, p < 0.01). The PLT values were significantly correlated with the PCT values (r = 0.687, p < 0.01). This study demonstrated that SE may lead to several alterations in platelet indices. Significantly lower values of MPV and higher values of PDW were detected in SE patients. However, the increase in the platelet counts and the decrease in the PCT values were not significant. PMID:26417303

  4. Examination of platelet function in whole blood under dynamic flow conditions with the cone and plate(let) analyzer: effect of erythrocytosis and thrombocytosis.

    PubMed

    Peerschke, Ellinor I; Silver, Richard T; Weksler, Babette B; Yin, Wei; Bernhardt, Bernard; Varon, David

    2007-03-01

    We studied cone and plate(let) analysis (CPA) for evaluating global platelet function in whole blood under arterial flow conditions (approximately 1,800 seconds(-1)). CPA allows direct visualization and quantitation of platelet adhesion (surface coverage [SC]) and determination of average aggregate size (AS) following brief shearing of a small blood sample (3.2% sodium citrate) in plastic wells. By using blood from healthy volunteers manipulated to alter platelet or RBC counts and blood from patients with myeloproliferative disorders (MPDs), quantitative and qualitative changes in SC and AS were observed. Thrombocytosis resulted in increased SC, whereas erythrocytosis increased AS. The RBC volume (mean corpuscular volume) had no effect. It is interesting that differences in CPA AS were discerned among subgroups of patients with MPD undergoing different treatment regimens. These studies suggest that CPA platelet deposition patterns may provide novel insight into global platelet function during hemodynamic flow. PMID:17276938

  5. Functional validation of platelet-activating factor receptor sites characterized biochemically by a specific and reproducible ( sup 3 H)platelet-activating factor binding in human platelets

    SciTech Connect

    Tahraoui, L.; Floch, A.; Cavero, I. )

    1990-03-01

    In human platelet membranes, (3H)platelet-activating factor(PAF)-C18 binding sites exhibited high affinity (Kd 0.074 +/- 0.005 nM, n = 28 healthy volunteers), saturability, elevated stereoselectivity, marked pharmacological specificity and small intersubject variability. The maximal binding capacity was 215 +/- 12 fmol/mg protein. Saturation of (3H)PAF binding was obtained with 0.3 nM ligand, and its isotherm was compatible with a single class of binding sites. The stereoselectivity for (3H)PAF was clearly indicated by the low displacing potency of enantio-PAF-C16 (the synthetic enantiomer of PAF) that was 5000-fold less potent than PAF. Specific (3H)PAF binding attained 65% with 0.1 nM ligand and was displaced fully not only by cold PAF but also by RP 59227 (Ki = 6.2 +/- 1.3 nM, n = 7), a novel, potent and specific PAF receptor antagonist in a pure enantiomeric form and several other antagonists such as CV-6209, WEB 2086, L-652,731 and BN 52021. Various classical pharmacological agents did not interfere with the (3H)PAF binding. In intact platelets, (3H)PAF binding shared the same properties as those just described for membrane preparations. A functional role for these binding sites was suggested by the high correlation (r = 0.94, P less than .001) between the Ki values for several known PAF antagonists determined in (3H)PAF binding and the IC50 values obtained against PAF-induced aggregation in whole platelets. Thus, the present (3H)PAF binding in human platelet membranes may be a useful pharmacological tool to study possible changes in (3H)PAF binding parameters induced by pathological states for which PAF may be directly or indirectly responsible.

  6. Lactodifucotetraose, a human milk oligosaccharide, attenuates platelet function and inflammatory cytokine release.

    PubMed

    Newburg, David S; Tanritanir, Ayse C; Chakrabarti, Subrata

    2016-07-01

    Human milk strongly quenches inflammatory processes in vitro, and breastfed infants have lower incidence of inflammatory diseases than those fed artificially. Platelets from neonates, in contrast to those from adults, are less responsive to platelet agonists such as collagen, thrombin, ADP, and epinephrine. Breastfed infants absorb oligosaccharides intact from the human milk in their gut to the circulation. This study was to determine whether these oligosaccharides can attenuate platelet function and platelet secretion of pro-inflammatory proteins, and to identify the active component. The natural mixture of oligosaccharides from human milk and pure individual human milk oligosaccharides were tested for their ability to modulate responses of platelets isolated from human blood following exposure to thrombin, ADP, and collagen. Human milk and the natural mixture of human milk oligosaccharides inhibited platelet release of inflammatory proteins. Of the purified human milk oligosaccharides tested, only lactodifucotetraose (LDFT) significantly inhibited thrombin induced release of the pro-inflammatory proteins RANTES and sCD40L. LDFT also inhibited platelet adhesion to a collagen-coated surface, as well as platelet aggregation induced by ADP or collagen. These data indicate that LDFT may help modulate hemostasis by suppressing platelet-induced inflammatory processes in breastfed infants. This activity suggests further study of LDFT for its potential as a therapeutic agent in infants and adults. PMID:26743063

  7. A shear gradient-activated microfluidic device for automated monitoring of whole blood haemostasis and platelet function

    PubMed Central

    Jain, Abhishek; Graveline, Amanda; Waterhouse, Anna; Vernet, Andyna; Flaumenhaft, Robert; Ingber, Donald E.

    2016-01-01

    Accurate assessment of blood haemostasis is essential for the management of patients who use extracorporeal devices, receive anticoagulation therapy or experience coagulopathies. However, current monitoring devices do not measure effects of haemodynamic forces that contribute significantly to platelet function and thrombus formation. Here we describe a microfluidic device that mimics a network of stenosed arteriolar vessels, permitting evaluation of blood clotting within small sample volumes under pathophysiological flow. By applying a clotting time analysis based on a phenomenological mathematical model of thrombus formation, coagulation and platelet function can be accurately measured in vitro in patient blood samples. When the device is integrated into an extracorporeal circuit in pig endotoxemia or heparin therapy models, it produces real-time readouts of alterations in coagulation ex vivo that are more reliable than standard clotting assays. Thus, this disposable device may be useful for personalized diagnostics and for real-time surveillance of antithrombotic therapy in clinic. PMID:26733371

  8. A shear gradient-activated microfluidic device for automated monitoring of whole blood haemostasis and platelet function.

    PubMed

    Jain, Abhishek; Graveline, Amanda; Waterhouse, Anna; Vernet, Andyna; Flaumenhaft, Robert; Ingber, Donald E

    2016-01-01

    Accurate assessment of blood haemostasis is essential for the management of patients who use extracorporeal devices, receive anticoagulation therapy or experience coagulopathies. However, current monitoring devices do not measure effects of haemodynamic forces that contribute significantly to platelet function and thrombus formation. Here we describe a microfluidic device that mimics a network of stenosed arteriolar vessels, permitting evaluation of blood clotting within small sample volumes under pathophysiological flow. By applying a clotting time analysis based on a phenomenological mathematical model of thrombus formation, coagulation and platelet function can be accurately measured in vitro in patient blood samples. When the device is integrated into an extracorporeal circuit in pig endotoxemia or heparin therapy models, it produces real-time readouts of alterations in coagulation ex vivo that are more reliable than standard clotting assays. Thus, this disposable device may be useful for personalized diagnostics and for real-time surveillance of antithrombotic therapy in clinic. PMID:26733371

  9. The Genetics of Common Variation affecting Platelet Development, Function and Pharmaceutical Targeting

    PubMed Central

    Johnson, Andrew D.

    2011-01-01

    Summary Common variant effects on human platelet function and response to anti-platelet treatment have traditionally been studied using candidate gene approaches involving a limited number of variants and genes. These studies have often been undertaken in clinically defined cohorts. More recently, studies have applied genome-wide scans in larger population samples than prior candidate studies, in some cases scanning relatively healthy individuals. These studies demonstrate synergy with some prior candidate gene findings (e.g., GP6, ADRA2A) but also uncover novel loci involved in platelet function. Here, I summarise findings on common genetic variation influencing platelet development, function and therapeutics. Taken together, candidate gene and genome-wide studies begin to account for common variation in platelet function and provide information that may ultimately be useful in pharmacogenetic applications in the clinic. More than 50 loci have been identified with consistent associations with platelet phenotypes in ≥2 populations. Several variants are under further study in clinical trials relating to anti-platelet therapies. In order to have useful clinical applications, variants must have large effects on a modifiable outcome. Regardless of clinical applications, studies of common genetic influences, even of small effect, offer additional insights into platelet biology including the importance of intracellular signalling and novel receptors. Understanding of common platelet-related genetics remains behind parallel fields (e.g., lipids, blood pressure) due to challenges in phenotype ascertainment. Further work is necessary to discover and characterise loci for platelet function, and to assess whether these loci contribute to disease aetiologies or response to therapeutics. PMID:21781261

  10. Single-cell RNA sequencing reveals molecular and functional platelet bias of aged haematopoietic stem cells.

    PubMed

    Grover, Amit; Sanjuan-Pla, Alejandra; Thongjuea, Supat; Carrelha, Joana; Giustacchini, Alice; Gambardella, Adriana; Macaulay, Iain; Mancini, Elena; Luis, Tiago C; Mead, Adam; Jacobsen, Sten Eirik W; Nerlov, Claus

    2016-01-01

    Aged haematopoietic stem cells (HSCs) generate more myeloid cells and fewer lymphoid cells compared with young HSCs, contributing to decreased adaptive immunity in aged individuals. However, it is not known how intrinsic changes to HSCs and shifts in the balance between biased HSC subsets each contribute to the altered lineage output. Here, by analysing HSC transcriptomes and HSC function at the single-cell level, we identify increased molecular platelet priming and functional platelet bias as the predominant age-dependent change to HSCs, including a significant increase in a previously unrecognized class of HSCs that exclusively produce platelets. Depletion of HSC platelet programming through loss of the FOG-1 transcription factor is accompanied by increased lymphoid output. Therefore, increased platelet bias may contribute to the age-associated decrease in lymphopoiesis. PMID:27009448

  11. Single-cell RNA sequencing reveals molecular and functional platelet bias of aged haematopoietic stem cells

    PubMed Central

    Grover, Amit; Sanjuan-Pla, Alejandra; Thongjuea, Supat; Carrelha, Joana; Giustacchini, Alice; Gambardella, Adriana; Macaulay, Iain; Mancini, Elena; Luis, Tiago C.; Mead, Adam; Jacobsen, Sten Eirik W.; Nerlov, Claus

    2016-01-01

    Aged haematopoietic stem cells (HSCs) generate more myeloid cells and fewer lymphoid cells compared with young HSCs, contributing to decreased adaptive immunity in aged individuals. However, it is not known how intrinsic changes to HSCs and shifts in the balance between biased HSC subsets each contribute to the altered lineage output. Here, by analysing HSC transcriptomes and HSC function at the single-cell level, we identify increased molecular platelet priming and functional platelet bias as the predominant age-dependent change to HSCs, including a significant increase in a previously unrecognized class of HSCs that exclusively produce platelets. Depletion of HSC platelet programming through loss of the FOG-1 transcription factor is accompanied by increased lymphoid output. Therefore, increased platelet bias may contribute to the age-associated decrease in lymphopoiesis. PMID:27009448

  12. Peroxiredoxin II Is an Antioxidant Enzyme That Negatively Regulates Collagen-stimulated Platelet Function*

    PubMed Central

    Jang, Ji Yong; Wang, Su Bin; Min, Ji Hyun; Chae, Yun Hee; Baek, Jin Young; Yu, Dae-Yeul; Chang, Tong-Shin

    2015-01-01

    Collagen-induced platelet signaling is mediated by binding to the primary receptor glycoprotein VI (GPVI). Reactive oxygen species produced in response to collagen have been found to be responsible for the propagation of GPVI signaling pathways in platelets. Therefore, it has been suggested that antioxidant enzymes could down-regulate GPVI-stimulated platelet activation. Although the antioxidant enzyme peroxiredoxin II (PrxII) has emerged as having a role in negatively regulating signaling through various receptors by eliminating H2O2 generated upon receptor stimulation, the function of PrxII in collagen-stimulated platelets is not known. We tested the hypothesis that PrxII negatively regulates collagen-stimulated platelet activation. We analyzed PrxII-deficient murine platelets. PrxII deficiency enhanced GPVI-mediated platelet activation through the defective elimination of H2O2 and the impaired protection of SH2 domain-containing tyrosine phosphatase 2 (SHP-2) against oxidative inactivation, which resulted in increased tyrosine phosphorylation of key components for the GPVI signaling cascade, including Syk, Btk, and phospholipase Cγ2. Interestingly, PrxII-mediated antioxidative protection of SHP-2 appeared to occur in the lipid rafts. PrxII-deficient platelets exhibited increased adhesion and aggregation upon collagen stimulation. Furthermore, in vivo experiments demonstrated that PrxII deficiency facilitated platelet-dependent thrombus formation in injured carotid arteries. This study reveals that PrxII functions as a protective antioxidant enzyme against collagen-stimulated platelet activation and platelet-dependent thrombosis. PMID:25802339

  13. Measurement of platelet aggregation functions using whole blood migration ratio in a microfluidic chip.

    PubMed

    Seo, Hong Seog; Choi, Sung Hyuk; Han, Miran; Kim, Kyeong Ah; Cho, Chi Hyun; An, Seong Soo A; Lim, Chae Seung; Shin, Sehyun

    2015-09-25

    Platelets play a major role in maintaining endothelial integrity and hemostasis. Of the various soluble agonists, ADP is an important in vivo stimulus for inducing platelet aggregation. In this study, a simple, rapid, and affordable method was designed for testing bleeding time (BT) and platelet aggregation with a two-channel microfluidic chip. Whole blood migration ratio (MR) from a microchip system was evaluated in comparison to the closure time (CT) from PFA-100 assays (Siemens, Germany) and CD62P expression on platelets. To induce platelet aggregation, a combination of collagen (1.84 mg/ml) and ADP (37.5 mg/ml) were used as agonists. After adding the agonists to samples, whole blood MR from the microchip system was measured. The outcome of the assessment depended on reaction time and agonist concentration. MR of whole blood from the microchip system was significantly correlated with CT from PFA-100 (r = 0.61, p <  0.05, n = 60). In addition, MR was negatively correlated with CD62P expression (r =-0.95, p <  0.05, n = 60). These results suggest that the measurement of MR using agonists is an easy, simple and efficient method for monitoring platelet aggregation in normal and ADP-receptors defective samples, along with the BT test. Thus, usage of the current microfluidic method could expand to diverse applications, including efficacy assessments in platelet therapy. PMID:26444593

  14. Changes in platelet morphology and function during 24 hours of storage.

    PubMed

    Braune, S; Walter, M; Schulze, F; Lendlein, A; Jung, F

    2014-01-01

    For in vitro studies assessing the interaction of platelets with implant materials, common and standardized protocols for the preparation of platelet rich plasma (PRP) are lacking, which may lead to non-matching results due to the diversity of applied protocols. Particularly, the aging of platelets during prolonged preparation and storage times is discussed to lead to an underestimation of the material thrombogenicity. Here, we study the influence of whole blood- and PRP-storage times on changes in platelet morphology and function. Blood from apparently healthy subjects was collected according to a standardized protocol and examined immediately after blood collection, four hours and twenty four hours later. The capability of platelets to adhere and form stable aggregates (PFA100, closure time) was examined in sodium citrate anticoagulated whole blood (WB) using the agonists equine type I collagen and epinephrine bitartrate (collagen/epinephrine) as well as equine type I collagen and adenosine-5'-diphosphate (collagen/ADP). Circulating platelets were quantified at each time point. Morphology of platelets and platelet aggregates were visualized microscopically and measured using an electric field multi-channel counting system (CASY). The percentage of activated platelets was assessed by means of P-selectin (CD62P) expression of circulating platelets. Furthermore, platelet factor 4 (PF4) release was measured in platelet poor plasma (PPP) at each time point. Whole blood PFA100 closure times increased after stimulation with collagen/ADP and collagen/epinephrine. Twenty four hours after blood collection, both parameters were prolonged pathologically above the upper limit of the reference range. Numbers of circulating platelets, measured in PRP, decreased after four hours, but no longer after twenty four hours. Mean platelet volumes (MPV) and platelet large cell ratios (P-LCR, 12 fL - 40 fL) decreased over time. Immediately after blood collection, no debris or platelet

  15. Platelet MAO and personality--function and dysfunction.

    PubMed

    Oreland, L; Hallman, J; Damberg, M

    2004-08-01

    Research on the association between platelet monoamine oxidase (MAO) activity and personality traits, such as sensation seeking and impulsiveness, is reviewed with an emphasis on early history and current situation. The effects of MAO-inhibiting compounds in cigarette smoke for the interpretation of this association are discussed and recent results confirming a true association between platelet MAO activity and personality and vulnerability, for e.g. type 2 alcoholism are presented. From a clinical point of view, the link between platelet MAO activity, which is highly genetically regulated and is stable in the individual, and personality traits, has had its greatest impact on the understanding of the nature of constitutional factors making individuals vulnerable, for e.g. substance abuse and other forms of sociopathic behaviour. The molecular mechanisms underlying the association between platelet MAO and behaviour are discussed and evidence that common transcriptional factors, e.g. within the AP-2 family, regulating both the expression of platelet MAO and components of the central monoaminergic systems, such as synthesising enzymes, receptors and transporters, are presented. A hypothesis is put forward, that such common transcription factors may not directly regulate platelet MAO expression, but rather mitochondrial density, or outer mitochondrial membrane surface. PMID:15279564

  16. The dimeric platelet collagen receptor GPVI-Fc reduces platelet adhesion to activated endothelium and preserves myocardial function after transient ischemia in mice.

    PubMed

    Schönberger, Tanja; Ziegler, Melanie; Borst, Oliver; Konrad, Ildiko; Nieswandt, Bernhard; Massberg, Steffen; Ochmann, Carmen; Jürgens, Tobias; Seizer, Peter; Langer, Harald; Münch, Götz; Ungerer, Martin; Preissner, Klaus T; Elvers, Margitta; Gawaz, Meinrad

    2012-10-01

    Platelets play a critical role in the pathophysiology of reperfusion, sepsis, and cardiovascular diseases. In a multiple step process, they adhere to activated endothelium and release proinflammatory cytokines thereby promoting the inflammatory process. Glycoprotein VI (GPVI) is the major collagen receptor on the platelet surface and triggers platelet activation and primary hemostasis. Activation of GPVI leads to stable platelet adhesion and degranulation of platelet granules. However, GPVI is critically involved in platelet adhesion to activated endothelium without exposure of subendothelial matrix. Earlier studies show that the soluble GPVI-Fc binds to collagen and protects mice from atherosclerosis and decreases neointima proliferation after arterial injury. Here, we show for the first time that recombinant GPVI-Fc binds to activated endothelium mainly via vitronectin and prevents platelet/endothelial interaction. Administration of GPVI-Fc reduced infarct size and preserved cardiac function in a mouse model of myocardial infarction. This process was associated with reduced GPVI-induced platelet degranulation and release of proinflammatory cytokines in vitro and in vivo. Taken together, administration of GPVI-Fc offers a novel strategy to control platelet-mediated inflammation and to preserve myocardial function following myocardial infarction. PMID:22814400

  17. Functional responses and molecular mechanisms involved in histone-mediated platelet activation.

    PubMed

    Carestia, A; Rivadeneyra, L; Romaniuk, M A; Fondevila, C; Negrotto, S; Schattner, M

    2013-11-01

    Histones are highly alkaline proteins found in cell nuclei and they can be released by either dying or inflammatory cells. The recent observations that histones are major components of neutrophil extracellular traps and promote platelet aggregation and platelet-dependent thrombin generation have shown that these proteins are potent prothrombotic molecules. Because the mechanism(s) of platelet activation by histones are not completely understood, we explored the ability of individual recombinant human histones H1, H2A, H2B, H3 and H4 to induce platelet activation as well as the possible molecular mechanisms involved. All histones were substrates for platelet adhesion and spreading and triggered fibrinogen binding, aggregation, von Willebrand factor release, P-selectin and phosphatidylserine (PS) exposure and the formation of platelet-leukocyte aggregates; however, H4 was the most potent. Histone-mediated fibrinogen binding, P-selectin and PS exposure and the formation of mixed aggregates were potentiated by thrombin. Histones induced the activation of ERK, Akt, p38 and NFκB. Accordingly, histone-induced platelet activation was significantly impaired by pretreatment of platelets with inhibitors of ERK (U 0126), PI3K/Akt (Ly 294002), p38 (SB 203580) and NFκB (BAY 11-7082 and Ro 106-9920). Preincubation of platelets with either aspirin or dexamethasone markedly decreased fibrinogen binding and the adhesion mediated by histones without affecting P-selectin exposure. Functional platelet responses induced by H3 and H4, but not H1, H2A and H2B, were partially mediated through interaction with Toll-like receptors -2 and -4. Our data identify histones as important triggers of haemostatic and proinflammatory platelet responses, and only haemostatic responses are partially inhibited by anti-inflammatory drugs. PMID:23965842

  18. A novel assay for the detection of anti-human platelet antigen antibodies (HPA-1a) based on peptide aptamer technology

    PubMed Central

    Thibaut, Julien; Mérieux, Yves; Rigal, Dominique; Gillet, Germain

    2012-01-01

    Background Neonatal alloimmune thrombocytopenia is mostly due to the presence of maternal antibodies against the fetal platelet antigen HPA-1a on the platelet integrin GPIIb-IIIa. Accurate detection of anti-HPA-1a antibodies in the mother is, therefore, critical. Current diagnostic assays rely on the availability of pools of human platelets that vary according to donors and blood centers. There is still no satisfactory standardization of these assays. Design and Methods Peptide aptamer was used to detect and identify HPA-1a-specific antibodies in human serum that do not require human platelets. A peptide aptamer library was screened using an anti-HPA-1a human monoclonal antibody as a bait to isolate an aptamer that mimics the human platelet antigen HPA-1a. Results This is the first report in platelet immunology of the use of a peptide aptamer for diagnostic purposes. This assay gives better results than the MAIPA currently in use, detecting around 90% of the expected alloantibodies. Conclusions This assay could help define a standard for the quantitation of anti-HPA antibodies. This report also demonstrates that peptide aptamers can potentially detect a variety of biomarkers in body fluids; this is of particular interest for diagnostic purposes. PMID:22133781

  19. Lyn and PECAM-1 function as interdependent inhibitors of platelet aggregation.

    PubMed

    Ming, Zhangyin; Hu, Yu; Xiang, Jizhou; Polewski, Peter; Newman, Peter J; Newman, Debra K

    2011-04-01

    Inhibition of platelet responsiveness is important to control pathologic thrombus formation. Platelet-endothelial cell adhesion molecule-1 (PECAM-1) and the Src family kinase Lyn inhibit platelet activation by the glycoprotein VI (GPVI) collagen receptor; however, it is not known whether PECAM-1 and Lyn function in the same or different inhibitory pathways. In these studies, we found that, relative to wild-type platelets, platelets derived from PECAM-1-deficient, Lyn-deficient, or PECAM-1/Lyn double-deficient mice were equally hyperresponsive to stimulation with a GPVI-specific agonist, indicating that PECAM-1 and Lyn participate in the same inhibitory pathway. Lyn was required for PECAM-1 tyrosine phosphorylation and subsequent binding of the Src homology 2 domain-containing phosphatase-2, SHP-2. These results support a model in which PECAM-1/SHP-2 complexes, formed in a Lyn-dependent manner, suppress GPVI signaling. PMID:21297004

  20. Effect in vitro on platelet function of two compounds developed from the pyrimido-pyrimidines

    PubMed Central

    Slater, S. D.; Turpie, A. G. G.; Douglas, A. S.; McNicol, G. P.

    1972-01-01

    VK 774 and VK 744, two new compounds developed from the pyrimido-pyrimidines, have been found to be powerful inhibitors of platelet function tested in vitro. They inhibit adenosine diphosphate (ADP)-induced platelet aggregation, and the release of platelet factor 3 by kaolin, and VK 774 also reduces platelet adhesiveness and inhibits platelet aggregation (`snowstorm' effect) in the Chandler tube system. Although measured percentage whole blood clot retraction was uninfluenced by these drugs the clot produced with VK 774 was friable and soft. VK 774 appears to be the most powerful of these compounds reported so far, being active in some test systems at 10−6M, and, if the results of toxicity testing are satisfactory, it should be an important agent for therapeutic trial. PMID:5046075

  1. Procoagulant and platelet-derived microvesicle absolute counts determined by flow cytometry correlates with a measurement of their functional capacity

    PubMed Central

    Ayers, Lisa; Harrison, Paul; Kohler, Malcolm; Ferry, Berne

    2014-01-01

    Background Flow cytometry is the most commonly used technology to measure microvesicles (MVs). Despite reported limitations of this technique, MV levels obtained using conventional flow cytometry have yielded many clinically relevant findings, such as associations with disease severity and ability to predict clinical outcomes. This study aims to determine if MV enumeration by flow cytometry correlates with a measurement of their functional capacity, as this may explain how flow cytometry generates clinically relevant results. Methods One hundred samples from healthy individuals and patients with obstructive sleep apnoea were analysed by conventional flow cytometry (FACSCalibur) and by three functional MV assays: Zymuphen MP-activity in which data were given as phosphatidylserine equivalent, STA® Phospholipid Procoag Assay expressed as clotting time and Endogenous Thrombin Potential (ETP) reflecting in vitro thrombin generation. Correlations were determined by Spearman correlation. Results Absolute counts of lactadherin+ procoagulant MVs generated by flow cytometry weakly correlated with the results obtained from the Zymuphen MP-activity (r=0.5370, p<0.0001); correlated with ETP (r=0.7444, p<0.0001); negatively correlated with STA® Phospholipid Procoag Assay clotting time (−0.7872, p<0.0001), reflecting a positive correlation between clotting activity and flow cytometry. Levels of Annexin V+ procoagulant and platelet-derived MVs were also associated with functional assays. Absolute counts of MVs derived from other cell types were not correlated with the functional results. Conclusions Quantitative results of procoagulant and platelet-derived MVs from conventional flow cytometry are associated with the functional capability of the MVs, as defined by three functional MV assays. Flow cytometry is a valuable technique for the quantification of MVs from different cellular origins; however, a combination of several analytical techniques may give the most comprehensive

  2. Platelet storage in PAS-2 or autologous plasma: impact on functional parameters.

    PubMed

    Bunescu, A; Hild, M; Lundahl, J; Egberg, N

    2001-04-01

    Currently, several platelet additive solutions for long-term platelet storage have been introduced. The aim of this study was to compare the deterioration of functional status of platelets stored for up to 5 days in autologous plasma (AP) only, with platelet stored in PAS-2, a salt solution containing acetate, citrate and sodium chloride. Change in platelet adhesion, aggregation and activation was measured by flow cytometric technique. In addition, beta-Thromboglobulin (beta-TG), lactate and glucose were determined. After 5 days of storage, the expression of P-Selectin was significantly higher, the production of lactate and the consumption of glucose were significantly lower, in platelets stored in PAS-2 than in autologous plasma. No significant differences were detected on day 5 between the two groups with regard to fibrinogen, von Willebrand factor binding capacity, or to beta-TG release. It can be concluded that neither storage medium was consistently better for the parameters tested. However, it must be emphasized that platelets stored in autologous plasma exhibited less lesion, in terms of P-Selectin expression compared with platelets stored in PAS-2. PMID:11299027

  3. Expression of the PlA2 allele of glycoprotein IIIa and its impact on platelet function

    PubMed Central

    Ferro, Albert; Warner, Timothy D

    2015-01-01

    Background The platelet fibrinogen receptor represents the final common pathway of platelet activation, and is formed from two glycoprotein (GP) subunits (GPIIb/IIIa). Carriage of the mutant PlA2 allele of GPIIIa has been shown to confer an increased risk of cardiovascular events, but published studies have disagreed as to the mechanism for this association. Objectives To assess whether carriage of the PlA2 allele conforms to Mendelian patterns of expression and to identify whether carriage of the mutant allele modulates platelet function. Methods Expression of the PlA2 allele was assessed in both healthy subjects (n = 25) and patients with known coronary artery disease (n = 90) through the development and validation of a liquid chromatography, tandem mass spectrometry (LC-MS/MS) assay. Platelet function was assessed in the patient cohort in response to multiple agonists, and these data were analysed in the context of the proteomic data. Results Expression of the wild-type PlA1 allele and mutant PlA2 alleles was readily quantifiable and conformed to Mendelian patterns in both healthy and patient cohorts. Patients who were homozygous for the mutant PlA2 allele had an increased aggregatory response to adenosine diphosphate, collagen, adrenaline, ristocetin, thrombin receptor-activating peptide 6 and U46619, when assessed using agonist-concentration response curves. Conclusions These findings support the hypothesis that carriage of the mutant PlA2 allele mediates an increased risk of cardiovascular events through the modulation of platelet reactivity. PMID:26858830

  4. Editorial Commentary: Platelet-Rich Plasma Improves Knee Pain and Function in Patients With Knee Osteoarthritis.

    PubMed

    Lubowitz, James H

    2015-11-01

    Systematic review of overlapping meta-analyses shows that platelet-rich plasma improves knee pain and function in patients with knee osteoarthritis. Ultimately, biologics hold promise for chondroprotection in addition to symptomatic relief. PMID:26542203

  5. Functionalized Nanofiber Meshes Enhance Immunosorbent Assays.

    PubMed

    Hersey, Joseph S; Meller, Amit; Grinstaff, Mark W

    2015-12-01

    Three-dimensional substrates with high surface-to-volume ratios and subsequently large protein binding capacities are of interest for advanced immunosorbent assays utilizing integrated microfluidics and nanosensing elements. A library of bioactive and antifouling electrospun nanofiber substrates, which are composed of high-molecular-weight poly(oxanorbornene) derivatives, is described. Specifically, a set of copolymers are synthesized from three 7-oxanorbornene monomers to create a set of water insoluble copolymers with both biotin (bioactive) and triethylene glycol (TEG) (antifouling) functionality. Porous three-dimensional nanofiber meshes are electrospun from these copolymers with the ability to specifically bind streptavidin while minimizing the nonspecific binding of other proteins. Fluorescently labeled streptavidin is used to quantify the streptavidin binding capacity of each mesh type through confocal microscopy. A simplified enzyme-linked immunosorbent assay (ELISA) is presented to assess the protein binding capabilities and detection limits of these nanofiber meshes under both static conditions (26 h) and flow conditions (1 h) for a model target protein (i.e., mouse IgG) using a horseradish peroxidase (HRP) colorimetric assay. Bioactive and antifouling nanofiber meshes outperform traditional streptavidin-coated polystyrene plates under flow, validating their use in future advanced immunosorbent assays and their compatibility with microfluidic-based biosensors. PMID:26551162

  6. Ovine platelet function is unaffected by extracorporeal membrane oxygenation within the first 24 h.

    PubMed

    Hayes, Rylan A; Foley, Samuel; Shekar, Kiran; Diab, Sara; Dunster, Kimble R; McDonald, Charles; Fraser, John F

    2015-10-01

    This study investigated platelet dysfunction during short-term extracorporeal membrane oxygenation (ECMO) and secondarily to determine if hyperoxaemia contributes to this dysfunction. Healthy sheep were anaesthetized and maintained on ECMO for either 2 or 24 h, with or without induction of smoke inhalation acute lung injury. A specialized animal-operating theatre was used to conduct the experimentation. Forty-three healthy female sheep were randomized into either a test or a control group. Following anaesthesia, test groups received ECMO ± smoke inhalation acute lung injury (SALI), whereas control groups were maintained with ventilation only ± SALI. Physiological, biochemical and coagulation data were obtained throughout via continuous monitoring and blood sampling. Platelet function was quantified through whole blood impedance aggregometry using Multiplate. Ovine platelet activity induced by adenosine diphosphate (ADP) and collagen was unaffected during the first 24 h of ECMO. However, progressive divergence of ADP-induced platelet activity was noted at cessation of the experiment. PaO2 was inversely related to ADP-dependent platelet activity in the ECMO groups--a relationship not identified in the control groups. ADP and collagen-dependent platelet activity are not significantly affected within the first 24 h of ECMO in sheep. However, dysfunction in ADP-dependent platelet activity may have continued to develop if observed beyond 24 h. Hyperoxaemia during ECMO does appear to affect how platelets react to ADP and may contribute to this developing dysfunction. Long-term animal models and investigation in clinical animals are warranted to fully investigate platelet function during ECMO. PMID:26196193

  7. Genomic landscape of megakaryopoiesis and platelet function defects

    PubMed Central

    Bianchi, Elisa; Norfo, Ruggiero; Pennucci, Valentina; Zini, Roberta

    2016-01-01

    Megakaryopoiesis is a complex, stepwise process that takes place largely in the bone marrow. At the apex of the hierarchy, hematopoietic stem cells undergo a number of lineage commitment decisions that ultimately lead to the production of polyploid megakaryocytes. On average, megakaryocytes release 1011 platelets per day into the blood that repair vascular injuries and prevent excessive bleeding. This differentiation process is tightly controlled by exogenous and endogenous factors, which have been the topics of intense research in the hematopoietic field. Indeed, a skewing of megakaryocyte commitment and differentiation may entail the onset of myeloproliferative neoplasms and other preleukemic disorders together with acute megakaryoblastic leukemia, whereas quantitative or qualitative defects in platelet production can lead to inherited platelet disorders. The recent advent of next-generation sequencing has prompted mapping of the genomic landscape of these conditions to provide an accurate view of the underlying lesions. The aims of this review are to introduce the physiological pathways of megakaryopoiesis and to present landmark studies on acquired and inherited disorders that target them. These studies have not only introduced a new era in the fields of molecular medicine and targeted therapies but may also provide us with a better understanding of the mechanisms underlying normal megakaryopoiesis and thrombopoiesis that can inform efforts to create alternative sources of megakaryocytes and platelets. PMID:26787733

  8. Oral administration of Bruton's tyrosine kinase inhibitors impairs GPVI-mediated platelet function.

    PubMed

    Rigg, Rachel A; Aslan, Joseph E; Healy, Laura D; Wallisch, Michael; Thierheimer, Marisa L D; Loren, Cassandra P; Pang, Jiaqing; Hinds, Monica T; Gruber, András; McCarty, Owen J T

    2016-03-01

    The Tec family kinase Bruton's tyrosine kinase (Btk) plays an important signaling role downstream of immunoreceptor tyrosine-based activation motifs in hematopoietic cells. Mutations in Btk are involved in impaired B-cell maturation in X-linked agammaglobulinemia, and Btk has been investigated for its role in platelet activation via activation of the effector protein phospholipase Cγ2 downstream of the platelet membrane glycoprotein VI (GPVI). Because of its role in hematopoietic cell signaling, Btk has become a target in the treatment of chronic lymphocytic leukemia and mantle cell lymphoma; the covalent Btk inhibitor ibrutinib was recently approved by the US Food and Drug Administration for treatment of these conditions. Antihemostatic events have been reported in some patients taking ibrutinib, although the mechanism of these events remains unknown. We sought to determine the effects of Btk inhibition on platelet function in a series of in vitro studies of platelet activation, spreading, and aggregation. Our results show that irreversible inhibition of Btk with two ibrutinib analogs in vitro decreased human platelet activation, phosphorylation of Btk, P-selectin exposure, spreading on fibrinogen, and aggregation under shear flow conditions. Short-term studies of ibrutinib analogs administered in vivo also showed abrogation of platelet aggregation in vitro, but without measurable effects on plasma clotting times or on bleeding in vivo. Taken together, our results suggest that inhibition of Btk significantly decreased GPVI-mediated platelet activation, spreading, and aggregation in vitro; however, prolonged bleeding was not observed in a model of bleeding. PMID:26659727

  9. Human CalDAG-GEFI gene (RASGRP2) mutation affects platelet function and causes severe bleeding

    PubMed Central

    Canault, Matthias; Ghalloussi, Dorsaf; Grosdidier, Charlotte; Guinier, Marie; Perret, Claire; Chelghoum, Nadjim; Germain, Marine; Raslova, Hana; Peiretti, Franck; Morange, Pierre E.; Saut, Noemie; Pillois, Xavier; Nurden, Alan T.; Cambien, François; Pierres, Anne; van den Berg, Timo K.; Kuijpers, Taco W.; Tregouet, David-Alexandre

    2014-01-01

    The nature of an inherited platelet disorder was investigated in three siblings affected by severe bleeding. Using whole-exome sequencing, we identified the culprit mutation (cG742T) in the RAS guanyl-releasing protein-2 (RASGRP2) gene coding for calcium- and DAG-regulated guanine exchange factor-1 (CalDAG-GEFI). Platelets from individuals carrying the mutation present a reduced ability to activate Rap1 and to perform proper αIIbβ3 integrin inside-out signaling. Expression of CalDAG-GEFI mutant in HEK293T cells abolished Rap1 activation upon stimulation. Nevertheless, the PKC- and ADP-dependent pathways allow residual platelet activation in the absence of functional CalDAG-GEFI. The mutation impairs the platelet’s ability to form thrombi under flow and spread normally as a consequence of reduced Rac1 GTP-binding. Functional deficiencies were confined to platelets and megakaryocytes with no leukocyte alteration. This contrasts with the phenotype seen in type III leukocyte adhesion deficiency caused by the absence of kindlin-3. Heterozygous did not suffer from bleeding and have normal platelet aggregation; however, their platelets mimicked homozygous ones by failing to undergo normal adhesion under flow and spreading. Rescue experiments on cultured patient megakaryocytes corrected the functional deficiency after transfection with wild-type RASGRP2. Remarkably, the presence of a single normal allele is sufficient to prevent bleeding, making CalDAG-GEFI a novel and potentially safe therapeutic target to prevent thrombosis. PMID:24958846

  10. The influence of herbal medicine on platelet function and coagulation: a narrative review.

    PubMed

    McEwen, Bradley J

    2015-04-01

    Cardiovascular disease (CVD) is the leading cause of death worldwide. Platelet activation and aggregation play a central role in hemostasis and thrombosis. Herbal medicines have been traditionally used in the management of CVD and can play a role in modifying CVD progression, particularly in platelet function, and have the potential of altering platelet function tests, as well as some coagulation parameters. Herbal medicines, such as feverfew, garlic, ginger, ginseng, motherwort, St John's wort, and willow bark, were found to reduce platelet aggregation. In vitro studies show promise in the reduction of platelet aggregation for Andrographis, feverfew, garlic, ginger, Ginkgo, ginseng, hawthorn, horse chestnut, and turmeric. In addition, cranberry, danshen, dong quai, Ginkgo, ginseng, green tea, and St John's wort were found to have potential interactions with warfarin. Furthermore, St John's wort interacted with clopidogrel and danshen with aspirin. Therefore, repeat testing of platelet function and coagulation studies, particularly for patients on warfarin therapy, may be required after exclusion of herbal medicines that could have possibly affected initial test results. PMID:25839871

  11. Multiwavelength UV/visible spectroscopy for the quantitative investigation of platelet quality

    NASA Astrophysics Data System (ADS)

    Mattley, Yvette D.; Leparc, German F.; Potter, Robert L.; Garcia-Rubio, Luis H.

    1998-04-01

    The quality of platelets transfused is vital to the effectiveness of the transfusion. Freshly prepared, discoid platelets are the most effective treatment for preventing spontaneous hemorrhage or for stopping an abnormal bleeding event. Current methodology for the routine testing of platelet quality involves random pH testing of platelet rich plasma and visual inspection of platelet rich plasma for a swirling pattern indicative of the discoid shape of the cells. The drawback to these methods is that they do not provide a quantitative and objective assay for platelet functionality that can be used on each platelet unit prior to transfusion. As part of a larger project aimed at characterizing whole blood and blood components with multiwavelength UV/vis spectroscopy, isolated platelets and platelet in platelet rich plasma have been investigated. Models based on Mie theory have been developed which allow for the extraction of quantitative information on platelet size, number and quality from multi-wavelength UV/vis spectra. These models have been used to quantify changes in platelet rich plasma during storage. The overall goal of this work is to develop a simple, rapid quantitative assay for platelet quality that can be used prior to platelet transfusion to ensure the effectiveness of the treatment. As a result of this work, the optical properties for isolated platelets, platelet rich plasma and leukodepleted platelet rich plasma have been determined.

  12. Effects of antihypertensive treatment on platelet function in essential hypertension.

    PubMed

    Gomi, T; Ikeda, T; Shibuya, Y; Nagao, R

    2000-11-01

    To evaluate the effect of antihypertensive therapy on platelet activation in essential hypertension, the plasma levels of beta-thromboglobulin (beta-TG) were examined in 45 patients with essential hypertension and 20 age-matched normotensive control subjects. Hypertensive patients were assigned to monotherapy with one of five different antihypertensive drugs for 6 months, and the change of plasma levels of beta-TG was reexamined after the completion of the monotherapy. The plasma beta-TG increased in hypertensive patients compared with levels in normotensive control subjects. Monotherapy with each drug resulted in sufficient blood pressure control in all hypertensive patients. The plasma beta-TG decreased significantly after monotherapy with an alpha-blocker or an angiotensin-converting enzyme inhibitor (ACEI). The plasma beta-TG increased with the use of a diuretic but did not change with the use of a beta-blocker or calcium antagonist. The platelet activation observed in patients with essential hypertension is reversed by monotherapy with an alpha-blocker or an ACEI. It is possible that these drugs reduce the development of hypertensive vascular complications due to suppression of platelet activation in patients with essential hypertension. PMID:11131267

  13. Evaluation of platelet function using the in vitro bleeding time and corrected count increment of transfused platelets. Comparison between platelet concentrates derived from pooled buffy coates and apheresis.

    PubMed

    Eriksson, L; Kristensen, J; Olsson, K; Bring, J; Högman, C F

    1996-01-01

    The functional capacity of transfused platelets was evaluated with in vitro bleeding time (IVBT) and corrected count increment (CCI) in order to compare platelet concentrates (PCs) derived from pooled buffy coats (BC-PCs) with PCs collected by apheresis (A-PCs). The suspension medium in the BC-PCs was 30% CPD plasma and 70% of an additive solution (containing sodium and potassium chloride, sodium citrate and phosphate, mannitol), and in the A-PCs the medium was 100% CPD plasma. IVBT was evaluated using a Thrombostat 4000/2. BC-PC and A-PC were transfused 57 and 41 times, respectively to 36 patients with chemotherapy-induced thrombocytopenia. PCs transfused within 2 days of donation were considered fresh, and those transfused within 3-5 days were considered stored. IVBT was determined before, as well as 10-30 min and 24 h after transfusion; CCI was determined 10-30 min and 24 h after transfusion. The median pretransfusion IVBT value was 486 s. It was measurable in 21 of 98 (21%) of the transfusions, i.e. below the cutoff limit of 486 s. Ten to 30 min after transfusion, the IVBT showed a measurable reduction in 90% of the transfusions with fresh BC-PCs, 92% of those with fresh a-PCs, 63% of those with stored BC-PCs and 79% of those with stored A-PCs. After 24 h, the corresponding values were 63% for fresh BC-PCs, 50% for fresh A-PCs, 26% for stored BC-PCs and 38% for stored A-PCs. The median value of CCI 10-30 min after transfusion was 20 for fresh BC-PCs, 17 for fresh A-PCs, 16 for stored BC-PCs and 14 for stored A-PCs. The difference in IVBT between fresh and stored BC-PCs was significant (p = 0.032), unlike that between fresh and stored A-PC. After 24 h the corresponding values were 7 for fresh BC-PCs, 4 for fresh A-PCs, 4 for stored BC-PCs and 3 for stored A-PCs. When all transfusions with fresh PCs (BC-PCs + A-PCs) were compared with all transfusions with stored PCs, a statistical difference was demonstrated in both CCI (p = 0.027) and IVBT (p = 0.043). Spearman

  14. Effects on platelet function of combination etoposide and carboplatin chemotherapy in pediatric oncology patients.

    PubMed

    Pignatelli, P; Properzi, E; Pisani, M; Clerico, A; Schiavetti, A; Lenti, L; Pulcinelli, F M; Ferroni, P; Gazzaniga, P P

    1998-01-01

    The effects of a therapeutic course of the combination of carboplatin and etoposide on platelet function have been evaluated in 10 pediatric patients with brain tumors. Platelet count, in vitro aggregation tests, P-selectin expression and agonist-induced ATP release were evaluated before, and 7 and 15 days after one cycle of chemotherapy. The analysis of the results demonstrated the presence of an in vitro platelet aggregation defect in response to collagen and arachidonic acid in all patients 7 days after therapy. A concomitant decrease of collagen- and arachidonic acid-induced ATP release was also observed. Both platelet aggregation and ATP release returned to baseline values 15 days after chemotherapy administration. Conversely, in vitro platelet aggregation and secretion induced by ADP and epinephrine were unaltered by carboplatin and etoposide administration. Furthermore, P-selectin expression was negative at baseline and did not change after chemotherapy. These results support the hypothesis that combination etoposide and carboplatin chemotherapy in pediatric patients is responsible for possible disturbances in biochemical pathways required for platelet secretion and aggregation. PMID:16793755

  15. Sulfasalazine and its metabolites inhibit platelet function in patients with inflammatory arthritis.

    PubMed

    MacMullan, Paul A; Madigan, Anne M; Paul, Nevin; Peace, Aaron J; Alagha, Ahmed; Nolan, Kevin B; McCarthy, Geraldine M; Kenny, Dermot

    2016-02-01

    The purpose of this study is to assess the effect of sulfasalazine and its metabolites on platelet function in patients with inflammatory arthritis (IA). One hundred thirty-five consecutive patients with an established diagnosis of IA were screened. Those with a history of cardiovascular disease (CVD), taking anti-platelet agents or non-steroidal anti-inflammatory drugs (NSAIDs) were excluded. A total of 32 patients were investigated, 15 taking sulfasalazine and 17 taking other disease-modifying anti-rheumatic drugs (DMARDs) and no sulfasalazine. These two cohorts were compared to 15 patients with stable CVD on long-term aspirin. The effect of sulfasalazine and its metabolites on arachidonic acid (AA)-induced platelet aggregation was also tested in vitro in samples from healthy donors (n = 18). Demographics, CVD risk factors and disease activity indices were similar in the sulfasalazine and other DMARD groups. AA-induced platelet aggregation was significantly inhibited in the sulfasalazine group (9 ± 7 %) and comparable to that in the aspirin group (10 ± 6 %). In contrast, there was no effect on AA-induced platelet aggregation in the other DMARDs group (77 ± 12 %) (p < 0.001). Furthermore, sulfasalazine therapy had no effect on platelet aggregation in response to multiple other agonists. Sulfasalazine and its metabolites (5-aminosalicylic acid and sulfapyridine) exerted an additive and dose-dependent inhibitory effect on AA-induced platelet aggregation in vitro (p < 0.001). The inhibition of AA-induced platelet aggregation by sulfasalazine is comparable to that achieved by aspirin and is dependent on both sulfasalazine and its metabolites. This represents a potential mechanism that may contribute to the known cardioprotective effect of sulfasalazine in patients with IA. PMID:25253538

  16. ARF6-dependent regulation of P2Y receptor traffic and function in human platelets.

    PubMed

    Kanamarlapudi, Venkateswarlu; Owens, Sian E; Saha, Keya; Pope, Robert J; Mundell, Stuart J

    2012-01-01

    Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y(1) and P2Y(12) purinoceptors. Recently, we demonstrated that P2Y(1) and P2Y(12) purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6) in the internalization and function of P2Y(1) and P2Y(12) purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y(1) or P2Y(12) purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP) kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function. PMID:22916275

  17. ARF6-Dependent Regulation of P2Y Receptor Traffic and Function in Human Platelets

    PubMed Central

    Kanamarlapudi, Venkateswarlu; Owens, Sian E.; Saha, Keya; Pope, Robert J.; Mundell, Stuart J.

    2012-01-01

    Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y1 and P2Y12 purinoceptors. Recently, we demonstrated that P2Y1 and P2Y12 purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6) in the internalization and function of P2Y1 and P2Y12 purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y1 or P2Y12 purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP) kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function. PMID:22916275

  18. Desmopressin in vitro effects on platelet function, monitored with Multiplate, ROTEM and Sonoclot.

    PubMed

    Pearson, Kevin; Jensen, Hanna; Kander, Thomas; Schött, Ulf

    2016-07-01

    Background and aims The vasopressin analogue desmopressin has demonstrated efficacy in decreasing bleeding time by increasing the circulating levels of coagulation factor VIII and von Willebrand factor, but also by direct effects on platelets. Previous studies have demonstrated contrasting results regarding the effect of desmopressin on platelets in vitro. The aim of this study was to investigate the dose-response effects of in vitro desmopressin in whole blood. Our hypothesis was that desmopressin could increase platelet function in anticoagulated whole blood being stored up to 4 hours. Methods Desmopressin was administered with up to four different concentrations to venous whole blood, sampled with standard vacutainer tubes from 10 healthy volunteers after consent. Platelet function was analyzed with three different point-of-care techniques: Multiplate platelet aggregometry with adenosine diphosphate, collagen, thrombin receptor activating peptide-6, ristocetin and arachidonic acid agonists, tissue factor-activated thromboelastometry and Sonoclot glass bead viscoelastic coagulation tests at baseline and 4 hours later using different activator reagents. Results Thromboelastometry and Sonoclot did not show any significant change between baseline and 4 h later. A significant decrease in area under curve (AUC) could be seen with the Multiplate between baseline and after 4 h. Desmopressin did not improve any of these tests at baseline or during a 4 h storage and incubation period. Conclusion In vitro administered desmopressin could not increase normal platelet function or coagulation being measured with thromboelastometry and Sonoclot. Multiplate indicated decreased platelet aggregation over time, without any effect of in vitro added desmopressin. PMID:26923171

  19. Comparison between urinary 11-dehydrothromboxane B2 detection and platelet Light Transmission Aggregometry (LTA) assays for evaluating aspirin response in elderly patients with coronary artery disease.

    PubMed

    Liu, Tengfei; Zhang, Jingwei; Chen, Xiahuan; Feng, Xueru; Fu, Sidney W; McCaffrey, Timothy A; Liu, Meilin

    2015-10-15

    Aspirin is widely used in the primary and secondary prevention of cardiovascular diseases. The aim of our study was to compare between two established methods of aspirin response, urinary 11-dehydrothromboxane B2 (11dhTXB2) and platelet Light Transmission Aggregometry (LTA) assays in elderly Chinese patients with coronary artery disease (CAD), and to investigate the clinical significance of both methods in predicting cardiovascular events. Urinary 11dhTxB2 assay and arachidonic acid-induced (AA, 0.5mg/ml) platelet aggregation by Light Transmission Aggregometry (LTAAA) assay were measured to evaluate aspirin responses. High-on aspirin platelet reactivity (HAPR) was defined as urinary 11dhTxB2>1500pg/mg or AA-induced platelet aggregation≥15.22%-the upper quartile of our enrolled population. The two tests showed a poor correlation for aspirin inhibition (r=0.063) and a poor agreement in classifying HAPR (kappa=0.053). With a mean follow-up time of 12months, cardiovascular events occurred more frequently in HAPR patients who were diagnosed by LTA assay as compared with no-HAPR patients (22.5% versus 10.6%, P=0.039, OR=2.45, 95% CI=1.06-5.63). However, the HAPR status, as determined by urinary 11dTXB2 measurement, did not show a significant correlation with outcomes. PMID:26095809

  20. Platelet function testing in transient ischaemic attack and ischaemic stroke: A comprehensive systematic review of the literature.

    PubMed

    Lim, Soon Tjin; Coughlan, Catherine A; Murphy, Stephen J X; Fernandez-Cadenas, Israel; Montaner, Joan; Thijs, Vincent; Marquardt, Lars; McCabe, Dominick J H

    2015-01-01

    The majority of patients with ischaemic cerebrovascular disease (CVD) are not protected from further vascular events with antiplatelet therapy. Measurement of inhibition of platelet function ex vivo on antiplatelet therapy, using laboratory tests that correlate with the clinical effectiveness of these agents, would potentially enable physicians to tailor antiplatelet therapy to suit individuals. A systematic review of the literature was performed to collate all available data on ex vivo platelet function/reactivity in CVD patients, especially those treated with aspirin, dipyridamole or clopidogrel. Particular emphasis was paid to information from commonly available whole blood platelet function analysers (PFA-100®, VerifyNow® and Multiplate®). Data on pharmacogenetic mechanisms potentially influencing high on-treatment platelet reactivity (HTPR) on antiplatelet therapy in CVD were reviewed. Two-hundred forty-nine potentially relevant articles were identified; 93 manuscripts met criteria for inclusion. The prevalence of ex vivo HTPR in CVD varies between 3-62% with aspirin monotherapy, 8-61% with clopidogrel monotherapy and 56-59% when dipyridamole is added to aspirin in the early, subacute or late phases after TIA/stroke onset. The prevalence of HTPR on aspirin was higher on the PFA-100 than on the VerifyNow in one study (p < 0.001). Furthermore, the prevalence of HTPR on aspirin was lower when one used 'novel longitudinal' rather than 'cross-sectional, case-control' definitions of HTPR on the PFA early after TIA or stroke (p = 0.003; 1 study). Studies assessing the influence of genetic polymorphisms on HTPR in CVD patients are limited, and need validation in large multicentre studies. Available data illustrate that an important proportion of CVD patients have ex vivo HTPR on their prescribed antiplatelet regimen, and that the prevalence varies depending on the definition and assay used. Large, adequately-sized, prospective multicentre collaborative studies are

  1. Platelet function during cardiopulmonary bypass using multiple electrode aggregometry: comparison of centrifugal and roller pumps.

    PubMed

    Kehara, Hiromu; Takano, Tamaki; Ohashi, Noburo; Terasaki, Takamitsu; Amano, Jun

    2014-11-01

    Blood trauma may be lower with centrifugal pumps (CPs) than with roller pumps (RPs) during cardiopulmonary bypass (CPB), because, unlike RPs, CPs do not compress the tubing, and shear stress is considered lower in CPs than in RPs. However, relative platelet function remains unclear. Using multiple electrode aggregometry (MEA), we compared platelet function with CP and RP. Ten swine underwent CPB for 3 h, with five weaned off using CP and five using RP. Platelet function was measured using MEA, as were hemoglobin concentration and platelet count, before sternotomy, after heparin infusion, 30 min and 3 h after starting CPB, after protamine infusion, and 60 min after stopping CPB. Platelet activation was initiated with adenosine diphosphate (ADP), arachidonic acid (AA), and thrombin receptor-activating protein 6 (TRAP). Fibrinogen, platelet factor 4 (PF4), and β-thromboglobin (β-TG) concentrations were measured before sternotomy and 60 min after stopping CPB. In the CP group and using ADP, aggregation was significantly reduced 30 min (P = 0.019) and 3 h (P = 0.027) after starting CPB, recovering to baseline 60 min after CPB was stopped. In the RP group, aggregation was significantly decreased 30 min (P = 0.007) and 3 h (P = 0.003) after starting CPB and after protamine administration (P = 0.028). With AA, aggregation significantly decreased 30 min after starting CPB in both the CP (P = 0.012) and RP (P = 0.016) groups, slightly increasing 3 h after starting CPB and after protamine infusion, and recovering to baseline 60 min after CPB cessation. With TRAP, aggregation in the CP and RP groups decreased 30 min after starting the pump, although changes were not significant; aggregation gradually recovered after 3 h and returned to baseline 60 min after the pumps were stopped. There were no significant differences at all sampling points of MEA. In both groups, fibrinogen, PF4, and β-TG concentrations were similar 60 min after pump cessation and before sternotomy

  2. Mice Lacking the ITIM-Containing Receptor G6b-B Exhibit Macrothrombocytopenia and Aberrant Platelet Function

    PubMed Central

    Mori, Jun; Bem, Danai; Finney, Brenda; Heising, Silke; Gissen, Paul; White, James G.; Berndt, Michael C.; Gardiner, Elizabeth E.; Nieswandt, Bernhard; Douglas, Michael R.; Campbell, Robert D.; Watson, Steve P.; Senis, Yotis A.

    2013-01-01

    Platelets are highly reactive cell fragments that adhere to exposed extracellular matrix (ECM) and prevent excessive blood loss by forming clots. Paradoxically, megakaryocytes, which produce platelets in the bone marrow, remain relatively refractory to the ECM-rich environment of the bone marrow despite having the same repertoire of receptors as platelets. These include the ITAM (immunoreceptor tyrosine–based activation motif)–containing collagen receptor complex, which consists of glycoprotein VI (GPVI) and the Fc receptor γ-chain, and the ITIM (immunoreceptor tyrosine–based inhibition motif)–containing receptor G6b-B. We showed that mice lacking G6b-B exhibited macrothrombocytopenia (reduced platelet numbers and the presence of enlarged platelets) and a susceptibility to bleeding as a result of aberrant platelet production and function. Platelet numbers were markedly reduced in G6b-B–deficient mice compared to those in wild-type mice because of increased platelet turnover. Furthermore, megakaryocytes in G6b-B–deficient mice showed enhanced metalloproteinase production, which led to increased shedding of cell-surface receptors, including GPVI and GPIba. In addition, G6b-B–deficient megakaryocytes exhibited reduced integrin-mediated functions and defective formation of proplatelets, the long filamentous projections from which platelets bud off. Together, these findings establish G6b-B as a major inhibitory receptor regulating megakaryocyte activation, function, and platelet production. PMID:23112346

  3. Diminished adhesion and activation of platelets and neutrophils with CD47 functionalized blood contacting surfaces.

    PubMed

    Finley, Matthew J; Rauova, Lubica; Alferiev, Ivan S; Weisel, John W; Levy, Robert J; Stachelek, Stanley J

    2012-08-01

    CD47 is a ubiquitously expressed transmembrane protein that, through signaling mechanisms mediated by signal regulatory protein alpha (SIRPα1), functions as a biological marker of 'self-recognition'. We showed previously that inflammatory cell attachment to polymeric surfaces is inhibited by the attachment of biotinylated recombinant CD47 (CD47B). We test herein the hypothesis that CD47 modified blood conduits can reduce platelet and neutrophil activation under clinically relevant conditions. We appended a poly-lysine tag to the C-terminus of recombinant CD47 (CD47L) allowing for covalent linkage to the polymer. SIRPα1 expression was confirmed in isolated platelets. We then compared biocompatibility between CD47B and CD47L functionalized polyvinyl chloride (PVC) surfaces and unmodified control PVC surfaces. Quantitative and Qualitative analysis of blood cell attachment to CD47B and CD47L surfaces, via scanning electron microscopy, showed strikingly fewer platelets attached to CD47 modified surfaces compared to control. Flow cytometry analysis showed that activation markers for neutrophils (CD62L) and platelets (CD62P) exposed to CD47 modified PVC were equivalent to freshly acquired control blood, while significantly elevated in the unmodified PVC tubing. In addition, ethylene oxide gas sterilization did not inhibit the efficacy of the CD47 modification. In conclusion, CD47 modified PVC inhibits both the adhesion and activation of platelets and neutrophils. PMID:22613135

  4. Functional Comparison of Induced Pluripotent Stem Cell- and Blood-Derived GPIIbIIIa Deficient Platelets

    PubMed Central

    Haas, Jessica; Sandrock-Lang, Kirstin; Gärtner, Florian; Jung, Christian Billy; Zieger, Barbara; Parrotta, Elvira; Kurnik, Karin; Sinnecker, Daniel; Wanner, Gerhard; Laugwitz, Karl-Ludwig; Massberg, Steffen; Moretti, Alessandra

    2015-01-01

    Human induced pluripotent stem cells (hiPSCs) represent a versatile tool to model genetic diseases and are a potential source for cell transfusion therapies. However, it remains elusive to which extent patient-specific hiPSC-derived cells functionally resemble their native counterparts. Here, we generated a hiPSC model of the primary platelet disease Glanzmann thrombasthenia (GT), characterized by dysfunction of the integrin receptor GPIIbIIIa, and compared side-by-side healthy and diseased hiPSC-derived platelets with peripheral blood platelets. Both GT-hiPSC-derived platelets and their peripheral blood equivalents showed absence of membrane expression of GPIIbIIIa, a reduction of PAC-1 binding, surface spreading and adherence to fibrinogen. We demonstrated that GT-hiPSC-derived platelets recapitulate molecular and functional aspects of the disease and show comparable behavior to their native counterparts encouraging the further use of hiPSC-based disease models as well as the transition towards a clinical application. PMID:25607928

  5. Thrombin-linked aptamer assay for detection of platelet derived growth factor BB on magnetic beads in a sandwich format.

    PubMed

    Guo, Limin; Zhao, Qiang

    2016-09-01

    Here we describe a thrombin-linked aptamer assay (TLAA) for protein by using thrombin as an enzyme label, harnessing enzyme activity of thrombin and aptamer affinity binding. TLAA converts detection of specific target proteins to the detection of thrombin by using a DNA sequence that consists of two aptamers with the first aptamer binding to the specific target protein and the second aptamer binding to thrombin. Through the affinity binding, the thrombin enzyme is labeled on the protein target, and thrombin catalyzes the hydrolysis of small peptide substrate into product, generating signals for quantification. As a proof of principle, we show a sandwich TLAA for platelet derived growth factor BB (PDGF-BB) by using anti-PDGF-BB antibody coated on magnetic beads and an oligonucleotide containing the aptamer for PDGF-BB and the aptamer for thrombin. The binding of PDGF-BB to both the antibody and the aptamer results in labeling the complex with thrombin. We achieved detection of PDGF-BB at 16 pM. This TLAA contributes a new application of thrombin and its aptamer in bioanalysis, and shows potentials in assay developments. PMID:27343590

  6. Poly (I:C) downregulates platelet production and function through type I interferon.

    PubMed

    Rivadeneyra, Leonardo; Pozner, Roberto Gabriel; Meiss, Roberto; Fondevila, Carlos; Gómez, Ricardo Martin; Schattner, Mirta

    2015-11-01

    Thrombocytopenia is a frequent complication of viral infections; the underlying mechanisms appear to depend on the identity of the virus involved. Previous research, including reports from our group, indicates that as well as having antiviral activity type I interferons (IFN I) selectively downregulate platelet production. In this study we extended understanding of the role of endogenous IFN I in megakaryo/thrombopoiesis by evaluating platelet and megakaryocyte physiology in mice treated with polyinosinic:polycytidylic acid [poly (I:C)], a synthetic analogue of double-stranded RNA, Toll-like receptor-3 ligand and strong IFNβ inducer. Mice-treated with poly (I:C) showed thrombocytopaenia, an increase in mean platelet volume and abnormal haemostatic and inflammatory platelet-mediated functionality, indicated by decreased fibrinogen binding and platelet adhesion, prolonged tail bleeding times and impaired P-Selectin externalisation, RANTES release and thrombin-induced platelet-neutrophil aggregate formation. These changes were associated with an increase in size and an abnormal distribution of bone marrow megakaryocytes within the vascular niche and were directly correlated with the plasmatic and bone marrow IFNβ levels. All these effects were absent in genetically modified mice lacking the IFN I receptor. Our results suggest that IFN I is the central mediator of poly (I:C)-induced thrombocytopenia and platelet dysfunction and indicate that these abnormalities are due to changes in the last stages of megakaryocyte development. These data provide new evidence for the role of IFN I in megakaryocyte distribution in the bone marrow niches and its influence on thrombopoiesis and haemostasis. PMID:26134179

  7. Glycoxidized HDL, HDL enriched with oxidized phospholipids and HDL from diabetic patients inhibit platelet function

    PubMed Central

    Lê, Quang Huy; El Alaoui, Meddy; Véricel, Evelyne; Ségrestin, Bérénice; Soulère, Laurent; Guichardant, Michel; Lagarde, Michel; Moulin, Philippe; Calzada, Catherine

    2015-01-01

    Context High-density lipoproteins (HDL) possess atheroprotective properties including anti-thrombotic and antioxidant effects. Very few studies relate to the functional effects of oxidized HDL on platelets in type 2 diabetes (T2D). Objective The objective of our study was to investigate the effects of in vitro glycoxidized HDL, and HDL from T2D patients on platelet aggregation and arachidonic acid signaling cascade. At the same time, the contents of hydroxylated fatty acids were assessed in HDL. Results Compared to control HDL, in vitro glycoxidized HDL had decreased proportions of linoleic (LA) and arachidonic (AA) acids in phospholipids and cholesteryl esters, and increased concentrations of hydroxy-octadecadienoic acids (9-HODE and 13-HODE) and 15-hydroxy-eicosatetraenoic acid (15-HETE), derived from LA and AA respectively, especially hydroxy derivatives esterified in phospholipids. Glycoxidized HDL dose-dependently decreased collagen-induced platelet aggregation by binding to SR-BI. Glycoxidized HDL prevented collagen-induced increased phosphorylation of platelet p38 MAPK and cytosolic phospholipase A2, as well as intracellular calcium mobilization. HDL enriched with oxidized phospholipids, namely PC(16:0/13-HODE) dose-dependently inhibited platelet aggregation. Increased concentrations of 9-HODE, 13-HODE and 15-HETE in phospholipids (2.1, 2.1 and 2.4-fold increase respectively) were found in HDL from patients with T2D, and these HDL also inhibited platelet aggregation via SR-BI. Conclusions Altogether, our results indicate that in vitro glycoxidized HDL as well as HDL from T2D patients inhibit platelet aggregation, and suggest that oxidized LA-containing phospholipids may contribute to the anti-aggregatory effects of glycoxidized HDL and HDL from T2D patients. PMID:25794249

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

    PubMed

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

    2001-01-01

    One of the mechanisms of action of non steroid antiinflammatory drugs (NSAIDs) consists of inhibition of prostaglandin synthesis. This explains many of the pharmacological effects and adverse events observed in medical practice. Administration of NSAIDs to patients with hemostatic disorders or perioperative conditions entails the risk of bleeding due to inhibition of platelet function. This study deals with platelet changes induced by lysine clonixinate vs diclofenac, ibuprofen and aspirin in classical tests such as platelet count, platelet factor 3 (PF3) activity and platelet aggregation with various inductors and more recent procedures such as P-selectin measurement by flow cytometry. Unlike control drugs, lysine clonixinate did not induce changes in platelet count or function when administered to healthy volunteers at the commonly used therapeutic doses. PMID:11474878

  9. Effect of Haemophilus influenzae infection and moxalactam on platelet function in children.

    PubMed Central

    Kaplan, S L; Courtney, J T; Kenal, K A

    1987-01-01

    In a prospective randomized study, children with Haemophilus influenzae type b meningitis received moxalactam or ampicillin or chloramphenicol. Of 41 children, 6 had prolonged bleeding times (greater than 6 min), and 7 of 9 tested had abnormal platelet aggregation at hospital admission. At the end of therapy, no children in the ampicillin-chloramphenicol group, compared with 5 of 22 moxalactam-treated children (23%) (P = 0.08), had prolonged bleeding times (6.5 to 7.5 min). Our data suggest that H. influenzae meningitis and treatment with moxalactam may each have an effect on platelet function in children. PMID:3579263

  10. Blood rheology and platelet function in untreated early-stage essential hypertensives complicated with metabolic syndrome.

    PubMed

    Sugimori, Hiroko; Tomoda, Fumihiro; Koike, Tsutomu; Kinuno, Hiroyuki; Kurosaki, Hiroko; Masutani, Toshitaka; Inoue, Hiroshi

    2012-01-01

    We examined whether hemorheology and platelet function are affected in essential hypertensives (EHTs) of the World Health Organization stage I when complicated with metabolic syndrome (Mets). In 156 untreated EHTs, blood viscosity and platelet surface markers were determined. Blood viscosity was significantly elevated in 54 subjects with Mets compared with 102 subjects without Mets. Hematocrit and plasma viscosity increased in the group with Mets, although red blood cell rigidity index "k" did not differ between groups. As a whole group, blood viscosity correlated positively with hematocrit and plasma viscosity. Additionally, plasma viscosity correlated positively with plasma leptin, triglyceride, homeostasis model assessment index, C-reactive protein, and plasma fibrinogen, but negatively with high-density lipoprotein cholesterol. In contrast, no differences were seen in platelet surface markers between groups. In conclusion, EHTs of the early stage complicated with Mets are characterized by increased blood viscosity due to hemoconcentration and increased plasma viscosity. PMID:22570768

  11. Inhibitory effects of Cyperus digitatus extract on human platelet function in vitro.

    PubMed

    Fuentes, Eduardo; Forero-Doria, Oscar; Alarcón, Marcelo; Palomo, Iván

    2015-01-01

    The purpose of this research was to investigate the mechanisms of antiplatelet action of Cyperus digitatus. The antiplatelet action of C. digitatus was studied on platelet function: secretion, adhesion, aggregation, and sCD40L release. The platelet ATP secretion and aggregation were significantly inhibited by CDA (ethyl acetate extract) at 0.1 mg/ml and after the incubation of whole blood with CDA, the platelet coverage was inhibited by 96 ± 3% (p < 0.001). At the same concentration, CDA significantly decreased sCD40L levels. The mechanism of antiplatelet action of CDA could be by NF-κB inhibition and that is cAMP independent. In conclusion, C. digitatus extract may serve as a new source of antiplatelet agents for food and nutraceutical applications. PMID:25548968

  12. Hemostatic function of apheresis platelets stored at 4 °C and 22 °C

    PubMed Central

    Reddoch, Kristin M.; Pidcoke, Heather F.; Montgomery, Robbie K.; Fedyk, Criselda G.; Aden, James K.; Ramasubramanian, Anand K.; Cap, Andrew P.

    2014-01-01

    Introduction Platelet refrigeration decreases the risk of bacterial contamination and may preserve function better than standard-of-care room temperature storage. Benefits could include lower transfusion-related complications, decreased costs, improved hemostasis in acutely bleeding patients, and extended shelf-life. In this study, we compared the effects of 22°C and 4°C storage on the functional and activation status of apheresis platelets (APs). Methods APs (n = 5 per group) were stored for 5 days at 22°C with agitation (RT) versus at 4°C with agitation (4C+AG) and without (4C). Measurements included platelet counts, mean platelet volume, blood gas analytes, aggregation response, thromboelastography, TxB2 and sCD40L release, activation markers and microparticle formation. Results Sample pH levels were within acceptable limits for storage products (pH 6.2-7.4). Platelet glucose metabolism (P < 0.05), aggregation response (ADP: RT 0; 4C+AG 5.0 ± 0.8; 4C 5.6 ± 0.9; P < 0.05), and clot strength (MA: RT 58 ± 2; 4C+AG 63 ± 2; 4C 67 ± 2; P < 0.05) were better preserved at 4°C compared to RT storage. Refrigerated samples were more activated compared to RT (P < 0.05), although TxB2 (P < 0.05) and sCD40L release (P < 0.05) were higher at RT. Agitation did not improve the quality of 4°C-stored samples. Conclusion AP stored at 4°C maintain more viable metabolic characteristics, are hemostatically more effective, and release fewer pro-inflammatory mediators than AP stored at RT over 5 days. Given the superior bacteriologic safety of refrigerated products, these data suggest that cold-stored platelets may improve outcomes for acutely bleeding patients. PMID:24169210

  13. Mechanism of activation and functional role of protein kinase Ceta in human platelets.

    PubMed

    Bynagari, Yamini S; Nagy, Bela; Tuluc, Florin; Bhavaraju, Kamala; Kim, Soochong; Vijayan, K Vinod; Kunapuli, Satya P

    2009-05-15

    The novel class of protein kinase C (nPKC) isoform eta is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKCeta using pharmacological and gene knock-out approaches. nPKCeta was phosphorylated (at Thr-512) in a time- and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y1 receptor antagonist, or YM-254890, a G(q) blocker, abolished 2MeSADP-induced phosphorylation of nPKCeta. Similarly, ADP failed to activate nPKCeta in platelets isolated from P2Y1 and G(q) knock-out mice. However, pretreatment of platelets with P2Y12 receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKCeta phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKCeta was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin alpha(IIb)beta3 mediated outside-in signaling. Moreover, in the presence of SC-57101, a alpha(IIb)beta3 receptor antagonist, nPKCeta dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1cgamma, a catalytic subunit of serine/threonine phosphatase, alpha(IIb)beta3 failed to dephosphorylate nPKCeta. Thus, we conclude that ADP activates nPKCeta via P2Y1 receptor and is subsequently dephosphorylated by PP1gamma phosphatase activated by alpha(IIb)beta3 integrin. In addition, pretreatment of platelets with eta-RACK antagonistic peptides, a specific inhibitor of nPKCeta, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKCeta positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation. PMID:19286657

  14. Cocoa flavanols and platelet and leukocyte function: recent in vitro and ex vivo studies in healthy adults.

    PubMed

    Heptinstall, Stan; May, Jane; Fox, Sue; Kwik-Uribe, Catherine; Zhao, Lian

    2006-01-01

    There is growing interest in possible beneficial effects of specific dietary components on cardiovascular health. Platelets and leukocytes contribute to arterial thrombosis and to inflammatory processes. Previous studies performed in vitro have demonstrated inhibition of platelet function by (-)-epicatechin and (+)-catechin, flavan-3-ols (flavanols) that are present in several foods including some cocoas. Also, some modest inhibition of platelet function has been observed ex vivo after the consumption of flavanol-containing cocoa products by healthy adults. So far there are no reports of effects of cocoa flavanols on leukocytes. This paper summarizes 2 recent investigations. The first was a study of the effects of cocoa flavanols on platelet and leukocyte function in vitro. The second was a study of the effects of consumption of a flavanol-rich cocoa beverage by healthy adults on platelet and leukocyte function ex vivo. Measurements were made of platelet aggregation, platelet-monocyte conjugate formation (P/M), platelet-neutrophil conjugate formation (P/N), platelet activation (CD62P on monocytes and neutrophils), and leukocyte activation (CD11b on monocytes and neutrophils) in response to collagen and/or arachidonic acid. In the in vitro study several cocoa flavanols and their metabolites were shown to inhibit platelet aggregation, P/M, P/N, and platelet activation. Their effects were similar to those of aspirin and the effects of a cocoa flavanol and aspirin did not seem to be additive. There was also inhibition of monocyte and neutrophil activation by flavanols, but this was not replicated by aspirin. 4'-O-methyl-epicatechin, 1 of the known metabolites of the cocoa flavanol (-)-epicatechin, was consistently effective as an inhibitor of platelet and leukocyte activation. The consumption of a flavanol-rich cocoa beverage also resulted in significant inhibition of platelet aggregation, P/M and P/N, and platelet activation induced by collagen. The inhibitory effects

  15. Moderate oral supplementation with docosahexaenoic acid improves platelet function and oxidative stress in type 2 diabetic patients.

    PubMed

    Véricel, E; Colas, R; Calzada, C; Lê, Q H; Feugier, N; Cugnet, C; Vidal, H; Laville, M; Moulin, P; Lagarde, M

    2015-08-01

    Platelets from patients with type 2 diabetes are characterised by hyperactivation and high level of oxidative stress. Docosahexaenoic acid (DHA) may have beneficial effects on platelet reactivity and redox status. We investigated whether moderate DHA supplementation, given as a triglyceride form, may correct platelet dysfunction and redox imbalance in patients with type 2 diabetes. We conducted a randomised, double-blind, placebo-controlled, two-period crossover trial (n=11 post-menopausal women with type 2 diabetes) to test the effects of 400 mg/day of DHA intake for two weeks on platelet aggregation, markers of arachidonic acid metabolism, lipid peroxidation status, and lipid composition. Each two week-period was separated from the other by a six-week washout. Daily moderate dose DHA supplementation resulted in reduced platelet aggregation induced by collagen (-46.5 %, p< 0.001), and decreased platelet thromboxane B2 (-35 %, p< 0.001), urinary 11-dehydro-thromboxane B2 (-13.2 %, p< 0.001) and F2-isoprostane levels (-19.6 %, p< 0.001) associated with a significant increase of plasma and platelet vitamin E concentrations (+20 % and +11.8 %, respectively, p< 0.001). The proportions of DHA increased both in plasma lipids and in platelet phospholipids. After placebo treatment, there was no effect on any parameters tested. Our findings support a significant beneficial effect of low intake of DHA on platelet function and a favourable role in reducing oxidative stress associated with diabetes. PMID:25832443

  16. Impact of an integrated treatment algorithm based on platelet function testing and clinical risk assessment: results of the TRIAGE Patients Undergoing Percutaneous Coronary Interventions To Improve Clinical Outcomes Through Optimal Platelet Inhibition study.

    PubMed

    Chandrasekhar, Jaya; Baber, Usman; Mehran, Roxana; Aquino, Melissa; Sartori, Samantha; Yu, Jennifer; Kini, Annapoorna; Sharma, Samin; Skurk, Carsten; Shlofmitz, Richard A; Witzenbichler, Bernhard; Dangas, George

    2016-08-01

    Assessment of platelet reactivity alone for thienopyridine selection with percutaneous coronary intervention (PCI) has not been associated with improved outcomes. In TRIAGE, a prospective multicenter observational pilot study we sought to evaluate the benefit of an integrated algorithm combining clinical risk and platelet function testing to select type of thienopyridine in patients undergoing PCI. Patients on chronic clopidogrel therapy underwent platelet function testing prior to PCI using the VerifyNow assay to determine high on treatment platelet reactivity (HTPR, ≥230 P2Y12 reactivity units or PRU). Based on both PRU and clinical (ischemic and bleeding) risks, patients were switched to prasugrel or continued on clopidogrel per the study algorithm. The primary endpoints were (i) 1-year major adverse cardiovascular events (MACE) composite of death, non-fatal myocardial infarction, or definite or probable stent thrombosis; and (ii) major bleeding, Bleeding Academic Research Consortium type 2, 3 or 5. Out of 318 clopidogrel treated patients with a mean age of 65.9 ± 9.8 years, HTPR was noted in 33.3 %. Ninety (28.0 %) patients overall were switched to prasugrel and 228 (72.0 %) continued clopidogrel. The prasugrel group had fewer smokers and more patients with heart failure. At 1-year MACE occurred in 4.4 % of majority HTPR patients on prasugrel versus 3.5 % of primarily non-HTPR patients on clopidogrel (p = 0.7). Major bleeding (5.6 vs 7.9 %, p = 0.47) was numerically higher with clopidogrel compared with prasugrel. Use of the study clinical risk algorithm for choice and intensity of thienopyridine prescription following PCI resulted in similar ischemic outcomes in HTPR patients receiving prasugrel and primarily non-HTPR patients on clopidogrel without an untoward increase in bleeding with prasugrel. However, the study was prematurely terminated and these findings are therefore hypothesis generating. PMID:27100112

  17. Platelet functional alterations in a Bernard-Soulier syndrome patient with filamin A mutation.

    PubMed

    Li, Jiaming; Dai, KeSheng; Wang, Zhaoyue; Cao, Lijuan; Bai, Xia; Ruan, Changgeng

    2015-01-01

    Defects in filamin A (FLNA) gene could lead to low platelet counts and decreased surface expression of glycoprotein (GP) Ibα. Here, we report and investigate the FLNA genomic alteration of a case with Bernard-Soulier syndrome (BSS), a rare hereditary bleeding disorder caused by quantitative or qualitative abnormalities in the GP Ib-IX-V receptor. DNA sequencing analysis reveals the presence of a GP Ibα c.987G > A mutation and a FLNA c.1582 G > A mutation in this patient. Transient transfection studies show that GP Ibα c.987G > A mutation abolishes the surface expression of GP Ibα on the transfected CHO cells. On the other hand, abnormal responses to collagen, including the platelet aggregation, secretion, and GP VI signaling pathways, are associated with FLNA c.1582G > A mutation. Our findings confirm a central role for FLNA in platelet-adhesive functions. The interaction between FLNA and GP Ibα in platelets deserves to be investigated. PMID:26133172

  18. Normal Platelet Integrin Function in Mice Lacking Hydrogen Peroxide-Induced Clone-5 (Hic-5)

    PubMed Central

    Popp, Michael; Thielmann, Ina; Nieswandt, Bernhard; Stegner, David

    2015-01-01

    Integrin αIIbβ3 plays a central role in the adhesion and aggregation of platelets and thus is essential for hemostasis and thrombosis. Integrin activation requires the transmission of a signal from the small cytoplasmic tails of the α or β subunit to the large extracellular domains resulting in conformational changes of the extracellular domains to enable ligand binding. Hydrogen peroxide-inducible clone-5 (Hic-5), a member of the paxillin family, serves as a focal adhesion adaptor protein associated with αIIbβ3 at its cytoplasmic tails. Previous studies suggested Hic-5 as a novel regulator of integrin αIIbβ3 activation and platelet aggregation in mice. To assess this in more detail, we generated Hic-5-null mice and analyzed activation and aggregation of their platelets in vitro and in vivo. Surprisingly, lack of Hic-5 had no detectable effect on platelet integrin activation and function in vitro and in vivo under all tested conditions. These results indicate that Hic-5 is dispensable for integrin αIIbβ3 activation and consequently for arterial thrombosis and hemostasis in mice. PMID:26172113

  19. New assay for measuring binding of platelet glycoprotein IIb/IIIa to unpurified von Willebrand factor.

    PubMed

    Veyradier, A; Jumilly, A L; Ribba, A S; Obert, B; Houllier, A; Meyer, D; Girma, J P

    1999-07-01

    Among the numerous variants of vWD, no patient with an abnormal vWF binding to GPIIb/IIIa has been described to date. To search for such potential variants, we developed a two-site assay for measuring the binding of purified GPIIb/IIIa to vWF in biological fluids and we used it to study a large series of plasmas from various types of von Willebrand disease (vWD) and recombinant vWF (rvWF). vWF in plasma or rvWF in culture medium was immobilized onto anti-vWF monoclonal antibodies (MoAb)-coated wells of microtiter plates. After incubation with either unlabeled GPIIb/IIIa and a 125I-anti-GPIIb/IIIa MoAb or 125I-GPIIb/IIIa, binding curves and binding isotherms were respectively established. Normal pool plasma and wild-type rvWF were used as reference samples. We tested plasmas from 85 normal subjects, 115 patients with different types of vWD (64 type 1, 2 type 3, 9 type 2A, 4 type 2M, 16 type 2B, 15 type 2N, 3 type IID and 2 acquired forms) and 50 patients with various bleeding disorders. Four mutated rvWF with 2A (Glu875Lys and Pro885Ser) or 2B (Dupl.Met540 and Val551Phe) substitutions and one rvWF mutated in the RGD domain of the C-terminal part of vWF-subunit (Asp1746Gly) were also studied. Among the various samples tested, only rvWF Asp1746Gly had no affinity for GPIIb/IIIa. In contrast, GPIIb/IIIa similarly bound to the other vWF, independently of the proteic environment, the factor VIII level, the degree of multimerization or the mutation of vWF. Our results indicate that subjects with an abnormal vWF binding to GPIIb/IIIa are probably rare and difficult to target for a specific screening. PMID:10456467

  20. Exosome poly-ubiquitin inhibits platelet activation, downregulates CD36, and inhibits pro-atherothombotic cellular functions

    PubMed Central

    Srikanthan, Sowmya; Li, Wei; Silverstein, Roy L.; McIntyre, Thomas M.

    2014-01-01

    Introduction Activated platelets shed microparticles from plasma membranes, but also release smaller exosomes from internal compartments. While microparticles participate in athero-thrombosis, little is known of exosomes in this process. Materials & Methods Ex vivo biochemical experiments with human platelets and exosomes, and FeCl3-induced murine carotid artery thrombosis. Results Both microparticles and exosomes were abundant in human plasma. Platelet-derived exosomes suppressed ex vivo platelet aggregation and reduced adhesion to collagen-coated microfluidic channels at high shear. Injected exosomes inhibited occlusive thrombosis in FeCl3-damaged murine carotid arteries. Control platelets infused into irradiated, thrombocytopenic mice reconstituted thrombosis in damaged carotid arteries, but failed to do so after prior ex vivo incubation with exosomes. CD36 promotes platelet activation, and exosomes dramatically reduced platelet CD36. CD36 is also expressed by macrophages where it binds and internalizes oxidized LDL and microparticles, supplying lipid to promote foam cell formation. Platelet exosomes inhibited oxidized-LDL binding and cholesterol loading into macrophages. Exosomes were not competitive CD36 ligands, but instead sharply reduced total macrophage CD36 content. Exosomal proteins, in contrast to microparticle or cellular proteins, were highly adducted by ubiquitin. Exosomes enhanced ubiquitination of cellular proteins, including CD36, and blockade of proteosome proteolysis with MG-132 rescued CD36 expression. Recombinant unanchored K48 poly-ubiquitin behaved similarly to exosomes, inhibiting platelet function, macrophage CD36 expression, and macrophage particle uptake. Conclusions Platelet-derived exosomes inhibit athero-thrombotic processes by reducing CD36-dependent lipid loading of macrophages and by suppressing platelet thrombosis. Exosomes increase protein ubiquitination, and enhance proteasome degradation of CD36. PMID:25163645

  1. A comparison of platelet function tests and thromboxane metabolites to evaluate aspirin response in healthy individuals and patients with coronary artery disease.

    PubMed

    Grove, Erik Lerkevang; Hvas, Anne-Mette; Johnsen, Helle Ladefoged; Hedegaard, Sofie Sommer; Pedersen, Susanne Bendesgaard; Mortensen, Jette; Kristensen, Steen Dalby

    2010-06-01

    Individualised antiplatelet therapy and platelet function testing have attracted considerable clinical interest, but several aspects of test performance have not been thoroughly evaluated. We investigated repeatability and concordance of light transmission aggregometry (LTA) induced with arachidonic acid (AA) 1.0 mM, PFA-100 induced with collagen/epinephrine, multiple electrode aggregometry (MEA) induced with AA 0.5 or 0.75 mM and VerifyNow Aspirin. Patients with stable coronary artery disease (n=43) and healthy individuals (n=21) were included. All tests were performed in duplicate at baseline in healthy individuals and in duplicate for four days in all study participants during aspirin treatment. Serum and urinary thromboxane metabolites were measured several times to evaluate cyclooxygenase-1 inhibition by aspirin. MEA was most sensitive for aspirin as treatment induced a 12-fold difference in AA-induced platelet aggregation. Coefficients of variation for duplicate measurements at baseline (0.4-12%), during aspirin treatment (3-46%) and for day-to-day variability (3-37%) differed markedly between tests and were lowest for VerifyNow. The prevalence of aspirin low-responsiveness also differed between tests (0-9%) and the agreement was low: kappaplatelet function test, whereas serum thromboxane correlated with VerifyNow Aspirin (r=0.41, p=0.001). Overall, repeatability was moderate and the correlation between tests was low. VerifyNow Aspirin proved most reproducible, and this was the only assay showing a significant positive correlation with serum thromboxane. This study demonstrated that conclusions based on platelet function testing strongly depend on the assay used. PMID:20352155

  2. Inhibition of human platelet function in vitro and ex vivo by acetaminophen.

    PubMed

    Lages, B; Weiss, H J

    1989-03-15

    The effects of acetaminophen (APAP) in vitro, or ex vivo following APAP ingestion, on human platelet aggregation, 14C-5HT secretion, and thromboxane B2 (TxB2) formation were assessed. APAP added in vitro to citrated platelet-rich plasma (PRP) inhibited aggregation, secretion, and TxB2 formation induced by collagen, epinephrine, arachidonate, and the ionophore A23187, but had no effect on the responses induced by the endoperoxide analog U44069. Arachidonate-induced responses were inhibited by lower concentrations of APAP than were the responses to the other agonists. In PRP obtained 1 hour after ingestion of 650 mg or 1000 mg APAP, arachidonate-induced TxB2 formation was inhibited by 40-99% in five subjects tested, whereas inhibition of collagen- or epinephrine-induced TxB2 formation was less consistent. Aggregation and secretion responses were not altered by APAP ingestion in 4 of the 5 subjects, but were inhibited in the remaining subject, who had the highest plasma APAP levels. In contrast to aspirin and indomethacin, APAP-induced inhibition of collagen-stimulated TxB2 formation could be partially overcome with increasing collagen concentrations. No such partial correction occurred with epinephrine, however. In washed platelet suspensions labeled with 3H-arachidonate, both APAP and aspirin inhibited the formation of labeled PGD2 and PGE2, as well as TxB2. These results suggest that APAP acts in human platelets as a reversible inhibitor of cyclo-oxygenase, as found previously in other tissues, and that recent APAP ingestion can, on occasion, produce inhibition of platelet functional responses measured in vitro. PMID:2499947

  3. Variability of residual platelet function despite clopidogrel treatment in patients with peripheral arterial occlusive disease.

    PubMed

    Linnemann, Birgit; Schwonberg, Jan; Toennes, Stefan W; Mani, Helen; Lindhoff-Last, Edelgard

    2010-04-01

    Residual platelet function despite treatment with clopidogrel may predict an unfavourable cardiovascular outcome. The majority of studies have investigated the effects of clopidogrel administration in conjunction with aspirin in patients undergoing percutaneous coronary intervention. The primary objective of the present study was to assess the platelet response to clopidogrel in the absence of aspirin in patients with peripheral arterial occlusive disease (PAOD) and to investigate whether non-responsiveness to clopidogrel is reproducible during long-term follow-up. Fifty-four clinically stable PAOD patients on a maintenance dose of 75 mg/d clopidogrel were enrolled in this study. Platelet function was assessed at baseline and after a median follow-up of 18 months using light transmittance aggregometry (LTA) with 2 microM ADP as an agonist. HPLC-coupled mass spectrometry was used to detect clopidogrel and clopidogrel carboxylic acid, the main metabolite of clopidogrel. Residual platelet function, as defined by late aggregation values within the reference range (i.e., >43%), was observed in 35.2% of patients at baseline and 17.6% during follow-up. During the observation period, 26.5% had switched from responder to non-responder status or vice versa. Among non-responders, either clopidogrel or its metabolite was detected in 89.5% and 83.3% of patients at baseline and at follow-up, respectively. We conclude that non-responsiveness to clopidogrel as determined by ADP-induced LTA is not stable over time. This phenomenon cannot be attributed to non-compliance alone. PMID:20153859

  4. Distinct roles for protein kinase C isoforms in regulating platelet purinergic receptor function.

    PubMed

    Mundell, Stuart J; Jones, Matthew L; Hardy, Adam R; Barton, Johanna F; Beaucourt, Stephanie M; Conley, Pamela B; Poole, Alastair W

    2006-09-01

    ADP is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors (GPCRs), P2Y1 and P2Y12. We have shown previously that the receptors are functionally desensitized, in a homologous manner, by distinct kinase-dependent mechanisms in which P2Y1 is regulated by protein kinase C (PKC) and P2Y12 by G protein-coupled receptor kinases. In this study, we addressed whether different PKC isoforms play different roles in regulating the trafficking and activity of these two GPCRs. Expression of PKCalpha and PKCdelta dominant-negative mutants in 1321N1 cells revealed that both isoforms regulated P2Y1 receptor signaling and trafficking, although only PKCdelta was capable of regulating P2Y12, in experiments in which PKC was directly activated by the phorbol ester phorbol 12-myristate 13-acetate (PMA). These results were paralleled in human platelets, in which PMA reduced subsequent ADP-induced P2Y1 and P2Y12 receptor signaling. PKC isoform-selective inhibitors revealed that novel, but not conventional, isoforms of PKC regulate P2Y12 function, whereas both novel and classic isoforms regulate P2Y1 activity. It is also noteworthy that we studied receptor internalization in platelets by a radioligand binding approach showing that both receptors internalize rapidly in these cells. ADP-induced P2Y1 receptor internalization is attenuated by PKC inhibitors, whereas that of the P2Y12 receptor is unaffected. Both P2Y1 and P2Y12 receptors can also undergo PMA-stimulated internalization, and here again, novel but not classic PKCs regulate P2Y12, whereas both novel and classic isoforms regulate P2Y1 internalization. This study therefore is the first to reveal distinct roles for PKC isoforms in the regulation of platelet P2Y receptor function and trafficking. PMID:16804093

  5. Luminal platelet aggregates in functional deficits in parenchymal vessels after subarachnoid hemorrhage

    PubMed Central

    Friedrich, Victor; Flores, Rowena; Muller, Artur; Sehba, Fatima A.

    2010-01-01

    The pathophysiology of early ischemic injury after aneurysmal subarachnoid hemorrhage (SAH) is not understood. This study examined the acute effect of endovascular puncture-induced SAH on parenchymal vessel function in rat, using intravascular fluorescent tracers to assess flow and vascular permeability and immunostaining to assess structural integrity and to visualize platelet aggregates. In sham-operated animals, vessels were well filled with tracer administered 10 seconds before sacrifice, and parenchymal escape of tracer was rare. At ten minutes and 3 hours after hemorrhage, patches of poor vascular filling were distributed throughout the forebrain. Close examination of these regions revealed short segments of narrowed diameter along many profiles. Most vascular profiles with reduced perfusion contained platelet aggregates and in addition showed focal loss of collagen IV, a principal component of basal lamina. In contrast, vessels were well filled at 24 hours post-hemorrhage, indicating that vascular perfusion had recovered. Parenchymal escape of intravascular tracer was detected at 10 minutes post-hemorrhage and later as plumes of fluorescence emanating into parenchyma from restricted microvascular foci. These data demonstrate that parenchymal microvessels are compromised in function by 10 minutes after SAH and identify focal microvascular constriction and local accumulation of luminal platelet aggregates as potential initiators of that compromise. PMID:20654597

  6. Modulation of platelet and leucocyte function by a Chinese herbal formulation as compared with conventional antiplatelet agents.

    PubMed

    Zhao, Lian; Gaudry, Leonie; Dunkley, Scott; Brighton, Tim; Guo, Zhi Xin; Ye, Zheng Liang; Luo, Run Zhi; Chesterman, Colin N

    2008-02-01

    Platelet and leucocyte activity are important in the acute development of thrombosis and in the pathogenesis of ischaemic vascular disease. Dan Shen Di Wan (DS, Cardiotonic Pill or Dantonic(R) Pill) is one of the most commonly used Chinese herbal formulations for treating patients with atherosclerotic disease in China and several Asian countries. We studied the effect of DS on platelet and leucocyte function and compared the effects with conventional antiplatelet agents, cangrelor (ADP P2Y(12) receptor antagonist) and aspirin (acetyl salicylic acid, ASA). Measurements were made by platelet aggregation (%) and activation (CD62P %), platelet-monocyte conjugate formation (P/M, CD42a median fluorescence, mf), platelet-neutrophil conjugate formation (P/N, mf), and leucocyte activation (CD11b median fluorescence on monocytes and neutrophils, mf) in response to 3.3 micromol/L adenosine diphosphate (ADP), 1.0 micromol/L platelet activating factor (PAF), 5.0 micromol/L adrenaline and 0.5 microg/mL collagen. We also evaluated the effect of its main component, water soluble extract of salvia miltiorrhiza (SME) on intracellular calcium mobilization in platelets triggered by 10 micromol/L ADP, 10 micromol/L PAF, 2 microg/mL collagen and 15 micromol/L thrombin receptor activating peptide (TRAP). Overall DS showed inhibition of platelet aggregation, platelet activation, platelet-leucocyte conjugate formation and leucocyte activation in response to all the agonists apart from adrenaline (all p < 0.01). DS showed inhibition of platelet aggregation and leucocyte activation equivalent to cangrelor 100 nmol/L and ASA 100 micromol/L. SME dose-dependently inhibited intracellular calcium mobilization in platelets following stimulation with all the platelet agonists with maximum effective at 0.36 mg/mL (all p < 0.01). When used at 0.18 mg/mL its inhibitory effect was equivalent to cangrelor and ASA. We conclude that DS is a potential inhibitor of both platelet and leucocyte activation

  7. The Hydraulic Permeability of Blood Clots as a Function of Fibrin and Platelet Density

    PubMed Central

    Wufsus, A.R.; Macera, N.E.; Neeves, K.B.

    2013-01-01

    Interstitial fluid flow within blood clots is a biophysical mechanism that regulates clot growth and dissolution. Assuming that a clot can be modeled as a porous medium, the physical property that dictates interstitial fluid flow is the hydraulic permeability. The objective of this study was to bound the possible values of the hydraulic permeability in clots formed in vivo and present relationships that can be used to estimate clot permeability as a function of composition. A series of clots with known densities of fibrin and platelets, the two major components of a clot, were formed under static conditions. The permeability was calculated by measuring the interstitial fluid velocity through the clots at a constant pressure gradient. Fibrin gels formed with a fiber volume fraction of 0.02–0.54 had permeabilities of 1.2 × 10−1–1.5 × 10−4μm2. Platelet-rich clots with a platelet volume fraction of 0.01–0.61 and a fibrin volume fraction of 0.03 had permeabilities over a range of 1.1 × 10−2–1.5 × 10−5μm2. The permeability of fibrin gels and of clots with platelet volume fraction of <0.2 were modeled as an array of disordered cylinders with uniform diameters. Clots with a platelet volume fraction of >0.2 were modeled as a Brinkman medium of coarse solids (platelets) embedded in a mesh of fine fibers (fibrin). Our data suggest that the permeability of clots formed in vivo can vary by up to five orders of magnitude, with pore sizes that range from 4 to 350 nm. These findings have important implications for the transport of coagulation zymogens/enzymes in the interstitial spaces during clot formation, as well as the design of fibrinolytic drug delivery strategies. PMID:23601328

  8. Formulation and Storage of Platelet-Rich Plasma Homemade Product

    PubMed Central

    Giraudo, Laurent; Veran, Julie; Magalon, Jeremy; Coudreuse, Jean-Marie; Magalon, Guy; Dubois, Christophe; Serratrice, Nicolas; Dignat-George, Françoise; Sabatier, Florence

    2012-01-01

    Abstract The platelet-rich plasma (PRP) is an autologous biotherapy based on platelet-healing properties. Here, we developed a simple and reproducible PRP purification protocol based on two successive centrifugations. We evaluated different centrifugation speeds and time-storage durations on the platelet quantity and quality. Sterility and stability of our PRP homemade product were also performed. We prepared PRP from 54 healthy volunteers. We tested activation state, reactivity, and stability of platelets by flow cytometry using basal and adenosine diphosphate (ADP)-induced P-selectin expression markers; growth factor release after platelet activation by an enzyme-linked immunosorbent assay (ELISA); platelet aggregation capacity by aggregrometry assays; clot formation and retraction by thromboelastography; and platelet morphology by ultrastructural analysis. About 130 and 250 g successive speed centrifugations further concentrated platelets while preserving their bioactivity during 6 h (after that, platelet functions were significantly altered). In these conditions, we obtained a highly concentrated pure PRP product (with a low leukocyte count) suitable to study platelet properties. To avoid the loss of efficacy, we recommend injecting PRP under 3 h after preparation. PMID:23516671

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

    PubMed

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

    2002-01-01

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

  10. The Catalytic Subunit of Protein Phosphatase 1 Gamma Regulates Thrombin-Induced Murine Platelet αIIbβ3 Function

    PubMed Central

    Gushiken, Francisca C.; Hyojeong, Han; Pradhan, Subhashree; Langlois, Kimberly W.; Alrehani, Nawaf; Cruz, Miguel A.; Rumbaut, Rolando E.; Vijayan, K. Vinod

    2009-01-01

    Background Hemostasis and thrombosis are regulated by agonist-induced activation of platelet integrin αIIbβ3. Integrin activation, in turn is mediated by cellular signaling via protein kinases and protein phosphatases. Although the catalytic subunit of protein phosphatase 1 (PP1c) interacts with αIIbβ3, the role of PP1c in platelet reactivity is unclear. Methodology/Principal Findings Using γ isoform of PP1c deficient mice (PP1cγ−/−), we show that the platelets have moderately decreased soluble fibrinogen binding and aggregation to low concentrations of thrombin or protease-activated receptor 4 (PAR4)-activating peptide but not to adenosine diphosphate (ADP), collagen or collagen-related peptide (CRP). Thrombin-stimulated PP1cγ−/− platelets showed decreased αIIbβ3 activation despite comparable levels of αIIbβ3, PAR3, PAR4 expression and normal granule secretion. Functions regulated by outside-in integrin αIIbβ3 signaling like adhesion to immobilized fibrinogen and clot retraction were not altered in PP1cγ−/− platelets. Thrombus formation induced by a light/dye injury in the cremaster muscle venules was significantly delayed in PP1cγ−/− mice. Phosphorylation of glycogen synthase kinase (GSK3)β-serine 9 that promotes platelet function, was reduced in thrombin-stimulated PP1cγ−/− platelets by an AKT independent mechanism. Inhibition of GSK3β partially abolished the difference in fibrinogen binding between thrombin-stimulated wild type and PP1cγ−/− platelets. Conclusions/Significance These studies illustrate a role for PP1cγ in maintaining GSK3β-serine9 phosphorylation downstream of thrombin signaling and promoting thrombus formation via fibrinogen binding and platelet aggregation. PMID:20016849

  11. Lymphocyte-mediated inhibition of platelet cytotoxic functions during Hymenoptera venom desensitization: characterization of a suppressive lymphokine.

    PubMed

    Tsicopoulos, A; Tonnel, A B; Vorng, H; Joseph, M; Wallaert, B; Kusnierz, J P; Pestel, J; Capron, A

    1990-06-01

    Recently, it has been shown that platelets, through a receptor for the Fc fragment of IgE, could be specially triggered by venom allergens in hypersensitivity to hymenoptera, generating cytocidal mediators toward Schistosoma mansoni larvae, and oxygen metabolites measured by chemiluminescence. After rush immunotherapy, a depressed platelet response was demonstrated to be associated with the production of lymphokine(s). Here we report the characterization of a factor present in supernatants of antigen-stimulated T cells from patients after hymenoptera venom desensitization which is able to inhibit platelet cytotoxic functions in a dose-dependent manner. The optimal inhibition was observed with supernatants obtained after T lymphocyte stimulated with 10(-5) micrograms venom allergen/ml. Once specifically produced the platelet-suppressive effect of lymphocyte supernatants was not antigen specific. The producing T cell subpopulation was identified as CD8+. This lymphokine had an approximate molecular mass of 25 kDa and a pI of 4.8. It was heat and acid stable and sensitive to trypsin and proteinase K but not to neuraminidase. This platelet inhibitory activity was absorbed by platelet membrane suggesting its binding to a receptor. These properties were very similar to a previously described platelet activity suppressive lymphokine, suggesting the participation of this lymphokine in the mechanisms of rush desensitization. PMID:2369915

  12. Alterations of platelet functions in children and adolescents with iron-deficiency anemia and response to therapy.

    PubMed

    Mokhtar, Galila M; Ibrahim, Wafaa E; Kassim, Nevine A; Ragab, Iman A; Saad, Abeer A; Abdel Raheem, Heba G

    2015-01-01

    Several changes in platelets have been reported in patients with iron-deficiency anemia (IDA), so a relationship between iron metabolism and thrombopoiesis should be considered. We aimed to study the alterations of platelet functions in patients with IDA by assessment of platelet aggregation with epinephrine, adenosine diphosphate (ADP) and ristocetin and by measuring platelet function analyzer-100 (PFA-100) closure time together with the effect of iron therapy on the same tests. A follow-up study was conducted in Ain Shams University Children's hospital in the period from June 2011 to June 2012 including 20 patients with confirmed IDA and 20 healthy age- and sex-matched control. Bleeding manifestations were reported. Laboratory analysis included complete blood count, assessment of iron status by measuring serum iron, TIBC and ferritin, assessment of platelet functions by PFA-100 closure time and platelet aggregation with collagen, ADP and ristocetin. Patients with IDA were treated by oral iron therapy 6 mg/kg/day of ferrous sulfate and post-therapeutic re-assessment was done. Mean age of IDA patients was 5.7 ± 4.2 years. Bleeding manifestations were more common in patients group. Mean PFA-100 closure times (with epinephrine) were significantly longer in patients (179.1 ± 86.4 seconds) compared to control group (115 ± 28.5 seconds) (p < 0.05). Platelet aggregation by ADP (38.1 ± 22.2%), epinephrine (19.7 ± 14.2%) and ristocetin (58.8 ± 21.4%) were significantly reduced in patients compared to control (62.7 ± 6.2, 63.3 ± 6.9, 73.8 ± 8.3, respectively; p < 0.001). After treatment platelet aggregation tests induced by ADP (64.78 ± 18.25%), and epinephrine (55.47 ± 24%) were significantly increased in patients with IDA compared to before treatment (39.44 ± 21.85%, 20.33 ± 14.58%; p < 0.001). PFA-100 closure time as well showed significant decreased after treatment (118.4 ± 27.242) compared to before treatment (186.2 ± 90.35; p < 0.05). A negative

  13. Examining Endothelial Function and Platelet Reactivity in Patients with Depression before and after SSRI Therapy.

    PubMed

    Dawood, Tye; Barton, David A; Lambert, Elisabeth A; Eikelis, Nina; Lambert, Gavin W

    2016-01-01

    Although it is recognized that patients with major depressive disorder (MDD) are at increased risk of developing cardiovascular disease (CVD) the mechanisms responsible remain unknown. Endothelial dysfunction is one of the first signs of CVD. Using two techniques, flow-mediated dilatation in response to reactive hyperemia and laser Doppler velocimetry with iontophoresis, we examined endothelial function in the forearm before and after serotonin-specific reuptake inhibitor (SSRI) treatment in 31 patients with MDD. Measurement of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, soluble P-selectin, and noradrenaline in plasma was also performed. Prior to treatment, markers of endothelial and vascular function and platelet reactivity were within the normal range. Following SSRI therapy (95 ± 5 days) symptoms of depression were reduced (paired difference between pre- and post-treatment Hamilton rating -18 ± 1, P < 0.001) with 19 patients recovered and 4 remitted. There occurred no significant change in markers of endothelial or vascular function following SSRI therapy. The improvement in Hamilton depression rating in response to therapy could be independently predicted by the baseline arterial plasma noradrenaline concentration (r (2) = 0.36, P = 0.003). In this cohort of patients with MDD, SSRI therapy did not influence endothelial function or markers of vascular or platelet reactivity. Patient response to SSRI therapy could be predicted by the initial circulating level of noradrenaline, with noradrenaline levels being lower in responders. PMID:26924994

  14. Examining Endothelial Function and Platelet Reactivity in Patients with Depression before and after SSRI Therapy

    PubMed Central

    Dawood, Tye; Barton, David A.; Lambert, Elisabeth A.; Eikelis, Nina; Lambert, Gavin W.

    2016-01-01

    Although it is recognized that patients with major depressive disorder (MDD) are at increased risk of developing cardiovascular disease (CVD) the mechanisms responsible remain unknown. Endothelial dysfunction is one of the first signs of CVD. Using two techniques, flow-mediated dilatation in response to reactive hyperemia and laser Doppler velocimetry with iontophoresis, we examined endothelial function in the forearm before and after serotonin-specific reuptake inhibitor (SSRI) treatment in 31 patients with MDD. Measurement of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, soluble P-selectin, and noradrenaline in plasma was also performed. Prior to treatment, markers of endothelial and vascular function and platelet reactivity were within the normal range. Following SSRI therapy (95 ± 5 days) symptoms of depression were reduced (paired difference between pre- and post-treatment Hamilton rating −18 ± 1, P < 0.001) with 19 patients recovered and 4 remitted. There occurred no significant change in markers of endothelial or vascular function following SSRI therapy. The improvement in Hamilton depression rating in response to therapy could be independently predicted by the baseline arterial plasma noradrenaline concentration (r2 = 0.36, P = 0.003). In this cohort of patients with MDD, SSRI therapy did not influence endothelial function or markers of vascular or platelet reactivity. Patient response to SSRI therapy could be predicted by the initial circulating level of noradrenaline, with noradrenaline levels being lower in responders. PMID:26924994

  15. Most Recent Evidence Behind Aggregometry and Genotyping Methods as Platelet Function Testing for Tailored Anti-Platelet Treatment Among PCI Patients.

    PubMed

    Gajda, Sylwia N; Kołtowski, Łukasz; Tomaniak, Mariusz

    2015-01-01

    Aggregometry and genotyping are methods of platelet function testing, which can be beneficial for high-risk patients undergoing invasive cardiac procedures. An optimal level of platelet reactivity (PR) should be maintained. There are discrepancies between individuals and their response to clopidogrel, accounting for the incidence of poor responders from 5% to 44%. This phenomenon predisposes the patients to increased risk of ischaemic events and thereby overall poorer clinical outcome. Prasugrel and tricagrelor are newer without genetic correlation to their action, however associated with increased bleeding risk. Aggregometry methods assess platelet reactivity at the exact moment of blood sampling. They reflect "phenotype" of the patient and vary after drug administration or dose change. The most popular tests are Light Transmission Aggregometry, Vasodilator-Stimulated Protein, VerifyNow, Multiple Electrode Aggregometry and Thrombelastography. There is proven genetic correlation between some cytochrome enzymes on clopidogrel response. The most widely tested is gene CYP2C19, which produces the enzyme transforming clopidogrel into an active metabolite. The CYP2C19*2 allele carriers have higher PR which can result in more thrombotic events. The manuscript shows the most recent evidence behind platelet function testing. Aggregometry is shown to be beneficial in 5 trials and 1 meta-analysis, while one paper was of different opinion. Ten studies show a positive clinical effect of genotyping on patients' outcome, while one does not support it. The best method of identifying high-risk individuals could be both methods and personalisation of antiplatelet therapy may decrease adverse ischaemic outcomes. PMID:26469115

  16. Structural functional and folding scenario of an anti platelet and thrombolytic enzyme crinumin.

    PubMed

    Singh, Kunwar Awaneesh; Singh, Sanjay; Jagannadham, Medicherla V

    2014-07-01

    A folding pattern, conformational stability and therapeutic role of a protein helps in developing a suitable drug. Crinumin, a thrombolytic and anti platelet agent, has been studied for its functional and conformational properties by equilibrium unfolding methods. The crinumin belongs to α+β class of protein and exhibits a non native structure and two molten globule states at different conditions. Two domains in the molecular structure of the protein with altered stability are present that unfold sequentially. The enzyme maintains activity as well as structural integrity even in adverse conditions. These observations provide an understanding of protein folding as well as facilitate the development of a potential drug. PMID:24726528

  17. Sulphate conjugation of p-hydroxytriamterene by platelet phenol sulphotransferase: assay conditions and correlation with metabolism in man.

    PubMed Central

    Reiter, C; Werness, P G; Van Loon, J; Smith, L H; Weinshilboum, R M

    1983-01-01

    1 Sulphate conjugation catalyzed by phenol sulphotransferase (PST) is an important pathway in the metabolism of many drugs including triamterene. Variations in PST activity in an easily obtained tissue such as the platelet might reflect individual differences in the sulphate conjugation in other organs and tissues. Human platelets contain at least two forms of PST, a thermolabile (TL) form for which dopamine is a substrate and a thermostable (TS) form for which low concentrations of p-nitrophenol serve as a substrate. 2 p-OH-triamterene, the major metabolite of triamterene, is conjugated with sulphate in vivo. p-OH-triamterene was a substrate for platelet PST with an apparent Michaelis-Menten value of 26 microM. Thermal stability studies indicated that p-OH-triamterene was a substrate for only the TS form of platelet PST. 3 When platelet homogenates from 29 individual subjects were tested, there was a significant correlation between PST activities measured with 4 microM p-nitrophenol and with p-OH-triamterene (r = 0.985, P less than 0.0001) but not between activities measured with dopamine and with p-OH-triamterene (r = 0.023, P greater than 0.2). These results confirmed that p-OH-triamterene was a substrate for only the TS form of human platelet PST. 4 The same 29 subjects were treated with 1 mg/kg of triamterene orally. 24-h urinary excretions of triamterene, p-OH-triamterene and p-OH-triamterene sulphate averaged 15.3%, 6.3% and 78.4%, respectively, of the total of triamterene plus measured metabolites excreted. The excretion of triamterene plus the two metabolites averaged 43.1 +/- 2.6% (mean +/- s.e. mean) of the ingested dose. There was not a significant correlation between the proportion of p-OH-triamterene excreted as sulphate conjugate and the activities of either the TS or TL forms of platelet PST activity. PMID:6573903

  18. Dose-dependent platelet stimulation and inhibition induced by anti-PIA1 IgG

    SciTech Connect

    Ryu, T.; Davis, J.M.; Schwartz, K.A. )

    1990-07-01

    The PIA1 antibody produces several clinically distinct and severe thrombocytopenias. Investigations have demonstrated divergent effects on platelet function; prior reports demonstrated inhibition, while a conflicting publication showed platelet activation. We have resolved this conflict using anti-PIA1 IgG produced by a patient with posttransfusion purpura. Relatively low concentrations stimulated platelet aggregation and release of adenosine triphosphate (ATP) whereas high concentrations inhibited platelet function, producing a thrombasthenia-like state. The number of molecules of platelet-associated IgG necessary to initiate aggregation and ATP release (2,086 +/- 556) or produce maximum aggregation (23,420 +/- 3,706) or complete inhibition (63,582 +/- 2654) were measured with a quantitative radiometric assay for bound anti-PIA1. Preincubation of platelets with high concentrations of PIA1 antibody inhibited platelet aggregation with 10 mumol/L adenosine diphosphate and blocked 125I-labeled fibrinogen platelet binding. Platelet activation with nonfibrinogen dependent agonist, 1 U/ml thrombin, was not inhibited by this high concentration of PIA1 IgG. In conclusion, anti-PIAI IgG produces (1) stimulation of platelet aggregation and ATP release that is initiated with 2000 molecules IgG per platelet and is associated with an increase of 125I-fibrinogen binding; (2) conversely, inhibition of platelet aggregation is observed with maximum antibody binding, 63,000 molecules IgG per platelet, and is mediated via a blockade of fibrinogen binding.

  19. Rhesus monkey platelets

    SciTech Connect

    Harbury, C.B.

    1986-03-01

    The purpose of this abstract is to describe the adenine nucleotide metabolism of Rhesus monkey platelets. Nucleotides are labelled with /sup 14/C-adenine and extracted with EDTA-ethanol (EE) and perchlorate (P). Total platelet ATP and ADP (TATP, TADP) is measured in the Holmsen Luciferase assay, and expressed in nanomoles/10/sup 8/ platelets. TR=TATP/TADP. Human platelets release 70% of their TADP, with a ratio of released ATP/ADP of 0.7. Rhesus platelets release 82% of their TADP, with a ratio of released ATP/ADP of 0.33. Thus, monkey platelets contain more ADP than human platelets. Thin layer chromatography of EE gives a metabolic ratio of 11 in human platelets and 10.5 in monkey platelets. Perchlorate extracts metabolic and actin bound ADP. The human and monkey platelets ratios were 5, indicating they contain the same proportion of actin. Thus, the extra ADP contained in monkey platelets is located in the secretory granules.

  20. Scalable Functionalized Graphene Nano-platelets as Tunable Cathodes for High-performance Lithium Rechargeable Batteries

    PubMed Central

    Kim, Haegyeom; Lim, Hee-Dae; Kim, Sung-Wook; Hong, Jihyun; Seo, Dong-Hwa; Kim, Dae-chul; Jeon, Seokwoo; Park, Sungjin; Kang, Kisuk

    2013-01-01

    High-performance and cost-effective rechargeable batteries are key to the success of electric vehicles and large-scale energy storage systems. Extensive research has focused on the development of (i) new high-energy electrodes that can store more lithium or (ii) high-power nano-structured electrodes hybridized with carbonaceous materials. However, the current status of lithium batteries based on redox reactions of heavy transition metals still remains far below the demands required for the proposed applications. Herein, we present a novel approach using tunable functional groups on graphene nano-platelets as redox centers. The electrode can deliver high capacity of ~250 mAh g−1, power of ~20 kW kg−1 in an acceptable cathode voltage range, and provide excellent cyclability up to thousands of repeated charge/discharge cycles. The simple, mass-scalable synthetic route for the functionalized graphene nano-platelets proposed in this work suggests that the graphene cathode can be a promising new class of electrode. PMID:23514953

  1. Correlation Between the CYP2C19 Phenotype Status and the Results of Three Different Platelet Function Tests in Cardiovascular Disease Patients Receiving Antiplatelet Therapy: An Emphasis on Newly Introduced Platelet Function Analyzer-200 P2Y Test

    PubMed Central

    Li, Shuhua; Choi, Jae-Lim; Guo, Long Zhe; Goh, Ri-Young; Kim, Bo-Ram; Woo, Kwang-Sook; Kim, Moo-Hyun

    2016-01-01

    Background An association has been reported between CYP2C19 polymorphism and the altered antiplatelet activity of clopidogrel. We investigated this association using the newly introduced platelet function analyzer (PFA)-200 (INNOVANCE PFA-200 System; Siemens Healthcare, Germany) P2Y test. Methods Polymorphisms of CYP2C19*2, *3, *17 and the degree of inhibition of platelet function were determined in 83 patients. Three different platelet function tests were used to evaluate the degree of platelet inhibition and to check the association with genotype. Results The post-procedure PFA-200 values of extensive metabolizers (EM) patients (285.3±38.8) were higher than those of intermediate metabolizers (IM) and poor metabolizers (PM) patients (227.7±98.3 and 133.7±99.2, respectively; P=0.024). Light transmittance aggregometry (LTA) and the VerifyNow system showed that the post-procedure values for EM patients were lower than those of IM and PM patients (LTA: 24.4±15.7, 34.1±17.6, and 42.2±16.9, respectively, P<0.001; VerifyNow: 133.2±60.5, 171.5±42.6, and 218.7±59.3, respectively, P<0.001). The high residual platelet reactivity (HPR) rates were significantly different among the EM, IM, and PM groups using PFA-200 (PM:IM:EM=82.4:40.6:11.8, P<0.001). Conclusions Approximately, 59.0% of Korean patients with cardiovascular disease receiving clopidogrel had CYP2C19 loss-of-function genotypes classified as IM or PM, and the frequency was similar to the data from Asian people. The PFA-200, LTA, and VerifyNow platelet function tests revealed evidence of a significant association between the efficacy of clopidogrel and CYP2C19 genotypes. PMID:26522758

  2. The role of peroxides as modulators of human blood platelet function

    SciTech Connect

    Jesse, R.L.; Hess, M.L. )

    1990-02-26

    The ability to peroxidize lipid in the vicinity of arterial walls has been demonstrated through oxidation of the LDL-particle by monocytes/macrophages, smooth muscle cells, and endothelial cells. The authors questioned what effect increased levels of peroxides might have on platelet function. Platelet aggregation, studied trubidometrically, was initiated by arachidonic acid (AA) alone or in the presence of a low concentration of H{sub 2}O{sub 2}, (which by itself could not initiate aggregation). Aggregation was analyzed quantitatively by measuring the time it took to reach 1/2 the maximal extent of aggregation (T1/2Max). Using the minimal concentration of AA still able to initiate aggregation (0.2-0.45 mM) the T1/2Max was 112{+-}35 (n=10). With 50% of the respective AA conc. plus 17 {mu}M H{sub 2}O{sub 2}, the T1/2Max was 52{+-}21. Under similar circumstances, the amount of AA able to initiate aggregation could be reduced by 80% with the addition of H{sub 2}O{sub 2}. These effects could be duplicated by glucose oxidase, 0.015-0.15 IU, and could be eliminated by the addition of catalase (<25 U/ml) at any time prior to the start of aggregation. With non-aggregating concentrations of AA alone, synthesis of Thromboxane B{sub 2} was negligible; with aggregation by the same AA plus H{sub 2}O{sub 2} it was 550{+-}79 pg/10{sup 3} platelets. Both aggregation and TxB{sub 2} synthesis were completely inhibited by aspirin or indomethacin, and by the antioxidants phenol and nor-dihydroguaretic acid, at concentrations known to inhibit cyclooxygenase. In platelets with a defect in the second wave of ADP induced aggregation, but having normal aggregation with added AA (indicating the cyclooxygenase-thromboxane axis was intact), sub-aggregating concentrations of AA, plus H{sub 2}O{sub 2} resulted in brisk aggregation.

  3. Targeted drug delivery to circulating tumor cells via platelet membrane-functionalized particles.

    PubMed

    Li, Jiahe; Ai, Yiwei; Wang, Lihua; Bu, Pengcheng; Sharkey, Charles C; Wu, Qianhui; Wun, Brittany; Roy, Sweta; Shen, Xiling; King, Michael R

    2016-01-01

    Circulating tumor cells (CTCs) are responsible for metastases in distant organs via hematogenous dissemination. Fundamental studies in the past decade have suggested that neutralization of CTCs in circulation could represent an effective strategy to prevent metastasis. Current paradigms of targeted drug delivery into a solid tumor largely fall into two main categories: unique cancer markers (e.g. overexpression of surface receptors) and tumor-specific microenvironment (e.g. low pH, hypoxia, etc.). While relying on a surface receptor to target CTCs can be greatly challenged by cancer heterogeneity, targeting of tumor microenvironments has the advantage of recognizing a broader spectrum of cancer cells regardless of genetic differences or tumor types. The blood circulation, however, where CTCs transit through, lacks the same tumor microenvironment as that found in a solid tumor. In this study, a unique "microenvironment" was confirmed upon introduction of cancer cells of different types into circulation where activated platelets and fibrin were physically associated with blood-borne cancer cells. Inspired by this observation, synthetic silica particles were functionalized with activated platelet membrane along with surface conjugation of tumor-specific apoptosis-inducing ligand cytokine, TRAIL. Biomimetic synthetic particles incorporated into CTC-associated micro-thrombi in lung vasculature and dramatically decreased lung metastases in a mouse breast cancer metastasis model. Our results demonstrate a "Trojan Horse" strategy of neutralizing CTCs to attenuate metastasis. PMID:26519648

  4. Study of a humanized inhibitory anti-platelet glycoprotein VI phage antibody from a phage antibody library.

    PubMed

    Liu, Qinghong; Zhang, Chunmei; Yu, Lingjia; Shi, Yongyu; Zhang, Liping; Peng, Jun; Ji, Xuebin; Hou, Ming

    2016-01-01

    Objective The aims of the study were to study the effect of anti-platelet glycoprotein (GP) VI auto-antibodies on platelet aggregation and use phage surface display technology to produce anti-platelet GPVI phage antibody fragment, which may be developed to inhibit platelet aggregation in the treatment of cardiovascular disease. Methods Plasma samples from patients with immune thrombocytopenia (ITP) were screened by monoclonal antibody immobilization of the platelet antigen assay and the platelet aggregation test for anti-platelet GPVI auto-antibody with an inhibitory effect. The humanized anti-platelet GPVI phage antibody was produced by phage surface display technology. The function of the phage antibody fragment against platelet aggregation was examined by the platelet aggregation test. Results Of 726 ITP patients, 2 (0.27%) patients' plasma significantly inhibited platelet aggregation induced by collagen-1. After five rounds of selection, enrichment, and purification, a soluble phage antibody fragment was produced, which can inhibit platelet aggregation induced by collagen-1. The results demonstrate that only a few of the screened anti-platelet GPVI auto-antibodies showed an inhibitory effect on platelet aggregation. Discussion A completely humanized anti-GPVI soluble phage antibody can be produced by phage surface display technology. The antibody was able to specifically block collagen-induced platelet aggregation without influencing the aggregation responses to other agonists. Conclusions Results of the present study suggest that very few anti-platelet GPVI auto-antibodies inhibit the aggregation function of platelet. The humanized anti-platelet GPVI produced by phage surface display technology is promising to be used to inhibit platelet aggregation in the treatment of cardiovascular disease. PMID:26330203

  5. Human platelet antigen 1 (HPA 1) genotyping with 5' nuclease assay and sequence-specific primers reveals a single nucleotide deletion in intron 2 of the HPA 1a allele of platelet glycoprotein IIIa.

    PubMed

    Kjaer, Killie Mette; Jaegtvik, Sissel; Husebekk, Anne; Skogen, Bjorn

    2002-05-01

    We have established a 5' nuclease assay (5' NA) for human platelet antigen (HPA) 1a/b allelic discrimination. The assay is based on the simultaneous amplification and detection of the two targets in a one-tube system. The results are read optically, immediately after termination of the polymerase chain reaction (PCR), and no post-PCR processing is necessary. This genotyping procedure is less time-consuming and cheaper than our conventional sequence-specific primer PCR (SSP-PCR), which is run as a two-tube test, with verification of the results after electrophoresis in agarose gel. The reduction of analytical steps, simplification of the procedure and potential for automation were important advantages for our choice of system. This test system is more suitable for large-scale testing and fits better for our screening programme for HPA 1bb determination. DNA from 1093 individuals were tested in parallel with the SSP-PCR and the 5' NA. One thousand and ninety-one samples gave identical results in SSP-PCR and 5' NA. Upon repeated testing, two samples consistently came out as HPA 1bb in SSP-PCR and HPA 1ab in 5' NA. DNA sequencing revealed a defect located in an intronic area that corresponds to the consensus primer used for the SSP-PCR HPA 1a typing. PMID:11972525

  6. Glutamate release from platelets: exocytosis versus glutamate transporter reversal.

    PubMed

    Kasatkina, Ludmila A; Borisova, Tatiana A

    2013-11-01

    Platelets express neuronal and glial glutamate transporters EAAT 1-3 in the plasma membrane and vesicular glutamate transporters VGLUT 1,2 in the membrane of secretory granules. This study is focused on the assessment of non-exocytotic glutamate release, that is, the unstimulated release, heteroexchange and glutamate transporter reversal in platelets. Using the glutamate dehydrogenase assay, the absence of unstimulated release of endogenous glutamate from platelets was demonstrated, even after inhibition of glutamate transporters and cytoplasmic enzyme glutamine synthetase by dl-threo-β-benzyloxyaspartate and methionine sulfoximine, respectively. Depolarization of the plasma membrane by exposure to elevated [K(+)] did not induce the release of glutamate from platelets that was shown using the glutamate dehydrogenase assay and radiolabeled l-[(14)C]glutamate. Glutamate efflux by means of heteroexchange with transportable inhibitor of glutamate transporters dl-threo-β-hydroxyaspartate (dl-THA) was not observed. Furthermore, the protonophore cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP) and inhibitor of V-type H(+)-ATPase bafilomycin A1 also failed to stimulate the release of glutamate from platelets. However, exocytotic release of glutamate from secretory granules in response to thrombin stimulation was not prevented by elevated [K(+)], dl-THA, FCCP and bafilomycin A1. In contrast to nerve terminals, platelets cannot release glutamate in a non-exocytotic manner. Heteroexchange, transporter-mediated and unstimulated release of glutamate are not inherent to platelets. Therefore, platelets may be used as a peripheral marker/model for the analysis of glutamate uptake by brain nerve terminals only (direct function of transporters), whereas the mechanisms of glutamate release are different in platelets and nerve terminals. Glutamate is released by platelets exclusively by means of exocytosis. Also, reverse function of vesicular glutamate transporters of platelets is

  7. The role of anticoagulation in the measurement of platelet volumes.

    PubMed

    Thompson, C B; Diaz, D D; Quinn, P G; Lapins, M; Kurtz, S R; Valeri, C R

    1983-09-01

    The effect of anticoagulation on platelet size stability was studied using blood collected in seven different anticoagulants and stored at room temperature for up to eight hours. The mean platelet volume (MPV) value was most stable in blood collected in 15% ACD and ACD/Na2EDTA. In blood collected in Na2EDTA, K3EDTA, or 11.9% ACD, there was an increase in MPV in the first two hours, after which the MPVs remained stable up to eight hours. Sodium citrate and heparin proved unreliable for the measurement of platelet volume. Platelet counts were stable (less than 5% variation) in all anticoagulants except heparin, which had 16% variation for the eight hours of study. Simultaneously, RBC counts and mean corpuscular volume (MCV) measurements were stable in all seven anticoagulants, with sodium citrate producing the most variation. A negative correlation was observed between MCV and pH of the anticoagulated blood. WBC counts showed less than 3% variation in all anticoagulants except sodium citrate and heparin. Separate experiments demonstrated that electrolyte composition, pH, tonicity, and method of calcium chelation all influenced the stability of the MPV. Of the anticoagulants studied, ACD/Na2 EDTA appeared to provide the best conditions of anticoagulation for both routine clinical and research laboratory measurement of the MPV. It inhibited platelet activation but left the platelets in their normal discoid shape. Platelets could be removed from the anticoagulant and studied in functional assays for up to eight hours after blood drawing. Both platelet counts and MPVs remained stable in blood collected in ACD/Na2 EDTA anticoagulant for up to eight hours at room temperature. In 52 volunteers studied, an inverse correlation (r = -0.72, P less than 0.001) was observed between platelet count and MPV, suggesting that the circulating platelet mass may be a more important indicator of platelet homeostasis than either the platelet count or the mean platelet volume alone. PMID

  8. The effects of haemodilution with hydroxyethyl starch 130/0.4 solution on coagulation as assessed by thromboelastography and platelet receptor function studies in vitro.

    PubMed

    Williams, P; Yang, K; Kershaw, G; Wong, G; Dunkley, S; Kam, P C A

    2015-11-01

    This study evaluated the effects of haemodilution with either 6% hydroxyethyl starch (HES) 130/0.4 (Voluven(®)) or 0.9% normal saline (NS) on blood coagulation in vitro. Haemodilution with 6% HES 130/0.4 impaired coagulation, as indicated by the changes in thromboelastographic parameters k-time, α-angle and maximum amplitude. Light transmission aggregometry and multiple electrode aggregometry demonstrated that impaired platelet receptor function occurred only at high levels of haemodilution (40%) with both fluids, but there was no significant difference between the two fluids (P=0.05). The thromboelastographic functional fibrinogen assay showed that the fibrinogen component of clot strength was significantly impaired with haemodilution with HES 130/0.4 compared with haemodilution with NS (whole blood [14.4 ± 4.6 mm] versus 40% HES dilution [3.7 ± 1.9], [P=0.001]; versus 40% NS dilution [10.4 ± 4.6], [P=0.129]). These findings suggest that there is little difference between HES or NS in relation to coagulation or platelet function during minor or moderate haemodilution, but at high levels of haemodilution with HES, fibrinogen activity is more impaired compared with NS. PMID:26603798

  9. Platelet-collagen adhesion enhances platelet aggregation induced by binding of VWF to platelets

    SciTech Connect

    Laduca, F.M.; Bell, W.R.; Bettigole, R.E. State Univ. of New York, Buffalo )

    1987-11-01

    Ristocetin-induced platelet aggregation (RIPA) was evaluated in the presence of platelet-collagen adhesion. RIPA of normal donor platelet-rich plasma (PRP) demonstrated a primary wave of aggregation mediated by the binding of von Willebrand factor (VWF) to platelets and a secondary aggregation wave, due to a platelet-release reaction, initiated by VWF-platelet binding and inhibitable by acetylsalicylic acid (ASA). An enhanced RIPA was observed in PRP samples to which collagen had been previously added. These subthreshold concentrations of collagen, which by themselves were insufficient to induce aggregation, caused measurable platelet-collagen adhesion. Subthreshold collagen did not cause microplatelet aggregation, platelet release of ({sup 3}H)serotonin, or alter the dose-responsive binding of {sup 125}I-labeled VWF to platelets, which occurred with increasing ristocetin concentrations. However, ASA inhibition of the platelet release reaction prevented collagen-enhanced RIPA. These results demonstrate that platelet-collagen adhesion altered the platelet-release reaction induced by the binding of VWF to platelets causing a platelet-release reaction at a level of VWF-platelet binding not normally initiating a secondary aggregation. These findings suggest that platelet-collagen adhesion enhances platelet function mediated by VWF.

  10. Highly active antiretroviral therapy-related mechanisms of endothelial and platelet function alterations.

    PubMed

    Gresele, Paolo; Falcinelli, Emanuela; Momi, Stefania; Francisci, Daniela; Baldelli, Franco

    2014-01-01

    Highly active antiretroviral therapy (HAART) has transformed human immunodeficiency virus (HIV) infection into a chronic condition, which has allowed the infected population to age and become prone to chronic degenerative diseases common to the general population, including atherosclerotic cardiovascular disease, and coronary artery disease (CAD). Possible causative mechanisms of HIV-associated CAD are related to classic cardiovascular risk factors, such as dyslipidemia, insulin resistance, and fat redistribution, which may be due to either HIV infection or to HAART-associated toxicity. However, other mechanisms are emerging as crucial for the cardiovascular complication of HIV and HAART. This article analyzes the effects of HIV and HAART on endothelial function, endothelium-leukocyte interactions, and platelets as possible mechanisms of enhanced cardiovascular risk. PMID:24987863

  11. Platelet-derived CXCL12 regulates monocyte function, survival, differentiation into macrophages and foam cells through differential involvement of CXCR4–CXCR7

    PubMed Central

    Chatterjee, M; von Ungern-Sternberg, S N I; Seizer, P; Schlegel, F; Büttcher, M; Sindhu, N A; Müller, S; Mack, A; Gawaz, M

    2015-01-01

    Platelets store and release CXCL12 (SDF-1), which governs differentiation of hematopoietic progenitors into either endothelial or macrophage-foam cells. CXCL12 ligates CXCR4 and CXCR7 and regulates monocyte/macrophage functions. This study deciphers the relative contribution of CXCR4–CXCR7 in mediating the effects of platelet-derived CXCL12 on monocyte function, survival, and differentiation. CXCL12 and macrophage migration inhibitory factor (MIF) that ligate CXCR4–CXCR7 induced a dynamic bidirectional trafficking of the receptors, causing CXCR4 internalization and CXCR7 externalization during chemotaxis, thereby influencing relative receptor availability, unlike MCP-1. In vivo we found enhanced accumulation of platelets and platelet-macrophage co-aggregates in peritoneal fluid following induction of peritonitis in mice. The relative surface expression of CXCL12, CXCR4, and CXCR7 among infiltrated monocytes was also enhanced as compared with peripheral blood. Platelet-derived CXCL12 from collagen-adherent platelets and recombinant CXCL12 induced monocyte chemotaxis specifically through CXCR4 engagement. Adhesion of monocytes to immobilized CXCL12 and CXCL12-enriched activated platelet surface under static and dynamic arterial flow conditions were mediated primarily through CXCR7 and were counter-regulated by neutralizing platelet-derived CXCL12. Monocytes and culture-derived-M1–M2 macrophages phagocytosed platelets, with the phagocytic potential of culture-derived-M1 macrophages higher than M2 involving CXCR4–CXCR7 participation. CXCR7 was the primary receptor in promoting monocyte survival as exerted by platelet-derived CXCL12 against BH3-mimetic induced apoptosis (phosphatidylserine exposure, caspase-3 activation, loss of mitochondrial transmembrane potential). In co-culture experiments with platelets, monocytes predominantly differentiated into CD163+ macrophages, which was attenuated upon CXCL12 neutralization and CXCR4/CXCR7 blocking antibodies

  12. York platelet syndrome is a CRAC channelopathy due to gain-of-function mutations in STIM1.

    PubMed

    Markello, Thomas; Chen, Dong; Kwan, Justin Y; Horkayne-Szakaly, Iren; Morrison, Alan; Simakova, Olga; Maric, Irina; Lozier, Jay; Cullinane, Andrew R; Kilo, Tatjana; Meister, Lynn; Pakzad, Kourosh; Bone, William; Chainani, Sanjay; Lee, Elizabeth; Links, Amanda; Boerkoel, Cornelius; Fischer, Roxanne; Toro, Camilo; White, James G; Gahl, William A; Gunay-Aygun, Meral

    2015-03-01

    Store-operated Ca(2+) entry is the major route of replenishment of intracellular Ca(2+) in animal cells in response to the depletion of Ca(2+) stores in the endoplasmic reticulum. It is primarily mediated by the Ca(2+)-selective release-activated Ca(2+) (CRAC) channel, which consists of the pore-forming subunits ORAI1-3 and the Ca(2+) sensors, STIM1 and STIM2. Recessive loss-of-function mutations in STIM1 or ORAI1 result in immune deficiency and nonprogressive myopathy. Heterozygous gain-of-function mutations in STIM1 cause non-syndromic myopathies as well as syndromic forms of miosis and myopathy with tubular aggregates and Stormorken syndrome; some of these syndromic forms are associated with thrombocytopenia. Increased concentration of Ca(2+) as a result of store-operated Ca(2+) entry is essential for platelet activation. The York Platelet syndrome (YPS) is characterized by thrombocytopenia, striking ultrastructural platelet abnormalities including giant electron-opaque organelles and massive, multilayered target bodies and deficiency of platelet Ca(2+) storage in delta granules. We present clinical and molecular findings in 7 YPS patients from 4 families, demonstrating that YPS patients have a chronic myopathy associated with rimmed vacuoles and heterozygous gain-of-function STIM1 mutations. These findings expand the phenotypic spectrum of STIM1-related human disorders and define the molecular basis of YPS. PMID:25577287

  13. The effects of siguazodan, a selective phosphodiesterase inhibitor, on human platelet function.

    PubMed Central

    Murray, K. J.; England, P. J.; Hallam, T. J.; Maguire, J.; Moores, K.; Reeves, M. L.; Simpson, A. W.; Rink, T. J.

    1990-01-01

    1. The effects of siguazodan (SK&F 94836) a selective phosphodiesterase (PDE) inhibitor with inotropic and vasodilator activity, were studied on human platelets. 2. Siguazodan selectively inhibited the major cyclic AMP-hydrolysing PDE in human platelet supernatants. The inhibited enzyme has been variously termed cyclic GMP-inhibited PDE or PDE-III. 3. In platelet-rich plasma (PRP), siguazodan inhibited U46619-induced aggregation more potently than that induced by ADP and collagen. Treatment of the PRP with aspirin had no effect on the potency of siguazodan. 4. In washed platelets, siguazodan increased cyclic AMP levels and reduced cytoplasmic free calcium [( Ca2+]i). ADP decreased the ability of siguazodan to raise cyclic AMP and this may explain its lower potency in inhibiting responses to ADP. 5. Siguazodan has anti-platelet actions over the same concentration range that it is an inotrope and vasodilator. PMID:2158847

  14. Investigation of function similarities between the sarcoplasmic reticulum and platelet calcium-dependent adenosinetriphosphatases with the inhibitors quercetin and calmidazolium

    SciTech Connect

    Fischer, T.H.; Campbell, K.P.; White, G.C. II

    1987-12-01

    The platelet and skeletal sarcoplasmic reticulum calcium-dependent adenosinetriphosphatases (Ca/sup 2 +/-ATPases) were functionally compared with respect to substrate activation by steady-state kinetic methods using the inhibitors quercetin and calmidazolium. Quercetin inhibited platelet and sarcoplasmic reticulum Ca/sup 2 +/-ATPase activities in a dose-dependent manner with IC/sub 50/ values of 25 and 10 ..mu..M, respectively. Calmidazolium also inhibited platelet and sarcoplasmic reticulum Ca/sup 2 +/-ATPase activities, with half-maximal inhibition measured at 5 and 4 ..mu..M, respectively. Both inhibitors also affected the (/sup 45/Ca) calcium transport activity of intact platelet microsomes at concentrations similar to those which reduced Ca/sup 2 +/-ATPase activity. These inhibitors were then used to examine substrate ligation by the platelet and sarcoplasmic reticulum calcium pump proteins. For both Ca/sup 2 +/-ATPase proteins, quercetin has an affinity for the E-Ca/sub 2/ (fully ligated with respect to calcium at the exterior high-affinity calcium binding sites, unligated with respect to ATP) conformational state of the protein that is approximately 10-fold grater than for other conformational states in the hydrolytic cycle. Quercetin can thus be considered a competitive inhibitor of the calcium pump proteins with respect to ATP. In contrast to the effect of quercetin, calmidazolium interacts with the platelet and sarcoplasmic reticulum Ca/sup 2 +/-ATPases in an uncompetitive manner. The dissociation constants for this inhibitor for the different conformational states of the calcium pump proteins were similar, indicating that calmidazolium has equal affinity for all of the reaction intermediates probed. These observations indicate that the substrate ligation processes are similar for the two pump proteins. This supports the concept that the hydrolytic cycles of the two proteins are comparable.

  15. Platelet kinetics with indium-111 platelets: comparison with chromium-51 platelets.

    PubMed

    Peters, A M; Lavender, J P

    1983-04-01

    The application of 111In-oxine to platelet labeling has contributed to the understanding of platelet kinetics along three lines: 1. It allows the measurement of new parameters of splenic function, such as the intrasplenic platelet transit time, which has shed new light on the physiology of splenic blood cell handling. 2. It facilitates the measurement of platelet life span in conditions, such as ITP, in which 51Cr may undergo undesirable elution from the platelet as a result of platelet-antibody interaction. 3. It allows the determination of the fate of platelets, that is, the site of platelet destruction in conditions in which reduced platelet life span is associated with abnormal platelet consumption, as a result of either premature destruction of "abnormal" platelets by the RE system, or the consumption (or destruction) of normal platelets after their interaction with an abnormal vasculature. Future research using 111In platelets may yield further valuable information on the control as well as the significance of intrasplenic platelet pooling, on the role of platelets in the development of chronic vascular lesions, and on the sites of platelet destruction in ITP. With regard to the latter, methods will have to be developed for harvesting sufficient platelets representative of the total circulating platelet population from severely thrombocytopenic patients for autologous platelet labeling. This would avoid the use of homologous platelets, which is likely to be responsible for some of the contradictory data relating to the use of radiolabeled platelet studies for the prediction of the response of patients with ITP to splenectomy. PMID:6346489

  16. Platelets and galectins

    PubMed Central

    2014-01-01

    A major function of platelets is keeping the vascular system intact. Platelet activation at sites of vascular injury leads to the formation of a hemostatic plug. Activation of platelets is therefore crucial for normal hemostasis; however, uncontrolled platelet activation may also lead to the formation of occlusive thrombi that can cause ischemic events. Although they are essential for proper hemostasis, platelet function extends to physiologic processes such as tissue repair, wound remodeling and antimicrobial host defense, or pathologic conditions such as thrombosis, atherosclerosis, chronic inflammatory diseases and cancer. Platelets can be activated by soluble molecules including thrombin, thromboxane A2 (TXA2), adenosine diphosphate (ADP), serotonin or by adhesive extracellular matrix (ECM) proteins such as von Willebrand factor (vWF) and collagen. Here we describe recent advances in the activation of platelets by non-canonical platelet agonists such as galectins. By acting either in soluble or immobilized form, these glycan-binding proteins trigger all platelet activation responses through modulation of discrete signaling pathways. We also offer new hypotheses and some speculations about the role of platelet-galectin interactions not only in hemostasis and thrombosis but also in inflammation and related diseases such as atherosclerosis and cancer. PMID:25405160

  17. Evaluation of participants with suspected heritable platelet function disorders including recommendation and validation of a streamlined agonist panel

    PubMed Central

    Lowe, Gillian C.; Lordkipanidzé, Marie; Bem, Danai; Daly, Martina E.; Makris, Mike; Mumford, Andrew; Wilde, Jonathan T.; Watson, Steve P.

    2012-01-01

    Light transmission aggregometry (LTA) is used worldwide for the investigation of heritable platelet function disorders (PFDs), but interpretation of results is complicated by the feedback effects of ADP and thromboxane A2 (TxA2) and by the overlap with the response of healthy volunteers. Over 5 years, we have performed lumi-aggregometry on 9 platelet agonists in 111 unrelated research participants with suspected PFDs and in 70 healthy volunteers. Abnormal LTA or ATP secretion test results were identified in 58% of participants. In 84% of these, the patterns of response were consistent with defects in Gi receptor signaling, the TxA2 pathway, and dense granule secretion. Participants with defects in signaling to Gq-coupled receptor agonists and to collagen were also identified. Targeted genotyping identified 3 participants with function-disrupting mutations in the P2Y12 ADP and TxA2 receptors. The results of the present study illustrate that detailed phenotypic analysis using LTA and ATP secretion is a powerful tool for the diagnosis of PFDs. Our data also enable subdivision at the level of platelet-signaling pathways and in some cases to individual receptors. We further demonstrate that most PFDs can be reliably diagnosed using a streamlined panel of key platelet agonists and specified concentrations suitable for testing in most clinical diagnostic laboratories. PMID:23002116

  18. The predictive value of the in vitro platelet toxicity assay (iPTA) for the diagnosis of hypersensitivity reactions to sulfonamides.

    PubMed

    Elzagallaai, Abdelbaset A; Koren, Gideon; Rieder, Michael J

    2013-06-01

    Drug hypersensitivity reactions (DHRs) are rare but potentially fatal adverse drug reactions (ADRs). A reliable test to diagnose DHRs would be a major advance in the clinical care for patients and in the evaluation of ADRs during drug development as well as for mechanistic studies of drug hypersensitivity. Available in vitro tests including the lymphocyte toxicity assay (LTA) have been used but are time-consuming, cumbersome, and expensive. We have developed a novel diagnostic test for DHRs, the in vitro platelet toxicity assay (iPTA). The aim of this study was to evaluate the predictive value of the iPTA in diagnosis of DHRs to sulfonamides. We recruited 66 individuals (36 DHS-sulfa patients and 30 healthy controls) to participate in the study. Blood samples were obtained and LTA and iPTA were performed in parallel. There was concentration-dependent toxicity in the cells of patients when incubated with the reactive hydroxylamine metabolite of sulfamethoxazole for both the LTA and iPTA (P < .05). The iPTA was more sensitive than conventional LTA test in detecting susceptibility of patient cells to in vitro toxicity (P < .05). The novel iPTA has considerable potential as an investigative tool for DHS as it is more sensitive and cheaper, requiring no special reagents. PMID:23670648

  19. Syncytiotrophoblast Extracellular Vesicles from Pre-Eclampsia Placentas Differentially Affect Platelet Function

    PubMed Central

    Tannetta, Dionne S.; Hunt, Kathryn; Jones, Chris I.; Davidson, Naomi; Coxon, Carmen H.; Ferguson, David; Redman, Christopher W.; Gibbins, Jonathan M.; Sargent, Ian L.; Tucker, Katherine L.

    2015-01-01

    Pre-eclampsia (PE) complicates around 3% of all pregnancies and is one of the most common causes of maternal mortality worldwide. The pathophysiology of PE remains unclear however its underlying cause originates from the placenta and manifests as raised blood pressure, proteinuria, vascular or systemic inflammation and hypercoagulation in the mother. Women who develop PE are also at significantly higher risk of subsequently developing cardiovascular (CV) disease. In PE, the failing endoplasmic reticulum, oxidative and inflammatory stressed syncytiotrophoblast layer of the placenta sheds increased numbers of syncytiotrophoblast extracellular vesicles (STBEV) into the maternal circulation. Platelet reactivity, size and concentration are also known to be altered in some women who develop PE, although the underlying reasons for this have not been determined. In this study we show that STBEV from disease free placenta isolated ex vivo by dual placental perfusion associate rapidly with platelets. We provide evidence that STBEV isolated from normal placentas cause platelet activation and that this is increased with STBEV from PE pregnancies. Furthermore, treatment of platelets with aspirin, currently prescribed for women at high risk of PE to reduce platelet aggregation, also inhibits STBEV-induced reversible aggregation of washed platelets. Increased platelet reactivity as a result of exposure to PE placenta derived STBEVs correlates with increased thrombotic risk associated with PE. These observations establish a possible direct link between the clotting disturbances of PE and dysfunction of the placenta, as well as the known increased risk of thromboembolism associated with this condition. PMID:26551971

  20. Optical Assay of Erythrocyte Function in Banked Blood

    NASA Astrophysics Data System (ADS)

    Bhaduri, Basanta; Kandel, Mikhail; Brugnara, Carlo; Tangella, Krishna; Popescu, Gabriel

    2014-09-01

    Stored red blood cells undergo numerous biochemical, structural, and functional changes, commonly referred to as storage lesion. How much these changes impede the ability of erythrocytes to perform their function and, as result, impact clinical outcomes in transfusion patients is unknown. In this study we investigate the effect of the storage on the erythrocyte membrane deformability and morphology. Using optical interferometry we imaged red blood cell (RBC) topography with nanometer sensitivity. Our time-lapse imaging quantifies membrane fluctuations at the nanometer scale, which in turn report on cell stiffness. This property directly impacts the cell's ability to transport oxygen in microvasculature. Interestingly, we found that cells which apparently maintain their normal shape (discocyte) throughout the storage period, stiffen progressively with storage time. By contrast, static parameters, such as mean cell hemoglobin content and morphology do not change during the same period. We propose that our method can be used as an effective assay for monitoring erythrocyte functionality during storage time.

  1. Optical Assay of Erythrocyte Function in Banked Blood

    PubMed Central

    Bhaduri, Basanta; Kandel, Mikhail; Brugnara, Carlo; Tangella, Krishna; Popescu, Gabriel

    2014-01-01

    Stored red blood cells undergo numerous biochemical, structural, and functional changes, commonly referred to as storage lesion. How much these changes impede the ability of erythrocytes to perform their function and, as result, impact clinical outcomes in transfusion patients is unknown. In this study we investigate the effect of the storage on the erythrocyte membrane deformability and morphology. Using optical interferometry we imaged red blood cell (RBC) topography with nanometer sensitivity. Our time-lapse imaging quantifies membrane fluctuations at the nanometer scale, which in turn report on cell stiffness. This property directly impacts the cell's ability to transport oxygen in microvasculature. Interestingly, we found that cells which apparently maintain their normal shape (discocyte) throughout the storage period, stiffen progressively with storage time. By contrast, static parameters, such as mean cell hemoglobin content and morphology do not change during the same period. We propose that our method can be used as an effective assay for monitoring erythrocyte functionality during storage time. PMID:25189281

  2. Functional co-operation between the subunits in heterodimeric platelet-derived growth factor receptor complexes.

    PubMed Central

    Emaduddin, M; Ekman, S; Rönnstrand, L; Heldin, C H

    1999-01-01

    To determine the importance of the phosphorylation capacity of receptor kinase as well as the ability to serve as docking sites for SH2-domain-containing signal transduction molecules, we established pig aortic endothelial cell lines stably expressing kinase-active platelet-derived growth factor (PDGF) alpha-receptors together with kinase-inactive beta-receptors, and vice versa. After stimulation with PDGF-AB, heterodimeric receptor complexes were formed in which the kinase-inactive receptor was phosphorylated by the kinase-active receptor, although less efficiently than in heterodimers of wild-type receptors. The kinase-active receptor was only minimally phosphorylated. Thus the phosphorylation within the receptor dimer occurred in trans between the components. Analyses of the abilities of heterodimeric receptor complexes of one kinase-active and one kinase-inactive receptor to mediate mitogenicity, chemotaxis and activation of mitogen-activated protein kinase revealed less efficient effects than those of heterodimers of wild-type receptors. Importantly, however, the fact that signalling capacities were retained illustrates a functional co-operation between the two receptor molecules in the dimer, where one receptor provides a functional kinase and the other acts as a substrate and provides docking sites for downstream signalling molecules. PMID:10417313

  3. Identification of anti-HPA-1a allo-antibodies using IgG platelet antibody detection and crossmatch system assay with Galileo Echo.

    PubMed

    Di Cristofaro, Julie; Frassati, Coralie; Montagnie, Rolande; Basire, Agnes; Merieux, Yves; Picard, Christophe

    2015-01-01

    Fetal/neonatal allo-immune thrombocytopenia is the most frequent and the most dangerous clinical condition involving anti-human platelet antigens (HPA)-1a allo-antibodies. Anti-HPA-1a allo-immunization requires rapid and accurate diagnosis to determine appropriate treatment. The Capture-P Ready-Screen assay (C-PRS) is a new qualitative immunoassay to detect IgG anti-human leukocyte antigen (HLA) and anti-HPA allo-antibodies. The aim of this study is to assess the identification of anti-HPA-1a allo-antibodies using the C-PRS assay, associated with HLA class I stripping reagents, on the automated benchtop analyzer Galileo Echo. Forty-nine sera were analyzed: without anti-HLA class I or anti-HPA allo-antibodies, with anti-HLA class I allo-antibodies, with anti-HPA-1a allo-antibodies, among which with anti-HLA class I allo-antibodies. None of the samples without allo-antibodies were reactive. Only anti-HLA antibodies, detected by cytotoxicity-dependent complement and not by Luminex, remained positive before and after stripping reagents. Of the 13 samples, anti-HPA-1a allo-antibodies that were correctly identified before and after incubation with HLA assassin reagent were 70% and 85%, respectively. Anti-glycoprotein auto-antibodies and anti-HLA allo-antibodies do not interfere with the detection of anti-HPA-1a antibodies. This preliminary study indicates that further improvement of the test will be helpful in developing a clinically useful assay in the future. PMID:25101933

  4. Mutational analysis clopidogrel resistance and platelet function in patients scheduled for coronary artery bypass grafting.

    PubMed

    Correll, Mick; Johnson, Christopher K; Ferrari, Giovanni; Brizzio, Mariano; Mak, Andrew W C; Quackenbush, John; Shaw, Richard E; Zapolanski, Alex; Grau, Juan B

    2013-06-01

    Clopidogrel is an oral antiplatelet pro-drug prescribed to 40 million patients worldwide who are at risk for thrombotic events or receiving percutaneous coronary intervention (PCI). However about a fifth of patients treated with clopidogrel do not respond adequately to the drug. From a cohort of 105 patients on whom we had functional data on clopidogrel response, we used ultra-high throughput sequencing to assay mutations in CYP2C19 and ABCB1, the two genes genetically linked to respond. Testing for mutations in CYP2C19, as recommended by the FDA, only correctly predicted if a patient would respond to clopidogrel 52.4% of the time. Similarly, testing of the ABCB1 gene only correctly foretold response in 51 (48.6%) patients. These results are clinically relevant and suggest that until additional genetic factors are discovered that predict response more completely, functional assays are more appropriate for clinical use. PMID:23462555

  5. Increased Platelet Concentration does not Improve Functional Graft Healing in Bio-Enhanced ACL Reconstruction

    PubMed Central

    Fleming, Braden C.; Proffen, Benedikt L.; Vavken, Patrick; Shalvoy, Matthew R.; Machan, Jason T.; Murray, Martha M.

    2014-01-01

    Purpose The use of an extra-cellular matrix scaffold (ECM) combined with platelets to enhance healing of an ACL graft (“bio-enhanced ACL reconstruction”) has shown promise in animal models. However, the effects of platelet concentration on graft healing remains unknown. The objectives of this study were to determine if increasing the platelet concentration in the ECM scaffold would; 1) improve the graft biomechanical properties, and 2) decrease cartilage damage after surgery. Methods Fifty-five adolescent minipigs were randomized to 5 treatment groups; untreated ACL transection (n=10), conventional ACL reconstruction (n=15), and bio-enhanced ACL reconstruction using 1X (n=10), 3X (n=10) or 5X (n=10) platelet-rich plasma. The graft biomechanical properties, anteroposterior (AP) knee laxity, graft histology and macroscopic cartilage integrity were measured at 15 weeks. Results The mean linear stiffness of the bio-enhanced ACL reconstruction procedure using the 1X preparation was significantly greater than traditional reconstruction while the 3X and 5X preparations were not. The failure loads of all the ACL reconstructed groups were equivalent but significantly greater than untreated ACL transection. There were no significant differences in the ligament maturity index or AP laxity between reconstructed knees. Macroscopic cartilage damage was relatively minor, though significantly less when the ECM-platelet composite was used. Conclusions Only the 1X platelet concentration improved healing over traditional ACL reconstruction. Increasing the platelet concentration from 1X to 5X in the ECM scaffold did not further improve the graft mechanical properties. The use of an ECM-platelet composite decreased the amount of cartilage damage seen after ACL surgery. PMID:24633008

  6. SDF-1α is a novel autocrine activator of platelets operating through its receptor CXCR4.

    PubMed

    Walsh, Tony G; Harper, Matthew T; Poole, Alastair W

    2015-01-01

    Platelets store and secrete the chemokine stromal cell-derived factor (SDF)-1α upon platelet activation, but the ability of platelet-derived SDF-1α to signal in an autocrine/paracrine manner mediating functional platelet responses relevant to thrombosis and haemostasis is unknown. We sought to explore the role of platelet-derived SDF-1α and its receptors, CXCR4 and CXCR7 in facilitating platelet activation and determine the mechanism facilitating SDF-1α-mediated regulation of platelet function. Using human washed platelets, CXCR4 inhibition, but not CXCR7 blockade significantly abrogated collagen-mediated platelet aggregation, dense granule secretion and thromboxane (Tx) A2 production. Time-dependent release of SDF-1α from collagen-activated platelets supports a functional role for SDF-1α in this regard. Using an in vitro whole blood perfusion assay, collagen-induced thrombus formation was substantially reduced with CXCR4 inhibition. In washed platelets, recombinant SDF-1α in the range of 20-100 ng/mL(-1) could significantly enhance platelet aggregation responses to a threshold concentration of collagen. These enhancements were completely dependent on CXCR4, but not CXCR7, which triggered TxA2 production and dense granule secretion. Rises in cAMP were significantly blunted by SDF-1α, which could also enhance collagen-mediated Ca2+ mobilisation, both of which were mediated by CXCR4. This potentiating effect of SDF-1α primarily required TxA2 signalling acting upstream of dense granule secretion, whereas blockade of ADP signalling could only partially attenuate SDF-1α-induced platelet activation. Therefore, this study supports a potentially novel autocrine/paracrine role for platelet-derived SDF-1α during thrombosis and haemostasis, through a predominantly TxA2-dependent and ADP-independent pathway. PMID:25283599

  7. Butyric acid stimulates bovine neutrophil functions and potentiates the effect of platelet activating factor.

    PubMed

    Carretta, M D; Hidalgo, A I; Burgos, J; Opazo, L; Castro, L; Hidalgo, M A; Figueroa, C D; Taubert, A; Hermosilla, C; Burgos, R A

    2016-08-01

    Increased short-chain fatty acid (SCFA) production is associated with subacute ruminal acidosis (SARA) and activation of inflammatory processes. In humans and rodents, SCFAs modulate inflammatory responses in the gut via free fatty acid receptor 2 (FFA2). In bovines, butyric acid is one of the most potent FFA2 agonists. Its expression in bovine neutrophils has recently been demonstrated, suggesting a role in innate immune response in cattle. This study aimed to evaluate if butyric acid modulates oxidative and non-oxidative functions or if it can potentiate other inflammatory mediators in bovine neutrophils. Our results showed that butyric acid can activate bovine neutrophils, inducing calcium (Ca(2+)) influx and mitogen-activated protein kinase (MAPK) phosphorylation, two second messengers involved in FFA2 activation. Ca(2+) influx induced by butyric acid was dependent on the extracellular and intracellular Ca(2+) source and phospholipase C (PLC) activation. Butyric acid alone had no significant effect on reactive oxygen species (ROS) production and chemotaxis; however, a priming effect on platelet-activating factor (PAF), a potent inflammatory mediator, was observed. Butyric acid increased CD63 expression and induced the release of neutrophil granule markers matrix metalloproteinase-9 (MMP-9) and lactoferrin. Finally, we observed that butyric acid induced neutrophil extracellular trap (NET) formation without affecting cellular viability. These findings suggest that butyric acid, a component of the ruminal fermentative process, can modulate the innate immune response of ruminants. PMID:27288853

  8. Effect of platelet-rich plasma (PRP) concentration on proliferation, neurotrophic function and migration of Schwann cells in vitro.

    PubMed

    Zheng, Canbin; Zhu, Qingtang; Liu, Xiaolin; Huang, Xijun; He, Caifeng; Jiang, Li; Quan, Daping; Zhou, Xiang; Zhu, Zhaowei

    2016-05-01

    Platelet-rich plasma (PRP) contains various growth factors and appears to have the potential to promote peripheral nerve regeneration, but evidence is lacking regarding its biological effect on Schwann cells (SCs). The present study was designed to investigate the effect of PRP concentration on SCs in order to determine the plausibility of using this plasma-derived therapy for peripheral nerve injury. PRP was obtained from rats by double-step centrifugation and was characterized by determining platelet numbers and growth factor concentrations. Primary cultures of rat SCs were exposed to various concentrations of PRP (40%, 20%, 10%, 5% and 2.5%). Cell proliferation assays and flow cytometry were performed to study to assess SC proliferation. Quantitative real-time PCR and ELISA analysis were performed to determine the ability of PRP to induce SCs to produce nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF). Microchemotaxis assay was used to analyse the cell migration capacity. The results obtained indicated that the platelet concentration and growth factors in our PRP preparations were significantly higher than in whole blood. Cell culture experiments showed that 2.5-20% PRP significantly stimulated SC proliferation and migration compared to untreated controls in a dose-dependent manner. In addition, the expression and secretion of NGF and GDNF were significantly increased. However, the above effects of SCs were suppressed by high PRP concentrations (40%). In conclusion, the appropriate concentration of PRP had the potency to stimulate cell proliferation, induced the synthesis of neurotrophic factors and significantly increased migration of SCs dose-dependently. Copyright © 2013 John Wiley & Sons, Ltd. PMID:23723151

  9. A functional assay-based strategy for nanomaterial risk forecasting.

    PubMed

    Hendren, Christine Ogilvie; Lowry, Gregory V; Unrine, Jason M; Wiesner, Mark R

    2015-12-01

    The study of nanomaterial impacts on environment, health and safety (nanoEHS) has been largely predicated on the assumption that exposure and hazard can be predicted from physical-chemical properties of nanomaterials. This approach is rooted in the view that nanoöbjects essentially resemble chemicals with additional particle-based attributes that must be included among their intrinsic physical-chemical descriptors. With the exception of the trivial case of nanomaterials made from toxic or highly reactive materials, this approach has yielded few actionable guidelines for predicting nanomaterial risk. This article addresses inherent problems in structuring a nanoEHS research strategy based on the goal of predicting outcomes directly from nanomaterial properties, and proposes a framework for organizing data and designing integrated experiments based on functional assays (FAs). FAs are intermediary, semi-empirical measures of processes or functions within a specified system that bridge the gap between nanomaterial properties and potential outcomes in complex systems. The three components of a functional assay are standardized protocols for parameter determination and reporting, a theoretical context for parameter application and reference systems. We propose the identification and adoption of reference systems where FAs may be applied to provide parameter estimates for environmental fate and effects models, as well as benchmarks for comparing the results of FAs and experiments conducted in more complex and varied systems. Surface affinity and dissolution rate are identified as two critical FAs for characterizing nanomaterial behavior in a variety of important systems. The use of these FAs to predict bioaccumulation and toxicity for initial and aged nanomaterials is illustrated for the case of silver nanoparticles and Caenorhabditis elegans. PMID:26188653

  10. Static platelet adhesion, flow cytometry and serum TXB2 levels for monitoring platelet inhibiting treatment with ASA and clopidogrel in coronary artery disease: a randomised cross-over study

    PubMed Central

    Eriksson, Andreas C; Jonasson, Lena; Lindahl, Tomas L; Hedbäck, Bo; Whiss, Per A

    2009-01-01

    Background Despite the use of anti-platelet agents such as acetylsalicylic acid (ASA) and clopidogrel in coronary heart disease, some patients continue to suffer from atherothrombosis. This has stimulated development of platelet function assays to monitor treatment effects. However, it is still not recommended to change treatment based on results from platelet function assays. This study aimed to evaluate the capacity of a static platelet adhesion assay to detect platelet inhibiting effects of ASA and clopidogrel. The adhesion assay measures several aspects of platelet adhesion simultaneously, which increases the probability of finding conditions sensitive for anti-platelet treatment. Methods With a randomised cross-over design we evaluated the anti-platelet effects of ASA combined with clopidogrel as well as monotherapy with either drug alone in 29 patients with a recent acute coronary syndrome. Also, 29 matched healthy controls were included to evaluate intra-individual variability over time. Platelet function was measured by flow cytometry, serum thromboxane B2 (TXB2)-levels and by static platelet adhesion to different protein surfaces. The results were subjected to Principal Component Analysis followed by ANOVA, t-tests and linear regression analysis. Results The majority of platelet adhesion measures were reproducible in controls over time denoting that the assay can monitor platelet activity. Adenosine 5'-diphosphate (ADP)-induced platelet adhesion decreased significantly upon treatment with clopidogrel compared to ASA. Flow cytometric measurements showed the same pattern (r2 = 0.49). In opposite, TXB2-levels decreased with ASA compared to clopidogrel. Serum TXB2 and ADP-induced platelet activation could both be regarded as direct measures of the pharmacodynamic effects of ASA and clopidogrel respectively. Indirect pharmacodynamic measures such as adhesion to albumin induced by various soluble activators as well as SFLLRN-induced activation measured by flow

  11. Non-Carriers of Reduced-Function CYP2C19 Alleles are Most Susceptible to Impairment of the Anti-Platelet Effect of Clopidogrel by Proton-Pump Inhibitors: A Pilot Study

    PubMed Central

    Lee, Jen-Kuang; Wu, Cho-Kai; Juang, Jyh-Ming; Tsai, Chia-Ti; Hwang, Juey-Jen; Lin, Jiuun-Lee; Chiang, Fu-Tien

    2016-01-01

    Background The phenomenon of CYP2C19 polymorphism affects the metabolism of both clopidogrel and proton-pump inhibitors (PPI). However, concomitant use of both drugs may reduce the desired therapeutic effects. In this study, we evaluated whether individuals with different numbers of reduced-function CYP2C19 alleles were equally affected and whether PPIs with different dependencies on CYP2C19 metabolism were equally involved. Methods Thirty healthy volunteers were recruited to a six-week regimen of clopidogrel. Three PPIs with different metabolic dependencies on CYP2C19 were included and separately administered in this order. Each PPI was given for a week, followed by a one-week washout period before the intervention of the next PPI. The anti-platelet effect was examined by Thromboelastography Platelet MappingTM (TEG®) and vasodilator-stimulated phosphoprotein (VASP) assays. Results Both TEG® and VASP tests showed the same general qualitative trend, but TEG® detected a statistically significant fluctuation of platelet aggregation in response to different drug interventions. The TEG® results also demonstrated that non-carriers experienced the most significant impairment of anti-platelet effect of clopidogrel after concomitant use of PPIs. This impairment was closely related to the metabolic dependence on CYP2C19 of PPI. Conclusions Our study indicated that non-carriers of reduced-function CYP2C19 alleles are most susceptible to impairment of the anti-platelet effect of clopidogrel after concomitant PPI use. Individual subjects are not equally affected, and PPIs are not equally involved. However, large-scale randomized clinical trials are needed to evaluate the clinical outcome. PMID:27122952

  12. A Study of Platelet Inhibition, Using a ‘Point of Care’ Platelet Function Test, following Primary Percutaneous Coronary Intervention for ST-Elevation Myocardial Infarction [PINPOINT-PPCI

    PubMed Central

    Johnson, Thomas W.; Mumford, Andrew D.; Scott, Lauren J.; Mundell, Stuart; Butler, Mark; Strange, Julian W.; Rogers, Chris A.; Reeves, Barnaby C.; Baumbach, Andreas

    2015-01-01

    Background Rapid coronary recanalization following ST-elevation myocardial infarction (STEMI) requires effective anti-platelet and anti-thrombotic therapies. This study tested the impact of door to end of procedure (‘door-to-end’) time and baseline platelet activity on platelet inhibition within 24hours post-STEMI. Methods and Findings 108 patients, treated with prasugrel and procedural bivalirudin, underwent Multiplate® platelet function testing at baseline, 0, 1, 2 and 24hours post-procedure. Major adverse cardiac events (MACE), bleeding and stent thrombosis (ST) were recorded. Baseline ADP activity was high (88.3U [71.8–109.0]), procedural time and consequently bivalirudin infusion duration were short (median door-to-end time 55minutes [40–70] and infusion duration 30minutes [20–42]). Baseline ADP was observed to influence all subsequent measurements of ADP activity, whereas door-to-end time only influenced ADP immediately post-procedure. High residual platelet reactivity (HRPR ADP>46.8U) was observed in 75% of patients immediately post-procedure and persisted in 24% of patients at 2hours. Five patients suffered in-hospital MACE (4.6%). Acute ST occurred in 4 patients, all were <120mins post-procedure and had HRPR. No significant bleeding was observed. In a post-hoc analysis, pre-procedural morphine use was associated with significantly higher ADP activity following intervention. Conclusions Baseline platelet function, time to STEMI treatment and opiate use all significantly influence immediate post-procedural platelet activity. PMID:26672598

  13. Platelet glycoprotein VI binds to polymerized fibrin and promotes thrombin generation.

    PubMed

    Mammadova-Bach, Elmina; Ollivier, Véronique; Loyau, Stéphane; Schaff, Mathieu; Dumont, Bénédicte; Favier, Rémi; Freyburger, Geneviève; Latger-Cannard, Véronique; Nieswandt, Bernhard; Gachet, Christian; Mangin, Pierre H; Jandrot-Perrus, Martine

    2015-07-30

    Fibrin, the coagulation end product, consolidates the platelet plug at sites of vascular injury and supports the recruitment of circulating platelets. In addition to integrin αIIbβ3, another as-yet-unidentified receptor is thought to mediate platelet interaction with fibrin. Platelet glycoprotein VI (GPVI) interacts with collagen and several other adhesive macromolecules. We evaluated the hypothesis that GPVI could be a functional platelet receptor for fibrin. Calibrated thrombin assays using platelet-rich plasma (PRP) showed that tissue factor-triggered thrombin generation was impaired in GPVI-deficient patients and reduced by the anti-GPVI Fab 9O12. Assays on reconstituted PRP and PRP from fibrinogen-deficient patients revealed a fibrinogen-dependent enhancement of thrombin generation, which relied on functional GPVI. The effect of GPVI was found to depend on fibrin polymerization. A binding assay showed a specific interaction between GPVI-Fc and fibrin, inhibited by the Fab 9O12. This Fab also reduced platelet adhesion to fibrin at low (300 s(-1)) and high (1500 s(-1)) wall shear rates. Platelets adherent to fibrin displayed shape change, exposure of procoagulant phospholipids, and the formation of small clots. When hirudinated blood was perfused at 1500 s(-1) over preformed fibrin-rich clots, the Fab 9O12 decreased the recruitment of platelets by up to 85%. This study identifies GPVI as a platelet receptor for polymerized fibrin with 2 major functions: (1) amplification of thrombin generation and (2) recruitment of circulating platelets to clots. These so-far-unrecognized properties of GPVI confer on it a key role in thrombus growth and stabilization. PMID:25977585

  14. Subpopulations in purified platelets adhering on glass.

    PubMed

    Donati, Alessia; Gupta, Swati; Reviakine, Ilya

    2016-01-01

    Understanding how platelet activation is regulated is important in the context of cardiovascular disorders and their management with antiplatelet therapy. Recent evidence points to different platelet subpopulations performing different functions. In particular, procoagulant and aggregating subpopulations have been reported in the literature in platelets treated with the GPVI agonists. How the formation of platelet subpopulations upon activation is regulated remains unclear. Here, it is shown that procoagulant and aggregating platelet subpopulations arise spontaneously upon adhesion of purified platelets on clean glass surfaces. Calcium ionophore treatment of the adhering platelets resulted in one platelet population expressing both the procoagulant and the adherent population markers phosphatidylserine and the activated form of GPIIb/IIIa, while all of the platelets expressed CD62P independently of the ionophore treatment. Therefore, all platelets have the capacity to express all three activation markers. It is concluded that platelet subpopulations observed in various studies reflect the dynamics of the platelet activation process. PMID:27338300

  15. Use of 8-methoxypsoralen and long-wavelength ultraviolet radiation for decontamination of platelet concentrates

    SciTech Connect

    Lin, L.; Wiesehahn, G.P.; Morel, P.A.; Corash, L. )

    1989-07-01

    Transmission of viral diseases through blood products remains an unsolved problem in transfusion medicine. We have developed a psoralen photochemical system for decontamination of platelet concentrates in which platelets are treated with long wavelength ultraviolet radiation (UVA, 320-400 nm) in the presence of 8-methoxypsoralen (8-MOP). Bacteria, RNA viruses, and DNA viruses ranging in genome size from 1.2 x 10(6) daltons, encompassing the size range of human pathogens, were inoculated into platelet concentrates and subjected to treatment. This system inactivated 25 to 30 logs/h of bacteria Escherichia coli or Staphylococcus aureus, 6 logs/h of bacteriophage fd, 0.9 log/h of bacteriophage R17 and 1.1 logs/h of feline leukemia virus (FeLV) in platelet concentrates maintained in standard storage bags. Platelet integrity and in vitro function before, immediately following photochemical treatment, and during prolonged storage after treatment, were evaluated by measuring: (1) extracellular pH; (2) platelet yields; (3) extracellular lactate dehydrogenase (LDH) levels; (4) platelet morphology; (5) platelet aggregation responsiveness; (6) thromboxane beta-2 (TXB-2) production; (7) dense body secretion; and (8) alpha granule secretion. These assays demonstrated that this photochemical inactivation system inactivated bacteria and viruses in platelet concentrates with minimal adverse effects on the in vitro function of platelets in comparison to untreated control concentrates maintained under current, standard blood bank conditions.

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

    PubMed Central

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

    2015-01-01

    Objective: Objective platelet function assessment after cardiac surgery can predict postoperative blood loss, guide transfusion requirements and discriminate the need for surgical re-exploration. We conducted this study to assess the predictive value of point-of-care testing platelet function using the Multiplate® device. Methods: Patients undergoing isolated coronary artery bypass grafting were prospectively recruited (n = 84). Group A (n = 42) patients were on anti-platelet therapy until surgery; patients in Group B (n = 42) stopped anti-platelet treatment at least 5 days preoperatively. Multiplate® and thromboelastography (TEG) tests were performed in the perioperative period. Primary end-point was excessive bleeding (>2.5 ml/kg/h) within first 3 h postoperative. Secondary end-points included transfusion requirements, re-exploration rates, intensive care unit and in-hospital stays. Results: Patients in Group A had excessive bleeding (59% vs. 33%, P = 0.02), higher re-exploration rates (14% vs. 0%, P < 0.01) and higher rate of blood (41% vs. 14%, P < 0.01) and platelet (14% vs. 2%, P = 0.05) transfusions. On multivariate analysis, preoperative platelet function testing was the most significant predictor of excessive bleeding (odds ratio [OR]: 2.3, P = 0.08), need for blood (OR: 5.5, P < 0.01) and platelet transfusion (OR: 15.1, P < 0.01). Postoperative “ASPI test” best predicted the need for transfusion (sensitivity - 0.86) and excessive blood loss (sensitivity - 0.81). TEG results did not correlate well with any of these outcome measures. Conclusions: Peri-operative platelet functional assessment with Multiplate® was the strongest predictor for bleeding and transfusion requirements in patients on anti-platelet therapy until the time of surgery. Study registration: ISRCTN43298975 (http://www.controlled-trials.com/ISRCTN43298975/). PMID:25566711

  17. Functional evaluation of paraplegin mutations by a yeast complementation assay.

    PubMed

    Bonn, Florian; Pantakani, Krishna; Shoukier, Moneef; Langer, Thomas; Mannan, Ashraf U

    2010-05-01

    An autosomal recessive form of hereditary spastic paraplegia (AR-HSP) is primarily caused by mutations in the SPG7 gene, which codes for paraplegin, a subunit of the hetero-oligomeric m-AAA protease in mitochondria. In the current study, sequencing of the SPG7 gene in the genomic DNA of 25 unrelated HSP individuals/families led to the identification of two HSP patients with compound heterozygous mutations (p.G349S/p.W583C and p.A510V/p.N739KfsX741) in the coding sequence of the SPG7 gene. We used a yeast complementation assay to evaluate the functional consequence of novel SPG7 sequence variants detected in the HSP patients. We assessed the proteolytic activity of hetero-oligomeric m-AAA proteases composed of paraplegin variant(s) and proteolytically inactive forms of AFG3L2 (AFG3L2(E575Q) or AFG3L2(K354A)) upon expression in m-AAA protease-deficient yeast cells. We demonstrate that the newly identified paraplegin variants perturb the proteolytic function of hetero-oligomeric m-AAA protease. Moreover, commonly occurring silent polymorphisms such as p.T503A and p.R688Q could be distinguished from mutations (p.G349S, p.W583C, p.A510V, and p.N739KfsX741) in our HSP cohort. The yeast complementation assay thus can serve as a reliable system to distinguish a pathogenic mutation from a silent polymorphism for any novel SPG7 sequence variant, which will facilitate the interpretation of genetic data for SPG7. PMID:20186691

  18. Phenotypic approaches to gene mapping in platelet function disorders - identification of new variant of P2Y12, TxA2 and GPVI receptors.

    PubMed

    Watson, S; Daly, M; Dawood, B; Gissen, P; Makris, M; Mundell, S; Wilde, J; Mumford, A

    2010-01-01

    Platelet number or function disorders cause a range of bleeding symptoms from mild to severe. Patients with platelet dysfunction but normal platelet number are the most prevalent and typically have mild bleeding symptoms. The study of this group of patients is particularly difficult because of the lack of a gold-standard test of platelet function and the variable penetrance of the bleeding phenotype among affected individuals. The purpose of this short review is to discuss the way in which this group of patients can be investigated through platelet phenotyping in combination with targeted gene sequencing. This approach has been used recently to identify patients with mutations in key platelet activation receptors, namely those for ADP, collagen and thromboxane A2 (TxA2). One interesting finding from this work is that for some patients, mild bleeding is associated with heterozygous mutations in platelet proteins that are co-inherited with other genetic disorders of haemostasis such as type 1 von Willebrand's disease. Thus, the phenotype of mild bleeding may be multifactorial in some patients and may be considered to be a complex trait. PMID:20162250

  19. Human endometrial stromal stem cells differentiate into megakaryocytes with the ability to produce functional platelets.

    PubMed

    Wang, Jinju; Chen, Shuzhen; Zhang, Cheng; Stegeman, Samantha; Pfaff-Amesse, Teresa; Zhang, Ying; Zhang, Wenfeng; Amesse, Lawrence; Chen, Yanfang

    2012-01-01

    Human endometrium is a high dynamic tissue that contains endometrial stromal stem cells (hESSCs). The hESSCs have been differentiated into a number of cell lineages. However, differentiation of hESSCs into megakaryocytes (MKs) has not yet been investigated. The aim of this study was to investigate the feasibility of MK generation from hESSCs and subsequent production of functional platelets (PLTs). In our study, hESSCs were cultured from endometrial stromal cells as confirmed by positive stromal cell specific markers (CD90 and CD29) and negative hematopoietic stem cell markers (CD45 and CD34) expression. Then, hESSCs were differentiated in a medium supplemented with thrombopoietin (TPO) for 18 days. The MK differentiation was analyzed by flow cytometry and confocal microscopy. The differentiation medium was collected for PLT production analysis by flow cytometry, transmission electron microscopy and functional measurements. Our results show: 1) MKs were successfully generated from hESSCs as identified by expression of specific markers (CD41a: 1 ± 0.09% and 39 ± 3.0%; CD42b: 1.2 ± 0.06% and 28 ± 2.0%, control vs. differentiation) accompanied with reduction of pluripotent transcription factors (Oct4 and Sox2) expression; 2) The level of PLTs in the differentiation medium was 16 ± 1 number/µl as determined by size (2-4 µm) and CD41a expression (CD41a: 1 ± 0.4% and 90±2.0%, control vs. differentiation); 3) Generated PLTs were functional as evidenced by the up-regulation of CD62p expression and fibrinogen binding following thrombin stimulation; 4) Released PLTs showed similar ultra-structure characteristics (alpha granules, vacuoles and dense tubular system) as PLTs from peripheral blood determined by electron microscopic analysis. Data demonstrate the feasibility of generating MKs from hESSCs, and that the generated MKs release functional PLTs. Therefore, hESSCs could be a potential new stem cell source for in vitro MK/PLT production. PMID:22952951

  20. Calmodulin antagonists induce platelet apoptosis.

    PubMed

    Wang, Zhicheng; Li, Suping; Shi, Quanwei; Yan, Rong; Liu, Guanglei; Dai, Kesheng

    2010-04-01

    Calmodulin (CaM) antagonists induce apoptosis in various tumor models and inhibit tumor cell invasion and metastasis, thus some of which have been extensively used as anti-cancer agents. In platelets, CaM has been found to bind directly to the cytoplasmic domains of several platelet receptors. Incubation of platelets with CaM antagonists impairs the receptors-related platelet functions. However, it is still unknown whether CaM antagonists induce platelet apoptosis. Here we show that CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7), tamoxifen (TMX), and trifluoperazine (TFP) induce apoptotic events in human platelets, including depolarization of mitochondrial inner transmembrane potential, caspase-3 activation, and phosphatidylserine exposure. CaM antagonists did not incur platelet activation as detected by P-selectin surface expression and PAC-1 binding. However, ADP-, botrocetin-, and alpha-thrombin-induced platelet aggregation, platelet adhesion and spreading on von Willebrand factor surface were significantly reduced in platelets pre-treated with CaM antagonists. Furthermore, cytosolic Ca(2+) levels were obviously elevated by both W7 and TMX, and membrane-permeable Ca(2+) chelator BAPTA-AM significantly reduced apoptotic events in platelets induced by W7. Therefore, these findings indicate that CaM antagonists induce platelet apoptosis. The elevation of the cytosolic Ca(2+) levels may be involved in the regulation of CaM antagonists-induced platelet apoptosis. PMID:20172594

  1. Assessment of omega-fatty-acid-supplemented human platelets for potential improvement in long-term storage.

    PubMed

    Krishnamurti, Chitra; Stewart, Michael W; Cutting, Mary A; Rothwell, Stephen W

    2002-01-15

    Uptake of omega (omega)-3 fatty acids can influence membrane stability and cell mobility. We investigated the effects of omega-3 and -6 fatty acids on the hemostatic efficacy of human platelets using an in vivo rabbit bleeding model. In vitro assays such as platelet aggregation, vWF bead-mediated ATP release and platelet adhesion to beads (measured by the residual platelet count [RPC] [free platelet count after reacting with the beads]/[baseline platelet count]x 100=%RPC; a high %RPC indicates reduced platelet function) were conducted on platelets treated with 1% fish oil (omega-3); 2% fish oil emulsion or 1% soy oil (omega-6). Oil treatment of platelets reduced the vWF bead-induced ATP release insignificantly. Addition of omega-3 agents reduced physical reactivity (%RPC) with the vWF beads by a factor of 1.2 (oil) and 1.9 (emulsion). The omega-6 oil enhanced reactivity by a factor of 1.7. After washing to remove excess reagent, platelet resuspension was most efficient with the omega-3 emulsion. Platelet function was higher with the omega-3-treated platelets (%RPC=52.3%, omega-3 oil; 63.3%, omega-3 emulsion vs. 85%, omega-6 oil; 82% untreated platelets). Ethyl-palmitate-treated thrombocytopenic rabbits were infused with human platelets. Survival times of the treated platelets, as monitored by flow cytometry (6.2-8.2 h) were comparable to untreated platelets (8.6 h). In the rabbit kidney injury model, blood loss after infusion of the treated platelets was similar to that of saline-infused rabbits (75.3+/-3.4 g). However, platelets washed prior to infusion reduced blood loss to a value comparable to that of fresh platelets (48.3+/-5 g). Furthermore, the presence of the infused platelets at the injury site was clearly visualized using FITC-tagged anti CD42a antibody. Thus, the omega-3-based agents protect the platelets from damage during the washing procedure as demonstrated in vitro by improved platelet resuspension, low %RPC, high stimulus-responsive ATP secretion

  2. Evaluation of platelet function during extended storage in additive solution, prepared in a new container that allows manual buffy-coat platelet pooling and leucoreduction in the same system

    PubMed Central

    Plaza, Eva María; Lozano, María Luisa; Guiu, Isabel Sánchez; Egea, José Manuel; Vicente, Vicente; de Terán, Laura Collantes; Rivera, José

    2012-01-01

    Background A novel and practical storage container designed for manual buffy-coat pooling and leucodepletion was evaluated to assess its filtration performance and to analyse the quality of stored leucoreduced buffy-coat-derived platelet pools. Materials and methods. To analyse the Grifols Leucored® Transfer PL system, blood was collected from random donors into standard triple bag systems, and fractionated using standard procedures to obtain buffy-coats. Ten leucodepleted platelet pools were prepared each from five units of buffy-coats in additive solution. Concentrates were stored for 10 days at 22 °C on an end-over-end agitator. On days 0, 5, 7, and 10 of storage, samples were tested using standard in vitro platelet parameters. Results The use of this novel system for volume reduction and leucodepletion of buffy-coats resuspended in additive solution led to platelet pools that met the European requirements. pH was maintained well, declining from an initial value of 7.11±0.04 to 6.88±0.08 after 10 days. Parameters of cell lysis, response to a hypotonic stimulus and aggregation induced by agonists (arachidonic acid, ristocetin, collagen or thrombin receptor activating peptide) were also well-preserved. During storage, the quality profile of the platelet pools remained very similar to that previously reported in platelet concentrates in terms of metabolism, platelet activation (CD62, CD63, sCD62), expression of glycoproteins Ib and IIb/IIIa, capacity of glycoprotein IIb/IIIa to become activated upon ADP stimulation, and release of biological response modifiers (sCD40L and RANTES). Discussion. This new system allows the preparation of leucodepleted buffy-coat platelet pools in additive solution with good preservation of platelet function. The logistics of the procedure are relatively simple and it results in good-quality components, which may reduce costs and ease the process of buffy-coat pooling and leucocyte reduction in transfusion services. PMID:22682335

  3. Approaches to synthetic platelet analogs.

    PubMed

    Modery-Pawlowski, Christa L; Tian, Lewis L; Pan, Victor; McCrae, Keith R; Mitragotri, Samir; Sen Gupta, Anirban

    2013-01-01

    Platelet transfusion is routinely used for treating bleeding complications in patients with hematologic or oncologic clotting disorders, chemo/radiotherapy-induced myelosuppression, trauma and surgery. Currently, these transfusions mostly use allogeneic platelet concentrates, while products like lyophilized platelets, cold-stored platelets and infusible platelet membranes are under investigation. These natural platelet-based products pose considerable risks of contamination, resulting in short shelf-life (3-5 days). Recent advances in pathogen reduction technologies have increased shelf-life to ~7 days. Furthermore, natural platelets are short in supply and also cause several biological side effects. Hence, there is significant clinical interest in platelet-mimetic synthetic analogs that can allow long storage-life and minimum side effects. Accordingly, several designs have been studied which decorate synthetic particles with motifs that promote platelet-mimetic adhesion or aggregation. Recent refinement in this design involves combining the adhesion and aggregation functionalities on a single particle platform. Further refinement is being focused on constructing particles that also mimic natural platelet's shape, size and elasticity, to influence margination and wall-interaction. The optimum design of a synthetic platelet analog would require efficient integration of platelet's physico-mechanical properties and biological functionalities. We present a comprehensive review of these approaches and provide our opinion regarding the future directions of this research. PMID:23092864

  4. Anti-platelet therapy and aspirin resistance - clinically and chemically relevant?

    PubMed

    Rafferty, M; Walters, M R; Dawson, J

    2010-01-01

    Platelets play a central role in the pathogenesis of the atherothrombosis which ultimately causes myocardial infarction, stroke and peripheral vascular disease. Commonly used oral anti-platelet drugs include aspirin (an irreversible inhibitor of cyclo-oxygenase), clopidogrel (an ADP receptor antagonist), other thienopyridines such as ticlopidine and prasgruel, and dipyridamole (an inhibitor of adenosine reuptake and platelet phosphodiesterase). Newer agents are in development and one, ticagrelor, a reversible ADP receptor antagonist has shown promise. Despite their proven benefit, recurrent vascular events still occur in those taking anti-platelet drugs. This has led to the concept of anti-platelet resistance, most commonly aspirin resistance as this drug is the cornerstone of most regimens. The causes of aspirin resistance are numerous but potential mechanisms include lack of patient adherence, non COX-1 mediated thromboxane A2 synthesis, increased activity of alternate platelet activation pathways, interference of aspirin action by other drugs and probably pharmacogenetic factors. Measurement of platelet response to aspirin is made possible using a number of in-vitro laboratory assays of platelet function which include measurement of thromboxane A2 metabolites as well as newer point-of-care assays of platelet aggregation. The phenomenon of aspirin resistance is important as it raises the possibility of developing strategies to identify those who respond best to a particular anti-platelet regimen, or to development of newer anti-platelet therapies to which more patients respond. This review discusses important aspects of aspirin resistance both in terms of clinical medicine, alternative anti-platelet strategies, and the potential to overcome its various causes. PMID:21062249

  5. Fluorescence-based assays for RGS box function.

    PubMed

    Willard, Francis S; Kimple, Randall J; Kimple, Adam J; Johnston, Christopher A; Siderovski, David P

    2004-01-01

    Ligand-activated, seven transmembrane-spanning receptors interact with inactive G-protein heterotrimers (Galphabetagamma) to catalyze GTP loading and, consequently, activation of Galpha subunits and the liberation of Gbetagamma. Galpha.GTP and Gbetagamma are then competent to regulate independent effector pathways. The duration of heterotrimeric G-protein signaling is determined by the lifetime of the Galpha subunit in the GTP-bound state. Signal termination is facilitated by the intrinsic guanosine triphosphatase (GTPase) activity of Galpha and subsequent reformation of the inactive heterotrimer. Regulators of G-protein signaling (RGS) proteins act enzymatically, via their hallmark "RGS box," as GTPase-accelerating proteins (GAPs) for Galpha subunits and thus function as negative regulators of G-protein signaling in vitro and in vivo. This article describes the use of fluorescence resonance energy transfer (FRET) to monitor the interaction between a Galpha subunit and an RGS box protein. Furthermore, this article describes optimization of this assay for high-throughput screening and the evaluation of mutant RGS box and Galpha proteins. Finally, this article describes the novel application of this FRET technique to measure the activity of RGS protein-derived GoLoco peptides that modulate Galpha activation by aluminum tetrafluoride. PMID:15313559

  6. The effect of Sildenafil on human platelet secretory function is controlled by a complex interplay between phosphodiesterases 2, 3 and 5.

    PubMed

    Dunkern, Torsten R; Hatzelmann, Armin

    2005-03-01

    Human platelets contain the cyclic nucleotide-hydrolyzing phosphodiesterases (PDEs) 2, 3 and 5. The cGMP-PDE5 inhibitors Sildenafil and Zaprinast have been demonstrated to potentiate the anti-platelet aggregatory effect of NO donors like sodium nitroprusside (SNP) in vitro but the mechanisms of Sildenafil's action on the secretory function of human platelets have not been analysed in detail. In the present paper, we show (1) that both compounds potentiate the SNP-induced increase in cGMP in human platelets concentration-dependently. (2) However, whereas Sildenafil plus SNP treatment only partially inhibits thrombin-induced release of serotonin, the less selective Zaprinast plus SNP cause a complete inhibition. (3) The inhibition mediated by Sildenafil plus SNP is limited to low compound concentrations at which cAMP levels are increased, probably due to cGMP-mediated inhibition of PDE3. (4) High concentrations of Sildenafil (plus SNP) neither affect cAMP levels, likely due to the activation of PDE2, nor inhibits the release of serotonin. Thus, increases in both cyclic nucleotides seem to control platelet function. (5) Accordingly, treatment with increasing concentrations of Sildenafil plus SNP and a selective PDE2 inhibitor, which by its own has no effect, induced a concentration-dependent increase in cAMP and complete inhibition of platelet activation. In summary, our data indicate that Sildenafil inhibits secretory function of human platelets at least in part due to the cGMP-mediated effects on intracellular cAMP and that entire inhibition of serotonin release from thrombin-activated platelets is controlled by both cyclic nucleotides. PMID:15567064

  7. Platelet Interaction with Innate Immune Cells

    PubMed Central

    Kral, Julia Barbara; Schrottmaier, Waltraud Cornelia; Salzmann, Manuel; Assinger, Alice

    2016-01-01

    Summary Beyond their traditional role in haemostasis and thrombosis, platelets are increasingly recognised as immune modulatory cells. Activated platelets and platelet-derived microparticles can bind to leukocytes, which stimulates mutual activation and results in rapid, local release of platelet-derived cytokines. Thereby platelets modulate leukocyte effector functions and contribute to inflammatory and immune responses to injury or infection. Platelets enhance leukocyte extravasation, differentiation and cytokine release. Platelet-neutrophil interactions boost oxidative burst, neutrophil extracellular trap formation and phagocytosis and play an important role in host defence. Platelet interactions with monocytes propagate their differentiation into macrophages, modulate cytokine release and attenuate macrophage functions. Depending on the underlying pathology, platelets can enhance or diminish leukocyte cytokine production, indicating that platelet-leukocyte interactions represent a fine balanced system to restrict excessive inflammation during infection. In atherosclerosis, platelet interaction with neutrophils, monocytes and dendritic cells accelerates key steps of atherogenesis by promoting leukocyte extravasation and foam cell formation. Platelet-leukocyte interactions at sites of atherosclerotic lesions destabilise atherosclerotic plaques and promote plaque rupture. Leukocytes in turn also modulate platelet function and production, which either results in enhanced platelet destruction or increased platelet production. This review aims to summarise the key effects of platelet-leukocyte interactions in inflammation, infection and atherosclerosis. PMID:27226790

  8. Platelet Interaction with Innate Immune Cells.

    PubMed

    Kral, Julia Barbara; Schrottmaier, Waltraud Cornelia; Salzmann, Manuel; Assinger, Alice

    2016-03-01

    Beyond their traditional role in haemostasis and thrombosis, platelets are increasingly recognised as immune modulatory cells. Activated platelets and platelet-derived microparticles can bind to leukocytes, which stimulates mutual activation and results in rapid, local release of platelet-derived cytokines. Thereby platelets modulate leukocyte effector functions and contribute to inflammatory and immune responses to injury or infection. Platelets enhance leukocyte extravasation, differentiation and cytokine release. Platelet-neutrophil interactions boost oxidative burst, neutrophil extracellular trap formation and phagocytosis and play an important role in host defence. Platelet interactions with monocytes propagate their differentiation into macrophages, modulate cytokine release and attenuate macrophage functions. Depending on the underlying pathology, platelets can enhance or diminish leukocyte cytokine production, indicating that platelet-leukocyte interactions represent a fine balanced system to restrict excessive inflammation during infection. In atherosclerosis, platelet interaction with neutrophils, monocytes and dendritic cells accelerates key steps of atherogenesis by promoting leukocyte extravasation and foam cell formation. Platelet-leukocyte interactions at sites of atherosclerotic lesions destabilise atherosclerotic plaques and promote plaque rupture. Leukocytes in turn also modulate platelet function and production, which either results in enhanced platelet destruction or increased platelet production. This review aims to summarise the key effects of platelet-leukocyte interactions in inflammation, infection and atherosclerosis. PMID:27226790

  9. A Functional Assay for GPR55: Envision Protocol.

    PubMed

    Anavi-Goffer, Sharon; Ross, Ruth A

    2016-01-01

    AlphaScreen(®) SureFire(®) assay is a novel technology that combines luminescent oxygen channeling technology, nano-beads, and monocloncal antibodies to detect the level of a selected protein in a volume lower than 5 μl. This method is more sensitive compared with the traditional enzyme-linked immunosorbent assays (ELISA), and can detect an increasing number of new targets. Here, we described a method for AlphaScreen(®) SureFire(®) assay that targets ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys activation of GPR55 by L-α-lysophosphatidylinositol (LPI) and certain cannabinoids. PMID:27245893

  10. A new fabrication route for PVA/graphene platelets composites with enhanced functionalities

    NASA Astrophysics Data System (ADS)

    Lavecchia, Teresa; Tamburri, Emanuela; Angjellari, Mariglen; Savi, Damiano; Terranova, Maria Letizia

    2016-05-01

    This work deals with the synthesis and characterization of composites made of poly(vinyl alcohol) (PVA) and oxidized graphene platelets obtained from an ad hoc treatment of graphite. The composite is produced by a modified solution mixing procedure in which the in situ crosslinking of PVA with maleic anhydride has been carried out in the presence of the carbon filler. A complete characterization of the material is presented carried out by SEM, DTGA, Raman spectroscopy and I-V characteristics analysis.

  11. Effects of single oral doses of lysine clonixinate and acetylsalicylic acid on platelet functions in man.

    PubMed

    Pallapies, D; Muhs, A; Bertram, L; Rohleder, G; Nagyiványi, P; Peskar, B A

    1996-01-01

    Lysine clonixinate is an analgesic drug with a so far unknown mechanism of action. We have determined its effect on platelet cyclooxygenase in man. Biosynthesis of thromboxane (TX)B2 and prostaglandin (PG)F2 alpha in clotting whole blood ex vivo as well as collagen-induced platelet aggregation measured before and at various time points after oral administration of 125 mg lysine clonixinate were compared to results obtained with 500 mg acetylsalicylic acid (ASA). While biosynthesis of both TXB2 and PGF2 alpha measured radioimmunologically was inhibited significantly 2.5 h, but not 6 h, after administration of lysine clonixinate, inhibition by ASA was much greater and still highly significant after 48 h. Similarly, collagen-induced aggregation of platelet-rich plasma was inhibited for a longer period and to a greater extent after administration of ASA than after lysine clonixinate. Our results indicate that lysine clonixinate is a cyclooxygenase inhibitor of moderate potency. It remains to be investigated whether mechanisms other than inhibition of cyclooxygenase contribute to the analgesic activity of lysine clonixinate. PMID:8866627

  12. [Diagnosis of inherited diseases of platelet function. Interdisciplinary S2K guideline of the Permanent Paediatric Committee of the Society of Thrombosis and Haemostasis Research (GTH e. V.)].

    PubMed

    Knöfler, R; Eberl, W; Schulze, H; Bakchoul, T; Bergmann, F; Gehrisch, S; Geisen, C; Gottstein, S; Halimeh, S; Harbrecht, U; Kappert, G; Kirchmaier, C; Kehrel, B; Lösche, W; Krause, M; Mahnel, R; Meyer, O; Pilgrimm, A K; Pillitteri, D; Rott, H; Santoso, S; Siegemund, A; Schambeck, C; Scheer, M; Schmugge, M; Scholl, T; Strauss, G; Zieger, B; Zotz, R; Hermann, M; Streif, W

    2014-01-01

    Congenital disorders of platelet function are a heterogeneous group of disorders that are often not detected until bleeding occurs. In clinical settings only a few methods have proven to be useful for identification and classification of inherited platelet disorders. For a rational diagnostic approach, a stepwise algorithm is recommended. Patient history and clinical investigation are mandatory. Von Willebrand disease and other coagulation disorders should always be ruled out prior to specific platelet testing. Platelet count, size, volume (MPV) and morphology may guide further investigations. The PFA-100® CT is suited for screening for severe platelet defects. Platelet aggregometry allows assessment of multiple aspects of platelet function. Flow cytometry enables diagnosis of thrombasthenia Glanzmann, Bernard-Soulier syndrome and storage pool defects. Molecular genetics may confirm a putative diagnosis or pave the way for identifying new defects. We present an unabridged version of the interdisciplinary guideline. PMID:24903476

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

    PubMed Central

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

    2015-01-01

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

  14. Platelet Adhesion under Flow

    PubMed Central

    Ruggeri, Zaverio M.

    2011-01-01

    Platelet adhesive mechanisms play a well-defined role in hemostasis and thrombosis, but evidence continues to emerge for a relevant contribution to other pathophysiological processes including inflammation, immune-mediated responses to microbial and viral pathogens, and cancer metastasis. Hemostasis and thrombosis are related aspects of the response to vascular injury, but the former protects from bleeding after trauma while the latter is a disease mechanism. In either situation, adhesive interactions mediated by specific membrane receptors support the initial attachment of single platelets to cellular and extracellular matrix constituents of the vessel wall and tissues. In the subsequent steps of thrombus growth and stabilization, adhesive interactions mediate platelet to platelet cohesion (aggregation) and anchoring to the fibrin clot. A key functional aspect of platelets is their ability to circulate in a quiescent state surveying the integrity of the inner vascular surface, coupled to a prompt reaction wherever alterations are detected. In many respects, therefore, platelet adhesion to vascular wall structures, to one another or to other blood cells are facets of the same fundamental biological process. The adaptation of platelet adhesive functions to the effects of blood flow is the main focus of this review. PMID:19191170

  15. Structural and Functional Analysis of a Platelet-Activating Lysophosphatidylcholine of Trypanosoma cruzi

    PubMed Central

    Gazos-Lopes, Felipe; Oliveira, Mauricio M.; Hoelz, Lucas V. B.; Vieira, Danielle P.; Marques, Alexandre F.; Nakayasu, Ernesto S.; Gomes, Marta T.; Salloum, Nasim G.; Pascutti, Pedro G.; Souto-Padrón, Thaïs; Monteiro, Robson Q.

    2014-01-01

    Background Trypanosoma cruzi is the causative agent of the life-threatening Chagas disease, in which increased platelet aggregation related to myocarditis is observed. Platelet-activating factor (PAF) is a potent intercellular lipid mediator and second messenger that exerts its activity through a PAF-specific receptor (PAFR). Previous data from our group suggested that T. cruzi synthesizes a phospholipid with PAF-like activity. The structure of T. cruzi PAF-like molecule, however, remains elusive. Methodology/Principal findings Here, we have purified and structurally characterized the putative T. cruzi PAF-like molecule by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Our ESI-MS/MS data demonstrated that the T. cruzi PAF-like molecule is actually a lysophosphatidylcholine (LPC), namely sn-1 C18:1(delta 9)-LPC. Similar to PAF, the platelet-aggregating activity of C18:1-LPC was abrogated by the PAFR antagonist, WEB 2086. Other major LPC species, i.e., C16:0-, C18:0-, and C18:2-LPC, were also characterized in all T. cruzi stages. These LPC species, however, failed to induce platelet aggregation. Quantification of T. cruzi LPC species by ESI-MS revealed that intracellular amastigote and trypomastigote forms have much higher levels of C18:1-LPC than epimastigote and metacyclic trypomastigote forms. C18:1-LPC was also found to be secreted by the parasite in extracellular vesicles (EV) and an EV-free fraction. A three-dimensional model of PAFR was constructed and a molecular docking study was performed to predict the interactions between the PAFR model and PAF, and each LPC species. Molecular docking data suggested that, contrary to other LPC species analyzed, C18:1-LPC is predicted to interact with the PAFR model in a fashion similar to PAF. Conclusions/Significance Taken together, our data indicate that T. cruzi synthesizes a bioactive C18:1-LPC, which aggregates platelets via PAFR. We propose that C18:1-LPC might be an important lipid mediator in the

  16. Studies of activated GPIIb/IIIa receptors on the luminal surface of adherent platelets. Paradoxical loss of luminal receptors when platelets adhere to high density fibrinogen.

    PubMed Central

    Coller, B S; Kutok, J L; Scudder, L E; Galanakis, D K; West, S M; Rudomen, G S; Springer, K T

    1993-01-01

    The accessibility of activated GPIIb/IIIa receptors on the luminal surface of platelets adherent to damaged blood vessels or atherosclerotic plaques is likely to play a crucial role in subsequent platelet recruitment. To define better the factors involved in this process, we developed a functional assay to assess the presence of activated, luminal GPIIb/IIIa receptors, based on their ability to bind erythrocytes containing a high density of covalently coupled RGD-containing peptides (thromboerythrocytes). Platelets readily adhered to wells coated with purified type I rat skin collagen and the adherent platelets bound a dense lawn of thromboerythrocytes. With fibrinogen-coated wells, platelet adhesion increased as the fibrinogen-coating concentration increased, reaching a plateau at about 11 micrograms/ml. Thromboerythrocyte binding to the platelets adherent to fibrinogen showed a paradoxical response, increasing at fibrinogen coating concentrations up to approximately 4-6 micrograms/ml and then dramatically decreasing at higher fibrinogen-coating concentrations. Scanning electron microscopy demonstrated that the morphology of platelets adherent to collagen was similar to that of platelets adherent to low density fibrinogen, with extensive filopodia formation and ruffling. In contrast, platelets adherent to high density fibrinogen showed a bland, flattened appearance. Immunogold staining of GPIIb/IIIa receptors demonstrated concentration of the receptors on the filopodia, and depletion of receptors on the flattened portion of the platelets. Thus, there is a paradoxical loss of accessible, activated GPIIb/IIIa receptors on the luminal surface of platelets adherent to high density fibrinogen. Two factors may contribute to this result: engagement of GPIIb/IIIa receptors with fibrinogen on the abluminal surface leading to the loss of luminal receptors, and loss of luminal filopodia that interact with thromboerythrocytes. These data provide insight into the differences

  17. [Genetic variants of platelet ADP receptor P2Y12 associated with changed platelet functional activity and development of cardiovascular diseases].

    PubMed

    Sirotkina, O V; Zabotina, A M; Berkovich, O A; Bazhenova, E A; Vavilova, T V; Shvartsman, A L

    2009-02-01

    The key role in platelet aggregation is played by the platelet ADP receptor P2Y12, which is the target for antiaggregant drugs, clopidogrel and ticlopidine. At present, only sporadic data on genetic variants of platelet ADP receptor P2Y12 are available from literature, and their association with thromboembolic and cardiovascular diseases still remains obscure. Analysis of the group of subjects with high platelet reactivity resulted in identification of two nucleotide substitutions, C18T and G36T, in the coding region of the P2Y12 gene. The frequency of the P2Y12 T1 8 allele was higher in control group than in the group of patients survived from myocardial infarction at the age under 45 years (39% versus 28%, respectively, P = 0.04). Moreover, in the T18 carriers, platelet aggregation activity was lower than in the carriers of the wild-type genotype (0.84 +/- 0.05% versus 1.01 +/- 0.08%, respectively, P = 0.03). In the group of patients with early myocardial infarctions, a tendency towards the increased frequency of 16T allele in comparison with control group (20 and 12%, respectively, P = 0.07) was observed. The rate of ADP-induced platelet aggregation in the carriers of 16T allele from the control group was somewhat higher than in the subjects with the GG36 genotype (1.31 +/- 0.16% versus 1.12 +/- 0.06%, respectively, P = 0.07). The nucleotide substitutions identified were in absolute disequilibrium, i.e., allele T18 conformed to allele G36. On the contrary, allele C18 conformed to allele T36. Haplotype T18G36 was found to be responsible for the decreased risk of myocardial infarction and decreased platelet reactivity. It is suggested that polymorphisms of the P2Y12 gene identified can be used for determination of the risk group for myocardial infarction in the young males. PMID:19334620

  18. A discrepancy between platelet alpha 2-receptor density and functional circulatory changes in hypertensives

    SciTech Connect

    Mores, N.; Martire, M.; Pistritto, G.; Cardillo, C.; Folli, G. )

    1990-09-01

    To investigate whether differences exist in peripheral alpha 2-adrenoceptors between normotensive and hypertensive subjects, we determined platelet alpha 2-adrenoceptor density in 10 (7 males) untreated essential hypertensives (mean age of 51.1 years, range of 44-59 years) and in 10 age- and sex-matched normotensive controls. Moreover, in hypertensive patients, we examined the relationship between receptor density and cardiovascular reactivity to mental arithmetic, static handgrip, and bicycle exercise, to verify the hypothesis that alpha 2-adrenoceptors might play a role in modulation of hemodynamic response to sympathetic stimuli. alpha 2-Adrenoceptor density, as calculated by binding of (3H)yohimbine to platelets, was significantly higher in essential hypertensives (314.8 +/- 38.7 fmol/mg) than in normotensive subjects (213.6 +/- 34.7 fmol/mg) (p less than 0.05), whereas receptor affinity was similar in both groups (4.0 +/- 0.5 nM hypertensives, 4.3 +/- 0.5 nM normotensives; p greater than 0.05). Mental arithmetic increased mean arterial pressure (MAP) by 21.5% from basal values and heart rate (HR) by 13.2%. During isometric exercise, MAP increased by 38.1% and HR by 24.7%, while during bicycle ergometry, mean increases in MAP and HR from baseline were of 27.2 and 54.3%, respectively. No correlation was found between platelet alpha 2-adrenoceptor density and percent changes in MAP induced by all tests, or between adrenoceptors and absolute basal and peak MAP values. Our findings suggest that in hypertensive patients, peripheral alpha 2-adrenoceptors are increased with respect to matched normotensives, but these receptors seem not to be involved in the modulation of cardiovascular adaptation to enhanced sympathetic activity.

  19. Impact of gender on platelet membrane functions of Alzheimer's disease patients.

    PubMed

    Vignini, Arianna; Giusti, Lucia; Raffaelli, Francesca; Giulietti, Alessia; Salvolini, Eleonora; Luzzi, Simona; Provinciali, Leandro; Mazzanti, Laura; Nanetti, Laura

    2013-03-01

    There are many evidences suggesting that oxidative stress is one of the earliest events in Alzheimer disease (AD) pathogenesis and plays a key role in the development of the AD pathology. The existence of substantial gender-related differences in the clinical features of AD has been recently confirmed (i.e. pathophysiologic features and epidemiologic trends). In addition, study results appear to indicate that the etiopathogenetic mechanisms of AD differ significantly in the 2 sexes. Based on previous results regarding changes in AD platelet plasma membrane, the purpose of the present study was to assess the impact of gender in the same model above reported. In particular we aimed at studying platelets from AD patients (M-AD and F-AD) and matched controls (M-C and F-C), divided into gender, by studying nitric oxide (NO) and peroxynitrite (ONOO(-)) production, the intracellular Ca(2+) concentration ([Ca(2+)]i), membrane Na(+)/K(+)-ATPase activity and fluidity. NO production was significantly elevated in platelets from both F-AD and M-AD compared to matched controls. M-AD showed NO production significantly higher than F-AD and it was the same between M-C and F-C. A similar trend was seen for ONOO(-). Platelets of both M-AD and F-AD had intracellular Ca(2+) concentrations significantly higher than F-C and M-C, while membrane Na(+)/K(+)-ATPase activity showed an opposite trend, but these differences are still significant. M-AD male subjects showed a significantly increased DPH fluorescence anisotropy (r) compared with controls, while for F-AD this discrepancy was not significant. The difference in DHP fluorescence anisotropy remained significant between M-AD and F-AD as well as between M-C and F-C. The TMA-DPH fluorescence anisotropy showed the same trend, but there were no significant differences between M-AD and F-AD, as well as between controls. The results of the current research support the conclusion that F-AD is not at greater risk than M-AD for oxidative stress

  20. Multiple and Contemporary Coronary Thrombosis inspite of Low Platelet Function Response

    PubMed Central

    Calcagno, Simone; Lucisano, Luigi; Pennacchi, Mauro; Sardella, Gennaro

    2014-01-01

    We are reporting a clinical case of a 78-year-old male who had oppressive chest pain at rest, which regressed with the intake of sublingual nitrates. Coronary angiography showed a chronic total occlusion (CTO) of the left anterior descending (LAD) artery, a normal circumflex, a hypoplasic right coronary artery and a Cardiac Magnetic Resonance showing vital tissue in anterior wall. During the procedure of CTO-PCI on LAD, patient developed multiple and contemporary coronary thrombosis in spite of low platelet reactivity, which was assessed by using Multiplate. A manual thrombectomy was performed with a good final result only after drug eluting stents (DES) implantation. PMID:25302230

  1. Acquisition and aggregation of canine blood platelets: basic mechanisms of function and differences because of breed origin.

    PubMed

    Clemmons, R M; Meyers, K M

    1984-01-01

    A method for obtaining reliable blood platelet yields in canine platelet-rich plasma, using increased sodium citrate concentration, is presented. Maintaining a quiet environment or anesthetizing the animals with thiamylal sodium aids in collection of platelets. Aggregation of platelets from 60 dogs of various breeds in response to arachidonic acid, collagen, adenosine diphosphate, epinephrine, and serotonin was monitored. Canine platelets reversibly or irreversibly aggregated to arachidonic acid. The percentage of arachidonate-irreversible platelets varied from 0% to 100% depending upon the breed of dogs examined. Arachidonate-irreversible platelets also aggregated irreversibly at lower concentrations of collagen and exhibited biphasic irreversible aggregation to adenosine diphosphate and serotonin. Serotonin-induced irreversible aggregation was dependent upon receptor activation and upon arachidonic acid metabolism. Irreversible aggregation to serotonin was associated with release of 3H-serotonin and thromboxane B2 formation, indicating that a couple between the serotonergic receptor and arachidonic acid metabolism may exist. PMID:6422804

  2. Functional domains on von Willebrand factor. Recognition of discrete tryptic fragments by monoclonal antibodies that inhibit interaction of von Willebrand factor with platelets and with collagen.

    PubMed Central

    Sixma, J J; Sakariassen, K S; Stel, H V; Houdijk, W P; In der Maur, D W; Hamer, R J; de Groot, P G; van Mourik, J A

    1984-01-01

    We have identified two functional domains on the von Willebrand factor (VWF) moiety of the Factor VIII-von Willebrand factor complex (FVIII-VWF), one interacting with blood platelets, and one interacting with vessel wall collagens, by means of two monoclonal antibodies directed against the VWF molecule, CLB-RAg 35 and CLB-RAg 201. The monoclonal antibody CLB-RAg 35 inhibited virtually all platelet adherence to artery subendothelium and to purified vessel wall collagen type III, at relatively high wall shear rates. CLB-RAg 35 also inhibited the ristocetin-induced platelet aggregation and the binding of FVIII-VWF to the platelet in the presence of ristocetin but did not affect the binding of FVIII-VWF to collagen. The monoclonal antibody CLB-RAg 201 inhibited the binding of FVIII-VWF to purified vessel wall collagen type I and III and all platelet adherence to collagen type III and the platelet adherence to subendothelium that was mediated by FVIII-VWF in plasma. The two functional domains on FVIII-VWF that were recognized by CLB-RAg 35 and CLB-RAg 201 were identified by means of immunoprecipitation studies of trypsin-digested FVIII-VWF. The domains resided on different polypeptide fragments, with a Mr of 48,000 for the collagen binding domain and a Mr of 116,000 for the platelet binding domain. The 116,000-mol wt fragment consisted of subunits of 52,000/56,000 mol wt and 14,000 mol wt after reduction. The 52,000/56,000-mol wt subunits possessed the epitope for CLB-RAg 35. Images PMID:6332119

  3. 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

  4. Platelet Immobilization on Supported Phospholipid Bilayers for Single Platelet Studies.

    PubMed

    Uhl, Eva; Donati, Alessia; Reviakine, Ilya

    2016-08-23

    The worldwide cardiovascular disease (CVD) epidemic is of grave concern. A major role in the etiology of CVDs is played by the platelets (thrombocytes). Platelets are anuclear cell fragments circulating in the blood. Their primary function is to catalyze clot formation, limiting traumatic blood loss in the case of injury. The same process leads to thrombosis in the case of CVDs, which are commonly managed with antiplatelet therapy. Platelets also have other, nonhemostatic functions in wound healing, inflammation, and tissue regeneration. They play a role in the early stages of atherosclerosis and the spread of cancer through metastases. Much remains to be learned about the regulation of these diverse platelet functions under physiological and pathological conditions. Breakthroughs in this regard are expected to come from single platelet studies and systems approaches. The immobilization of platelets at surfaces is advantageous for developing such approaches, but platelets are activated when they come in contact with foreign surfaces. In this work, we develop and validate a protocol for immobilizing platelets on supported lipid bilayers without activation due to immobilization. Our protocol can therefore be used for studying platelets with a wide variety of surface-sensitive techniques. PMID:27438059

  5. A new variant of Glanzmann's thrombasthenia (Strasbourg I). Platelets with functionally defective glycoprotein IIb-IIIa complexes and a glycoprotein IIIa 214Arg----214Trp mutation.

    PubMed Central

    Lanza, F; Stierlé, A; Fournier, D; Morales, M; André, G; Nurden, A T; Cazenave, J P

    1992-01-01

    We describe a new variant of Glanzmann's thrombasthenia (variant Strasbourg I). The patient (M.S.) showed an absence of platelet aggregation to ADP, thrombin, and collagen, and a decreased clot retraction. Platelet fibrinogen was approximately 20% of normal levels. ADP-stimulated platelets bound markedly reduced amounts of soluble fibrinogen and platelet adhesion to surface-bound fibrinogen was defective. Normal to subnormal amounts of glycoprotein (GP) IIb-IIIa (alpha IIb beta 3) complexes, the platelet fibrinogen receptor, were revealed by SDS-PAGE, crossed immunoelectrophoresis, and antibody binding. However, the complexes were unusually sensitive to dissociation with EDTA at room temperature. Furthermore, flow cytometry showed that the platelets failed to bind the activation-dependent monoclonal antibody, PAC-1, after stimulation. In contrast, an RGDS-containing peptide induced significant binding of the anti-ligand-induced binding site antibody, D3GP3, suggesting the presence of a functional RGD binding domain on the patient's GPIIb-IIIa complex. Sequence analysis was performed after polymerase chain reaction amplification of selected patient's GPIIIa exons, and of the patient's platelet GPIIb and GPIIIa mRNAs. A point mutation (C to T) was localized in exon D (iv) of GPIIIa that resulted in an 214Arg to 214Trp amino acid substitution. The defect has been inherited from the parents who are heterozygous for the same mutation. This substitution points to an essential amino acid in a region of GPIIIa involved in the binding of fibrinogen and influencing the Ca(2+)-dependent stability of the GPIIb-IIIa complex. Images PMID:1602006

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

    PubMed

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

    2016-09-01

    Decompression from elevated ambient pressure is associated with platelet activation and decreased platelet counts. Standard treatment for decompression sickness (DCS) is hyperbaric oxygen therapy. Intravenous perfluorocarbon (PFC) emulsion is a nonrecompressive therapy being examined that improves mortality in animal models of DCS. However, PFC emulsions are associated with a decreased platelet count. We used a swine model of DCS to study the effect of PFC therapy on platelet count, function, and hemostasis. Castrated male swine (n = 50) were fitted with a vascular port, recovered, randomized, and compressed to 180 feet of sea water (fsw) for 31 min followed by decompression at 30 fsw/min. Animals were observed for DCS, administered 100% oxygen, and treated with either emulsified PFC Oxycyte (DCS-PFC) or isotonic saline (DCS-NS). Controls underwent the same procedures, but were not compressed (Sham-PFC and Sham-NS). Measurements of platelet count, thromboelastometry, and coagulation were obtained 1 h before compression and 1, 24, 48, 96, 168 and 192 h after treatment. No significant changes in normalized platelet counts were observed. Prothrombin time was elevated in DCS-PFC from 48 to 192 h compared with DCS-NS, and from 96 to 192 h compared with Sham-PFC. Normalized activated partial thromboplastin time was also elevated in DCS-PFC from 168 to 192 h compared with Sham-PFC. No bleeding events were noted. DCS treated with PFC (Oxycyte) does not impact platelet numbers, whole blood clotting by thromboelastometry, or clinical bleeding. Late changes in prothrombin time and activated partial thromboplastin time associated with PFC use in both DCS therapy and controls warrant further investigation. PMID:26650458

  7. Radioimmunoassay of factor V in human plasma and platelets

    SciTech Connect

    Tracy, P.B.; Eide, L.L.; Bowie, E.J.W.; Mann, K.G.

    1982-07-01

    Homogeneous, single-chain human factor V was used to develop a double antibody competition radioimmunoassay to measure factor V concentrations in plasma and platelets. Standard curves were constructed that allow for the detection of as little as 20 ng factor V/ml of plasma. Normal factor V concentrations range from 4 to 14 ..mu..g/ml of plasma with an average value of 7.0 +/- 2.0 ..mu..g/ml (n = 64). No correlation was observed between antigen levels and age or sex. The radioimmunoassay data are consistent with factor V clotting assays, providing freshly drawn plasma is used in the bioassay. Radioimmunoassay of washed platelets indicate that 0.63-1.93 ..mu..g of factor V is present per 2.5 X 10/sup 8/ platelets (6412-14128 molecules of factor V per platelet). When normalized to individual hematocrits and platelet count, the data indicated that platelets contribute approximately 18%-25% of the factor V found in whole blood. In addition, two individuals with functionally deficient factor V were examined and found to be deficient in both antigen and activity.

  8. Platelet dysfunction-eosinophilia syndrome in parasitized Venezuelan children.

    PubMed

    Ruiz-Sáez, Arlette; Sifontes, Luz Núñez; Feijoo, Rosa; Certad, Gabriela; Arenas-Pinto, Alejandro; Pocaterra, Leonor; Ferrara, Guiseppe; Giménez, Rita; Torres, Obdulita; Goldstein, Carlos; Bosch, Norma

    2005-08-01

    Platelet dysfunction was detected in six children with purpura and eosinophilia. We conducted clinical evaluations, hematologic and platelet function tests, clotting studies (bleeding time, prothrombin time, partial thromboplastin time, thrombin time, factor XIII, factor VIII, and von Willebrand factor), assays for IgG and IgM antibodies to platelets, and a search for stool parasites. Mild bleeding phenomena (ecchymoses, petechiae, epistaxis, and gingival) were transient. All children showed intestinal parasites and marked eosinophilia (mean count = 2,615.2 cells/muL, 95% confidence interval = 1,259.6-5,429.8). Main abnormalities included prolonged bleeding times (50%) and defective aggregation with collagen (100%) adrenaline (66%), or ADP (66%). Antibodies to platelets were not detected. Anti-parasite therapy reversed the hemorrhagic manifestations and normalized eosinophil counts and platelet alterations. No relationship could be established between excess eosinophils, intensity of bleeding, or type and degree of platelet abnormalities. Thrombocytopathic features mimicked the intrinsic defect of storage pool disease. The possible pathogenic roles of eosinophilia and parasitism are reviewed. This is the first report of this pathologic combination in Latin American children. PMID:16103609

  9. pH-responsive drug delivery system based on AIE luminogen functionalized layered zirconium phosphate nano-platelets

    NASA Astrophysics Data System (ADS)

    Li, Dongdong; Zhang, Yuping; Zhou, Bingbing

    2015-05-01

    Aggregation-induced emission (AIE) luminogen, quaternary tetraphenylethene cation (TPEN), was successfully incorporated into layered α-zirconium phosphate (α-ZrP) by using co-precipitation method to form inorganic-organic hybrid materials. The obtained materials show the characteristic hexagonal platelet shape with the interlayer distance did not reveal significant difference compared with pure α-ZrP. In addition, the obtained hybrid materials emit strong blue emission centered at 476 nm in aqueous media due to the electrostatic interactions of TPEN with the anionic framework of α-ZrP, which largely restrict their intramolecular rotation. More importantly, the materials provide a pH dependent release of doxorubicin (DOX), suggesting that AIE luminogen functionalized α-ZrP may be used as an imaging guided and pH-responsive delivery system for targeting therapy.

  10. Platelets and diabetes mellitus.

    PubMed

    Santilli, Francesca; Simeone, Paola; Liani, Rossella; Davì, Giovanni

    2015-07-01

    Platelet activation plays a key role in atherothrombosis in type 2 diabetes mellitus (T2DM) and increased in vivo platelet activation with enhanced thromboxane (TX) biosynthesis has been reported in patients with impairment of glucose metabolism even in the earlier stages of disease and in the preclinical phases. In this regards, platelets appear as addresses and players carrying and transducing metabolic derangement into vascular injury. The present review critically addresses key pathophysiological aspects including (i) hyperglycemia, glycemic variability and insulin resistance as determinants and predictors of platelet activation, (ii) inflammatory mediators derived from platelets, such as soluble CD40 ligand, soluble CD36, Dickkopf-1 and probably soluble receptor for advanced glycation-end-products (sRAGE), which expand the functional repertoire of platelets from players of hemostasis and thrombosis to powerful amplifiers of inflammation by promoting the release of cytokines and chemokines, cell activation, and cell-cell interactions; (iii) molecular mechanisms underpinning the less-than-expected antithrombotic protection by aspirin (ASA), despite regular antiplatelet prophylaxis at the standard dosing regimen, and (iv) stratification of patients deserving different antiplatelet strategies, based on the metabolic phenotype. Taken together, these pathophysiological aspects may contribute to the development of promising mechanism-based therapeutic strategies to reduce the progression of atherothrombosis in diabetic subjects. PMID:25986598

  11. Metalloproteolytic receptor shedding…platelets "acting their age".

    PubMed

    Andrews, Robert K; Gardiner, Elizabeth E

    2016-09-01

    Whilst significant effort has been focused on development of tools and approaches to clinically modulate activation processes that consume platelets, the platelet receptors that initiate activation processes remain untargeted. The modulation of receptor levels is also linked to underlying platelet aging processes which influence normal platelet lifespan and also the functionality and survival of stored platelets that are used in transfusion. In this review, we will focus on platelet adhesion receptors initiating thrombus formation, and discuss how regulation of levels of these receptors impact platelet function and platelet survival. PMID:27459696

  12. PLATELET FORMATION

    PubMed Central

    Thon, Jonathan N.; Italiano, Joseph E.

    2010-01-01

    Thrombocytopenia is the underlying cause of a number of major clinical conditions and genetic disorders worldwide. While therapeutic agents that bind and stimulate the thrombopoietin receptor are currently available, the development of drugs that directly stimulate megakaryocytes to generate platelets has lagged behind. To improve the management of thrombocytopenia, we will need to define the cell biological pathways that drive the production of platelets from megakaryocytes. This review integrates the latest research of platelet biogenesis and focuses on the molecular pathways that power and regulate proplatelet production. PMID:20620432

  13. Platelets in Inflammation and Atherogenesis

    PubMed Central

    Nording, Henry M.; Seizer, Peter; Langer, Harald F.

    2015-01-01

    Platelets contribute to processes beyond thrombus formation and may play a so far underestimated role as an immune cell in various circumstances. This review outlines immune functions of platelets in host defense, but also how they may contribute to mechanisms of infectious diseases. A particular emphasis is placed on the interaction of platelets with other immune cells. Furthermore, this article outlines the features of atherosclerosis as an inflammatory vascular disease highlighting the role of platelet crosstalk with cellular and soluble factors involved in atheroprogression. Understanding, how platelets influence these processes of vascular remodeling will shed light on their role for tissue homeostasis beyond intravascular thrombosis. Finally, translational implications of platelet-mediated inflammation in atherosclerosis are discussed. PMID:25798138

  14. Myeloperoxidase induces the priming of platelets.

    PubMed

    Kolarova, H; Klinke, A; Kremserova, S; Adam, M; Pekarova, M; Baldus, S; Eiserich, J P; Kubala, L

    2013-08-01

    The release of myeloperoxidase (MPO) from polymorphonuclear neutrophils is a hallmark of vascular inflammation and contributes to the pathogenesis of vascular inflammatory processes. However, the effects of MPO on platelets as a contributory mechanism in vascular inflammatory diseases remain unknown. Thus, MPO interaction with platelets and its effects on platelet function were examined. First, dose-dependent binding of MPO (between 1.7 and 13.8nM) to both human and mouse platelets was observed. This was in direct contrast to the absence of MPO in megakaryocytes. MPO was localized both on the surface of and inside platelets. Cytoskeleton inhibition did not prevent MPO localization inside the three-dimensional platelet structure. MPO peroxidase activity was preserved upon the MPO binding to platelets. MPO sequestered in platelets catabolized NO, documented by the decreased production of NO (on average, an approximately 2-fold decrease). MPO treatment did not affect the viability of platelets during short incubations; however, it decreased platelet viability after long-term storage for 7 days (an approximately 2-fold decrease). The activation of platelets by MPO was documented by an MPO-mediated increase in the expression of surface platelet receptors P-selectin and PECAM-1 (of about 5 to 20%) and the increased formation of reactive oxygen species (of about 15 to 200%). However, the activation was only partial, as MPO did not induce the aggregation of platelets nor potentiate platelet response to classical activators. Nor did MPO induce a significant release of the content of granules. The activation of platelets by MPO was connected with increased MPO-treated platelet interaction with polymorphonuclear leukocytes (an approximately 1.2-fold increase) in vitro. In conclusion, it can be suggested that MPO can interact with and activate platelets, which can induce priming of platelets, rather than the classical robust activation of platelets. This can contribute to the

  15. Platelet aggregability and in vivo platelet deposition in patients with ischemic cerebrovascular disease--evaluation by indium-111-platelet scintigraphy

    SciTech Connect

    Isaka, Y.; Kimura, K.; Uehara, A.; Hashikawa, K.; Mieno, M.; Matsumoto, M.; Handa, N.; Nakabayashi, S.; Imaizumi, M.; Kamada, T. )

    1989-12-15

    In ischemic cerebrovascular disease, it is not clear whether platelet function in vitro actually reflects the situation in vivo. Using indium-111 platelet scintigraphy as a method for detecting platelet activation in vivo, we tried to elucidate this problem. Twenty eight patients with chronic stage of ischemic cerebrovascular disease (CVD) and 17 control subjects were examined. Platelet scintigrams were positive in 9 of 28 patients in CVD, while all were negative in control. A comparison of the results obtained from qualitative platelet imaging and platelet aggregability was performed to evaluate whether threshold aggregation concentration (TAC) grade differed across the three groups (control, CVD patients without platelet deposition and CVD patients with platelet deposition). CVD patients with platelet deposition showed a higher TAC than those patients who did not show platelet deposition (P less than 0.05) or control subjects without platelet deposition (P less than 0.05). These results suggest that some patients in chronic stages of CVD may have active platelet deposition on carotid atheromatous lesions, and presence of platelet deposition in vivo could contribute to reduce platelet reactivity in peripheral blood.

  16. Platelet aggregability and in vivo platelet deposition in patients with ischemic cerebrovascular disease--evaluation by indium-111-platelet scintigraphy.

    PubMed

    Isaka, Y; Kimura, K; Uehara, A; Hashikawa, K; Mieno, M; Matsumoto, M; Handa, N; Nakabayashi, S; Imaizumi, M; Kamada, T

    1989-12-15

    In ischemic cerebrovascular disease, it is not clear whether platelet function in vitro actually reflects the situation in vivo. Using indium-111 platelet scintigraphy as a method for detecting platelet activation in vivo, we tried to elucidate this problem. Twenty eight patients with chronic stage of ischemic cerebrovascular disease (CVD) and 17 control subjects were examined. Platelet scintigrams were positive in 9 of 28 patients in CVD, while all were negative in control. A comparison of the results obtained from qualitative platelet imaging and platelet aggregability was performed to evaluate whether threshold aggregation concentration (TAC) grade differed across the three groups (control, CVD patients without platelet deposition and CVD patients with platelet deposition). CVD patients with platelet deposition showed a higher TAC than those patients who did not show platelet deposition (P less than 0.05) or control subjects without platelet deposition (P less than 0.05). These results suggest that some patients in chronic stages of CVD may have active platelet deposition on carotid atheromatous lesions, and presence of platelet deposition in vivo could contribute to reduce platelet reactivity in peripheral blood. PMID:2633402

  17. Compartmentation and compartment-specific regulation of PDE5 by protein kinase G allows selective cGMP-mediated regulation of platelet functions.

    PubMed

    Wilson, Lindsay S; Elbatarny, Hisham S; Crawley, Scott W; Bennett, Brian M; Maurice, Donald H

    2008-09-01

    It is generally accepted that nitric oxide (NO) donors, such as sodium nitroprusside (SNP), or phosphodiesterase 5 (PDE5) inhibitors, including sildenafil, each impact human platelet function. Although a strong correlation exists between the actions of NO donors in platelets and their impact on cGMP, agents such as sildenafil act without increasing global intra-platelet cGMP levels. This study was undertaken to identify how PDE5 inhibitors might act without increasing cGMP. Our data identify PDE5 as an integral component of a protein kinase G1beta (PKG1beta)-containing signaling complex, reported previously to coordinate cGMP-mediated inhibition of inositol-1, 4, 5-trisphosphate receptor type 1 (IP(3)R1)-mediated Ca(2+)-release. PKG1beta and PDE5 did not interact in subcellular fractions devoid of IP(3)R1 and were not recruited to IP(3)R1-enriched membranes in response to cGMP-elevating agents. Activation of platelet PKG promoted phosphorylation and activation of the PDE5 fraction tethered to the IP(3)R1-PKG complex, an effect not observed for the nontethered PDE5. Based on these findings, we elaborate a model in which PKG selectively activates PDE5 within a defined microdomain in platelets and propose that this mechanism allows spatial and temporal regulation of cGMP signaling in these cells. Recent reports indicate that sildenafil might prove useful in limiting in-stent thrombosis and the thrombotic events associated with the acute coronary syndromes (ACS), situations poorly regulated with currently available therapeutics. We submit that our findings may define a molecular mechanism by which PDE5 inhibition can differentially impact selected cellular functions of platelets, and perhaps of other cell types. PMID:18757735

  18. Cytokine and Nitric Oxide Levels in Patients with Sepsis – Temporal Evolvement and Relation to Platelet Mitochondrial Respiratory Function

    PubMed Central

    Sjövall, Fredrik; Morota, Saori; Åsander Frostner, Eleonor; Hansson, Magnus J.; Elmér, Eskil

    2014-01-01

    Background The levels of nitric oxide (NO) and various cytokines are known to be increased during sepsis. These signaling molecules could potentially act as regulators and underlie the enhancement of mitochondrial function described in the later phase of sepsis. Therefore, we investigated the correlation between observed changes in platelet mitochondrial respiration and a set of pro- and anti-inflammatory cytokines as well as NO plasma levels in patients with sepsis. Methods and Results Platelet mitochondrial respiration and levels of TNFα, MCP-1 (monocyte chemotactic protein-1), INFγ (interferon-γ), IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10 and IL-17 and NO were analyzed in 38 patients with severe sepsis or septic shock at three time points during one week following admission to the ICU. Citrate synthase, mitochondrial DNA and cytochrome c were measured as markers of cellular mitochondrial content. All mitochondrial respiratory states increased over the week analyzed (p<0.001). IL-8 levels correlated with maximal mitochondrial respiration on day 6–7 (p = 0.02, r2 = 0.22) and was also higher in non-survivors compared to survivors on day 3–4 and day 6–7 (p = 0.03 respectively). Neither NO nor any of the other cytokines measured correlated with respiration or mortality. Cytochrome c levels were decreased at day 1–2 by 24±5% (p = 0.03) and returned towards values of the controls at the last two time points. Citrate synthase activity and mitochondrial DNA levels were similar to controls and remained constant throughout the week. Conclusions Out of ten analyzed cytokines and nitric oxide, IL-8 correlated with the observed increase in mitochondrial respiration. This suggests that cytokines as well as NO do not play a prominent role in the regulation of platelet mitochondrial respiration in sepsis. Further, the respiratory increase was not accompanied by an increase in markers of mitochondrial content, suggesting a possible role for post

  19. Platelet count

    MedlinePlus

    ... reactions Cancer Certain medicines Bone marrow disease called polycythemia vera Bone marrow making too many platelets without a ... leukemia (CML) Hemolytic anemia Idiopathic thrombocytopenic purpura (ITP) Polycythemia vera Thrombocytopenia Patient Instructions Deep vein thrombosis - discharge Update ...

  20. Platelet Count

    MedlinePlus

    ... rash Small purplish spots on the skin called purpura, caused by bleeding under the skin Testing may ... Idiopathic thrombocytopenia (ITP), also known as immune thrombocytopenic purpura, is the result of antibody production against platelets. ...

  1. Structural and Functional Properties of Platelet-Derived Growth Factor and Stem Cell Factor Receptors

    PubMed Central

    Heldin, Carl-Henrik; Lennartsson, Johan

    2013-01-01

    The receptors for platelet-derived growth factor (PDGF) and stem cell factor (SCF) are members of the type III class of PTK receptors, which are characterized by five Ig-like domains extracellularly and a split kinase domain intracellularly. The receptors are activated by ligand-induced dimerization, leading to autophosphorylation on specific tyrosine residues. Thereby the kinase activities of the receptors are activated and docking sites for downstream SH2 domain signal transduction molecules are created; activation of these pathways promotes cell growth, survival, and migration. These receptors mediate important signals during the embryonal development, and control tissue homeostasis in the adult. Their overactivity is seen in malignancies and other diseases involving excessive cell proliferation, such as atherosclerosis and fibrotic diseases. In cancer, mutations of PDGF and SCF receptors—including gene fusions, point mutations, and amplifications—drive subpopulations of certain malignancies, such as gastrointestinal stromal tumors, chronic myelomonocytic leukemia, hypereosinophilic syndrome, glioblastoma, acute myeloid leukemia, mastocytosis, and melanoma. PMID:23906712

  2. Platelet Activation Following Implant of the Levitronix PediVAS in the Ovine Model

    PubMed Central

    Johnson, Carl A.; Shankarraman, Venkat; Wearden, Peter D.; Kocyildirim, Ergin; Maul, Timothy M.; Marks, John D.; Richardson, J. Scott; Gellman, Barry N.; Borovetz, Harvey S.; Dasse, Kurt A.; Wagner, William R.

    2011-01-01

    The Levitronix PediVAS is an extracorporeal magnetically levitated pediatric ventricular assist system with an optimal flow rate range of 0.3 – 1.5 L/min. The system is being tested in preclinical studies to assess hemodynamic performance and biocompatibility. The PediVAS was implanted in 9 ovines for 30 days duration using either commercially available cannulae (N = 3) or customized Levitronix cannulae (N = 6). Blood biocompatibility in terms of circulating activated platelets was measured by flow cytometric assays to detect P-selectin. Platelet activation was further examined following exogenous agonist stimulation. Platelet activation rose following surgery and eventually returned to baseline in animal studies where minimal kidney infarcts were observed. Platelet activation remained elevated for the duration of the study in animals where a moderate number of kidney infarcts with or without thrombotic deposition in the cannulae were observed. When platelet activation did return to baseline, platelets appropriately responded to agonist stimulation signifying conserved platelet function following PediVAS implant. Platelet activation returned to baseline in the majority of studies, representing a promising biocompatibility result for the Levitronix PediVAS. PMID:21989419

  3. Platelet Proteome Analysis Reveals Integrin-dependent Aggregation Defects in Patients with Myelodysplastic Syndromes*

    PubMed Central

    Fröbel, Julia; Cadeddu, Ron-Patrick; Hartwig, Sonja; Bruns, Ingmar; Wilk, Christian M.; Kündgen, Andrea; Fischer, Johannes C.; Schroeder, Thomas; Steidl, Ulrich G.; Germing, Ulrich; Lehr, Stefan; Haas, Rainer; Czibere, Akos

    2013-01-01

    Bleeding complications are a significant clinical problem in patients with myelodysplastic syndromes even at sufficient platelet counts (>50,000/μl). However, the underlying pathology of this hemorrhagic diathesis is still unknown. Here, we analyzed the platelet proteome of patients with myelodysplastic syndromes by quantitative two-dimensional difference gel electrophoresis followed by mass spectrometric protein identification. Proteins identified with lower concentrations, such as Talin-1, Vinculin, Myosin-9, Filmain-A, and Actin play critical roles in integrin αIIbβ3 signaling and thus platelet aggregation. Despite normal agonist receptor expression, calcium flux, and granule release upon activation, the activation capacity of integrin αIIbβ3 was diminished in myelodysplastic syndrome platelets. Förster resonance energy transfer analysis showed a reduced co-localization of Talin-1 to the integrin's β3-subunit, which is required for receptor activation and fibrinogen binding. In addition, platelet spreading on immobilized fibrinogen was incomplete, and platelet aggregation assays confirmed a general defect in integrin-dependent platelet aggregation in patients with myelodysplastic syndromes. Our data provide novel aspects on the molecular pathology of impaired platelet function in myelodysplastic syndromes and suggest a mechanism of defective integrin αIIbβ3 signaling that may contribute to the hemorrhagic diathesis observed in these patients. PMID:23382103

  4. Assessment of blood clot formation and platelet receptor function ex vivo in patients with primary Sjögren's syndrome

    PubMed Central

    Collins, K S; Balasubramaniam, K; Viswanathan, G; Natasari, A; Tarn, J; Lendrem, D; Mitchell, S; Zaman, A; Ng, W F

    2013-01-01

    Objectives Primary Sjögren's syndrome (pSS) shares clinical features and pathogenetic mechanisms with systemic lupus erythematosus (SLE). SLE is associated with an increased thromboembolic risk; however, it is unclear whether pSS patients are susceptible to thromboembolic diseases. In this study, we examined ex vivo blood clot formation (clot strength, rates of clot formation and lysis) in pSS using thromboelastography (TEG) and platelet aggregation to common agonists using multiple electrode aggregometry (MEA). We also investigated the relationship between TEG/MEA parameters and clinical/laboratory features of pSS. Design Case control. Setting Secondary care, single centre. Participants 34 pSS patients, 11 SLE patients and 13 healthy volunteers (all women) entered and completed the study. Primary and secondary outcome measures Primary outcomes: TEG and MEA parameters between three subject groups. Secondary outcomes: The relationships between TEG/MEA and clinical/laboratory parameters analysed using bivariate correlation analysis with corrections for multiple testing. Results All TEG and MEA parameters were similar for the three subject groups. After corrections for multiple testing, interleukin (IL)-1α and Macrophage inflammatory proteins (MIP)-1α remain correlated inversely with clot strength (r=−0.686, p=0.024 and r=−0.730, p=0.012, respectively) and overall coagulability (r=−0.640, p=0.048 and r=−0.648, p=0.048). Stepwise regression analysis revealed that several cytokines such as MIP-1α, IL-17a, IL-1α and Interferon (IFN)-γ may be key predictors of clot strength and overall coagulability in pSS. Conclusions Clot kinetics and platelet receptor function are normal in pSS. Several cytokines correlate with clot strength and overall coagulability in pSS. PMID:23793707

  5. Repurposing CRISPR/Cas9 for in situ functional assays

    PubMed Central

    Malina, Abba; Mills, John R.; Cencic, Regina; Yan, Yifei; Fraser, James; Schippers, Laura M.; Paquet, Marilène; Dostie, Josée; Pelletier, Jerry

    2013-01-01

    RNAi combined with next-generation sequencing has proven to be a powerful and cost-effective genetic screening platform in mammalian cells. Still, this technology has its limitations and is incompatible with in situ mutagenesis screens on a genome-wide scale. Using p53 as a proof-of-principle target, we readapted the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR associated 9) genome-editing system to demonstrate the feasibility of this methodology for targeted gene disruption positive selection assays. By using novel “all-in-one” lentiviral and retroviral delivery vectors heterologously expressing both a codon-optimized Cas9 and its synthetic guide RNA (sgRNA), we show robust selection for the CRISPR-modified Trp53 locus following drug treatment. Furthermore, by linking Cas9 expression to GFP fluorescence, we use an “all-in-one” system to track disrupted Trp53 in chemoresistant lymphomas in the Eμ-myc mouse model. Deep sequencing analysis of the tumor-derived endogenous Cas9-modified Trp53 locus revealed a wide spectrum of mutants that were enriched with seemingly limited off-target effects. Taken together, these results establish Cas9 genome editing as a powerful and practical approach for positive in situ genetic screens. PMID:24298059

  6. Flavonoids inhibit the platelet TxA2 signalling pathway and antagonize TxA2 receptors (TP) in platelets and smooth muscle cells

    PubMed Central

    Guerrero, José A; Navarro-Nuñez, Leyre; Lozano, María L; Martínez, Constantino; Vicente, Vicente; Gibbins, Jonathan M; Rivera, José

    2007-01-01

    What is already known about this subject Flavonoids are largely recognized as potential inhibitors of platelet function, through nonspecific mechanisms such as antioxidant activity and/or inhibition of several enzymes and signalling proteins. In addition, we, and few others, have shown that certain antiaggregant flavonoids may behave as specific TXA2 receptor (TP) ligands in platelets. Whether flavonoids interact with TP isoforms in other cell types is not known, and direct evidence that flavonoid–TP interaction inhibits signalling downstream TP has not been shown. What this study adds This study first demonstrates that certain flavonoids behave as ligands for both TP isoforms, not only in platelets, but also in human myometrium and in TP-transfected HEK 293T cells. Differences in the effect of certain flavonoids in platelet signalling, induced by either U46619 or thrombin, suggest that abrogation of downstream TP signalling is related to their specific blockage of the TP, rather than to a nonspecific effect on tyrosine kinases or other signalling proteins. Aims Flavonoids may affect platelet function by several mechanisms, including antagonism of TxA2 receptors (TP). These TP are present in many tissues and modulate different signalling cascades. We explored whether flavonoids affect platelet TP signalling, and if they bind to TP expressed in other cell types. Methods Platelets were treated with flavonoids, or other selected inhibitors, and then stimulated with U46619. Similar assays were performed in aspirinized platelets activated with thrombin. Effects on calcium release were analysed by fluorometry and changes in whole protein tyrosine phosphorylation and activation of ERK 1/2 by Western blot analysis. The binding of flavonoids to TP in platelets, human myometrium and TPα- and TPβ-transfected HEK 293T cells was explored using binding assays and the TP antagonist 3H-SQ29548. Results Apigenin, genistein, luteolin and quercetin impaired U46619-induced calcium

  7. A point mutation in the EGF-4 domain of β3 integrin is responsible for the formation of the Seca platelet alloantigen and affects receptor function

    PubMed Central

    Sachs, Ulrich J.; Bakchoul, Tamam; Eva, Olga; Giptner, Astrid; Bein, Gregor; Aster, Richard H.; Gitter, Maria; Peterson, Julie; Santoso, Sentot

    2013-01-01

    Summary Neonatal alloimmune thrombocytopenia (NAIT) is caused by fetomaternal platelet incompatibility with maternal antibodies crossing the placenta and destroying fetal platelets. Antibodies against human platelet antigen-1a (HPA-1a) and HPA-5b are responsible for the majority of NAIT cases. We observed a suspected NAIT in a newborn with a platelet count of 25 G/l and petechial haemorrhages. Serological analysis of maternal serum revealed an immunisation against αIIbβ3 on paternal platelets only, indicating the presence of an antibody against a new rare alloantigen (Seca) residing on αIIbβ3. The location of Seca on αIIbβ3 was confirmed by immunoprecipitation. Nucleotide sequence analysis of paternal β3 revealed a single nucleotide exchange (G1818T) in exon 11 of the β3 gene (ITGB3), changing Lys580 (wild-type) to Asn580 (Seca). Two additional members of the family Sec were typed Seca positive, but none of 300 blood donors. Chinese hamster ovary cells expressing Asn580, but not Lys580 αIIbβ3, bound anti-Seca, which was corroborated by immunoprecipitation. Adhesion of transfected cells onto immobilised fibrinogen showed reduced binding of the Asn580 variant compared to wild-type αIIbβ3. Analysis of transfected cells with anti-LIBS and PAC-1 antibody showed reduced binding when compared to the wild-type. No such effects were observed with Seca positive platelets, which, however, are heterozygous for the Lys580Asn mutation. In this study, we describe a NAIT case caused by maternal alloimmunisation against a new antigen on αIIbβ3. Analysis with mutant transfected cells showed that the Lys580Asn mutation responsible for the formation of the Seca antigenic determinant affects αIIbβ3 receptor function. PMID:22116617

  8. The effects of cytochalasin D and abciximab on hemostasis in canine whole blood assessed by thromboelastography and the PFA-100® platelet function analyzer system.

    PubMed

    Brainard, Benjamin M; Abed, Janan M; Koenig, Amie

    2011-07-01

    The selective inhibition of platelet function in whole blood coagulation testing may allow insights into the nature of hypercoagulability in dogs with critical illness. To determine the effects of cytochalasin D and abciximab on hemostatic parameters in canine citrated whole blood, an in-vitro study was designed using thromboelastography (TEG) and a platelet function analyzer (PFA-100®). 8 clinically healthy mixed breed dogs donated blood that was anticoagulated with 3.2% sodium citrate in a 9:1 blood-to-citrate ratio. Addition of cytochalasin D to citrated whole blood from 6 dogs at concentrations ranging from 0 µg/ml to 10 µg/ml caused a maximal reduction of TEG maximum amplitude (MA) at a concentration of 7.5 µg/ml (52.7 ± 4.3 to 14.3 ± 7.8 mm). Addition of abciximab to canine citrated whole blood at concentrations of either 20 µg/ml or 40 µg/ml did not affect the TEG tracing; however, addition of abciximab to citrated canine whole blood at concentrations of 10 µg/ml and 20 µg/ml significantly prolonged PFA-100 closure times (72.5 ± 15 to 149.2 ± 91 sec and 275.6 ± 54 sec, respectively, P < 0.04). Inhibition of canine platelet function by cytochalasin D is demonstrated by TEG, but abciximab did not change TEG tracings. Abciximab does, however, inhibit platelet aggregation under shear stress as measured by the PFA-100. Inhibition of canine platelet function with cytochalasin D may allow further TEG studies in dogs with clinical disease. PMID:21908311

  9. Nanosecond pulsed platelet-rich plasma (nsPRP) improves mechanical and electrical cardiac function following myocardial reperfusion injury.

    PubMed

    Hargrave, Barbara; Varghese, Frency; Barabutis, Nektarios; Catravas, John; Zemlin, Christian

    2016-02-01

    Ischemia and reperfusion (I/R) of the heart is associated with biochemical and ionic changes that result in cardiac contractile and electrical dysfunction. In rabbits, platelet-rich plasma activated using nanosecond pulsed electric fields (nsPRP) has been shown to improve left ventricular pumping. Here, we demonstrate that nsPRP causes a similar improvement in mouse left ventricular function. We also show that nsPRP injection recovers electrical activity even before reperfusion begins. To uncover the mechanism of nsPRP action, we studied whether the enhanced left ventricular function in nsPRP rabbit and mouse hearts was associated with increased expression of heat-shock proteins and altered mitochondrial function under conditions of oxidative stress. Mouse hearts underwent 30 min of global ischemia and 1 h of reperfusion in situ. Rabbit hearts underwent 30 min of ischemia in vivo and were reperfused for 14 days. Hearts treated with nsPRP expressed significantly higher levels of Hsp27 and Hsp70 compared to hearts treated with vehicle. Also, pretreatment of cultured H9c2 cells with nsPRP significantly enhanced the "spare respiratory capacity (SRC)" also referred to as "respiratory reserve capacity" and ATP production in response to the uncoupler FCCP. These results suggest a cardioprotective effect of nsPRP on the ischemic heart during reperfusion. PMID:26908713

  10. Alterations in plasma membrane promote overexpression and increase of sodium influx through epithelial sodium channel in hypertensive platelets.

    PubMed

    Cerecedo, D; Martínez-Vieyra, Ivette; Sosa-Peinado, Alejandro; Cornejo-Garrido, Jorge; Ordaz-Pichardo, Cynthia; Benítez-Cardoza, Claudia

    2016-08-01

    Platelets are small, anucleated cell fragments that activate in response to a wide variety of stimuli, triggering a complex series of intracellular pathways leading to a hemostatic thrombus formation at vascular injury sites. However, in essential hypertension, platelet activation contributes to causing myocardial infarction and ischemic stroke. Reported abnormalities in platelet functions, such as platelet hyperactivity and hyperaggregability to several agonists, contribute to the pathogenesis and complications of thrombotic events associated with hypertension. Platelet membrane lipid composition and fluidity are determining for protein site accessibility, structural arrangement of platelet surface, and response to appropriate stimuli. The present study aimed to demonstrate whether structural and biochemical abnormalities in lipid membrane composition and fluidity characteristic of platelets from hypertensive patients influence the expression of the Epithelial Sodium Channel (ENaC), fundamental for sodium influx during collagen activation. Wb, cytometry and quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) assays demonstrated ENaC overexpression in platelets from hypertensive subjects and in relation to control subjects. Additionally, our results strongly suggest a key role of β-dystroglycan as a scaffold for the organization of ENaC and associated proteins. Understanding of the mechanisms of platelet alterations in hypertension should provide valuable information for the pathophysiology of hypertension. PMID:27137675

  11. Hepatic sinusoidal endothelium avidly binds platelets in an integrin-dependent manner, leading to platelet and endothelial activation and leukocyte recruitment.

    PubMed

    Lalor, Patricia F; Herbert, John; Bicknell, Roy; Adams, David H

    2013-03-01

    Platelets have recently been shown to drive liver injury in murine models of viral hepatitis and promote liver regeneration through the release of serotonin. Despite their emerging role in inflammatory liver disease, little is known about the mechanisms by which platelets bind to the hepatic vasculature. Therefore, we referenced public expression data to determine the profile of potential adhesive receptors expressed by hepatic endothelium. We then used a combination of tissue-binding and flow-based endothelial-binding adhesion assays to show that resting platelets bind to human hepatic sinusoidal endothelial cells and that the magnitude of adhesion is greatly enhanced by thrombin-induced platelet activation. Adhesion was mediated by the integrins Gp1b, αIIbβIII, and αvβ3, as well as immobilized fibrinogen. Platelet binding to hepatic endothelial cells resulted in NF-κB activation and increased chemokine secretion. The functional relevance of platelet binding was confirmed by experiments that showed markedly increased binding of neutrophils and lymphocytes to hepatic endothelial cells under shear conditions replicating those found in the hepatic sinusoid, which was in part dependent on P-selectin expression. Thus the ability of platelets to activate endothelium and promote leukocyte adhesion may reflect an additional mechanism through which they promote liver injury. PMID:23257923

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

    SciTech Connect

    Nygaard, Gyrid; Herfindal, Lars; Kopperud, Reidun; Aragay, Anna M.; Holmsen, Holm; Døskeland, Stein Ove; Kleppe, Rune; Selheim, Frode

    2014-07-04

    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 investigated 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.

  13. pH-responsive drug delivery system based on AIE luminogen functionalized layered zirconium phosphate nano-platelets

    SciTech Connect

    Li, Dongdong Zhang, Yuping; Zhou, Bingbing

    2015-05-15

    Aggregation-induced emission (AIE) luminogen, quaternary tetraphenylethene cation (TPEN), was successfully incorporated into layered α-zirconium phosphate (α-ZrP) by using co-precipitation method to form inorganic–organic hybrid materials. The obtained materials show the characteristic hexagonal platelet shape with the interlayer distance did not reveal significant difference compared with pure α-ZrP. In addition, the obtained hybrid materials emit strong blue emission centered at 476 nm in aqueous media due to the electrostatic interactions of TPEN with the anionic framework of α-ZrP, which largely restrict their intramolecular rotation. More importantly, the materials provide a pH dependent release of doxorubicin (DOX), suggesting that AIE luminogen functionalized α-ZrP may be used as an imaging guided and pH-responsive delivery system for targeting therapy. - Graphical abstract: AIE luminogen was successfully incorporated into layered α-zirconium phosphate by a co-precipitation method to form inorganic–organic hybrid materials, showing a pH dependent release of DOX. - Highlights: • AIE luminogen cation was incorporated into layered α-ZrP by co-precipitation method. • The obtained material emits strong blue emission upon UV irradiation. • The material exhibits pH dependent release of DOX. • The AIE functionalized α-ZrP has potential applications in imaging guided therapy.

  14. Effect of citalopram treatment on relationship between platelet serotonin functions and the Karolinska scales of personality in panic patients.

    PubMed

    Neuger, Jolanta; Wistedt, Börje; Aberg-Wistedt, Anna; Stain-Malmgren, Rigmor

    2002-08-01

    Using the Karolinska Scales of Personality (KSP), we investigated the effect of the selective serotonin reuptake inhibitor citalopram on personality traits and the relationship between personality traits and peripheral indexes for central serotonergic function in patients with panic disorder at baseline and after 6 months of treatment. The degree of anxiety and depression was assessed using the Beck Anxiety Inventory, the Beck Depression Inventory, the Clinical Anxiety Scale, and the Montgomery Asberg Depression Rating Scale. A reduction in anxiety and depression scores of 75% was observed after treatment in two thirds of the patients. Mean changes of 12% in the direction of normalization were observed in all KSP anxiety-related items (Somatic Anxiety, Muscular Tension, Psychic Anxiety, and Psychasthenia), the aggression and hostility related items (Inhibition of Aggression, Irritability, and Guilt) and the item of Socialisation. A positive correlation was found between Vmax for the platelet [14C]-serotonin uptake and Inhibition of Aggression before treatment, and a negative correlation was found between the affinity of serotonin uptake and Inhibition of Aggression after treatment. Negative childhood experiences influenced enhanced scores on some KSP items but not the serotonergic function. In panic patients treated with citalopram, effects were seen on personality traits, confirming an association between serotonergic activity and aggression. PMID:12172340

  15. Utrophins compensate for Dp71 absence in mdx3cv in adhered platelets.

    PubMed

    Cerecedo, Doris; Mondragón, Ricardo; Candelario, Aurora; García-Sierra, Francisco; Mornet, Dominique; Rendón, Alvaro; Martínez-Rojas, Dalila

    2008-01-01

    Platelet adhesion is a critical step due to its hemostatic role in stopping bleeding after vascular damage. Short dystrophins are the most abundant dmd gene products in nonmuscle tissues, and in association with cytoskeleton proteins contribute to their intrinsic function; while utrophins are dystrophin-homologous related family proteins with structural and functional similarities. We previously demonstrated the presence of Dp71 isoforms, utrophins, and various dystrophin-associated proteins and their participation in cytoskeleton re-organization, filopodia and lamellipodia extension, and in centralizing cytoplasmic granules during the adhesion process of human platelets. To evaluate the morphologic changes and actin-based structures of mdx(3cv) platelets during the adhesion process, we compared the topographic distribution of Dp71d/Dp71Delta110(m) and dystrophin-associated protein in adhered platelets from dystrophic mdx(3cv) mouse. By confocal microscopy, we showed that absence of Dp71 isoforms in platelets from this animal model disrupted dystrophin-associated protein expression and distribution without modifying the platelet morphology displayed during the glass-adhesion process. By immunoprecipitation assays, we proved that up-regulated utrophins were associated with dystrophin-associated proteins to conform the dystrophin-associated protein complex corresponding to utrophins, which might compensate for Dp71 absence in mdx(3cv) platelets. PMID:18180614

  16. Platelets: production, morphology and ultrastructure.

    PubMed

    Thon, Jonathan N; Italiano, Joseph E

    2012-01-01

    Platelets are anucleate, discoid cells, roughly 2-3 μm in diameter that function primarily as regulators of hemostasis, but also play secondary roles in angiogensis and innate immunity. Although human adults contain nearly one trillion platelets in circulation that are turned over every 8-10 days, our understanding of the mechanisms involved in platelet production is still incomplete. Platelets stem from large (30-100 μm) nucleated cells called megakaryocytes that reside primarily in the bone marrow. During maturation megakaryocytes extend long proplatelet elongations into sinusoidal blood vessels from which platelets ultimately release. During this process, platelets develop a number of distinguishable structural elements including: a delimited plasma membrane; invaginations of the surface membrane that form the open canalicular system (OCS); a closed-channel network of residual endoplasmic reticulum that form the dense tubular system (DTS); a spectrin-based membrane skeleton; an actin-based cytoskeletal network; a peripheral band of microtubules; and numerous organelles including α-granules, dense-granules, peroxisomes, lysosomes, and mitochondria. Proplatelet elongation and platelet production is an elaborate and complex process that defines the morphology and ultrastructure of circulating platelets, and is critical in understanding their increasingly numerous and varied biological functions. PMID:22918725

  17. Effects of very low versus standard dose acetyl salicylic acid, dipyridamole and sulfinpyrazone on platelet function and thromboxane formation in man.

    PubMed

    Lorenz, R; Siess, W; Weber, P C

    1981-04-01

    The effects of three conventional antiplatelet regimes, dipyridamole 3 X 75 mg/day (D), sulfinpyrazone 4 X 200 mg/day (S), acetylsalicylic acid 3 X 330 mg/day combined with dipyridamole 3 X 75 mg/day (ASA + D), and very low dose acetylsalicylic acid 100 mg/day (ASA) on platelet function were studied in man following 4 days of treatment. D and S slightly increased mean minimal ADP concentration for irreversible aggregation (n.s.), S reduced aggregation and thromboxane (TXB2) formation on low dose collagen (2P less than or equal to 0.05), ASA and ASA + D increased platelet count (2P less than or equal to 0.05), increased bleeding time (n.s. for ASA, 2P less than or equal to 0.05 for ASA + D), abolished irreversible aggregation on ADP, suppressed TXB2 formation on all (2P less than or equal to 0.001) and aggregation on lower concentrations of collagen (2P less than or equal to 0.01) and abolished aggregation and TXB2 formation on arachidonic acid (2P less than or equal to 0.001). Very low dose ASA suppresses platelet aggregation and TXB2 formation on several stimuli of possible physiologic significance. In the light of a recently proposed critical balance of vascular antiaggregatory prostacyclin and platelet proaggregatory TXA2 very low dose ASA might offer advantages over conventional dosage of ASA and should be evaluated in thromboembolic disorders. PMID:7238575

  18. [STRUCTURAL CHARACTERIZATION OF PLATELETS AND PLATELET-DERIVED MICROVESICLES].

    PubMed

    Ponomareva, A A; Nevzorova, T A; Mordakhanova, E R; Andrianova, I A; Litvinov, R I

    2016-01-01

    Platelets are the anucleated blood cells, wich together with the fibrin stop bleeding (hemostasis). Cellular microvesicles are membrane-surrounded microparticles released into extracellular space upon activation and/or apoptosis of various cells. Platelet-derived macrovesicles from the major population of circulating blood microparticles that play an important role in hemostasis and thrombosis. Despite numerous studies on the pathophysiology of platelet-derived macrovesicles, mechanisms of their formation and structural details remain poorly understood. Here we investigated the ultrastructure of parental platelets and platelet-derived microvesicles formed in vitro by quiescent cells as well as by cells stimulated with one of the following activators: arachidonic acid, ADP, thrombin, calcium ionophore A23187. Using transmission electron microscopy of human platelets and isolated microvesicles, we analyzed the intracellular origin, steps of formation, structural diversity, and size distributions of the subcellular particles. We have revealed that thrombin, unlike other stimuli, not only induced vesiculation of the plasma membrane but also caused break-up of the cells followed by formation of microparticles that are comparable with microvesicles by size. A fraction of these microparticles contained cellular organelles surrounded by a thin membrane. The size of platelet-derived macrovesicles varied from 30 nm to 500 nm, however, the size distributions depended on the nature of a cell-activating stimulus. The results obtained provide new information about the formation of platelet-derived macrovesicles and their structural diversity, wich is important to understand their multiple functions in normal and disease states. PMID:27228656

  19. Rupture Forces among Human Blood Platelets at different Degrees of Activation.

    PubMed

    Nguyen, Thi-Huong; Palankar, Raghavendra; Bui, Van-Chien; Medvedev, Nikolay; Greinacher, Andreas; Delcea, Mihaela

    2016-01-01

    Little is known about mechanics underlying the interaction among platelets during activation and aggregation. Although the strength of a blood thrombus has likely major biological importance, no previous study has measured directly the adhesion forces of single platelet-platelet interaction at different activation states. Here, we filled this void first, by minimizing surface mediated platelet-activation and second, by generating a strong adhesion force between a single platelet and an AFM cantilever, preventing early platelet detachment. We applied our setup to measure rupture forces between two platelets using different platelet activation states, and blockade of platelet receptors. The rupture force was found to increase proportionally to the degree of platelet activation, but reduced with blockade of specific platelet receptors. Quantification of single platelet-platelet interaction provides major perspectives for testing and improving biocompatibility of new materials; quantifying the effect of drugs on platelet function; and assessing the mechanical characteristics of acquired/inherited platelet defects. PMID:27146004

  20. Rupture Forces among Human Blood Platelets at different Degrees of Activation

    PubMed Central

    Nguyen, Thi-Huong; Palankar, Raghavendra; Bui, Van-Chien; Medvedev, Nikolay; Greinacher, Andreas; Delcea, Mihaela

    2016-01-01

    Little is known about mechanics underlying the interaction among platelets during activation and aggregation. Although the strength of a blood thrombus has likely major biological importance, no previous study has measured directly the adhesion forces of single platelet-platelet interaction at different activation states. Here, we filled this void first, by minimizing surface mediated platelet-activation and second, by generating a strong adhesion force between a single platelet and an AFM cantilever, preventing early platelet detachment. We applied our setup to measure rupture forces between two platelets using different platelet activation states, and blockade of platelet receptors. The rupture force was found to increase proportionally to the degree of platelet activation, but reduced with blockade of specific platelet receptors. Quantification of single platelet-platelet interaction provides major perspectives for testing and improving biocompatibility of new materials; quantifying the effect of drugs on platelet function; and assessing the mechanical characteristics of acquired/inherited platelet defects. PMID:27146004

  1. The nature of interactions between tissue-type plasminogen activator and platelets

    SciTech Connect

    Torr, S.R.; Winters, K.J.; Santoro, S.A.; Sobel, B.E. )

    1990-07-15

    To elucidate interactions responsible for inhibition of aggregation of platelets in platelet-rich plasma (PRP) harvested from whole blood preincubated with t-PA, experiments were performed with PRP and washed platelets under diverse conditions of preincubation. Both ADP and collagen induced aggregation were inhibited in PRP unless aprotinin had been added to the preincubated whole blood concomitantly with t-PA. However, in washed platelets prepared after the same exposure aggregation was intact. When washed platelets were supplemented with fibrinogen degradation products (FDPs) in concentrations simulating those in whole blood preincubated with t-PA, aggregation induced with either ADP or collagen was inhibited. Thus, the inhibition in PRP depended on generation of FDPs by activated plasminogen. The functional integrity of surface glycoprotein (GP) IIb/IIIa receptors in washed platelets was documented by autoradiography after SDS-PAGE of surface labeled GPs and by fibrinogen binding despite preincubation of the whole blood or washed platelets themselves with t-PA and plasminogen as long as exogenous calcium (greater than or equal to 0.1 microM) was present. In contrast, when calcium was absent, the platelet GP IIb/IIIa receptor was rendered susceptible to degradation by plasmin, and aggregation was inhibited by preincubation at 37 degrees C even if aprotinin was present when aggregation was being assayed. These observations reconcile disparate results in the literature from studies in vivo and in vitro by demonstrating that inhibition of aggregation of platelets in PRP and in whole blood reflects indirect effects of plasminogen activation rather than direct effects of t-PA or plasmin on the platelets themselves.

  2. Biologic nanoparticles and platelet reactivity

    PubMed Central

    Miller, Virginia M; Hunter, Larry W; Chu, Kevin; Kaul, Vivasvat; Squillace, Phillip D; Lieske, John C; Jayachandran, Muthuvel

    2009-01-01

    Aim Nanosized particles (NPs) enriched in hydroxyapatite and protein isolated from calcified human tissue accelerate occlusion of endothelium-denuded arteries when injected intravenously into rabbits. Since platelet aggregation and secretory processes participate in normal hemostasis, thrombosis and vascular remodeling, experiments were designed to determine if these biologic NPs alter specific platelet functions in vitro. Methods Platelet-rich plasma was prepared from citrate anticoagulated human blood. Platelet aggregation and ATP secretion were monitored in response to thrombin receptor agonists peptide (10 μM) or convulxin (50 μg/ml) prior to and following 15 min incubation with either control solution, human-derived NPs, bovine-derived NPs or crystals of hydroxyapatite at concentrations of 50 and 150 nephelometric turbidity units. Results Incubation of platelets for 15 min with either human- or bovine-derived NPs reduced aggregation induced by thrombin receptor activator peptide and convulxin in a concentration-dependent manner. Hydroxyapatite caused a greater inhibition than either of the biologically derived NPs. Human-derived NPs increased ATP secretion by unstimulated platelets during the 15 min incubation period. Conclusion Effects of bovine-derived and hydroxyapatite NPs on basal release of ATP were both time and concentration dependent. These results suggest that biologic NPs modulate both platelet aggregation and secretion. Biologically derived NPs could modify platelet responses within the vasculature, thereby reducing blood coagulability and the vascular response to injury. PMID:19839809

  3. IgG platelet antibodies in EDTA-dependent pseudothrombocytopenia bind to platelet membrane glycoprotein IIb.

    PubMed

    Fiorin, F; Steffan, A; Pradella, P; Bizzaro, N; Potenza, R; De Angelis, V

    1998-08-01

    EDTA-dependent pseudothrombocytopenia (PTCP) consists of an inappropriate low platelet count caused by autoantibodies present in the serum samples reacting with platelets only in EDTA-anticoagulated blood. By using immunoprecipitation and Western blot techniques, we studied the immunochemical specificity of platelet agglutinating autoantibodies in the serum samples of 10 patients with PTCP. Furthermore, to evaluate a possible role of PTCP-associated IgG autoantibodies in increased platelet turnover, we assayed the plasma glycocalicin (GC) level and calculated the GC index for every patient. Our results provide direct evidence that an epitope located on platelet membrane glycoprotein IIb is recognized by PTCP-associated IgG antibodies; moreover GC levels in patients with EDTA-dependent PTCP were similar to control levels, thus excluding an increased platelet turnover. We conclude that antiplatelet antibodies directed against platelet cryptantigens are unlikely to have a major role in the increased removal of cells from circulation. PMID:9704616

  4. Reticulated platelets: analytical aspects and clinical utility.

    PubMed

    Hoffmann, Johannes J M L

    2014-08-01

    Reticulated platelets are immature platelets circulating in blood; they reflect the activity of megakaryopoiesis in the bone marrow. Therefore, they can be used as a non-invasive test in patients with thrombocytopenia in various clinical conditions. The preferred method of analysis is by flow cytometry. However, there is an evident lack of analytical standardization, making it difficult to compare results obtained in different laboratories. Currently, two types of hematology analyzers are on the market offering fully automated measurement of reticulated or immature platelets: the high end analyzers manufactured by Sysmex (XE- and XN-series) and Abbott (CELL-DYN Sapphire). Although the methods are essentially different and cannot be used interchangeably, both have been proven to have clinical utility. Reticulated or immature platelet assays are useful for the differential diagnosis of thrombocytopenia and for monitoring bone marrow recovery after chemotherapy or stem cell transplantation. These assays may aid clinicians in platelet transfusion decisions when recovery from thrombocytopenia is imminent. In addition, preliminary findings indicate that there is a rationale for reticulated or immature platelets for risk stratification in acute coronary syndromes and for monitoring the effect of treatment with antiplatelet drugs in patients with coronary artery diseases. The aim of this paper is to present the present technology available for measuring reticulated platelets as well as an overview of the current status of clinical application. This overview also indicates that more research is needed before reticulated or immature platelet assays can be applied in other clinical conditions than thrombocytopenia and after transplantation. PMID:24807169

  5. The role of point-of-care platelet function testing in predicting postoperative bleeding following cardiac surgery: a systematic review and meta-analysis.

    PubMed

    Corredor, C; Wasowicz, M; Karkouti, K; Sharma, V

    2015-06-01

    This systematic review and meta-analysis appraises the utility of point-of-care platelet function tests for predicting blood loss and transfusion requirements in cardiac surgical patients, and analyses whether their use within a transfusion management algorithm is associated with improved patient outcomes. We included 30 observational studies incorporating 3044 patients in the qualitative assessment, and nine randomised controlled trials including 1057 patients in the meta-analysis. Platelet function tests demonstrated significant variability in their ability to predict blood loss and transfusion requirements. Their use within a blood transfusion algorithm demonstrated a reduction in blood loss at longest follow-up (mean difference -102.9 ml (95% CI -149.9 to -56.1 ml), p < 0.001), and transfusion of packed red cells (RR 0.86 (95% CI 0.78-0.94), p = 0.001) and fresh frozen plasma (RR 0.42 (95% CI 0.30-0.59), p < 0.001). Viscoelastic methods used in combination with other platelet function tests achieved greater reduction in blood loss (mean difference -111.8 ml (95% CI -174.9 to -49.1 ml), p = 0.0005) compared with their use alone (mean difference -90.6 ml (95% CI 166.1-15.0 ml), p = 0.02). We conclude that incorporation of point-of-care platelet function tests into transfusion management algorithms is associated with a reduction in blood loss and transfusion requirements in cardiac surgery patients. PMID:25916344

  6. Platelet adhesion to decorin but not collagen I correlates with the integrin α2 dimorphism E534K, the basis of the human platelet alloantigen (HPA)-5 system

    PubMed Central

    Kunicki, Thomas J.; Williams, Shirley A.; Diaz, Daniel; Farndale, Richard W.; Nugent, Diane J.

    2012-01-01

    A single nucleotide polymorphism in the integrin α2 gene ITGA2 (rs1801106; G1600A) creates the non-conservative amino acid substitution E534K, the basis of the human platelet alloantigen system HPA-5. Yet HPA-5 alleles do not influence binding of α2β1 to its primary ligand collagen I, and the effect of HPA-5 on platelet function has not been determined. We used a direct platelet adhesion assay to evaluate whether differential inheritance of HPA-5 alleles influences platelet adhesion to collagen I or an alternative ligand, decorin. Platelets from donors bearing one or more minor allele HPA-5b showed attenuated adhesion to purified decorin but not collagen I. Adhesion to decorin was significantly inhibited by human alloantibodies specific for HPA-5a but not by the collagen I sequence GFOGER or α2-specific inhibitory monoclonal antibodies. The minor allele 534K attenuates platelet adhesion to decorin but not collagen I, providing the first evidence of a functional effect of HPA-5 alleles. PMID:22133774

  7. Mechanisms of platelet-mediated liver regeneration.

    PubMed

    Lisman, Ton; Porte, Robert J

    2016-08-01

    Platelets have multiple functions beyond their roles in thrombosis and hemostasis. Platelets support liver regeneration, which is required after partial hepatectomy and acute or chronic liver injury. Although it is widely assumed that platelets stimulate liver regeneration by local excretion of mitogens stored within platelet granules, definitive evidence for this is lacking, and alternative mechanisms deserve consideration. In-depth knowledge of mechanisms of platelet-mediated liver regeneration may lead to new therapeutic strategies to treat patients with failing regenerative responses. PMID:27297793

  8. A Flow Cytometric Analysis of the Inhibition of Platelet Reactivity Due to Nitrite Reduction by Deoxygenated Erythrocytes

    PubMed Central

    Akrawinthawong, Krittapoom; Park, Ji Won; Piknova, Barbora; Sibmooh, Nathawut; Fucharoen, Suthat; Schechter, Alan N.

    2014-01-01

    Nitric oxide (NO), a small gas molecule, has long been known to be a potent inhibitor of platelet function but the physiological and pathological implications of platelet inhibition by NO have not been well clarified. We recently showed that the addition of nitrite to platelet-rich plasma in the presence of erythrocytes could inhibit platelet aggregation and this inhibitory effect of nitrite + erythrocytes was enhanced by deoxygenation of erythrocytes as measured by P-selectin expression and cGMP production. In order to study the nitrite effect on platelets at different oxygen levels, we used the flow cytometric assays to detect platelet membrane surface markers upon activation. The P-selectin and activated gpIIb/IIIa expression on platelet membranes in response to ADP, collagen and thrombin stimulation was measured at various hematocrit and oxygen levels. Nitrite (0.1 to 1.0 μM) significantly decreased the percentage of these surface markers on the platelet membrane at the hematocrit values above 23% and oxygen levels lower than 49 mmHg. The inhibitory effect of nitrite was augmented by increasing hematocrit values and decreasing oxygen saturation. C-PTIO (an NO scavenger) prevented the platelet inhibition by nitrite + erythrocytes whereas the inhibitors of NO synthase and xanthine oxidoreductase had no effect. These results support the proposal that circulating nitrite decreases platelet reactivity in the presence of partially deoxygenated erythrocytes through its reduction to NO, which may also explain certain differences between arterial and venous thrombosis and support directly the role of deoxyhemoglobin in this process. We believe that our flow cytometric assays offer a possibility to identify the individual molecular process involved in these effects. PMID:24642865

  9. Identification and Functional Characterization of Glycosylation of Recombinant Human Platelet-Derived Growth Factor-BB in Pichia pastoris

    PubMed Central

    Dai, Mengmeng; Yu, Changming; Fang, Ting; Fu, Ling; Wang, Jing; Zhang, Jun; Ren, Jun; Xu, Junjie; Zhang, Xiaopeng; Chen, Wei

    2015-01-01

    Yeast Pichia pastoris is a widely used system for heterologous protein expression. However, post-translational modifications, especially glycosylation, usually impede pharmaceutical application of recombinant proteins because of unexpected alterations in protein structure and function. The aim of this study was to identify glycosylation sites on recombinant human platelet-derived growth factor-BB (rhPDGF-BB) secreted by P. pastoris, and investigate possible effects of O-linked glycans on PDGF-BB functional activity. PDGF-BB secreted by P. pastoris is very heterogeneous and contains multiple isoforms. We demonstrated that PDGF-BB was O-glycosylated during the secretion process and detected putative O-glycosylation sites using glycosylation staining and immunoblotting. By site-directed mutagenesis and high-resolution LC/MS analysis, we, for the first time, identified two threonine residues at the C-terminus as the major O-glycosylation sites on rhPDGF-BB produced in P. pastoris. Although O-glycosylation resulted in heterogeneous protein expression, the removal of glycosylation sites did not affect rhPDGF-BB mitogenic activity. In addition, the unglycosylated PDGF-BBΔGly mutant exhibited the immunogenicity comparable to that of the wild-type form. Furthermore, antiserum against PDGF-BBΔGly also recognized glycosylated PDGF-BB, indicating that protein immunogenicity was unaltered by glycosylation. These findings elucidate the effect of glycosylation on PDGF-BB structure and biological activity, and can potentially contribute to the design and production of homogeneously expressed unglycosylated or human-type glycosylated PDGF-BB in P. pastoris for pharmaceutical applications. PMID:26701617

  10. Platelet-type von Willebrand disease: a rare, often misdiagnosed and underdiagnosed bleeding disorder.

    PubMed

    Othman, Maha

    2011-07-01

    Platelet-type von Willebrand disease (PT-VWD) is an autosomal dominant rare bleeding disorder characterized by hyperresponsive platelets. This inherent platelet function defect is due to a gain-of-function mutation within the GP1BA gene coding for the platelet surface glycoprotein Ib alpha protein, the receptor for the adhesive protein von Willebrand factor (VWF). The defect results in excessive and unnecessary platelet-VWF interaction with subsequent removal of the hemostatically efficient high molecular weight VWF as well as platelets from the circulation, leading to thrombocytopenia and bleeding diathesis. Patients with PT-VWD present with mild to moderate mucocutaneous bleeding, which becomes more pronounced during pregnancy and following aspirin ingestion or drugs that have antiplatelet activity. Laboratory testing shows low VWF:ristocetin cofactor and low or normal VWF:antigen and characteristically an enhanced ristocetin-induced platelet agglutination (RIPA). These laboratory features are also indicators of the closely similar and more common bleeding disorder type 2B VWD. Simplified RIPA mixing assays, cryoprecipitate challenge, and flow cytometry can differentiate between the two disorders. However, the gold standard is to identify mutations within the VWF gene (indicating type 2B VWD) or the platelet GP1BA gene (confirming PT-VWD). Treatment is based on making a correct diagnosis of PT-VWD where platelet concentrates instead of VWF/factor VIII preparations should be administered. A recent fairly large retrospective/prospective registry-based international study showed that PT-VWD is very rare, likely to be misdiagnosed as type 2B VWD or idiopathic thrombocytopenic purpura, and represents 15% of type 2B VWD diagnoses. PMID:22102188

  11. Functional behavior of vessels from pigs with von Willebrand disease. Values of platelet deposition are identical to those obtained on normal vessels

    SciTech Connect

    Badimon, L.; Badimon, J.J.; Turitto, V.T.; Rand, J.; Fuster, V.

    1989-03-01

    Vessels from normal pigs and pigs with severe von Willebrand disease were exposed for up to 30 minutes to both nonanticoagulated and heparinized blood from normal pigs in an ex vivo perfusion system. Shear rates at the vessel surface were varied over a broad physiological range, gamma w = 212 to 3380 sec-1. The deposition of /sup 111/In-labeled platelets was determined by radiometric counting. For all shear rates and exposure times investigated, the levels of platelet deposition on de-endothelialized thoracic aorta of normal and von Willebrand disease pigs were not significantly different. Thus, the functional activity of the vessels correlated with the results obtained previously by immunofluorescence. Namely, the von Willebrand factor protein in the thoracic subendothelium of normal pigs is significantly diminished or absent and is comparable to the levels observed in von Willebrand disease pigs.

  12. Adiponectin, ghrelin, and leptin differentially influence human platelet and human vascular endothelial cell functions: implication in obesity-associated cardiovascular diseases.

    PubMed

    Elbatarny, Hisham S; Netherton, Stuart J; Ovens, Jeffrey D; Ferguson, Alastair V; Maurice, Donald H

    2007-03-01

    A very strong epidemiological link exists between obesity, the metabolic syndrome, diabetes and diabetes-associated cardiovascular pathologies. For this reason the peripheral effects of the centrally-acting satiety adipokines, adiponectin and leptin, and of non-adipose-derived hormones with similar effects, like ghrelin, have received considerable attention. In this report, we have extended our previous studies of the pro-thrombotic effects of leptin and determined the effects of adiponectin or ghrelin on human platelet activation. Thus, while leptin stimulated human platelet aggregation and adhesion, addition of adiponectin or of ghrelin did not affect either aggregation or adhesion of these cells; even at supra-physiological concentrations. In addition, we compared the impact of these three important hormones on microvascular endothelial cell permeability, an important parameter of endothelial function that when impaired contributes to several vascular pathologies. While physiologically relevant concentrations of either leptin or adiponectin increased the integrity of the diffusion barrier formed by a monolayer of human microvascular endothelial cells, only supra-physiological concentrations of ghrelin had this effect. None of these agents reduced microvascular endothelial barrier function. Taken together, our data are consistent with the ideas that leptin activates human platelets and limits transendothelial cell diffusion but that adiponectin only influences endothelial cell permeability. In contrast, ghrelin had neither of these effects. We propose that these data identify important differences in the effects of leptin, adiponectin or ghrelin on microvascular endothelial cells and platelets and may provide a basis on which to pharmacologically manipulate the selective effects of these peptides on these cell types in human cardiovascular or thrombotic diseases associated with obesity. PMID:17207790

  13. Increases in ambient particulate matter air pollution, acute changes in platelet function, and effect modification by aspirin and omega-3 fatty acids: A panel study.

    PubMed

    Becerra, Adan Z; Georas, Steve; Brenna, J Thomas; Hopke, Philip K; Kane, Cathleen; Chalupa, David; Frampton, Mark W; Block, Robert; Rich, David Q

    2016-01-01

    Increased particulate matter (PM) air pollutant concentrations have been associated with platelet activation. It was postulated that elevated air pollutant concentrations would be associated with increases in measures of platelet function and that responses would be blunted when taking aspirin and/or fish oil. Data from a sequential therapy trial (30 subjects with type 2 diabetes mellitus), with 4 clinic visits (first: no supplements, second: aspirin, third: omega-3 fatty acid supplements, fourth: aspirin and omega-3 fatty acids) per subject, were utilized. Using linear mixed models, adjusted for relative humidity, temperature, visit number, and season, changes in three platelet function measures including (1) aggregation induced by adenosine diphosphate (ADP), (2) aggregation induced by collagen, and (3) thromboxane B2 production were associated with interquartile range (IQR) increases in mean concentrations of ambient PM2.5, black carbon, ultrafine particles (UFP; 10-100 nm), and accumulation mode particles (AMP; 100-500 nm) in the previous 1-96 h. IQR increases in mean UFP and AMP concentrations were associated with significant decreases in platelet response, with the largest being a -0.43 log(pg/ml) decrease in log(thromboxane B2) (95% CI = -0.8, -0.1) associated with each 582-particles/cm(3) increase in AMP, and a -1.7 ohm reduction in collagen-induced aggregation (95% CI = -3.1, -0.3) associated with each 2097-particles/cm(3) increase in UFP in the previous 72 h. This UFP effect on thromboxane B2 was significantly muted in diabetic subjects taking aspirin (-0.01 log[pg/ml]; 95% CI = -0.4, 0.3). The reason for this finding remains unknown, and needs to be investigated in future studies. PMID:27029326

  14. Fast Functional Germline and Epigenetic Assays in the Nematode Caenorhabditis elegans.

    PubMed

    Lundby, Zachary; Camacho, Jessica; Allard, Patrick

    2016-01-01

    Germ cells are unique in their ability to transfer traits and genetic information from one generation to the next. The proper development and integrity of their genome are therefore of utmost importance for the health of organisms and survival of species. Many features of mammalian germ cells, including their long development span and difficulty of access, present challenges for their study in the context of toxicity assays. In light of these barriers, the model system Caenorhabditis elegans shows great potential given its ease of manipulation and genetic tractability which can be easily adapted for high-throughput analysis. In this chapter, we discuss the advantages of examining germ cell processes in C. elegans, and describe three functional germline assays for the examination of chemical impact on germline maintenance and function including assays probing germ cell differentiation, germline apoptosis, and germline epigenetic regulation. PMID:27518628

  15. [Therapy of inherited diseases of platelet function. Interdisciplinary S2K guideline of the Permanent Paediatric Committee of the Society of Thrombosis and Haemostasis Research (GTH e. V.)].

    PubMed

    Streif, W; Knöfler, R; Eberl, W; Andres, O; Bakchoul, T; Bergmann, F; Beutel, K; Dittmer, R; Gehrisch, S; Gottstein, S; Halimeh, S; Haselböck, J; Hassenpflug, W-A; Heine, S; Holzhauer, S; King, S; Kirchmaier, C M; Krause, M; Kreuz, W; Lösche, W; Mahnel, R; Maurer, M; Nimtz-Talaska, A; Olivieri, M; Rott, H; Schambeck, Ch M; Schedel, A; Schilling, F H; Schmugge, M; Schneppenheim, R; Scholz, U; Scholz, T; Schulze, H; Siegemund, A; Strauß, G; Sykora, K-W; Wermes, C; Wiegering, V; Wieland, I; Zieger, B; Zotz, R B

    2014-01-01

    Inherited disorders of platelet function are a heterogeneous group. For optimal prevention and management of bleeding, classification and diagnosis of the underlying defect are highly recommended. An interdisciplinary guideline for a diagnostic approach has been published (AWMF # 086-003 S2K; Hämostaseologie 2014; 34: 201-212). Underlying platelet disorder, platelet count, age and clinical situation modify treatment. Exclusive transfusion of platelet concentrates may be inappropriate as potentially adverse effects can outweigh its benefit. A stepwise and individually adjusted approach for restitution and maintenance of haemostasis is recommended. Administration of antifibrinolytics is generally endorsed, but is of particular use in Quebec disease. Restricted to older children, desmopressin is favourable in storage pool disease and unclassified platelet disorders. Although licensed only for patients with Glanzmann thrombasthenia and alloantibodies, in clinical practice rFVIIa is widely used in inherited platelet disorders with severe bleeding tendency. This guideline aims at presenting the best available advice for the management of patients with inherited platelet function disorders. PMID:25370176

  16. Hemostatic Function and Transfusion Efficacy of Apheresis Platelet Concentrates Treated with Gamma Irradiation in Use for Thrombocytopenic Patients

    PubMed Central

    Zhu, Mei; Xu, Wei; Wang, Bao-Long; Su, Hong

    2014-01-01

    Summary Background During the transfusion of blood components, the transfer of allogeneic donor white blood cells (WBCs) can mediate transfusion-associated graft-versus-host disease (TA-GVHD). To minimize the reaction, exposure of blood products to gamma irradiation is currently the standard of care. The aim of our study was to evaluate and compare hemostatic function, transfusion efficacy, and safety of gamma-irradiated single-donor apheresis platelet concentrates (PCs) and of conventional non-irradiated PCs in patients with chemotherapy-induced thrombocytopenia. Methods 20 double-dose single-donor leukoreduced PCs were split in two identical units; one was gamma-irradiated with 25 Gy (study arm A) and the other remains non-irradiated (study arm B). Both units were stored under equal conditions. Hematologic patients were randomly assigned to receive gamma-irradiated or conventional non-irradiated PCs. Hemostatic function was evaluated by thrombelastography (TEG). TEG measurements were taken pre transfusion and 1 and 24 h post transfusion. TEG profiles were measured, noting the time to initiate clotting (R), the angle of clot formation (α), and the maximum amplitude (clot strength (MA)). Whole blood samples were collected from these thrombocytopenic patients at 1 and 24 h for PLT count increments (CIs) and corrected count increments (CCIs) with assessments of transfusion efficacy. Time to next PLT transfusion, transfusion requirement of RBCs, active bleeding, and adverse events (AEs), were analyzed. Results No differences could be found in hemostatic function parameters (MA, R, and α) between study arms A and B (all p values > 0.096) pre transfusion as well as 1 and 24 h post transfusion. No differences between study arms A and B were observed for mean (± standard deviation (SD)) 1-hour CCI (12.83 ± 6.33 vs. 11.59 ± 5.97) and 24-hour CCI (6.56 ± 4.10 vs. 5.76 ± 4.05). Mean 1-hour CI and 24-hour CI were not significantly different in both study arms (p = 0

  17. Manipulating megakaryocytes to manufacture platelets ex vivo

    PubMed Central

    Karagiannis, P; Eto, K

    2015-01-01

    Historically, platelet transfusion has proven a reliable way to treat patients suffering from thrombocytopenia or similar ailments. An undersupply of donors, however, has demanded alternative platelet sources. Scientists have therefore sought to recapitulate the biological events that convert hematopoietic stem cells into platelets in the laboratory. Such platelets have shown good function and potential for treatment. Yet the number manufactured ex vivo falls well short of clinical application. Part of the reason is the remarkable gaps in our understanding of the molecular mechanisms driving platelet formation. Using several stem cell sources, scientists have progressively clarified the chemical signaling and physical microenvironment that optimize ex vivo platelets and reconstituted them in synthetic environments. Key advances in cell reprogramming and the ability to propagate self-renewal have extended the lifetime of megakaryocytes to increase the pool of platelet progenitors. PMID:26149050

  18. Role of platelets in allergic airway inflammation.

    PubMed

    Idzko, Marco; Pitchford, Simon; Page, Clive

    2015-06-01

    Increasing evidence suggests an important role for platelets and their products (e.g., platelet factor 4, β-thromboglobulin, RANTES, thromboxane, or serotonin) in the pathogenesis of allergic diseases. A variety of changes in platelet function have been observed in patients with asthma, such as alterations in platelet secretion, expression of surface molecules, aggregation, and adhesion. Moreover, platelets have been found to actively contribute to most of the characteristic features of asthma, including bronchial hyperresponsiveness, bronchoconstriction, airway inflammation, and airway remodeling. This review brings together the current available data from both experimental and clinical studies that have investigated the role of platelets in allergic airway inflammation and asthma. It is anticipated that a better understanding of the role of platelets in the pathogenesis of asthma might lead to novel promising therapeutic approaches in the treatment of allergic airway diseases. PMID:26051948

  19. Procoagulant activity on platelets adhered to collagen or plasma clot.

    PubMed

    Ilveskero, S; Siljander, P; Lassila, R

    2001-04-01

    In a new 2-stage assay of platelet procoagulant activity (PCA), we first subjected gel-filtered platelets to adhesion on collagen (as a model of primary hemostasis) or plasma clots (as a model of preformed thrombus) for 30 minutes, and then the adherent platelets were supplemented with pooled, reptilase-treated, diluted plasma. Defibrinated plasma provided coagulation factors for assembly on platelet membranes without uncontrolled binding of thrombin to fibrin(ogen). Platelet adhesion to both surfaces showed modest individual variation, which increased at platelet densities that allowed aggregation. However, adhesion-induced PCA varied individually and surface-independently >3-fold, suggesting a uniform platelet procoagulant mechanism. Permanently adhered platelets showed markedly enhanced PCA when compared with the platelet pool in suspension, even after strong activation. The rate of thrombin generation induced by clot-adherent platelets was markedly faster than on collagen-adherent platelets during the initial phase of coagulation, whereas collagen-induced PCA proceeded slowly, strongly promoted by tissue thromboplastin. Therefore at 10 minutes, after adjustment for adhered platelets, collagen supported soluble thrombin formation as much as 5 times that of the thrombin-retaining clots. Activation of platelets by their firm adhesion was accompanied by formation of microparticles, representing about one third of the total soluble PCA. Collagen-adhered platelets provide soluble thrombin and microparticles, whereas the preformed clot serves to localize and accelerate hemostasis at the injury site, with the contribution of retained thrombin and microparticles. PMID:11304482

  20. Real-time monitoring of the functional status of platelets treated by Infukoll using a computer-aided laser phase microscope

    NASA Astrophysics Data System (ADS)

    Vasilenko, Irina A.; Babakova, Svetlana; Kiseleva, Elena; Dyugeev, Adyan; Konradov, Alexander A.; Shabalin, Vladimir

    1999-02-01

    Combined analysis of optic-geometrical characteristics makes it possible to comprehensively evaluate the morphological and functional state of the cytological object, which can not be done during visual observation. The technique is discussed for real-time monitoring of the functional status of platelets using computer-aided phase microscope (CPM) 'Cytoscan'. High accuracy and sensitivity of CPM with respect to determination of local temporal phase make it possible to register the dynamic processes in the voluntarily chosen points and sections of micro-objects, to obtain the Fourier's spectra and other characteristics suitable for statistical analysis. Human platelets were prepared from venous blood of healthy donors and pregnant women by standard methods, suspended in culture medium 199 and treated by different doses of 6% Infukoll HES. Nonfixating and nonstaining cells were studied with CPM: height accuracy 0.5 nm, magnification 1000, acquisition time 4 - 30 s. In our experiments we used time resolution about 0.03 s and 30 x lens with numerical aperture 0.65. During investigations of temporal processes a certain section was chosen in the topogram of cell image and local values for the phase of scattered wave in each of the points of the chosen cell's profile were measured. On the basis of the results of automated phase image analysis of optic-geometrical characteristics of living cells, the new quantitative express-method for evaluating of the functional status of human platelets was developed and tested. The structural changes of cells were visualized in alteration of 3-D images, phase profiles, in the decrease of mean cell phase diameters, heights, volumes, in disturbance of histograms of phase heights distribution by cell image points. New data on the behavior of platelets treated by Infukoll in vitro and in vivo were obtained. Analysis of intracellular dynamics was allowed to characterize the cell's regions of maximal activity, but the intensity of processes

  1. Immune Monitoring in Cancer Vaccine Clinical Trials: Critical Issues of Functional Flow Cytometry-Based Assays

    PubMed Central

    Urbani, Francesca; Proietti, Enrico

    2013-01-01

    The development of immune monitoring assays is essential to determine the immune responses against tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs) and their possible correlation with clinical outcome in cancer patients receiving immunotherapies. Despite the wide range of techniques used, to date these assays have not shown consistent results among clinical trials and failed to define surrogate markers of clinical efficacy to antitumor vaccines. Multiparameter flow cytometry- (FCM-) based assays combining different phenotypic and functional markers have been developed in the past decade for informative and longitudinal analysis of polyfunctional T-cells. These technologies were designed to address the complexity and functional heterogeneity of cancer biology and cellular immunity and to define biomarkers predicting clinical response to anticancer treatment. So far, there is still a lack of standardization of some of these immunological tests. The aim of this review is to overview the latest technologies for immune monitoring and to highlight critical steps involved in some of the FCM-based cellular immune assays. In particular, our laboratory is focused on melanoma vaccine research and thus our main goal was the validation of a functional multiparameter test (FMT) combining different functional and lineage markers to be applied in clinical trials involving patients with melanoma. PMID:24195078

  2. Signaling during platelet adhesion and activation

    PubMed Central

    Li, Zhenyu; Delaney, M. Keegan; O’Brien, Kelly A.; Du, Xiaoping

    2011-01-01

    Upon vascular injury, platelets are activated by adhesion to adhesive proteins like von Willebrand factor and collagen, or by soluble platelet agonists like ADP, thrombin, and thromboxane A2. These adhesive proteins and soluble agonists induce signal transduction via their respective receptors. The various receptor-specific platelet activation signaling pathways converge into common signaling events, which stimulate platelet shape change, granule secretion, and ultimately induce the “inside-out” signaling process leading to activation of the ligand binding function of integrin αIIbβ3. Ligand binding to integrin αIIbβ3 mediates platelet adhesion and aggregation and triggers “outside-in” signaling, resulting in platelet spreading, additional granule secretion, stabilization of platelet adhesion and aggregation, and clot retraction. It has become increasingly evident that agonist-induced platelet activation signals also crosstalk with integrin “outside-in” signals to regulate platelet responses. Platelet activation involves a series of rapid positive feedback loops that greatly amplify initial activation signals, and enable robust platelet recruitment and thrombus stabilization. Recent studies have provided novel insight into the molecular mechanisms of these processes. PMID:21071698

  3. Platelet aggregation test

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003669.htm Platelet aggregation test To use the sharing features on this page, please enable JavaScript. The platelet aggregation blood test checks how well platelets , a ...

  4. Targeting Phosphodiesterases in Anti-platelet Therapy

    PubMed Central

    Rondina, Matthew T.; Weyrich, Andrew S.

    2013-01-01

    There are two primary modes of platelet inhibition: blockade of membrane receptors or neutralization of intracellular pathways. Both means of inhibition have proven benefits in the prevention and resolution of atherothrombotic events. With regard to intracellular inhibition, phosphodiesterases (PDEs) are fundamental for platelet function. Platelets possess several PDEs (PDE2, PDE3 and PDE5) that catalyze the hydrolysis of cyclic adenosine 3′-5′-monophosphate (cAMP) and cyclic guanosine 3′-5′-monophosphate (cGMP), thereby limiting the levels of intracellular nucleotides. PDE inhibitors, such as cilostazol and dipyridamole, dampen platelet function by increasing cAMP and cGMP levels. This review focuses on the roles of PDE inhibitors in modulating platelet function, with particular attention paid to drugs that have anti-platelet clinical indications. PMID:22918733

  5. Impaired responsiveness of platelets to epinephrine due to α2A adrenoreceptor deficiency in Male Chinese.

    PubMed

    Lin, Tsun-Mei; Lin, Jih-Shyan; Tseng, Jen-Yu; Wu, Shang-Yin; Chen, Tsai-Yun

    2016-01-01

    Epinephrine is known as a weak, but important, agonist for platelet activation. It has been reported that the responsiveness of platelets to epinephrine was markedly impaired in 6% of Caucasians and in 16% of Japanese. The purpose of this study was to screen and characterize this abnormality in healthy Taiwanese Chinese volunteers. We used aggregometry, flow cytometry and platelet function analyzer (PFA)-100 system to assess in 50 healthy male volunteers the responsiveness of platelets to epinephrine stimulation. Using α2A adrenoceptor antagonist BRL44408 maleate competition and a [(3)H]yohimbin binding assay, we evaluated α2A adrenoceptors on platelets. The aggregation of platelets after stimulation with 10 μM of epinephrine indicated two distinct groups of study participants: 24 (48.0%) good- and 26 (52.0%) impaired-responders to epinephrine. Flow cytometric analysis of platelets after stimulated with 1 μM epinephrine showed that glycoprotein (GP) IIb/IIIa and P-selectin expression of epinephrine good- and impaired-responders were 27.1 ± 11.0% vs. 9.9 ± 5.4% (p = 0.003) and 12.2 ± 6.2% vs. 3.6 ± 3.5% (p < 0.001), respectively. The PFA-100 system showed that epinephrine-impaired-responders had a longer collagen-epinephrine induced closure time. Good-responder platelets incubated with BRL44408 maleate had an impaired response to epinephrine stimulation. [(3)H]yohimbine binding studies showed fewer α2A adrenoreceptors on the platelets of epinephrine-impaired-responders than on those of good-responders. The prevalence of impaired responsiveness to epinephrine was high and probably due to α2A adrenoreceptor deficiency in male Taiwanese Chinese. PMID:26083800

  6. Platelet antibody: review of detection methods

    SciTech Connect

    Schwartz, K.A.

    1988-10-01

    The driving force behind development of in vitro methods for platelet antibodies is identification of plasma factors causing platelet destruction. Early methods relied on measurement of platelet activation. Current methods are more specific and use a purified antibody against immunoglobulin or complement, which is usually labeled with /sup 125/I or tagged with an enzyme or fluorescein. Comparisons of quantitation of platelet-associated IgG show wide variability between different methods. The disparate results can be related both to differences in binding of secondary antibodies to immunoglobulin in solution compared to immunoglobulins attached to platelets and to the improper assumption that the binding ratio between the secondary detecting and primary antiplatelet antibody is one. Most assays can 1) identify neonatal isoimmune thrombocytopenia and posttransfusion purpura, 2) help to differentiate between immune and nonimmune thrombocytopenias, 3) help to sort out the offending drug when drug-induced thrombocytopenia is suspected, and 4) identify platelet alloantibodies and potential platelet donors via a cross match assay for refractory patients. However, the advantages of quantitative assays over qualitative methods with respect to predictions of patients clinical course and response to different treatments remain to be investigated. 61 references.

  7. Platelet depletion and severity of streptococcal endocarditis

    PubMed Central

    Dall, Lawrence; Miller, Todd; Herndon, Betty; Diez, Ireneo; Dew, Michelle

    1998-01-01

    OBJECTIVE: To evaluate the importance of thrombocytopenia in streptococcal endocarditis using an animal model. DESIGN: A model of human septic endocarditis was established in rats (polyethylene catheters across the aortic valve and administration of Streptococcus sanguis, 5×107 colony forming units [cfu] intravenous). Thrombocytopenia at four levels was produced by antiplatelet serum. Secondary methods of producing thrombocytopenia were also evaluated. At sacrifice (96 h after platelet depletion and 72 h after infection), vegetations were removed, weighed, diluted, plated and counted. Potential mechanisms of the dose-response relationship between vegetation density and platelet count were evaluated. SETTING: Controlled research laboratory experiments. POPULATION STUDIED: Animal models of streptococcal endocarditis. MAIN RESULTS: The bacterial density of the aortic valve vegetations significantly increased as the platelet count decreased (P=0.0007). In severely thrombocytopenic animals (two-dose antiplatelet serum), data suggest increased vegetation embolism. Platelet depletion, which was minimal with chemical methods, was produced most effectively by antithrombocyte serum. Platelet surfaces in endocarditis were found to express elevated CD62p proteins (72.7% endocarditis, 34.7% control). Platelet protein fractions were evaluated in vitro by both streptocidal (P=0.19) and phagocytosis-stimulating assays. Platelet presence in mature aortic valve vegetations averaged only about 2%. CONCLUSIONS: In platelet depletion experiments using a rat model, a dose-response relationship of peripheral circulating platelet depletion to aortic valve vegetation density was found. The mechanism relating thrombocytopenia to endocarditis severity remains unresolved. PMID:22346555

  8. A Human Platelet Receptor Protein Microarray Identifies the High Affinity Immunoglobulin E Receptor Subunit α (FcεR1α) as an Activating Platelet Endothelium Aggregation Receptor 1 (PEAR1) Ligand*

    PubMed Central

    Sun, Yi; Vandenbriele, Christophe; Kauskot, Alexandre; Verhamme, Peter; Hoylaerts, Marc F.; Wright, Gavin J.

    2015-01-01

    Genome-wide association studies to identify loci responsible for platelet function and cardiovascular disease susceptibility have repeatedly identified polymorphisms linked to a gene encoding platelet endothelium aggregation receptor 1 (PEAR1), an “orphan” cell surface receptor that is activated to stabilize platelet aggregates. To investigate how PEAR1 signaling is initiated, we sought to identify its extracellular ligand by creating a protein microarray representing the secretome and receptor repertoire of the human platelet. Using an avid soluble recombinant PEAR1 protein and a systematic screening assay designed to detect extracellular interactions, we identified the high affinity immunoglobulin E (IgE) receptor subunit α (FcεR1α) as a PEAR1 ligand. FcεR1α and PEAR1 directly interacted through their membrane-proximal Ig-like and 13th epidermal growth factor domains with a relatively strong affinity (KD ∼ 30 nm). Precomplexing FcεR1α with IgE potently inhibited the FcεR1α-PEAR1 interaction, and this was relieved by the anti-IgE therapeutic omalizumab. Oligomerized FcεR1α potentiated platelet aggregation and led to PEAR1 phosphorylation, an effect that was also inhibited by IgE. These findings demonstrate how a protein microarray resource can be used to gain important insight into the function of platelet receptors and provide a mechanistic basis for the initiation of PEAR1 signaling in platelet aggregation. PMID:25713122

  9. Function of glycoprotein VI and integrin alpha2beta1 in the procoagulant response of single, collagen-adherent platelets.

    PubMed

    Heemskerk, J W; Siljander, P; Vuist, W M; Breikers, G; Reutelingsperger, C P; Barnes, M J; Knight, C G; Lassila, R; Farndale, R W

    1999-05-01

    Various collagen-based materials were used to assess the structural requirements of collagen for inducing the procoagulant response of adhering platelets, as well as the collagen receptors involved. Cross-linked or monomeric collagen-related peptide (CRP), Gly-Cys-Hyp-(Gly-Pro-Hyp)10-Gly-Cys-Hyp-Gly was highly adhesive for platelets in a glycoprotein VI-(GpVI-)dependent manner. Adhesion was followed by a prolonged increase in cytosolic [Ca2+]i, formation of membrane blebs, exposure of phosphatidylserine (PS) and generation of prothrombinase-stimulating activity. Fibrils of type-I collagen were less adhesive but, once adhered, many of the platelets presented a full procoagulant response. Monomeric type-I collagen was unable to support adhesion, unless Mg(2+)-dependent integrin alpha2beta1 interactions were facilitated by omission of Ca2+ ions. With all surfaces, however, post-addition of CaCl2 to adhering platelets resulted in a potent Ca(2+)-influx signal, followed by PS exposure and bleb formation. The procoagulant response elicited by binding to CRP was inhibited by anti-GpVI Fab fragments, but not by impeding integrin alpha2beta1-mediated events. With fibrillar collagen, it was inhibited by blocking either the GpVI- or integrin alpha2beta1-mediated interactions. This suggests that the triple-helical Gly-Pro-Hyp repeat in CRP and analogous sequences in fibrillar collagen stimulate the procoagulant response of adherent platelets by acting as ligands for GpVI. Influx of Ca2+ is required for this response, and adhesion via integrin alpha2beta1 serves to potentiate the signaling effects of GpVI. PMID:10365754

  10. An extended set of yeast-based functional assays accurately identifies human disease mutations.

    PubMed

    Sun, Song; Yang, Fan; Tan, Guihong; Costanzo, Michael; Oughtred, Rose; Hirschman, Jodi; Theesfeld, Chandra L; Bansal, Pritpal; Sahni, Nidhi; Yi, Song; Yu, Analyn; Tyagi, Tanya; Tie, Cathy; Hill, David E; Vidal, Marc; Andrews, Brenda J; Boone, Charles; Dolinski, Kara; Roth, Frederick P

    2016-05-01

    We can now routinely identify coding variants within individual human genomes. A pressing challenge is to determine which variants disrupt the function of disease-associated genes. Both experimental and computational methods exist to predict pathogenicity of human genetic variation. However, a systematic performance comparison between them has been lacking. Therefore, we developed and exploited a panel of 26 yeast-based functional complementation assays to measure the impact of 179 variants (101 disease- and 78 non-disease-associated variants) from 22 human disease genes. Using the resulting reference standard, we show that experimental functional assays in a 1-billion-year diverged model organism can identify pathogenic alleles with significantly higher precision and specificity than current computational methods. PMID:26975778

  11. An extended set of yeast-based functional assays accurately identifies human disease mutations

    PubMed Central

    Sun, Song; Yang, Fan; Tan, Guihong; Costanzo, Michael; Oughtred, Rose; Hirschman, Jodi; Theesfeld, Chandra L.; Bansal, Pritpal; Sahni, Nidhi; Yi, Song; Yu, Analyn; Tyagi, Tanya; Tie, Cathy; Hill, David E.; Vidal, Marc; Andrews, Brenda J.; Boone, Charles; Dolinski, Kara; Roth, Frederick P.

    2016-01-01

    We can now routinely identify coding variants within individual human genomes. A pressing challenge is to determine which variants disrupt the function of disease-associated genes. Both experimental and computational methods exist to predict pathogenicity of human genetic variation. However, a systematic performance comparison between them has been lacking. Therefore, we developed and exploited a panel of 26 yeast-based functional complementation assays to measure the impact of 179 variants (101 disease- and 78 non-disease-associated variants) from 22 human disease genes. Using the resulting reference standard, we show that experimental functional assays in a 1-billion-year diverged model organism can identify pathogenic alleles with significantly higher precision and specificity than current computational methods. PMID:26975778

  12. A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity

    PubMed Central

    Israeli, Ma’ayan; Rotem, Shahar; Elia, Uri; Bar-Haim, Erez; Cohen, Ofer; Chitlaru, Theodor

    2016-01-01

    Edema Factor (EF), the toxic sub-unit of the Bacillus anthracis Edema Toxin (ET) is a calmodulin-dependent adenylate cyclase whose detrimental activity in the infected host results in severe edema. EF is therefore a major virulence factor of B. anthracis. We describe a simple, rapid and reliable functional adenylate-cyclase assay based on inhibition of a luciferase-mediated luminescence reaction. The assay exploits the efficient adenylate cyclase-mediated depletion of adenosine tri-phosphate (ATP), and the strict dependence on ATP of the light-emitting luciferase-catalyzed luciferin-conversion to oxyluciferin, which can be easily visualized. The assay exhibits a robust EF-dose response decrease in luminescence, which may be specifically reverted by anti-EF antibodies. The application of the assay is exemplified in: (a) determining the presence of EF in B. anthracis cultures, or its absence in cultures of EF-defective strains; (b) evaluating the anti-EF humoral response in experimental animals infected/vaccinated with B. anthracis; and (c) rapid discrimination between EF producing and non-producing bacterial colonies. Furthermore, the assay may be amenable with high-throughput screening for EF inhibitory molecules. PMID:27548219

  13. A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity.

    PubMed

    Israeli, Ma'ayan; Rotem, Shahar; Elia, Uri; Bar-Haim, Erez; Cohen, Ofer; Chitlaru, Theodor

    2016-01-01

    Edema Factor (EF), the toxic sub-unit of the Bacillus anthracis Edema Toxin (ET) is a calmodulin-dependent adenylate cyclase whose detrimental activity in the infected host results in severe edema. EF is therefore a major virulence factor of B. anthracis. We describe a simple, rapid and reliable functional adenylate-cyclase assay based on inhibition of a luciferase-mediated luminescence reaction. The assay exploits the efficient adenylate cyclase-mediated depletion of adenosine tri-phosphate (ATP), and the strict dependence on ATP of the light-emitting luciferase-catalyzed luciferin-conversion to oxyluciferin, which can be easily visualized. The assay exhibits a robust EF-dose response decrease in luminescence, which may be specifically reverted by anti-EF antibodies. The application of the assay is exemplified in: (a) determining the presence of EF in B. anthracis cultures, or its absence in cultures of EF-defective strains; (b) evaluating the anti-EF humoral response in experimental animals infected/vaccinated with B. anthracis; and (c) rapid discrimination between EF producing and non-producing bacterial colonies. Furthermore, the assay may be amenable with high-throughput screening for EF inhibitory molecules. PMID:27548219

  14. Acquired platelet function defect

    MedlinePlus

    ... on the skin Gastrointestinal bleeding resulting in bloody, dark black, or tarry bowel movements ; or vomiting blood ... to the principles of the Health on the Net Foundation (www.hon.ch). The information provided herein ...

  15. Comparison of PFA-100 testing and bleeding time for detecting platelet hypofunction and von Willebrand disease in clinical practice.

    PubMed

    Posan, Emoke; McBane, Robert D; Grill, Diane E; Motsko, Cheri L; Nichols, William L

    2003-09-01

    The PFA-100 instrument (Platelet Function Analyzer, Dade Behring) has been reported to be superior to the bleeding time (BT) as a screening test of primary hemostasis. However evaluation of this device has been principally limited to selected populations. The study's aim was to determine testing performance in clinical practice, by comparing the PFA-100 to the BT for the identification of von Willebrand disease (VWD) and intrinsic platelet hypofunction. From 1998-2000, PFA-100 closure time (CT) for epinephrinecollagen (EPI) and ADP-collagen (ADP) cartridges and modified Ivy BTs were performed on outpatients referred for testing for suspected or known hemorrhagic diathesis (n = 346). Evaluation included assays of von Willebrand factor and platelet aggregometry in addition to platelet flow cytometry and electron microscopy when indicated. The normal distribution of PFA-100 CTs was determined using blood samples from 61 normal donors studied on 155 occasions. Results show that thirty-four patients met the diagnostic criteria for VWD and 31 patients were diagnosed with congenital or acquired intrinsic platelet hypofunction. The sensitivity of the PFA-100 for identification of VWD was significantly better (p < 0.01) than the BT with similar specificity. In contrast, the PFA-100 was comparable, but not superior to the BT for detecting platelet hypofunction. We conclude that the PFA-100 performance compares favorably to the BT for the identification of intrinsic platelet hypofunction in clinical practice with superior sensitivity for detecting VWD. Therefore, the PFA-100 could replace the BT for purposes of screening for VWD and intrinsic platelet hypofunction. When clinical suspicion is strong, testing should be supplemented with assays of von Willebrand factor and platelet aggregometry. PMID:12958618

  16. Functional assays for analysis of variants of uncertain significance in BRCA2

    PubMed Central

    Guidugli, Lucia; Carreira, Aura; Caputo, Sandrine M.; Ehlen, Asa; Galli, Alvaro; Monteiro, Alvaro N.A.; Neuhausen, Susan L.; Hansen, Thomas V.O.; Couch, Fergus J.; Vreeswijk, Maaike P.G.

    2014-01-01

    Missense variants in the BRCA2 gene are routinely detected during clinical screening for pathogenic mutations in patients with a family history of breast and ovarian cancer. These subtle changes frequently remain of unknown clinical significance because of the lack of genetic information that may help establish a direct correlation with cancer predisposition. Therefore alternative ways of predicting the pathogenicity of these variants are urgently needed. Since BRCA2 is a protein involved in important cellular mechanisms such as DNA repair, replication and cell cycle control, functional assays have been developed that exploit these cellular activities to explore the impact of the variants on protein function. In this review we summarize assays developed and currently utilized for studying missense variants in BRCA2. We specifically depict details of each assay, including VUS analyzed, and describe a validation set of (genetically) proven pathogenic and neutral missense variants to serve as a golden standard for the validation of each assay. Guidelines are proposed to enable implementation of laboratory-based methods to assess the impact of the variant on cancer risk. PMID:24323938

  17. Mutation in the gene encoding the. alpha. chain of platelet glycoprotein Ib in platelet-type von Willebrand disease

    SciTech Connect

    Miller, J.L.; Cunningham, D.; Lyle, V.A.; Finch, C.N. )

    1991-06-01

    Platelet-type von Willebrand disease (PT-vWD) is an autosomal dominant bleeding disorder characterized by abnormally enhanced binding of von Willebrand factor (vWF) by patient platelets. Although the platelet glycoprotein (GP) Ib/IX complex is known to constitute the platelet's ristocetin-dependent receptor for vWF, a unique structural abnormality within this complex has not previously been identified in PT-vWD. Using the poly merase chain reaction to amplify genomic DNA coding for the {alpha} chain of GP Ib (GP IB{alpha}) and then sequencing the amplified DNA following cloning into M13mp18 and M13mp19 phage vectors, the authors have found a single point mutation in the GP Ib{alpha} coding region of PT-vWD DNA resulting in the substitution of valine for glycine at residue 233. This substitution within the vWF-binding region of GP Ib{alpha} is likely to exert a significant influence on the conformation of the resulting protein. Competitive oligonucleotide primer assay for this mutation showed a homozygous wild-type pattern in genomic DNA from the 161 normal volunteers studied and from 6 phenotypically normal members of a PT-vWD family. All 7 affected members of this family studied were heterozygous for the mutant allele. Platelet GP Ib{alpha} mRNA reverse-transcribed and studied by competitive oligonucleotide primer assay showed similar expression of the mutant and wild-type alleles in the affected PT-vWD patients. Absence in the normal population, tight linkage with phenotypic expression of disease, and absence of any additional abnormality of GP Ib{alpha} in these patients identify the glycine-to-valine substitution as a point mutation underlying functional abnormality of the vWF receptor in PT-vWD.

  18. A Novel Flow Cytometric HTS Assay Reveals Functional Modulators of ATP Binding Cassette Transporter ABCB6

    PubMed Central

    Chavan, Hemantkumar; Young, Susan; Ma, Xiaochao; Waller, Anna; Garcia, Matthew; Perez, Dominique; Chavez, Stephanie; Strouse, Jacob J.; Haynes, Mark K.; Bologa, Cristian G.; Oprea, Tudor I.; Tegos, George P.; Sklar, Larry A.; Krishnamurthy, Partha

    2012-01-01

    ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6’s ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity. PMID:22808084

  19. Platelet utilization: a Canadian Blood Services research and development symposium.

    PubMed

    Webert, Kathryn E; Alam, Asim Q; Chargé, Sophie B; Sheffield, William P

    2014-04-01

    Considerable progress has been made in recent years in understanding platelet biology and in strengthening the clinical evidence base around platelet transfusion thresholds and appropriate platelet dosing. Platelet alloimmunization rates have also declined. Nevertheless, controversies and uncertainties remain that are relevant to how these products can best be used for the benefit of platelet transfusion recipients. Platelets are unique among the blood products directly derived from whole blood or apheresis donations in requiring storage, with shaking, at ambient temperature. Storage is accordingly constrained between the need to limit the growth of any microbes in the product and the need to minimize losses in platelet function associated with storage. Proteomic and genomic approaches are being applied to the platelet storage lesion. Platelet inventory management is made challenging by these constraints. Although bacterial screening has enhanced the safety of platelet transfusions, pathogen reduction technology may offer further benefits. Continuing clinical investigations are warranted to understand the value of transfusing platelets prophylactically or only in response to bleeding in different patient groups and how best to manage the most grievously injured trauma patients. Patients refractory to platelet transfusions also require expert clinical management. The engineering of platelet substitute products is an active area of research, but considerable hurdles remain before any clinical uses may be contemplated. Roles for platelets in biological areas distinct from hemostasis are also emerging. Platelet utilization is variably affected by all of the above factors, by demographic changes, by new medications, and by new patient care approaches. PMID:24629305

  20. Structure-guided creation of AcAP5-derived and platelet targeted factor Xa inhibitors.

    PubMed

    Zhu, Yuanjun; Lin, Yuan; Liu, Aihua; Shui, Mengyang; Li, Ruyi; Liu, Xiaoyan; Hu, Wenhui; Wang, Yinye

    2015-06-15

    Anticoagulants and anti-platelet agents are simultaneously administrated in clinical practice (i.e. percutaneous coronary intervention), which cause significant risk of systemic bleeding. Targeted delivery of anticoagulants to the activated platelets at sites of vascular injuries may condense the site-specific anticoagulant effect and reduce the hemorrhage side effects in uninjured vessels. To this end, we prepared three ancylostoma caninum anticoagulant peptide 5 (AcAP5) variants NR1, NR2 and NR3 engineered with a platelet-binding Arg-Gly-Asp (RGD) motif and evaluated their anti-Factor Xa (FXa) and platelet-binding effects. These RGD-containing AcAP5 variants were capable of interacting with platelet receptor αIIbβ3 as shown in computational analysis. All variants, especially NR2 and NR3, retained entirely the anti-FXa function of parent AcAP5. Moreover, they prevented the formation of occlusive thrombi in rat carotid artery injury model, suggesting that they inhibit platelet aggregation in vivo. Further functional investigation of NR3 demonstrated that NR3 inhibited platelet aggregation in vitro and FXa activity in vivo, and prolonged the coagulation time, all in a dose-dependent manner. Through flow cytometry assay, we confirmed the binding of NR3 to αIIbβ3 receptor. In mouse model of carotid artery endothelium injury, NR3-treated mice showed less tail bleeding time than AcAP5-treated mice, and aspirin plus NR3 treatment exhibited moderate reduction of blood loss compared with aspirin plus AcAP5 treatment. These results indicate the feasibility to engineer a novel FXa inhibitor specifically targeting the activated platelets, which centralizes its anticoagulation efficacy in the injured vascular endothelium and reduces the risk of systemic bleeding. PMID:25887920

  1. Platelet mimicry: The emperor's new clothes?

    PubMed

    Moghimi, Seyed Moein; Hunter, Alan Christy; Peer, Dan

    2016-01-01

    Here we critically examine whether coating of nanoparticles with platelet membranes can truly disguise them against recognition by elements of the innate immune system. We further assess whether the "cloaking technology" can sufficiently equip nanoparticles with platelet-mimicking functionalities to include in vivo targeting of damaged blood vessels and binding to platelet-adhering opportunistic pathogens. We present views for improved, and pharmaceutically viable nanoparticle design strategies. PMID:26409192

  2. Platelet Activation Test in Unprocessed Blood (Pac-t-UB) to Monitor Platelet Concentrates and Whole Blood of Thrombocytopenic Patients

    PubMed Central

    Roest, Mark; van Holten, Thijs C.; Fleurke, Ger-Jan; Remijn, Jasper A.

    2013-01-01

    Summary Background Platelet concentrate transfusion is the standard treatment for hemato-oncology patients to compensate for thrombocytopenia. We have developed a novel platelet activation test in anticoagulated unprocessed blood (pac-t-UB) to determine platelet function in platelet concentrates and in blood of thrombocytopenic patients. Methods We have measured platelet activity in a platelet concentrate and in anticoagulated unprocessed blood of a post-transfusion thrombocytopenic patient. Results Our data show time-dependent platelet activation by GPVI agonist (collagen related peptide; CRP), PAR-1 agonist (SFLLRN), P2Y12 agonist (ADP), and thromboxane receptor agonist (U46619) in a platelet concentrate. Furthermore, pac-t-UB showed time-dependent platelet activation in unprocessed blood of a post-transfusion patient with thrombocytopenia. Testing platelet function by different agonists in relation to storage show that 3-day-old platelet concentrates are still reactive to the studied agonists. This reactivity rapidly drops for each agonists during longer storage. Discussion Pac-t-UB is a novel tool to estimate platelet function by different agonists in platelet concentrates and in unprocessed blood of thrombocytopenic patients. In the near future, we will validate whether pac-t-UB is an adequate test to monitor the quality of platelet concentrates and whether pac-t-UB predicts the bleeding risk of transfused thrombocytopenic patients. PMID:23652405

  3. Toll-Like Receptor Signalling Is Not Involved in Platelet Response to Streptococcus pneumoniae In Vitro or In Vivo

    PubMed Central

    Schaap, Marianne C. L.; Hou, Baidong; van der Poll, Tom; Nieuwland, Rienk; van ‘t Veer, Cornelis

    2016-01-01

    Streptococcus (S.) pneumoniae strains vary considerably in their ability to cause invasive disease in humans, which is at least in part determined by the capsular serotype. Platelets have been implicated as sentinel cells in the circulation for host defence. One of their utensils for this function is the expression of Toll-like receptors (TLRs). We here aimed to investigate platelet response to S. pneumoniae and a role for TLRs herein. Platelets were stimulated using four serotypes of S. pneumonia including an unencapsulated mutant strain. In vitro aggregation and flow cytometry assays were performed using blood of healthy volunteers, or blood of TLR knock out and WT mice. For in vivo pneumonia experiments, platelet specific Myd88 knockout (Plt-Myd88-/-) mice were used. We found that platelet aggregation was induced by unencapsulated S. pneumoniae only. Whole blood incubation with all S. pneumoniae serotypes tested resulted in platelet degranulation and platelet-leukocyte complex formation. Platelet activation was TLR independent, as responses were not inhibited by TLR blocking antibodies, not induced by TLR agonists and were equally induced in wild-type and Tlr2-/-, Tlr4-/-, Tlr2/4-/-, Tlr9-/- and Myd88-/- blood. Plt-Myd88-/- and control mice displayed no differences in bacterial clearance or immune response to pneumonia by unencapsulated S. pneumoniae. In conclusion, S. pneumoniae activates platelets through a TLR-independent mechanism that is impeded by the bacterial capsule. Additionally, platelet MyD88-dependent TLR signalling is not involved in host defence to unencapsulated S. pneumoniae in vivo. PMID:27253707

  4. Calreticulin-independent regulation of the platelet integrin alphaIIbbeta3 by the KVGFFKR alphaIIb-cytoplasmic motif.

    PubMed

    Reilly, Dermot; Larkin, Deirdre; Devocelle, Marc; Fitzgerald, Desmond J; Moran, Niamh

    2004-02-01

    The platelet integrin alphaIIbbeta3 alters conformation in response to platelet activation and ligand binding, although the molecular mechanisms involved are not known. We previously showed that a lipid modified peptide, corresponding to the membrane proximal 989KVGFFKR995 portion of the alphaIIb cytoplasmic tail, independently activates platelet alphaIIbbeta3. Calreticulin (CRT) is a potential integrin regulatory protein based on its interaction with the highly conserved alpha-integrin sequence KxGFFKR. We therefore examined the possible interaction of calreticulin and alphaIIbbeta3 in human platelets. We demonstrate that calreticulin in platelets is localised to the granulomere. In contrast, the known integrin-binding protein talin accumulates at the periphery of spreading platelets and colocalises with alphaIIbbeta3 during the process of adhesion. An interaction between calreticulin and alphaIIbbeta3 could not be demonstrated using co-immunoprecipitation techniques under various platelet activation states, even in the presence of covalent chemical crosslinkers. Thus, calreticulin does not functionally interact with the major integrin in human platelets. In order to identify proteins that interact with the integrin KVGFFKR motif we then used a peptide 'pull-down' assay from platelet lysates with biotinylated peptides and demonstrate that only the alphaIIb and beta3 subunits selectively and individually interact with this sequence. This interaction is divalent cation-dependent, has high-affinity, and occurs both with purified alphaIIbbeta3 complex and with electroeluted alpha and beta subunits. Thus, our data show that the conserved integrin KVGFFKR domain interacts primarily with the alpha and beta cytoplasmic tails and not with CRT in human platelets. PMID:14985176

  5. Impact of experimental haemodilution on platelet function, thrombin generation and clot firmness: effects of different coagulation factor concentrates

    PubMed Central

    Caballo, Carolina; Escolar, Gines; Diaz-Ricart, Maribel; Lopez-Vílchez, Irene; Lozano, Miguel; Cid, Joan; Pino, Marcos; Beltrán, Joan; Basora, Misericordia; Pereira, Arturo; Galan, Ana M.

    2013-01-01

    Background Haemodilution during resuscitation after massive haemorrhage may worsen the coagulopathy and perpetuate bleeding. Materials and methods Blood samples from healthy donors were diluted (30 and-60%) using crystalloids (saline, Ringer’s lactate, PlasmalyteTM) or colloids (6% hydroxyethylstarch [HES130/0.4], 5% human albumin, and gelatin). The effects of haemodilution on platelet adhesion (Impact R), thrombin generation (TG), and thromboelastometry (TEM) parameters were analysed as were the effects of fibrinogen, prothrombin complex concentrates (PCC), activated recombinant factor VII (FVIIa), and cryoprecipates on haemodilution. Results Platelet interactions was already significantly reduced at 30% haemodilution. Platelet reactivity was not improved by addition of any of the concentrates tested. A decrease in TG and marked alterations of TEM parameters were noted at 60% haemodilution. HES130/0.4 was the expander with the most deleterious action. TG was significantly enhanced by PCC whereas rFVIIa only caused a mild acceleration of TG initiation. Fibrinogen restored the alterations of TEM parameters caused by haemodilution including those caused by HES 130/0.4. Cryoprecipitates significantly improved the alterations caused by haemodilution on TG and TEM parameters; the effects on TG disappeared after ultracentrifugation of the cryoprecipitates. Discussion The haemostatic alterations caused by haemodilution are multifactorial and affect both blood cells and coagulation. In our in vitro approach, HES 130/0.4 had the most deleterious effect on haemostasis parameters. Coagulation factor concentrates did not improve platelet interactions in the Impact R, but did have favourable effects on coagulation parameters measured by TG and TEM. Fibrinogen notably improved TEM parameters without increasing thrombin generation, suggesting that this concentrate may help to preserve blood clotting abilities during haemodilution without enhancing the prothrombotic risk. PMID

  6. Life-threatening postpartum hemolysis, elevated liver functions tests, low platelets syndrome versus thrombocytopenic purpura – Therapeutic plasma exchange is the answer

    PubMed Central

    Nasa, Prashant; Dua, J. M.; Kansal, Sudha; Chadha, Geeta; Chawla, Rajesh; Manchanda, Manav

    2011-01-01

    The differential diagnosis of life-threatening microangiopathic disorders in a postpartum female includes severe preeclampsia–eclampsia, hemolysis, elevated liver functions tests, low platelets syndrome and thrombotic thrombocytopenic purpura. There is considerable overlapping in the clinical and laboratory findings between these conditions, and hence an exact diagnosis may not be always possible. However, there is considerable maternal mortality and morbidity associated with these disorders. This case underlines the complexity of pregnancy-related microangiopathies regarding their differential diagnosis, multiple organ dysfunction and role of therapeutic plasma exchange in their management. PMID:21814380

  7. beta-Dystroglycan modulates the interplay between actin and microtubules in human-adhered platelets.

    PubMed

    Cerecedo, Doris; Cisneros, Bulmaro; Suárez-Sánchez, Rocío; Hernández-González, Enrique; Galván, Iván

    2008-05-01

    To maintain the continuity of an injured blood vessel, platelets change shape, secrete granule contents, adhere, aggregate, and retract in a haemostatic plug. Ordered arrays of microtubules, microfilaments, and associated proteins are responsible for these platelet responses. In full-spread platelets, microfilament bundles in association with other cytoskeleton proteins are anchored in focal contacts. Recent studies in migrating cells suggest that co-ordination and direct physical interaction of microtubules and actin network modulate adhesion development. In platelets, we have proposed a feasible association between these two cytoskeletal systems, as well as the participation of the dystrophin-associated protein complex, as part of the focal adhesion complex. The present study analysed the participation of microtubules and actin during the platelet adhesion process. Confocal microscopy, fluorescence resonance transfer energy and immunoprecipitation assays were used to provide evidence of a cross-talk between these two cytoskeletal systems. Interestingly, beta-dystroglycan was found to act as an interplay protein between actin and microtubules and an additional communication between these two cytoskeleton networks was maintained through proteins of focal adhesion complex. Altogether our data are indicative of a dynamic co-participation of actin filaments and microtubules in modulating focal contacts to achieve platelet function. PMID:18341635

  8. Longitudinal platelet reactivity to acute psychological stress among older men and women.

    PubMed

    Aschbacher, Kirstin; von Känel, Roland; Mills, Paul J; Roepke, Susan K; Hong, Suzi; Dimsdale, Joel E; Mausbach, Brent T; Patterson, Thomas L; Ziegler, Michael G; Ancoli-Israel, Sonia; Grant, Igor

    2009-09-01

    Platelet reactivity to acute stress is associated with increased cardiovascular disease risk; however, little research exists to provide systematic methodological foundations needed to generate strong longitudinal research designs. Study objectives were: 1) to evaluate whether markers of platelet function increase in response to an acute psychological stress test among older adults, 2) to establish whether reactivity remains robust upon repeated administration (i.e. three occasions approximately 1 year apart), and 3) to evaluate whether two different acute speech stress tasks elicit similar platelet responses. The 149 subjects (mean age 71 years) gave a brief impromptu speech on one of two randomly assigned topics involving interpersonal conflict. Blood samples drawn at baseline and post-speech were assayed using flow cytometry for platelet responses on three outcomes (% aggregates, % P-selectin expression, and % fibrinogen receptor expression). Three-level hierarchical linear modeling analyses revealed significant stress-induced increases in platelet activation on all outcomes (p < 0.001). No significant habituation on any measure was found. Additional reactivity differences were associated with male gender, history of myocardial infarction, and use of aspirin, statins, and antidepressants. The results demonstrate that laboratory acute stress tests continued to produce robust platelet reactivity on three activation markers among older adults over 3 years. PMID:19096987

  9. Reduced platelet-mediated and enhanced leukocyte-mediated fibrinolysis in experimentally induced diabetes in rats

    SciTech Connect

    Winocour, P.D.; Colwell, J.A.

    1985-05-01

    Studies of fibrinolytic activity in diabetes mellitus have produced conflicting results. This may be a result of methodologic insensitivity or of variable contributions of the different blood components to whole blood fibrinolysis. To explore these two possibilities, the authors used a sensitive solid-phase radiometric assay to examine the fibrinolytic activity of whole blood, platelet-rich plasma, leukocytes, and platelet- and leukocyte-poor plasma prepared from control rats and rats with streptozocin-induced diabetes at various times after induction of diabetes. Fibrinolytic activity of whole blood from diabetic rats after 7 days was significantly reduced, and remained reduced after longer durations of diabetes up to 28 days. Platelet-rich plasma from diabetic rats had decreased fibrinolytic activity, which followed the same time course of changes as in whole blood. The platelet contribution to whole blood fibrinolysis was further reduced in vivo after 14 days of diabetes by a reduced whole blood platelet count. In contrast, fibrinolytic activity of leukocytes from diabetic rats became enhanced after 7 days of diabetes. After 49 days of diabetes, the whole blood leukocyte count was reduced, and in vivo would offset the enhanced activity. Plasma fibrinolytic activity was small compared with that of whole blood and was unaltered in diabetic rats. The authors conclude that altered platelet function contributes to decreased fibrinolytic activity of whole blood in diabetic rats, and that this may be partially offset by enhanced leukocyte-mediated fibrinolysis.

  10. 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.

  11. 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

  12. Characteristics of platelet gels combined with silk.

    PubMed

    Pallotta, Isabella; Kluge, Jonathan A; Moreau, Jodie; Calabrese, Rossella; Kaplan, David L; Balduini, Alessandra

    2014-04-01

    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

  13. Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function

    PubMed Central

    Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J.; Smithgall, Thomas E.

    2015-01-01

    The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important

  14. Preparation of washed platelets from non-anticoagulated human blood.

    PubMed

    Bowry, S K; Müller-Berghaus, G

    1986-09-15

    Sodium citrate is almost always used to anticoagulate blood for the preparation of washed platelet suspensions. Several adverse effects of citrate on platelet functional responses have been reported. We investigated the extent of activation of platelets and plasmatic coagulation during the preparation of washed platelets from human blood to which no anticoagulant was added. Washed platelets from native blood (PNB) were prepared by passing freshly-drawn human blood rapidly through a mixed Sephadex G-25/G-50 column to remove divalent cations. Gel-filtered blood (GFB), diluted by the elution medium containing 0.35% albumin and 2U/ml apyrase, was obtained within 5 minutes of venepuncture. Using CaCl2, the GFB was found to contain a mean of 1.65 X 10 mM calcium. Fibrinopeptide A measurements indicated less activation of plasmatic coagulation in GFB than in citrated blood. Measurements of beta-thromboglobulin and platelet factor 4 during the various stages of the preparation of PNB showed no platelet activation. No platelet aggregates, as measured by the platelet aggregate ratio method, were observed in GFB. Transmission electron microscopy showed intact discoid platelets similar to those in platelet-rich plasma. The reduced activation of platelets and of plasmatic coagulation was due to the more effective removal of calcium ions from the blood by gel filtration than chelation by citrate. PNB may thus provide a model for studying the requirements for calcium in platelet function in the absence of any citrate-platelet interactions. PMID:2945282

  15. Mean platelet size related to glycoprotein-specific autoantibodies and platelet-associated IgG.

    PubMed

    Javela, K; Kekomäki, R

    2007-12-01

    Recent evidence suggests that platelet-associated glycoprotein-specific (GP) antibodies represent true positive autoantibodies and can therefore be taken as the gold standard. Earlier tests, which aimed at detecting platelet-associated IgG (PA-IgG), might have been hampered, e.g. by the variation of platelet size in thrombocytopenic patients. In this study, 206 samples with increased PA-IgG from consecutive thrombocytopenic patients were tested further for GP-specific antibodies with a monoclonal antibody immobilized platelet antigen test (MAIPA) using a combination of a GP IIbIIIa-specific and a GP IbIX-specific antibody for immobilization or, in a separate assay, GP V-specific antibody. Mean platelet size was recorded as forward scatter (FSC) of platelets in flow cytometric analysis of PA-IgG. GP-specific antibodies were detected in 49 (24%) of the 206 patient samples. Their presence correlated well with increased PA-IgG (R = 0.769). The mean platelet size and mean fluorescence intensity (MFI) of PA-IgG were both significantly increased in patients compared with healthy controls (n = 112; P < 0.0001). Notably, PA-IgG was associated with platelet size within the platelet population of both healthy controls and patients (R = 0.999). Further, the probability of GP IIbIIIa and/or IbIX and GP V-specific PA-IgG tended to increase with the mean platelet size of the patients (P = 0.045). In conclusion, large platelets bound more IgG than platelets of normal size, which may explain at least in part the reported low specificity of total PA-IgG measurement. As the PA-IgG displays low specificity compared with the gold standard, its use as such may be abandoned and replaced by tests for platelet-associated GP-specific autoantibodies. PMID:17988298

  16. Mean platelet size related to glycoprotein-specific autoantibodies and platelet-associated IgG

    PubMed Central

    JAVELA, K; KEKOMÄKI, R

    2007-01-01

    Recent evidence suggests that platelet-associated glycoprotein-specific (GP) antibodies represent true positive autoantibodies and can therefore be taken as the gold standard. Earlier tests, which aimed at detecting platelet-associated IgG (PA-IgG), might have been hampered, e.g. by the variation of platelet size in thrombocytopenic patients. In this study, 206 samples with increased PA-IgG from consecutive thrombocytopenic patients were tested further for GP-specific antibodies with a monoclonal antibody immobilized platelet antigen test (MAIPA) using a combination of a GP IIbIIIa-specific and a GP IbIX-specific antibody for immobilization or, in a separate assay, GP V-specific antibody. Mean platelet size was recorded as forward scatter (FSC) of platelets in flow cytometric analysis of PA-IgG. GP-specific antibodies were detected in 49 (24%) of the 206 patient samples. Their presence correlated well with increased PA-IgG (R = 0.769). The mean platelet size and mean fluorescence intensity (MFI) of PA-IgG were both significantly increased in patients compared with healthy controls (n = 112; P < 0.0001). Notably, PA-IgG was associated with platelet size within the platelet population of both healthy controls and patients (R = 0.999). Further, the probability of GP IIbIIIa and/or IbIX and GP V-specific PA-IgG tended to increase with the mean platelet size of the patients (P = 0.045). In conclusion, large platelets bound more IgG than platelets of normal size, which may explain at least in part the reported low specificity of total PA-IgG measurement. As the PA-IgG displays low specificity compared with the gold standard, its use as such may be abandoned and replaced by tests for platelet-associated GP-specific autoantibodies. PMID:17988298

  17. Mouse ES-cell-based functional assay to evaluate mutations in BRCA2

    PubMed Central

    Kuznetsov, Sergey G.; Liu, Pentao; Sharan, Shyam K.

    2009-01-01

    Individuals with mutations in breast cancer susceptibility genes, BRCA1 and BRCA2 have up to 80% risk of developing breast cancer by the age of 70. Sequencing based genetic tests are now available to identify mutation carriers in effort to reduce mortality through prevention and early diagnosis. However, lack of a suitable functional assay hinders risk assessment of more than 1900 BRCA1 and BRCA2 variants in the Breast Cancer Information Core database that do not clearly disrupt the gene product. We have established a simple, versatile and reliable assay to test for functional significance of mutations in BRCA2 using mouse embryonic stem cells (ES-cells) and bacterial artificial chromosomes (BACs) and have used it to classify 17 sequence variants. The assay is based on the ability of human BRCA2 to complement the loss of endogenous Brca2 in mouse ES-cells. This technique may also serve as a paradigm for functional analysis of mutations found in other human disease genes. PMID:18607349

  18. Association of membrane/lipid rafts with the platelet cytoskeleton and the caveolin PY14: participation in the adhesion process.

    PubMed

    Cerecedo, Doris; Martínez-Vieyra, Ivette; Maldonado-García, Deneb; Hernández-González, Enrique; Winder, Steve J

    2015-11-01

    Platelets are the most prominent elements of blood tissue involved in hemostasis at sites of blood vessel injury. Platelet cytoskeleton is responsible for their shape modifications observed during activation and adhesion to the substratum; therefore the interactions between cytoskeleton and plasma membrane are critical to modulate blood platelet functions. Several cytoskeletal components and binding partners, as well as enzymes that regulate the cytoskeleton, localize to membrane/lipid rafts (MLR) and regulate lateral diffusion of membrane proteins and lipids. Resting, thrombin-activated, and adherent human platelets were processed for biochemical studies including western-blot and immunprecipitation assays and confocal analysis were performed to characterize the interaction of MLR with the main cytoskeleton elements and β-dystroglycan as well as with the association of caveolin-1 PY14 with focal adhesion proteins. We transfected a megakaryoblast cell line (Meg-01) to deplete β-dystroglycan, subsequent to their differentiation to the platelet progenitors. Our data showed a direct interaction of the MLR with cytoskeleton to regulate platelet shape, while an association of caveolin-1 PY14 with vinculin is needed to establish focal adhesions, which are modulated for β-dystroglycan. In conclusion, caveolin-1 PY14 in association with platelet cytoskeleton participate in focal adhesions dynamics. PMID:26085308

  19. Breaking the Mold: Transcription Factors in the Anucleate Platelet and Platelet-Derived Microparticles

    PubMed Central

    Lannan, Katie L.; Sahler, Julie; Kim, Nina; Spinelli, Sherry L.; Maggirwar, Sanjay B.; Garraud, Olivier; Cognasse, Fabrice; Blumberg, Neil; Phipps, Richard P.

    2015-01-01

    Platelets are small anucleate blood cells derived from megakaryocytes. In addition to their pivotal roles in hemostasis, platelets are the smallest, yet most abundant, immune cells and regulate inflammation, immunity, and disease progression. Although platelets lack DNA, and thus no functional transcriptional activities, they are nonetheless rich sources of RNAs, possess an intact spliceosome, and are thus capable of synthesizing proteins. Previously, it was thought that platelet RNAs and translational machinery were remnants from the megakaryocyte. We now know that the initial description of platelets as “cellular fragments” is an antiquated notion, as mounting evidence suggests otherwise. Therefore, it is reasonable to hypothesize that platelet transcription factors are not vestigial remnants from megakaryocytes, but have important, if only partly understood functions. Proteins play multiple cellular roles to minimize energy expenditure for maximum cellular function; thus, the same can be expected for transcription factors. In fact, numerous transcription factors have non-genomic roles, both in platelets and in nucleated cells. Our lab and others have discovered the presence and non-genomic roles of transcription factors in platelets, such as the nuclear factor kappa β (NFκB) family of proteins and peroxisome proliferator-activated receptor gamma (PPARγ). In addition to numerous roles in regulating platelet activation, functional transcription factors can be transferred to vascular and immune cells through platelet microparticles. This method of transcellular delivery of key immune molecules may be a vital mechanism by which platelet transcription factors regulate inflammation and immunity. At the very least, platelets are an ideal model cell to dissect out the non-genomic roles of transcription factors in nucleated cells. There is abundant evidence to suggest that transcription factors in platelets play key roles in regulating inflammatory and hemostatic

  20. Platelets contribute to growth and metastasis in hepatocellular carcinoma.

    PubMed

    Bihari, Chhagan; Rastogi, Archana; Shasthry, Saggere Muralikrishna; Bajpai, Meenu; Bhadoria, Ajeet Singh; Rajesh, S; Mukund, Amar; Kumar, Anupam; Sarin, Shiv K

    2016-09-01

    To determine the association of platelets with hepatocellular carcinoma (HCC) growth and its metastasis. We examined platelets, laboratory, and radiological data of consecutive 420 HCC and 1008 cirrhosis cases. Follow-up information of platelet count in cirrhosis to HCC, pre- to post-therapy, and post-therapy to HCC outcome was analyzed. Cytokine profiling was performed in HCC and cirrhosis (n = 10 each). On the basis of imaging, HCC was divided into six subgroups. Cytosmears of HCC were assessed for platelet clustering around tumor cells. An in vitro Matrigel invasion assay was performed on human HCC cell lines using graded concentration of platelets. Baseline platelet numbers and platelet/lymphocyte ratios (PLRs) were significantly higher (p < 0.001) in HCC than cirrhosis. IL-1, IL-6, FGF, G-CSF, thrombopoietin, and VEGF were higher in HCC than cirrhosis. Platelet counts were increased after HCC conversion of cirrhosis (p < 0.001) and decreased (p < 0.001) after therapy. Platelets and PLR in recurrence cases were higher than in responders at baseline. AFP, PIVKAII, platelets, and PLR increase (p < 0.001 each) with advancement in HCC growth. Multivariate analysis showed platelets (p = 0.002), PLR (p = 0.004), and AFP (p < 0.001) associated with distant metastasis. Platelet clustering seen in 75.7% of HCC group 3, 45% in group 2, and 12.5% in group 1 cases (p < 0.001). Invaded cells in Matrigel assay positively correlated with platelet concentration. Platelets can contribute to the development, growth, invasion, and metastasis of HCC. Rising platelet count after HCC therapy is indicative of incomplete response or recurrence. PMID:27457354

  1. Fluorometric functional assay for ion channel proteins in lipid nanovesicle membranes

    NASA Astrophysics Data System (ADS)

    Patti, J. T.; Montemagno, C. D.

    2007-08-01

    Voltage-gated membrane proteins function as biomolecular transistors, making them attractive components for biologically based nanodevices. A functional assay for purified channel proteins is described and demonstrated with sodium selective, voltage-gated NaChBac ion channels. Purified NaChBac proteins were incorporated into a nanovesicle system utilizing oxonol VI, a fluorescent indicator of trans-membrane voltage. The ionophore valinomycin was used to trigger a change in membrane potential, allowing the observation of sodium permeability using a fluorometer. This method is suitable for concurrently testing a large population of purified proteins prior to incorporation in nanodevices.

  2. Evaluation of four methods for platelet compatibility testing

    SciTech Connect

    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 donor 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.

  3. A novel surface acoustic wave-based biosensor for highly sensitive functional assays of olfactory receptors.

    PubMed

    Wu, Chunsheng; Du, Liping; Wang, Di; Wang, Le; Zhao, Luhang; Wang, Ping

    2011-04-01

    Olfactory receptors, which are responsible for sensing odor molecules, form the largest G protein-coupled receptor (GPCR) family in mammalian animals. These proteins play an important role in the detection of chemical signals and signal transduction to the brain. Currently, only a limited number of olfactory receptors have been characterized, which is mainly due to the lack of sensitive and efficient tools for performing functional assays of these receptors. This paper describes a novel surface acoustic wave (SAW)-based biosensor for highly sensitive functional assays of olfactory receptors. An olfactory receptor of Caenorhabditis elegans, ODR-10, was expressed on the plasma membrane of human breast cancer MCF-7 cells, which was used as a model system for this study. For specific odorant response assays, the membrane fraction of MCF-7 cells containing ODR-10 was extracted and integrated with our SAW sensors. The response of ODR-10 to various odorants was monitored by recording the resonance frequency shifts of SAWs applied to the sensor. Our results show that heterologously expressed ODR-10 receptors can specifically respond to diacetyl, its natural ligand. Dose-dependent responses were obtained by performing measurements using various concentrations of diacetyl. The sensitivity of this biosensor is 2kHz/ng and can detect concentrations as low as 10(-10)mM, which is 10× lower than what has previously been reported. This biosensor can be used to characterize odorant response profiles of olfactory receptors and provide information rich data for functional assays of olfactory receptors. In addition to providing a greater understanding of the biological mechanisms of GPCRs, such data holds great potential in many other fields such as food industry, biomedicine, and environmental protection. PMID:21333624

  4. The routine measurement of platelet size using sodium citrate alone as the anticoagulant.

    PubMed

    Bath, P M

    1993-10-18

    Mean platelet volume (MPV), a measure of platelet size, is becoming recognised as an important marker of platelet function. However, platelets swell in edetic acid (EDTA), the standard haematology anticoagulant, in a time-dependent manner making such measurements potentially unreliable. The effect of incubation time on MPV, and platelet distribution width (PDW), as measured in EDTA, low (1:9 volume/volume with blood) or high (1:4 v/v with blood) concentration sodium citrate was studied. MPV measured in high concentration sodium citrate did not change with time in contrast to MPV measured in either low concentration sodium citrate or EDTA which both increased in an inverse exponential fashion. MPV and PDW, measured in high concentration sodium citrate, had similar within-assay and between-assay coefficients of variation as other platelet, red cell and white cell haematology variables measured in EDTA: MPV 1.4%, 2.1%; PDW 1.4%, 1.5%; MCV 0.4%, 0.7%; PC 3.1%, 6.1%; WCC 1.5%, 7.3%; Hb 2.1%, 2.4% respectively. MPV measured in EDTA and corrected for incubation time approximated to, but was higher than, the MPV measured in high concentration sodium citrate. PDW correlated inversely with platelet count (r = -0.415, 2p < 0.001). MPV may be measured in sodium citrate (at 1:4 v/v with blood) alone with a better accuracy and reproducibility than similar measurements made in EDTA. Furthermore, such measurements are not influenced by incubation time, unlike for EDTA. PMID:8115997

  5. Long-term untreated streptozotocin-diabetes leads to increased expression and elevated activity of prostaglandin H2 synthase in blood platelets.

    PubMed

    Siewiera, Karolina; Kassassir, Hassan; Talar, Marcin; Wieteska, Lukasz; Watala, Cezary

    2016-05-01

    In diabetes-related states of chronic hyperglycaemia elevated concentrations of glucose may alter the functioning of platelet enzymes involved in arachidonic acid metabolism, including prostaglandin H2 synthase (cyclooxygenase) (PGHS, COX). Therefore, the principal aim of this study was to assess the effects of experimental chronic hyperglycaemia on platelet PGHS-1 (COX-1) expression and activity. Blood platelet activation and reactivity were assessed in Sprague-Dawley rats with the 5-month streptozotocin (STZ) diabetes. The PGHS-1 abundance in platelets was evaluated with flow cytometry and Western blotting, while its activity monitored using a high resolution respirometry and the peroxidase fluorescent assay. The production of prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) in platelets were assayed immunoenzymatically. Circulating platelets from diabetic were characterised by increased size, elevated 'priming' and altered reactivity, compared to non-diabetic animals. Both Western blot analysis and flow cytometry revealed significantly elevated expressions of platelet PGHS-1 in STZ-diabetic rats (p < 0.05). We also observed significantly elevated platelet PGHS-1-related arachidonic acid metabolism in diabetic vs. non-diabetic animals, with the use of polarographic (p < 0.05) and total activity assay (p < 0.001). Such increases were accompanied by the elevated production of PGE2 (p < 0.001) and TXB2 (p < 0.05) in diabetic animals. The increased PGHS-1-dependent oxygen consumption and the total activity of PGHS-1 in diabetic animals remained very significant (p < 0.001) also upon adjusting for blood platelet PGHS-1 abundance. Therefore, our results further contribute to the explanation of the increased metabolism of arachidonic acid observed in diabetes. PMID:26325148

  6. Hematopoietic transcription factor mutations and inherited platelet dysfunction

    PubMed Central

    Songdej, Natthapol

    2015-01-01

    The molecular and genetic mechanisms in most patients with inherited platelet dysfunction are unknown. There is increasing evidence that mutations in hematopoietic transcription factors are major players in the pathogenesis of defective megakaryopoiesis and platelet dysfunction in patients with inherited platelet disorders. These hematopoietic transcription factors include RUNX1, FLI1, GATA-1, and GFI1B. Mutations involving these transcription factors affect diverse aspects of platelet production and function at the genetic and molecular levels, culminating in clinical manifestations of thrombocytopenia and platelet dysfunction. This review focuses on these hematopoietic transcription factors in the pathobiology of inherited platelet dysfunction. PMID:26097739

  7. A TR-FRET-based functional assay for screening activators of CARM1.

    PubMed

    Zeng, Hao; Wu, Jiacai; Bedford, Mark T; Sbardella, Gianluca; Hoffmann, F Michael; Bi, Kun; Xu, Wei

    2013-05-10

    Epigenetics is an emerging field that demands selective cell-permeable chemical probes to perturb, especially in vivo, the activity of specific enzymes involved in modulating the epigenetic codes. Coactivator-associated arginine methyltransferase 1 (CARM1) is a coactivator of estrogen receptor α (ERα), the main target in human breast cancer. We previously showed that twofold overexpression of CARM1 in MCF7 breast cancer cells increased the expression of ERα-target genes involved in differentiation and reduced cell proliferation, thus leading to the hypothesis that activating CARM1 by chemical activators might be therapeutically effective in breast cancer. Selective, potent, cell-permeable CARM1 activators will be essential to test this hypothesis. Here we report the development of a cell-based, time-resolved (TR) FRET assay that uses poly(A) binding protein 1 (PABP1) methylation to monitor cellular activity of CARM1. The LanthaScreen TR-FRET assay uses MCF7 cells expressing GFP-PABP1 fusion protein through BacMam gene delivery system, methyl-PABP1 specific antibody, and terbium-labeled secondary antibody. This assay has been validated as reflecting the expression and/or activity of CARM1 and optimized for high throughput screening to identify CARM1 allosteric activators. This TR-FRET platform serves as a generic tool for functional screening of cell-permeable, chemical modulators of CARM1 for elucidation of its in vivo functions. PMID:23585185

  8. A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

    PubMed Central

    Viczian, Andrea S.; Zuber, Michael E.

    2014-01-01

    Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once. PMID:24962702

  9. The acute extracellular flux (XF) assay to assess compound effects on mitochondrial function.

    PubMed

    Wang, Ruolan; Novick, Steven J; Mangum, James B; Queen, Kennedy; Ferrick, David A; Rogers, George W; Stimmel, Julie B

    2015-03-01

    Numerous investigations have linked mitochondrial dysfunction to adverse health outcomes and drug-induced toxicity. The pharmaceutical industry is challenged with identifying mitochondrial liabilities earlier in drug development and thereby reducing late-stage attrition. Consequently, there is a demand for reliable, higher-throughput screening methods for assessing the impact of drug candidates on mitochondrial function. The extracellular flux (XF) assay described here is a plate-based method in which galactose-conditioned HepG2 cells were acutely exposed to test compounds, then real-time changes in the oxygen consumption rate and extracellular acidification rate were simultaneously measured using a Seahorse Bioscience XF-96 analyzer. The acute XF assay was validated using marketed drugs known to modulate mitochondrial function, and data analysis was automated using a spline curve fitting model developed at GlaxoSmithKline. We demonstrate that the acute XF assay is a robust, sensitive screening platform for evaluating drug-induced effects on mitochondrial activity in whole cells. PMID:25381255

  10. A simple behavioral assay for testing visual function in Xenopus laevis.

    PubMed

    Viczian, Andrea S; Zuber, Michael E

    2014-01-01

    Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once. PMID:24962702

  11. Influenza Neuraminidase Subtype N1: Immunobiological Properties and Functional Assays for Specific Antibody Response

    PubMed Central

    Changsom, Don; Lerdsamran, Hatairat; Wiriyarat, Witthawat; Chakritbudsabong, Warunya; Siridechadilok, Bunpote; Prasertsopon, Jarunee; Noisumdaeng, Pirom; Masamae, Wanibtisam; Puthavathana, Pilaipan

    2016-01-01

    Influenza neuraminidase (NA) proteins expressed in TK− cells infected with recombinant vaccinia virus carrying NA gene of highly pathogenic avian influenza H5N1 virus or 2009 pandemic H1N1 (H1N1pdm) virus were characterized for their biological properties, i.e., cell localization, molecular weight (MW), glycosylation and sialidase activity. Immune sera collected from BALB/c mice immunized with these recombinant viruses were assayed for binding and functional activities of anti-NA antibodies. Recombinant NA proteins were found localized in cytoplasm and cytoplasmic membrane of the infected cells. H1N1pdm NA protein had MW at about 75 kDa while it was 55 kDa for H5N1 NA protein. Hyperglycosylation was more pronounced in H1N1pdm NA compared to H5N1 NA according to N-glycosidase F treatment. Three dimensional structures also predicted that H1N1 NA globular head contained 4 and that of H5N1 contained 2 potential glycosylation sites. H5N1 NA protein had higher sialidase activity than H1N1pdm NA protein as measured by both MUNANA-based assay and fetuin-based enzyme-linked lectin assay (ELLA). Plaque reduction assay demonstrated that anti-NA antibody could reduce number of plaques and plaque size through inhibiting virus release, not virus entry. Assay for neuraminidase-inhibition (NI) antibody by ELLA showed specific and cross reactivity between H5N1 NA and H1N1pdm NA protein derived from reverse genetic viruses or wild type viruses. In contrast, replication-inhibition assay in MDCK cells showed that anti-H1N1 NA antibody moderately inhibited viruses with homologous NA gene only, while anti-H5N1 NA antibody modestly inhibited the replication of viruses containing homologous NA gene and NA gene derived from H1N1pdm virus. Anti-H1N1 NA antibody showed higher titers of inhibiting virus replication than anti-H5N1 NA antibody, which are consistent with the results on reduction in plaque numbers and sizes as well as in inhibiting NA enzymatic activity. No assay showed cross

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

    PubMed

    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; Poncz, Mortimer

    2015-06-01

    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

  13. Platelets: covert regulators of lymphatic development.

    PubMed

    Bertozzi, Cara C; Hess, Paul R; Kahn, Mark L

    2010-12-01

    The field of platelet biology has rapidly expanded beyond the classical role of platelets in preventing blood loss and orchestrating clot formation. Despite the lack of transcriptional ability of these anuclear cell fragments, platelet function is now thought to encompass such diverse contexts as tissue repair, immune activation, primary tumor formation, and metastasis. Recent studies from multiple groups have turned the spotlight on an exciting new role for platelets in the formation of lymphatic vessels during embryonic development. Genetic experiments demonstrate that podoplanin, a transmembrane protein expressed on lymphatic endothelial cells, engages the platelet C-type lectin-like receptor 2 (CLEC-2) when exposed to blood, leading to SYK-SLP-76-dependent platelet activation. When components of this pathway are disrupted, aberrant vascular connections form, resulting in blood-lymphatic mixing. Furthermore, platelet-null embryos manifest identical blood-lymphatic mixing. The identification of platelets as the critical cell type mediating blood-lymphatic vascular separation raises new questions in our understanding of lymphatic development and platelet biology. PMID:21071706

  14. Unscheduled DNA Synthesis: The Clinical and Functional Assay for Global Genomic DNA Nucleotide Excision Repair

    PubMed Central

    Latimer, Jean J.; Kelly, Crystal M.

    2016-01-01

    The unscheduled DNA synthesis (UDS) assay measures the ability of a cell to perform global genomic nucleotide excision repair (NER). This chapter provides instructions for the application of this technique by creating 6-4 photoproducts and pyrimidine dimers using UV-C irradiation. This procedure is designed specifically for quantification of the 6-4 photoproducts. Repair is quantified by the amount of radioactive thymidine incorporated during repair synthesis after this insult, and radioactivity is evaluated by grain counting after autoradiography. The results are used to clinically diagnose human DNA repair deficiency disorders and provide a basis for investigation of repair deficiency in human tissues or tumors. No other functional assay is available that directly measures the capacity to perform NER on the entire genome without the use of specific antibodies. Since live cells are required for this assay, explant culture techniques must be previously established. Host cell reactivation (HCR), as discussed in Chapter 37, is not an equivalent technique, as it measures only transcription-coupled repair (TCR) at active genes, a small subset of total NER. PMID:24623250

  15. NMR Binding and Functional Assays for Detecting Inhibitors of S. aureus MnaA.

    PubMed

    Hou, Yan; Mayhood, Todd; Sheth, Payal; Tan, Christopher M; Labroli, Marc; Su, Jing; Wyss, Daniel F; Roemer, Terry; McCoy, Mark A

    2016-07-01

    Nonessential enzymes in the staphylococcal wall teichoic acid (WTA) pathway serve as highly validated β-lactam potentiation targets. MnaA (UDP-GlcNAc 2-epimerase) plays an important role in an early step of WTA biosynthesis by providing an activated form of ManNAc. Identification of a selective MnaA inhibitor would provide a tool to interrogate the contribution of the MnaA enzyme in the WTA pathway as well as serve as an adjuvant to restore β-lactam activity against methicillin-resistant Staphylococcus aureus (MRSA). However, development of an epimerase functional assay can be challenging since both MnaA substrate and product (UDP-GlcNAc/UDP-ManNAc) share an identical molecular weight. Herein, we developed a nuclear magnetic resonance (NMR) functional assay that can be combined with other NMR approaches to triage putative MnaA inhibitors from phenotypic cell-based screening campaigns. In addition, we determined that tunicamycin, a potent WTA pathway inhibitor, inhibits both S. aureus MnaA and a functionally redundant epimerase, Cap5P. PMID:27028606

  16. The utility of International Society on Thrombosis and Haemostasis-Bleeding Assessment Tool and other bleeding questionnaires in assessing the bleeding phenotype in two platelet function defects.

    PubMed

    Kaur, Harmanpreet; Borhany, Munira; Azzam, Hanan; Costa-Lima, Carolina; Ozelo, Margareth; Othman, Maha

    2016-07-01

    The main objective of this study is to investigate the utility of International Society on Thrombosis and Haemostasis-Bleeding Assessment Tool (ISTH-BAT) in comparison with the condensed form of Molecular and Clinical Markers for the Diagnosis and Management of type 1 and WHO BATs, in assessing bleeding in two well known and clinically significant platelet function defects. Thirty-eight patients previously diagnosed with Glanzmann's thrombasthenia and 10 with Bernard-Soulier syndrome (BSS) were analyzed. Bleeding scores were significantly higher than that of controls using both electronic bleeding questionnaire (eBQ) and ISTH-BAT with no significant difference between both tools. ISTH-BAT had a sensitivity, specificity, positive predictive value and negative predictive value of 100%, 76.2%, 0.9 and 1. This was closely similar to eBQ. Both ISTH-BAT and eBQ are efficient in BSS and Glanzmann's thrombasthenia. However, given the ISTH recommendation, ISTH-BAT should be adopted. Larger study including other platelet defects will enhance its utility and support the integration of bleeding scores with standardized laboratory testing to allow for a universal diagnostic approach to patients with suspected bleeding disorders. PMID:27100304

  17. Aspirin sensitivity of platelet aggregation in diabetes mellitus.

    PubMed

    Albert, Stewart G; Hasnain, Bibi I; Ritter, Detlef G; Joist, J Heinrich; Mooradian, Arshag D

    2005-11-01

    Although aspirin is cardioprotective in high-risk populations, many with diabetes mellitus (DM) are unresponsive to these benefits. We questioned whether cardiovascular unresponsiveness might be demonstrated by lack of aspirin sensitivity to in vitro platelet functions especially in subjects with poorly controlled diabetes. Six women and 4 men (48+/-8 years [mean+/-S.D.]), selected for poor control (glycohemoglobin 11.9+/-2.2%) and 10 sex-age (+/-5 years) matched controls received 81 mg aspirin daily. There was a 2-week washout from aspirin and related drugs. After the aspirin dose on day-7, blood for platelet aggregation assays, and 24-h urine for 2,3 dinor thromboxane B2 (TxB2) and 2,3 dinor 6-keto (PGF1alpha) were obtained. Aspirin sensitivity was defined as inhibition (i.e., lower than expected) platelet aggregation after exposure to an agonist. Those with diabetes and controls were sensitive to aspirin inhibition of platelet aggregation induced by 1.6 mM arachidonic acid (9.5+/-3.9% versus 9.1+/-3.1%, normal range 40-100%) and by 0.83 microg/mL collagen (17.4+/-13.9% versus 13.2+/-9.3%, normal range 60-93%), respectively. Aspirin sensitivity to 2 microM ADP was present in five with diabetes and five controls. Urinary prostaglandin metabolites were suppressed below reference ranges, without differences between those with DM or controls for TxB2 (350+/-149 pg/mg versus 348+/-93 pg/mg creatinine) and PGF1alpha (255+/-104 pg/mg versus 222+/-88 pg/mg creatinine). In conclusion, in poorly controlled diabetes, there was no differential lack of aspirin sensitivity to platelet aggregation, or lack of aspirin suppression of urinary TxB2 or PGF1alpha, compared with controls on aspirin. Despite suppression of urinary prostaglandin metabolites, aspirin resistance was most apparent to ADP-mediated platelet aggregation. It is not known what level of inhibition of in vitro tests is necessary for the cardioprotective benefits of aspirin in diabetes mellitus. Thus, the lack of

  18. Isolation of Mouse Hair Follicle Bulge Stem Cells and Their Functional Analysis in a Reconstitution Assay.

    PubMed

    Zheng, Ying; Hsieh, Jen-Chih; Escandon, Julia; Cotsarelis, George

    2016-01-01

    The hair follicle (HF) is a dynamic structure readily accessible within the skin, and contains various pools of stem cells that have a broad regenerative potential during normal homeostasis and in response to injury. Recent discoveries demonstrating the multipotent capabilities of hair follicle stem cells and the easy access to skin tissue make the HF an attractive source for isolating stem cells and their subsequent application in tissue engineering and regenerative medicine. Here, we describe the isolation and purification of hair follicle bulge stem cells from mouse skin, and hair reconstitution assays that allows the functional analysis of multipotent stem cells. PMID:27431247

  19. Isolation, Culture, Functional Assays, and Immunofluorescence of Myofiber-Associated Satellite Cells.

    PubMed

    Vogler, Thomas O; Gadek, Katherine E; Cadwallader, Adam B; Elston, Tiffany L; Olwin, Bradley B

    2016-01-01

    Adult skeletal muscle stem cells, termed satellite cells, regenerate and repair the functional contractile cells in adult skeletal muscle called myofibers. Satellite cells reside in a niche between the basal lamina and sarcolemma of myofibers. Isolating single myofibers and their associated satellite cells provides a culture system that partially mimics the in vivo environment. We describe methods for isolating and culturing intact individual myofibers and their associated satellite cells from the mouse extensor digitorum longus muscle. Following dissection and isolation of individual myofibers we provide protocols for myofiber transplantation, satellite cell transfection, immune detection of satellite cell antigens, and assays to examine satellite cell self-renewal and proliferation. PMID:27492171

  20. Current and emerging approaches for evaluating platelet disorders.

    PubMed

    Podda, G; Femia, E A; Cattaneo, M

    2016-05-01

    Platelets play a central role in physiological hemostasis and also in pathological thrombosis. It is well established that congenital or acquired abnormalities of platelet function are associated with a heightened risk of bleeding of variable severity and excessive hemorrhage after surgery or trauma. Several kinds of different platelet function tests have been developed over the years to identify or diagnose platelet function disorders. The use of these tests for the assessment of thrombotic risk or for monitoring the effects of drugs inhibiting platelet function is not well established. Light transmission aggregometry (LTA) is the gold standard for the study of patients with defects of platelet function. Its results are affected by several pre-analytical and analytical variables. The Subcommittee on Platelet Physiology of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis published official guidelines for the standardization of the variables affecting LTA, which should be followed to harmonize the procedures across different laboratories worldwide. The lumi-aggregometer, a modification of LTA that measures platelet secretion in parallel with aggregation, is preferable to LTA for diagnosing inherited defects of platelet function, because it is more sensitive to the most common disorders, which are characterized by abnormalities of platelet secretion. LTA (or lumi-aggregometry) is useful as a first screening test of patients with the clinical suspicion of defects of platelet function, because it helps to provide an interim diagnostic hypothesis, which can then be confirmed or discounted using appropriate and specific tests. PMID:27426860

  1. Platelet generation in vivo and in vitro.

    PubMed

    Wang, Biao; Zheng, Jiansheng

    2016-01-01

    Platelet (PLT) transfusion, which is the primary cell therapy for thrombocytopenia, has been a source of concern in recent years due to its limitations of donor-dependent supply and soaring costs. In vitro platelet generation on an industrial scale is a possible solution requiring exploration. The technology of platelet generation ex vivo has been widely studied across the world, though the mechanisms of physiological thrombopoiesis and platelet biology function in vivo still remain elusive today. Various culture systems have been studied, most of which proved quite inefficient in generating functional platelets ex vivo, so there is still a long way to reach our ultimate goal of generating a fully functional platelet in vitro on an industrial scale. This review integrates the latest research into physiological platelet biogenesis and ex vivo-platelet/megakaryocyte (MK) generation protocols with a focus on the ability to generate PLT/MK in large quantities, summarizes current culture systems based on induced human pluripotent stem cells and adipose-derived stem cells, and discusses significant challenges that must be overcome for these approaches to be perfected. PMID:27390629

  2. Platelets and their interactions with other immune cells

    PubMed Central

    Lam, Fong W.; Vijayan, K. Vinod; Rumbaut, Rolando E.

    2015-01-01

    Platelets are anucleate blood cells, long known to be critically involved in hemostasis and thrombosis. In addition to their role in blood clots, increasing evidence reveals significant roles for platelets in inflammation and immunity. However, the notion that platelets represent immune cells is not broadly recognized in the field of Physiology. This manuscript reviews the role of platelets in inflammation and immune responses, and highlights their interactions with other immune cells, including examples of major functional consequences of these interactions. PMID:26140718

  3. Functional Analysis of Protein Tyrosine Phosphatases in Thrombosis and Hemostasis.

    PubMed

    Rahmouni, Souad; Hego, Alexandre; Delierneux, Céline; Wéra, Odile; Musumeci, Lucia; Tautz, Lutz; Oury, Cécile

    2016-01-01

    Platelets are small blood cells derived from cytoplasmic fragments of megakaryocytes and play an essential role in thrombosis and hemostasis. Platelet activation depends on the rapid phosphorylation and dephosphorylation of key signaling molecules, and a number of kinases and phosphatases have been identified as major regulators of platelet function. However, the investigation of novel signaling proteins has suffered from technical limitations due to the anucleate nature of platelets and their very limited levels of mRNA and de novo protein synthesis. In the past, experimental methods were restricted to the generation of genetically modified mice and the development of specific antibodies. More recently, novel (phospho)proteomic technologies and pharmacological approaches using specific small-molecule inhibitors have added additional capabilities to investigate specific platelet proteins.In this chapter, we report methods for using genetic and pharmacological approaches to investigate the function of platelet signaling proteins. While the described experiments focus on the role of the dual-specificity phosphatase 3 (DUSP3) in platelet signaling, the presented methods are applicable to any signaling enzyme. Specifically, we describe a testing strategy that includes (1) aggregation and secretion experiments with mouse and human platelets, (2) immunoprecipitation and immunoblot assays to study platelet signaling events, (3) detailed protocols to use selected animal models in order to investigate thrombosis and hemostasis in vivo, and (4) strategies for utilizing pharmacological inhibitors on human platelets. PMID:27514813

  4. Comparison of Aggregometry with Flow Cytometry for the Assessment of Agonists´-Induced Platelet Reactivity in Patients on Dual Antiplatelet Therapy

    PubMed Central

    Gremmel, Thomas; Koppensteiner, Renate; Panzer, Simon

    2015-01-01

    patients with HRPR by LTA AA and MEA AA (both p<0.02). In contrast, P-selectin expression in response to AA was similar in patients without and with HRPR by the VerifyNow aspirin assay (p = 0.5), and platelet surface activated GPIIb/IIIa in response to AA did not differ significantly between patients without and with HRPR to AA by all test systems (all p>0.1). In conclusion, ADP-induced platelet reactivity by aggregometry translates partly into flow cytometry. In contrast, AA-induced platelet reactivity correlates poorly between different platelet aggregation tests, and between aggregometry and flow cytometry. Overall, both approaches capture different aspects of platelet function and are therefore not interchangeable in the assessment of agonists´-induced platelet reactivity. Clinical outcome data are needed to determine which test systems and settings are associated with different in vivo consequences. PMID:26058047

  5. Multi-parameter assessment of platelet inhibition and its stability during aspirin and clopidogrel therapy

    PubMed Central

    Timur, A. Anil; Murugesan, Gurunathan; Zhang, Li; Barnard, John; Bhatt, Deepak L.; Kottke-Marchant, Kandice

    2014-01-01

    Introduction Poor response to antiplatelet drugs is associated with adverse outcomes. We assessed platelet inhibition and its stability and tested correlation and agreement between platelet function assays. Methods Peripheral blood from 58 patients on both aspirin and clopidogrel who underwent percutaneous coronary intervention (PCI) was collected at hospital discharge (visit-1) and at 30–90 days (visit-2). Platelet function was measured using light transmission aggregometry (LTA-AA and LTA-ADP), VerifyNow (Aspirin; ARU and P2Y12; PRU), ex vivo TxB2, urinary 11dhTxB2, and VASP (PRI) assays. Data were analyzed as continuous, quartiles and binary. Patients were defined as aspirin poor responder (PR) with ARU ≥550, LTA-AA maximum ≥20%, TxB2 ≥1 ng/mL or 11dhTxB2 ≥1,500 pg/mg of creatinine and as clopidogrel PR with PRU ≥240, PRU ≥208, LTA-ADP maximum ≥40%, PRI ≥50%, or PRI ≥66%. Results Aspirin PR was 3–33% and clopidogrel PR was 10–35% in visit-1. LTA-AA, 11dhTxB2, and all clopidogrel-response measures showed correlation and agreement between visit-1 and visit-2. The highest agreement between two visits was revealed by PRU ≥240 and PRI ≥66% (PRU-κ=0.7, 95% CI=0.47, 0.93; PRI-κ=0.69, 95% CI=0.42, 0.95, p-values<0.001). Comparison of platelet function assays in a single visit (Visit-1) revealed a poor correlation between LTA-AA and 11dhTxB2 assays and no agreement among aspirin-response assays. The highest correlation and agreement were obtained between VerifyNow P2Y12 and VASP assays (rho=0.7, p-value<0.001 and PRU ≥208-PRI-κ=0.41–0.42, 95% CI=0.13, 0.69, p-values<0.001). Conclusions Platelet inhibition is stable during aspirin and clopidogrel treatment. Clopidogrel-response assays correlate and agree with each other better than aspirin-response assays. PMID:24852729

  6. Micro-Arrayed Human Embryonic Stem Cells-Derived Cardiomyocytes for In Vitro Functional Assay

    PubMed Central

    Serena, Elena; Cimetta, Elisa; Zatti, Susi; Zaglia, Tania; Zagallo, Monica; Keller, Gordon; Elvassore, Nicola

    2012-01-01

    Introduction The heart is one of the least regenerative organs in the body and any major insult can result in a significant loss of heart cells. The development of an in vitro-based cardiac tissue could be of paramount importance for many aspects of the cardiology research. In this context, we developed an in vitro assay based on human cardiomyocytes (hCMs) and ad hoc micro-technologies, suitable for several applications: from pharmacological analysis to physio-phatological studies on transplantable hCMs. We focused on the development of an assay able to analyze not only hCMs viability, but also their functionality. Methods hCMs were cultured onto a poly-acrylamide hydrogel with tunable tissue-like mechanical properties and organized through micropatterning in a 20×20 array. Arrayed hCMs were characterized by immunofluorescence, GAP-FRAP analyses and live and dead assay. Their functionality was evaluated monitoring the excitation-contraction coupling. Results Micropatterned hCMs maintained the expression of the major cardiac markers (cTnT, cTnI, Cx43, Nkx2.5, α-actinin) and functional properties. The spontaneous contraction frequency was (0.83±0.2) Hz, while exogenous electrical stimulation lead to an increase up to 2 Hz. As proof of concept that our device can be used for screening the effects of pathological conditions, hCMs were exposed to increasing levels of H2O2. Remarkably, hCMs viability was not compromised with exposure to 0.1 mM H2O2, but hCMs contractility was dramatically suppressed. As proof of concept, we also developed a microfluidic platform to selectively treat areas of the cell array, in the perspective of performing multi-parametric assay. Conclusions Such system could be a useful tool for testing the effects of multiple conditions on an in vitro cell model representative of human heart physiology, thus potentially helping the processes of therapy and drug development. PMID:23152776

  7. Interactions of human blood-platelets with vaccinia

    SciTech Connect

    Vernon, C.E.B.

    1989-01-01

    These investigations were conducted to determine whether vaccinia (strain WR) adsorbs to the human platelet and alters specific platelet activities, namely, the uptake of {sup 14}C-serotonin, the release of {sup 14}C-serotonin and also the release of {sup 14}C-serotonin stimulated by thrombin. Vaccinia did not alter the platelet uptake of {sup 14}C-serotonin. To determine if vaccinia induces a release of {sup 14}C-serotonin from platelets, vaccinia was added to washed or unwashed {sup 14}C-serotonin labeled platelets, and the release of {sup 14}C-Serotonin into the supernatant was measured. Less than 8% of the {sup 14}C-Serotonin was released. The action of vaccinia to alter the platelet release of {sup 14}C-serotonin induced by thrombin was monitored by measuring the radioactivity released from thrombin stimulated {sup 14}C-serotonin labeled platelets incubated with or without vaccinia. Vaccinia inhibited the thrombin induced release of {sup 14}C-serotonin from platelets at a virus to platelet ratios of 5 through 80 plaque forming units (p.f.u.)/platelet. The inhibition was dose dependent. The binding of virus to platelets was determined by a plaque assay of a washed mixture of vaccinia virus and platelets. After inoculation of mixture onto a monolayer of BSC40 cells at a virus to platelet ratio of 0.1 p.f.u./platelet, 50 cell-bound-virus per 130,000-150,000 platelets were enumerated. Vaccinia was observed to inhibit the thrombin induced clot formation of plasma by a thrombin clotting time test. Scanning electron micrographs of the clot formed in the presence of vaccinia revealed a close packed fibrous structure lacking the cross-linked mesh-like pattern seen in a normal clot. Transmission electron micrographs showed an increase in the length and a close packing of the fibrin threads.

  8. Platelet antibody in idiopathic thrombocytopenic purpura and other thrombocytopenias

    SciTech Connect

    Sugiura, K.; Steiner, M.; Baldini, M.G.

    1980-10-01

    Platelet-associated immunoglobulin was measured by the use of fluorescent anti-1gG antibody. The method is simple, rapid, and sensitive and provides a precise quantitive assay of bound (direct) and free (indirect) 1gG with platelet specificity. We have evaluated this test in 30 normal volunteers and in 50 patients with immune and nonimmune, treated and untreated thrombocytopenias. All patients with immune thrombocytopenias (acute and chronic idiopathic thrombocytopenic purpura and systemic lupus erythematosus) having platelet counts < 100,000/..mu..l had elevated levels of platelet-bound 1gG and 86% had also positive results in the indirect assay. All patients with nonimmunological thrombocytopenias showed normal results in the direct and indirect assay of platelet-associated immunoglobulin. In patients studied repeatedly during the course of their illness, an inverse relation was found between platelet count and level of platelet-bound 1gG. Patients with systemic lupus erythematosus presented clear exceptions to this rule. Investigations of the absorbability of platelet autoantibodies and alloantibodies showed that this assay can readily differentiate between these two antibody species and can also identify specificities of alloantibodies.

  9. Aminopropyltriethoxysilane-mediated surface functionalization of hydroxyapatite nanoparticles: synthesis, characterization, and in vitro toxicity assay

    PubMed Central

    Wang, Shige; Wen, Shihui; Shen, Mingwu; Guo, Rui; Cao, Xueyan; Wang, Jianhua; Shi, Xiangyang

    2011-01-01

    Background We report on aminopropyltriethoxysilane (APTS)-mediated surface modification of nanohydroxyapatite with different surface functional groups for potential biomedical applications. In this study, nanohydroxyapatite covalently linked with APTS (n-HA-APTS) was reacted with acetic anhydride or succinic anhydride to produce neutralized (n-HA-APTS. Ac) or negatively charged (n-HA-APTS.SAH) nanohydroxyapatite, respectively. Nanohydroxyapatite formed with amine, acetyl, and carboxyl groups was extensively characterized using Fourier transform infrared spectroscopy, transmission electron microscopy, 1H nuclear magnetic resonance spectroscopy, X-ray diffraction, inductively coupled plasma-atomic emission spectroscopy, and zeta potential measurements. Results In vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay revealed that the slight toxicity of the amine-functionalized n-HA-APTS could be eliminated by post-functionalization of APTS amines to form acetyl and carboxyl groups. Blood compatibility assessment demonstrated that the negligible hemolytic activity of the pristine nanohydroxyapatite particles did not appreciably change after APTS-mediated surface functionalization. Conclusion APTS-mediated functionalization of nanohydroxyapatite with different surface groups may be useful for further functionalization of nanohydroxyapatite with biologically active materials, thereby providing possibilities for a broad range of biomedical applications. PMID:22267929

  10. Clinical application of radiolabelled platelets

    SciTech Connect

    Kessler, C. )

    1990-01-01

    This book presents papers on the clinical applications of radiolabelled platelets. The papers are grouped into six sections on platelet labelling techniques, radiolabelled platelets in cardiology, monitoring of antiplatelet therapy, platelet scintigraphy in stroke patients, platelet scintigraphy in angiology, and platelet scintigraphy in hematology and other clinical applications, including renal transplant rejection.

  11. Relative performance of a TGS for the assay of drummed waste as function of collimator opening

    SciTech Connect

    Kane, S.C.; Croft, S.; McClay, P.; Venkataraman, R.; Villani, M.F.

    2007-07-01

    , one may wish to widen the repertoire but there is currently little guidance as to the trade-off involved. In this work, we address this weakness by studying the performance of a typical TGS arrangement as a function of collimator opening, scan pattern and scan time for a representative selection of simulated waste forms. Our focus is on assessing the impact on the precision and accuracy of the quantitative assay result but we also report the utility of the imaging information in confirming acceptable knowledge about the packages. (authors)

  12. Surveys, simulation and single-cell assays relate function and phylogeny in a lake ecosystem.

    PubMed

    Preheim, Sarah P; Olesen, Scott W; Spencer, Sarah J; Materna, Arne; Varadharajan, Charuleka; Blackburn, Matthew; Friedman, Jonathan; Rodríguez, Jorge; Hemond, Harold; Alm, Eric J

    2016-01-01

    Much remains unknown about what drives microbial community structure and diversity. Highly structured environments might offer clues. For example, it may be possible to identify metabolically similar species as groups of organisms that correlate spatially with the geochemical processes they carry out. Here, we use a 16S ribosomal RNA gene survey in a lake that has chemical gradients across its depth to identify groups of spatially correlated but phylogenetically diverse organisms. Some groups had distributions across depth that aligned with the distributions of metabolic processes predicted by a biogeochemical model, suggesting that these groups performed biogeochemical functions. A single-cell genetic assay showed, however, that the groups associated with one biogeochemical process, sulfate reduction, contained only a few organisms that have the genes required to reduce sulfate. These results raise the possibility that some of these spatially correlated groups are consortia of phylogenetically diverse and metabolically different microbes that cooperate to carry out geochemical functions. PMID:27562262

  13. Platelet activation during angiotensin II infusion in healthy volunteers.

    PubMed

    Larsson, P T; Schwieler, J H; Wallén, N H

    2000-01-01

    The present study was undertaken to evaluate the effects of an intravenous infusion of angiotensin II (10 ng/kg per min) on platelet function and coagulation in vivo in 18 healthy males. The infusion increased mean arterial pressure by 23+/-2 mm Hg. Plasma beta-thromboglobulin levels, reflecting platelet secretion, increased by 66+/-24% during the infusion, as did also platelet surface expression of P-selectin as measured by flow cytometry. Platelet fibrinogen binding increased, whereas platelet aggregability, assessed by ex vivo filtragometry, was unaltered. Angiotensin II caused mild activation of the coagulation cascade with increases in plasma levels of thrombin-antithrombin complex and prothrombin fragment F1 + 2. In conclusion, angiotensin II has mild platelet-activating effects in vivo and also enhances coagulation. Markers of platelet secretion are significantly increased, whereas markers of platelet aggregability are less affected. The clinical relevance of these findings remains to be clarified. PMID:10691100

  14. Detection of platelet isoantibodies by (3H)serotonin platelet release and its clinical application to the problem of platelet matching.

    PubMed Central

    Gockerman, J P; Bowman, R P; Conrad, M E

    1975-01-01

    The detection of platelet isoantibodies by the release of (3H)serotonin from platelets has been evaluated. The conditions for optimal release of (3H)serotonin with platelet isoantibodies using a microtechnique have been defined. A group of cardiac surgery patients were followed pre- and post-transfusions, with 48percent developing a positive serotonin release assay. Of these patients, 16percent also had a platelet complement-fixing and/or lymphocytotoxic isoantibody. There was variation in the degree of correlation between (3H)serotonin release and lymphocytotoxicity using individual National Institutes of Health typing serum. The matching obtained between family members by both techniques showed a close correlation when each technique was evaluated separately using the same NIH typing serum. The detection of iso-antibodies in patients with hematological malignancies correlated with the unresponsiveness to unmatched platelet transfusions in 15 out of 17 cases. The use of the patient's isoantibody to matched platelets of family members by (3H)serotonin release correlated well with the clinical response to transfusion with these platelets. The data suggest that (a) platelet isoantibodies can be detected with increased frequency by (3H)serotonin release; (b) (3H)serotonin release is a specific reaction depending on the surface antigen of the platelet; and (c) the method can be used to match compatible family members for platelet transfusions. PMID:1109183

  15. Cross-match-compatible platelets improve corrected count increments in patients who are refractory to randomly selected platelets

    PubMed Central

    Elhence, Priti; Chaudhary, Rajendra K.; Nityanand, Soniya

    2014-01-01

    Background Cross-match-compatible platelets are used for the management of thrombocytopenic patients who are refractory to transfusions of randomly selected platelets. Data supporting the effectiveness of platelets that are compatible according to cross-matching with a modified antigen capture enzyme-linked immunosorbent assay (MAC-ELISA or MACE) are limited. This study aimed to determine the effectiveness of cross-match-compatible platelets in an unselected group of refractory patients. Materials and methods One hundred ABO compatible single donor platelet transfusions given to 31 refractory patients were studied. Patients were defined to be refractory if their 24-hour corrected count increment (CCI) was <5×109/L following two consecutive platelet transfusions. Platelets were cross-matched by MACE and the CCI was determined to monitor the effectiveness of platelet transfusions. Results The clinical sensitivity, specificity, positive predictive value and negative predictive value of the MACE-cross-matched platelets for post-transfusion CCI were 88%, 54.6%, 39.3% and 93.2%, respectively. The difference between adequate and inadequate post-transfusion 24-hour CCI for MACE cross-matched-compatible vs incompatible single donor platelet transfusions was statistically significant (p=0.000). The 24-hour CCI (mean±SD) was significantly higher for cross-match-compatible platelets (9,250±026.6) than for incompatible ones (6,757.94±2,656.5) (p<0.0001). Most of the incompatible cross-matches (73.2%) were due to anti-HLA antibodies, alone (55.3% of cases) or together with anti-platelet glycoprotein antibodies (17.9%). Discussion The clinical sensitivity and negative predictive value of platelet cross-matching by MACE were high in this study and such tests may, therefore, be used to select compatible platelets for refractory patients. A high negative predictive value demonstrates the greater chance of an adequate response with cross-matched-compatible platelets. PMID

  16. Platelet peripheral benzodiazepine receptors are decreased in Parkinson's disease

    SciTech Connect

    Bonuccelli, U.; Nuti, A.; Del Dotto, P.; Piccini, P.; Martini, C.; Giannacccini, G.; Lucacchini, A.; Muratorio, A. )

    1991-01-01

    Peripheral benzodiazepine (BDZ) receptors are located in a variety of tissues, including platelets, in the nuclear and/or mitochondrial membranes. The authors studied the density of peripheral BDZ receptors in platelets of 10 de novo Parkinson's disease (PD) patients, 18 PD patients treated with a levodopa/carbidopa combination, and in 15 healthy subjects matched for sex and age. The binding assay was conducted using ({sup 3}H)PK 11195, a specific ligand for peripheral BDZ receptors. A significant decrease in the density of ({sup 3}H)PK 11195 binding sites has been observed in PD patients with respect to controls but not between de novo and treated PD patients. No correlation has been found between the decrease in density of ({sup 3}H)PK 11195 binding sites in platelets and either the duration or severity of PD. Peripheral BDZ receptors are implicated in the regulation of mitochondrial respiratory function. Thus, their decrease in PD might parallel the abnormalities in mitochondrial function recently found in this neurologic disease.

  17. A Functional Assay to Assess Connexin 43-Mediated Cell-to-Cell Communication of Second Messengers in Cultured Bone Cells.

    PubMed

    Stains, Joseph P; Civitelli, Roberto

    2016-01-01

    Cell-to-cell transfer of small molecules is a fundamental way by which multicellular organisms coordinate function. Recent work has highlighted the complexity of biologic responses downstream of gap junctions. As the connexin-regulated effectors are coming into focus, there is a need to develop functional assays that allow specific testing of biologically relevant second messengers. Here, we describe a modification of the classic gap junction parachute assay to assess biologically relevant molecules passed through gap junctions. PMID:27207296

  18. Northern elephant seal platelets: analysis of shape change and response to platelet agonists.

    PubMed

    Field, C L; Walker, N J; Tablin, F

    2001-02-15

    Blood platelets have a vital role in the maintenance of normal mammalian hemostasis. Rapid pressure changes and temperatures lower than 20 degrees C cause activation of human and terrestrial mammal platelets. Elephant seals are routinely subjected to pressures as high as 150 atm, yet do not suffer from the thrombotic effects of platelet activation associated with rapid decompression. We examined the ultrastructure of Northern elephant seal (Mirounga angustirostris) platelets and their functional and morphological response to various platelet agonists. Unstimulated elephant seal platelets are discoid cells, with a microtubule coil, randomly dispersed alpha and dense granules, and glycogen granules. There are well-defined areas of membranous invaginations that indicate the presence of an open canalicular system (OCS). Aggregometry was used to determine the response of elephant seal platelets to various platelet agonists. Dose-dependent curves were generated for thrombin, collagen, and adenosine diphosphate (ADP). Platelet response to thrombin was dose-dependent and was maximal at 2.5 U/ml. Platelets collected in sodium citrate had blunted responses to both ADP and collagen. ADP stimulation caused only reversible, primary activation (shape change) at > or = 5 microM, while platelets did not aggregate in response to any concentration of collagen. Platelets collected in sodium heparin did respond fully to both to ADP and collagen. There was small, reversible shape change in response to ristocetin, but no response to epinephrine. Decreased sensitivity of elephant seal platelets to agonists may be a protective mechanism developed in response to rapid pressure changes and cold temperatures associated with adaptation to an extreme environment. PMID:11248288

  19. Platelet glycoprotein Ibα supports experimental lung metastasis

    PubMed Central

    Jain, Shashank; Zuka, Masahiko; Liu, Jungling; Russell, Susan; Dent, Judith; Guerrero, José A.; Forsyth, Jane; Maruszak, Brigid; Gartner, T. Kent; Felding-Habermann, Brunhilde; Ware, Jerry

    2007-01-01

    The platelet paradigm in hemostasis and thrombosis involves an initiation step that depends on platelet membrane receptors binding to ligands on a damaged or inflamed vascular surface. Once bound to the surface, platelets provide a unique microenvironment supporting the accumulation of more platelets and the elaboration of a fibrin-rich network produced by coagulation factors. The platelet-specific receptor glycoprotein (GP) Ib-IX, is critical in this process and initiates the formation of a platelet-rich thrombus by tethering the platelet to a thrombogenic surface. A role for platelets beyond the hemostasis/thrombosis paradigm is emerging with significant platelet contributions in both tumorigenesis and inflammation. We have established congenic (N10) mouse colonies (C57BL/6J) with dysfunctional GP Ib-IX receptors in our laboratory that allow us an opportunity to examine the relevance of platelet GP Ib-IX in syngeneic mouse models of experimental metastasis. Our results demonstrate platelet GP Ib-IX contributes to experimental metastasis because a functional absence of GP Ib-IX correlates with a 15-fold reduction in the number of lung metastatic foci using B16F10.1 melanoma cells. The results demonstrate that the extracellular domain of the α-subunit of GP Ib is the structurally relevant component of the GP Ib-IX complex contributing to metastasis. Our results support the hypothesis that platelet GP Ib-IX functions that support normal hemostasis or pathologic thrombosis also contribute to tumor malignancy. PMID:17494758

  20. Nanoparticle biointerfacing by platelet membrane cloaking.

    PubMed

    Hu, Che-Ming J; Fang, Ronnie H; Wang, Kuei-Chun; Luk, Brian T; Thamphiwatana, Soracha; Dehaini, Diana; Nguyen, Phu; Angsantikul, Pavimol; Wen, Cindy H; Kroll, Ashley V; Carpenter, Cody; Ramesh, Manikantan; Qu, Vivian; Patel, Sherrina H; Zhu, Jie; Shi, William; Hofman, Florence M; Chen, Thomas C; Gao, Weiwei; Zhang, Kang; Chien, Shu; Zhang, Liangfang

    2015-10-01

    Development of functional nanoparticles can be encumbered by unanticipated material properties and biological events, which can affect nanoparticle effectiveness in complex, physiologically relevant systems. Despite the advances in bottom-up nanoengineering and surface chemistry, reductionist functionalization approaches remain inadequate in replicating the complex interfaces present in nature and cannot avoid exposure of foreign materials. Here we report on the preparation of polymeric nanoparticles enclosed in the plasma membrane of human platelets, which are a unique population of cellular fragments that adhere to a variety of disease-relevant substrates. The resulting nanoparticles possess a right-side-out unilamellar membrane coating functionalized with immunomodulatory and adhesion antigens associated with platelets. Compared to uncoated particles, the platelet membrane-cloaked nanoparticles have reduced cellular uptake by macrophage-like cells and lack particle-induced complement activation in autologous human plasma. The cloaked nanoparticles also display platelet-mimicking properties such as selective adhesion to damaged human and rodent vasculatures as well as enhanced binding to platelet-adhering pathogens. In an experimental rat model of coronary restenosis and a mouse model of systemic bacterial infection, docetaxel and vancomycin, respectively, show enhanced therapeutic efficacy when delivered by the platelet-mimetic nanoparticles. The multifaceted biointerfacing enabled by the platelet membrane cloaking method provides a new approach in developing functional nanoparticles for disease-targeted delivery. PMID:26374997

  1. Nanoparticle biointerfacing via platelet membrane cloaking

    PubMed Central

    Hu, Che-Ming J.; Fang, Ronnie H.; Wang, Kuei-Chun; Luk, Brian T.; Thamphiwatana, Soracha; Dehaini, Diana; Nguyen, Phu; Angsantikul, Pavimol; Wen, Cindy H.; Kroll, Ashley V.; Carpenter, Cody; Ramesh, Manikantan; Qu, Vivian; Patel, Sherrina; Zhu, Jie; Shi, William; Hofman, Florence M.; Chen, Thomas C.; Gao, Weiwei; Zhang, Kang; Chien, Shu; Zhang, Liangfang

    2015-01-01

    Development of functional nanoparticles can be encumbered by unanticipated material properties and biological events, which can negatively impact nanoparticle effectiveness in complex, physiologically relevant systems1–3. Despite the advances in bottom-up nanoengineering and surface chemistry, reductionist functionalization approaches remain inadequate in replicating the complex interfaces present in nature and cannot avoid exposure of foreign materials. Here we report on the preparation of polymeric nanoparticles enclosed in the plasma membrane of human platelets, which are a unique population of cellular fragments that adhere to a variety of disease-relevant substrates4–7. The resulting nanoparticles possess a right-side-out unilamellar membrane coating functionalized with immunomodulatory and adhesion antigens associated with platelets. As compared to uncoated particles, the platelet membrane-cloaked nanoparticles have reduced cellular uptake by macrophage-like cells and are absent of particle-induced complement activation in autologous human plasma. The cloaked nanoparticles also display platelet-mimicking properties such as selective adhesion to damaged human and rodent vasculatures as well as enhanced binding to platelet-adhering pathogens. In an experimental rat model of coronary restenosis and a mouse model of systemic bacterial infection, docetaxel and vancomycin, respectively, show enhanced therapeutic efficacy when delivered by the platelet-mimetic nanoparticles. The multifaceted biointerfacing enabled by the platelet membrane cloaking method provides a new approach in developing functional nanoparticles for disease-targeted delivery. PMID:26374997

  2. Arf6 plays an early role in platelet activation by collagen and convulxin.

    PubMed

    Choi, Wangsun; Karim, Zubair A; Whiteheart, Sidney W

    2006-04-15

    Small GTPases play critical roles in hemostasis, though the roster of such molecules in platelets is not complete. In this study, we report the presence of Ras-related GTPases of the ADP-ribosylation factor (Arf) family. Platelets contain Arf1 or 3 and Arf6, with the latter being predominantly membrane associated. Using effector domain pull-down assays, we show, counter to other GTPases, that Arf6-GTP is present in resting platelets and decreases rapidly upon activation with collagen or convulxin. This decrease does not completely rely on secondary agonists (ADP and thromboxane A2) or require integrin signaling. The decrease in free Arf6-GTP temporally precedes activation of Rho family GTPases (RhoA, Cdc42, and Rac1). Using a membrane-permeant, myristoylated peptide, which mimics the N-terminus of Arf6, we show that the Arf6-GTP decrease is essential for collagen- and convulxin-induced aggregation, platelet adherence, and spreading on collagen-coated glass. Treatment with this peptide also affects the activation of Rho family GTPases, but has little effect on RalA and Rap1 or on agonist-induced calcium mobilization. These data show that Arf6 is a key element in activation through GPVI, and is required for activation of the Rho family GTPases and the subsequent cytoskeletal rearrangements needed for full platelet function. PMID:16352809

  3. Platelet hyperaggregability in obesity: is there a role for nitric oxide impairment and oxidative stress?

    PubMed

    Leite, Natália Rodrigues Pereira; Siqueira de Medeiros, Mariana; Mury, Wanda Vianna; Matsuura, Cristiane; Perszel, Monique Bandeira Moss; Noronha Filho, Gerson; Brunini, Tatiana Mc; Mendes-Ribeiro, Antônio Claúdio

    2016-08-01

    Epidemiological evidence has shown that platelet activation markers are consistently elevated in obesity, contributing to its prothrombotic state. In order to improve the understanding of the regulation of platelet function in obesity, the aim of this study was to investigate the l-arginine-nitric oxide (NO) pathway in obese adults without other cardiovascular risk factor. Seventeen obese (body mass index [BMI] 35.9±1.0 kg/m(2) ) and eighteen age-matched normal weight subjects (BMI 22.0±0.6 kg/m(2) ) were included in this study. l-arginine influx was measured with incubation of l-[(3) H]-arginine. NO synthase (NOS) and arginase activities were determined by the citrulline assay and the conversion of l-[(14) C]-arginine to [(14) C]-urea, respectively. Cyclic guanosine monophosphate (cGMP) content was evaluated by enzyme-linked immunosorbent assay. In addition, the study analyzed: platelet aggregation; intraplatelet antioxidant enzymes, via superoxide dismutase (SOD) and catalase activities; and systemic levels of l-arginine, fibrinogen, and C-reactive protein (CRP). Obese patients presented a significant decrease of platelet l-arginine influx, NOS activity, and cGMP levels, along with platelet hyperaggregability. On the presence of NO donor, platelet aggregation was similar between the groups. The fibrinogen and CRP systemic levels were significantly higher and SOD activity was reduced in obesity. No significant differences were observed in plasma levels of l-arginine and intraplatelet arginase and catalase activities between groups. The diminished NO bioavailability associated with inflammatory status and impaired enzymatic antioxidant defence may contribute to future cardiovascular complications in obesity. PMID:27145241

  4. Outcomes of an automated procedure for the selection of effective platelets for patients refractory to random donors based on cross-matching locally available platelet products.

    PubMed

    Rebulla, Paolo; Morelati, Fernanda; Revelli, Nicoletta; Villa, Maria Antonietta; Paccapelo, Cinzia; Nocco, Angela; Greppi, Noemi; Marconi, Maurizio; Cortelezzi, Agostino; Fracchiolla, Nicola; Martinelli, Giovanni; Deliliers, Giorgio Lambertenghi

    2004-04-01

    In 1999, we implemented an automated platelet cross-matching (XM) programme to select compatible platelets from the local inventory for patients refractory to random donor platelets. In this study, we evaluated platelet count increments in 40 consecutive refractory patients (8.3% of 480 consecutive platelet recipients) given 569 cross-match-negative platelets between April 1999 and December 2001. XM was performed automatically with a commercially available immunoadherence assay. Pre-, 1- and 24-h post-transfusion platelet counts (mean +/- SD) for the 569 XM-negative platelet transfusions containing 302 +/- 71 x 109 platelets were 7.7 +/- 5.5, 32.0 +/- 21.0 and 16.8 +/- 15.5 x 109/l respectively. Increments were significantly higher (P < 0.05, t-test) than those observed in the same patients given 303 random platelet pools (dose = 318 +/- 52 x 109 platelets) during the month before refractoriness was detected, when pre-, 1- and 24-h post-transfusion counts were 7.0 +/- 8.6, 15.9 +/- 16.1 and 9.6 +/- 12.8 x 109/l respectively. The cost of the platelet XM disposable kit per transfusion to produce 1-h post-transfusion platelet count increments >10 x 109/l was euro 447. This programme enabled the rapid selection of effective platelets for refractory patients, from the local inventory. PMID:15015974

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

    SciTech Connect

    Asmis, Lars; Tanner, Felix C.; Sudano, Isabella; Luescher, Thomas F.; Camici, Giovanni G.

    2010-01-22

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

  6. Identification of APC mutations and evaluation of their expression level using a functional screening assay

    SciTech Connect

    Varesco, L.; Gismondi, V.; Bafico, A.

    1994-09-01

    A functional screen for chain-terminating mutations in the APC gene recently has been developed. It is based on the PCR and cloning of a segment of the gene in-frame with a colorimetric marker gene (lacz) followed by screening for the level of activity of the marker polypeptide (beta-galactosidase). This method scores colony number with different blue colors that are produced by bacteria containing normal and mutant APC segments. In the present work this method was used to screen the entire APC coding region by using eight primer pairs. DNA segments with known APC mutations at different positions in the gene were used as controls and were clearly identifiable with this assay. In addition, the entire APC coding region has been examined in 21 APC patients in whom PCR-SSCP did not identify an APC mutation. Novel mutations (n=14) were identified by the blue/white assay and were all confirmed by sequence analysis. This method also was used to quantitate the expression of paternal and maternal APC alleles taking advantage of an RsaI site polymorphism at position 1458 in a small number of informative individuals. Differential expression of some known mutant APC mRNAs was observed.

  7. Platelets Direct Monocyte Differentiation Into Epithelioid-Like Multinucleated Giant Foam Cells With Suppressive Capacity Upon Mycobacterial Stimulation

    PubMed Central

    Feng, Yonghong; Dorhoi, Anca; Mollenkopf, Hans-Joachim; Yin, Hongyun; Dong, Zhengwei; Mao, Ling; Zhou, Jun; Bi, Aixiao; Weber, Stephan; Maertzdorf, Jeroen; Chen, Gang; Chen, Yang; Kaufmann, Stefan H. E.

    2014-01-01

    Background. Epithelioid, foam, and multinucleated giant cells (MNGCs) are characteristics of tuberculosis granulomas, yet the precise genesis and functions of these transformed macrophages are unclear. We evaluated the role of platelets as drivers of macrophage transformation in mycobacterial infection. Methods. We employed flow cytometry and microscopy to assess cellular phenotype and phagocytosis. Immune assays allowed quantification of cytokines and chemokines, whereas gene microarray technology was applied to estimate global transcriptome alterations. Immunohistochemical investigations of tuberculosis granulomas substantiated our findings at the site of infection. Results. Monocytes differentiated in presence of platelets (MP-Macs) acquired a foamy, epithelioid appearance and gave rise to MNGCs (MP-MNGCs). MP-Macs up-regulated activation markers, phagocytosed mycobacteria, and released abundant interleukin 10. Upon extended culture, MP-Macs shared transcriptional features with epithelioid cells and M2 macrophages and up-regulated CXCL5 transcripts. In line with this, CXCL5 concentrations were significantly increased in airways of active tuberculosis patients. The platelet-specific CD42b antigen was detected in MP-Macs, likewise in macrophages, MNGCs, and epithelioid cells within tuberculosis granulomas, along with the platelet aggregation-inducing factor PDPN. Conclusions. Platelets drive macrophage differentiation into MNGCs with characteristics of epithelioid, foam, and giant cells observed in tuberculosis granulomas. Our data define platelets as novel participants in tuberculosis pathogenesis. PMID:24987031

  8. [Determination of tolerance ability of platelet to the change of solution osmotic pressure and its significance].

    PubMed

    Ouyang, Xi-Lin; Liu, Jing-Han; Gao, Dayong

    2003-02-01

    In order to determine the tolerance ability of platelet to change of osmotic pressure in solution, the isotonic fresh platelets were exposed to a series of crystal salt solutions with osmotic pressure range from 47 to 611 mOsm for 15 minutes. Then the platelets were returned to isotonic condition and kept for 15 minutes. The expressions of phosphatidylserine and CD62p were assayed in platelets. The results showed that the phosphatidylserine and CD62p expressions were increased when the osmotic pressure of solution was below 238 mOsm, but no significant rise was detected when the platelets were exposed to 611 mOsm solution. No increases of positive rate of CD62p and phosphatidylserine were detected in platelets returned to isotonic condition. It is concluded that platelets are sensitive to hypoosmotic solution and tolerated to hyperosmotic solution. Exceeding the platelet safe volume limitation may lead to injure of platelet osmosis in crystal salt solution. PMID:12667298

  9. Platelets in Lung Biology

    PubMed Central

    Weyrich, Andrew S.; Zimmerman, Guy A.

    2013-01-01

    Platelets and the lungs have an intimate relationship. Platelets are anucleate mammalian blood cells that continuously circulate through pulmonary vessels and that have major effector activities in hemostasis and inflammation. The lungs are reservoirs for megakaryocytes, the requisite precursor cell in thrombopoiesis, which is the intricate process by which platelets are generated. Platelets contribute to basal barrier integrity of the alveolar capillaries, which selectively restricts the transfer of water, proteins, and red blood cells out of the vessels. Platelets also contribute to pulmonary vascular repair. Although platelets bolster hemostatic and inflammatory defense of the healthy lung, experimental evidence and clinical evidence indicate that these blood cells are effectors of injury in a variety of pulmonary disorders and syndromes. Newly discovered biological capacities of platelets are being explored in the context of lung defense, disease, and remodeling. PMID:23043249

  10. A slot blot procedure for the measurement of yessotoxins by a functional assay.

    PubMed

    Pierotti, Silvia; Albano, Clara; Milandri, Anna; Callegari, Federica; Poletti, Roberto; Rossini, Gian Paolo

    2007-01-01

    We originally developed a functional assay for the detection of yessotoxins (YTX) based on its capacity to induce dose-dependent changes in cellular levels of two marker proteins, consisting of E-cadherin and an E-cadherin fragment (ECRA100) in epithelial cells. The procedure is time-consuming and we have shortened it by a slot blot format, using antibodies recognizing two different epitopes of E-cadherin (HECD-1 and C20820), thereby discriminating those markers. The best performing membrane under our conditions, in terms of binding capacity and even absorption of proteins, was a positively charged nylon membrane. Treatment of the membrane with 0.5mug of Ab/ml was appropriate for maximal detection of antigens by our slot blot procedure with both HECD-1 and C20820 antibodies. The treatment of cells with YTX, resulting in a relative increase in the cellular levels of ECRA100, led to a dose-dependent increase of the signal detected by Ab HECD-1 without a concomitant increase in the signal detected by Ab C20820 in our slot blot format, and the concentrations of YTX were correlated to both the increase of the signal detected through Ab HECD-1 and to the decrease in the ratio of the signals obtained with the two Abs (C20820 over HECD-1). Upon analyses of extracts from cells treated with shellfish samples, we could detect and quantify YTX in naturally contaminated materials. The slot blot format of our functional assay allows a substantial shortening of its analytical step (about seven hr, as compared to the two working days of the original method), providing YTX measurements that are accurate but show large standard deviations. PMID:17055548

  11. Gut Microbial Metabolite TMAO Enhances Platelet Hyperreactivity and Thrombosis Risk.

    PubMed

    Zhu, Weifei; Gregory, Jill C; Org, Elin; Buffa, Jennifer A; Gupta, Nilaksh; Wang, Zeneng; Li, Lin; Fu, Xiaoming; Wu, Yuping; Mehrabian, Margarete; Sartor, R Balfour; McIntyre, Thomas M; Silverstein, Roy L; Tang, W H Wilson; DiDonato, Joseph A; Brown, J Mark; Lusis, Aldons J; Hazen, Stanley L

    2016-03-24

    Normal platelet function is critical to blood hemostasis and maintenance of a closed circulatory system. Heightened platelet reactivity, however, is associated with cardiometabolic diseases and enhanced potential for thrombotic events. We now show gut microbes, through generation of trimethylamine N-oxide (TMAO), directly contribute to platelet hyperreactivity and enhanced thrombosis potential. Plasma TMAO levels in subjects (n > 4,000) independently predicted incident (3 years) thrombosis (heart attack, stroke) risk. Direct exposure of platelets to TMAO enhanced sub-maximal stimulus-dependent platelet activation from multiple agonists through augmented Ca(2+) release from intracellular stores. Animal model studies employing dietary choline or TMAO, germ-free mice, and microbial transplantation collectively confirm a role for gut microbiota and TMAO in modulating platelet hyperresponsiveness and thrombosis potential and identify microbial taxa associated with plasma TMAO and thrombosis potential. Collectively, the present results reveal a previously unrecognized mechanistic link between specific dietary nutrients, gut microbes, platelet function, and thrombosis risk. PMID:26972052

  12. RhoGAPs and Rho GTPases in platelets.

    PubMed

    Elvers, Margitta

    2016-08-01

    Platelet cytoskeletal reorganization is essential for platelet adhesion and thrombus formation in hemostasis and thrombosis. The Rho GTPases RhoA, Rac1 and Cdc42 are the main players in cytoskeletal dynamics of platelets responsible for the formation of filopodia and lamellipodia to strongly increase the platelet surface upon activation. They are involved in platelet activation and aggregate formation including platelet secretion, integrin activation and arterial thrombus formation. The activity of Rho GTPases is tightly controlled by different proteins such as GTPase-activating proteins (GAPs). GAPs stimulate GTP hydrolysis to terminate Rho signaling. The role and impact of GAPs in platelets is not well-defined and many of the RhoGAPs identified are not known to be present in platelets or to have any function in platelets. The recently identified RhoGAPs Oligophrenin1 (OPHN1) and Nadrin regulate the activity of RhoA, Rac1 and Cdc42 and subsequent platelet cytoskeletal reorganization, platelet activation and thrombus formation. In the last years, the analysis of genetically modified mice helped to gain the understanding of Rho GTPases and their regulators in cytoskeletal rearrangements and other Rho mediated cellular processes in platelets. PMID:25639730

  13. Platelet Interaction with Bacteria

    PubMed Central

    Clawson, C. C.

    1973-01-01

    The interaction of several common strains of bacteria with rabbit or human platelets in vitro has been examined sequentially with scanning and transmission electron microscopy. Bacteria were added to platelets in their native plasma or to washed platelets in a balanced salt solution at ratios of about 1:1 or at low bacteria to platelet ratios (down to 1:100). The platelet-bacterial interaction (PBI) was studied with recording nephelometry. Matched samples were fixed for microscopy at various points in the aggregation response. The results support these conclusions: a) Bacteria stimulate platelet aggregation by direct contact and adhesion with the platelet surface. b) Adhesion between the two cell types requires divalent cations, occurs through fusion of normal cell-surface coats and appears identical in the presence or absence of extracellular plasma protein. c) The morphologic transformation of platelets during PBI is identical to that produced by collagen. d) During PBI the bacteria are incorporated into the forming platelet aggregates and reside predominantly intercellularly. e) Phagocytosis of bacteria by a single platelet is very rare. f) Bacteria which have resided within platelet aggregates for one hour are unaltered morphologically. g) PBI occurs even at very low bacterial numbers and produces platelet-bacterial aggregates in small numbers without stimulating generalized platelet aggregation. Methods for concentration of thrombocytopenic plasma and washing human platelets are presented. ImagesFig 6Fig 7Fig 8Fig 9Fig 10Fig 11Fig 1Fig 2Fig 12Fig 13Fig 3Fig 14Fig 4Fig 5 PMID:4632008

  14. Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypic and functional comparison of umbilical cord blood- and bone marrow-derived progenitors

    PubMed Central

    Avanzini, Maria Antonietta; Bernardo, Maria Ester; Cometa, Angela Maria; Perotti, Cesare; Zaffaroni, Nadia; Novara, Francesca; Visai, Livia; Moretta, Antonia; Del Fante, Claudia; Villa, Raffaella; Ball, Lynne M.; Fibbe, Willem E.; Maccario, Rita; Locatelli, Franco

    2009-01-01

    Background Mesenchymal stromal cells are employed in various different clinical settings in order to modulate immune response. However, relatively little is known about the mechanisms responsible for their immunomodulatory effects, which could be influenced by both the cell source and culture conditions. Design and Methods We tested the ability of a 5% platelet lysate-supplemented medium to support isolation and ex vivo expansion of mesenchymal stromal cells from full-term umbilical-cord blood. We also investigated the biological/functional properties of umbilical cord blood mesenchymal stromal cells, in comparison with platelet lysate-expanded bone marrow mesenchymal stromal cells. Results The success rate of isolation of mesenchymal stromal cells from umbilical cord blood was in the order of 20%. These cells exhibited typical morphology, immunophenotype and differentiation capacity. Although they have a low clonogenic efficiency, umbilical cord blood mesenchymal stromal cells may possess high proliferative potential. The genetic stability of these cells from umbilical cord blood was demonstrated by a normal molecular karyotype; in addition, these cells do not express hTERT and telomerase activity, do express p16ink4a protein and do not show anchorage-independent cell growth. Concerning alloantigen-specific immune responses, umbilical cord blood mesenchymal stromal cells were able to: (i) suppress T- and NK-lymphocyte proliferation, (ii) decrease cytotoxic activity and (iii) only slightly increase interleukin-10, while decreasing interferon-γ secretion, in mixed lymphocyte culture supernatants. While an indoleamine 2,3-dioxygenase-specific inhibitor did not reverse mesenchymal stromal cell-induced suppressive effects, a prostaglandin E2-specific inhibitor hampered the suppressive effect of both umbilical cord blood- and bone marrow-mesenchymal stromal cells on alloantigen-induced cytotoxic activity. Mesenchymal stromal cells from both sources expressed HLA

  15. Functional gold nanoparticles coupled with microporous membranes: a flow controlled assay for colorimetric visualization of proteins.

    PubMed

    Chen, Yu-Yuan; Unnikrishnan, Binesh; Li, Yu-Jia; Huang, Chih-Ching

    2014-11-21

    We report a rapid and simple assay for colorimetric visualization of thrombin at nanomolar levels using functional gold nanoparticles (FAuNPs) coupled with microporous membranes. We used a 29-mer thiolated-thrombin-binding-aptamer (TBA29) to prepare TBA29 functionalized AuNPs (TBA29-AuNPs) for the selective detection of human thrombin. The sensing mechanism is based on the principle of TBA29-AuNPs flowing down through the nitrocellulose membrane (NCM) pores at different flow rates after binding to thrombin. Compared with free TBA29-AuNPs, when thrombin-TBA29-AuNPs were dropped on the NCM, the particles flowed down more easily through the NCM pores along with the buffer solution due to the increase in the gravity of particles. Therefore, color intensities of TBA29-AuNPs on the NCM depended on the concentration of thrombin; the color intensity was lighter when the concentration of thrombin was higher. Thrombin can be detected at the nanomolar level with the naked eye using this colorimetric probe. A protein G modified AuNP based probe (PG-AuNPs/NCM) was employed to detect human immunoglobulin G (hIgG) in plasma samples to demonstrate the practicality of our sensing system. Also, fibrinogen modified Au NPs were analyzed to demonstrate that this concept of detection could be extended to other proteins or systems, by functionalizing with suitable molecules. PMID:25267979

  16. Platelets and plasma stimulate sheep rotator cuff tendon tenocytes when cultured in an extracellular matrix scaffold.

    PubMed

    Kelly, Brian A; Proffen, Benedikt L; Haslauer, Carla M; Murray, Martha M

    2016-04-01

    The addition of platelet-rich plasma (PRP) to rotator cuff repair has not translated into improved outcomes after surgery. However, recent work stimulating ligament healing has demonstrated improved outcomes when PRP or whole blood is combined with an extracellular matrix carrier. The objective of this study was to evaluate the effect of three components of blood (plasma, platelets, and macrophages) on the in vitro activity of ovine rotator cuff cells cultured in an extracellular matrix environment. Tenocytes were obtained from six ovine infraspinatus tendons and cultured over 14 days in an extracellular matrix scaffold with the following additives: (1) plasma (PPP), (2) plasma and platelets (PAP), (3) plasma and macrophages (PPPM), (4) plasma, platelets and macrophages (PAPM), (5) phosphate buffered saline (PBS), and (6) PBS with macrophages (PBSM). Assays measuring cellular metabolism (AlamarBlue), proliferation (Quantitative DNA assay), synthesis of collagen and cytokines (SIRCOL, TNF-α and IL-10 ELISA, and MMP assay), and collagen gene expression (qPCR) were performed over the duration of the experiment, as well as histology at the conclusion. Plasma was found to stimulate cell attachment and spreading on the scaffold, as well as cellular proliferation. Platelets also stimulated cell proliferation, cellular metabolism, transition of cells to a myofibroblast phenotype, and contraction of the scaffolds. The addition of macrophages did not have any significant effect on the sheep rotator cuff cells in vitro. In vivo studies are needed to determine whether these changes in cellular function will translate into improved tendon healing. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:623-629, 2016. PMID:26419602

  17. A central role of GPIb-IX in the procoagulant function of platelets that is independent of the 45-kDa GPIbalpha N-terminal extracellular domain.

    PubMed

    Ravanat, Catherine; Strassel, Catherine; Hechler, Béatrice; Schuhler, Simone; Chicanne, Gaëtan; Payrastre, Bernard; Gachet, Christian; Lanza, François

    2010-08-19

    Activated platelets become procoagulant and efficiently promote the conversion of prothrombin to thrombin. A role of the GPIb-V-IX complex has long been postulated in view of the decreased prothrombin consumption in Bernard-Soulier patients. We evaluated the impact of GPIb-V-IX deficiency and the requirement for the GPIbalpha extracellular domain. In GPIbbeta(-/-) mice, thrombin generation was profoundly decreased in tissue factor- or collagen-related peptide (CRP)-activated platelet-rich plasma and in washed platelets supplemented with normal plasma or with FVa, FXa, and prothrombin. Phosphatidylserine (PS) exposure was similarly decreased in response to thrombin, CRP, or CRP + PAR4 peptide despite a normal platelet phospholipid composition. The hypothesis that these defects originate from lack of the GPIbalpha N-terminal domain was evaluated after its removal from normal mouse and human platelets with Nk protease or O-sialoglycoprotein endopeptidase. Unexpectedly, the treated platelets exhibited normal thrombin generation and PS exposure, indicating that GPIb-V-IX regulates procoagulant activity independently of its GPIbalpha-binding region. These results suggested a more general structuring role through intracellular cytoskeleton-anchoring portions regulating responses leading to PS exposure. This hypothesis was supported by the decreased calcium mobilization observed in GPIbbeta(-/-) platelets in response to several agonists, some acting independently of GPIb, in contrast to the normal calcium responses in Nk protease-treated platelets. PMID:20457869

  18. Bleeding risk assessment in patients undergoing elective cardiac surgery using ROTEM(®) platelet and Multiplate(®) impedance aggregometry.

    PubMed

    Petricevic, M; Konosic, S; Biocina, B; Dirkmann, D; White, A; Mihaljevic, M Z; Ivancan, V; Konosic, L; Svetina, L; Görlinger, K

    2016-06-01

    Impaired platelet function is a major risk factor for peri-operative bleeding and transfusion. This prospective, observational study enrolled 101 consecutive patients undergoing elective cardiac surgery with cardiopulmonary bypass. Platelet function was assessed by two whole blood impedance aggregometers (ROTEM(®) platelet and Multiplate(®) ), using three different activators (arachidonic acid, adenosine diphosphate and thrombin receptor-activating peptide-6), at three peri-operative time points (before anaesthesia, after aortic declamping and 5-10 min after protamine administration). Platelet function was impaired over the time-course in all assays. Results after protamine administration demonstrated the best correlation with postoperative chest tube drainage. Patients with a chest tube drainage exceeding the 75th percentile of the entire study population, during the first 24 postoperative hours, were characterised to have excessive bleeding. Both devices provided similar predictability for postoperative chest tube drainage and red blood cell transfusion requirements. The latter was associated with the degree of platelet inhibition and the number of pathways inhibited determined respective cut-off values. PMID:26763378

  19. A functional variant in promoter region of platelet-derived growth factor-D is probably associated with intracerebral hemorrhage

    PubMed Central

    2012-01-01

    Background Platelet-derived growth factor D (PDGF-D) plays an important role in angiogenesis, vessel remodeling, inflammation and repair in response to injury. We hypothesized that genetic variation in PDGFD gene might alter the susceptibility to stroke. Findings We determined the genotypes of a single nucleotide polymorphism (SNP) (-858A/C, rs3809021) in 1484 patients with stroke (654 cerebral thrombosis, 419 lacunar infarction, 411 intracerebral hemorrhage [ICH]) and 1528 control subjects from an unrelated Chinese Han population and followed the stroke patients up for a median of 4.5 years. The -858AA genotype showed significantly increased risk of ICH (dominant model: odds ratio [OR] 1.29, 95% confidence interval [CI] 1.00-1.68, P = 0.05; additive model: OR 1.24, 95% CI 1.01-1.52, P = 0.04) than wild-type genotype. Further analyses showed that -858AA genotype conferred about 2-fold increase in risk of non-hypertensive ICH (dominant model: OR 2.1, 95%CI 1.34-3.29, P = 0.001; additive model: OR 1.75, 95% CI 1.24-2.46, P = 0.001). After a median follow-up of 4.5 years, -858AA genotype was associated with a reduced risk of ICH recurrence (dominant model: adjusted hazard ratio [HR] 0.09, 95%CI 0.01-0.74, P = 0.025; additive model: HR 0.21, 95% CI 0.04-1.16, P = 0.073) in non-hypertensive patients. Conclusions The -858AA genotype is probably associated with risk for non-hypertensive ICH. Further studies should be conducted to reveal the role of PDGF-D at various stages of ICH development--beneficial, or deleterious. PMID:22289441

  20. A novel agglutination test for antigen-specific detection of platelet antibodies.

    PubMed

    Meyer, Oliver; Agaylan, Ashraf; Borchert, Hans-Hubert; Aslan, Tunay; Bombard, Stéphane; Kiesewetter, Holger; Salama, Abdulgabar

    2006-10-15

    A simple and rapid antigen-specific assay for the identification antibodies to platelets is lacking, yet. Red-dyed polystyrene microbeads were coated with monoclonal antibodies to various platelet glycoprotein complexes, and used for the detection of platelet autoantibodies and alloantibodies. The results were largely identical with those obtained by monoclonal antibody-specific immobilization of platelet antigen assay (MAIPA). The new test is reliable yet less complex and time-consuming than the currently available assays, and it can be implemented in any routine laboratory. PMID:16933262

  1. Measuring stem cell frequency in epidermis: A quantitative in vivo functional assay for long-term repopulating cells

    NASA Astrophysics Data System (ADS)

    Schneider, T. E.; Barland, C.; Alex, A. M.; Mancianti, M. L.; Lu, Y.; Cleaver, J. E.; Lawrence, H. J.; Ghadially, R.

    2003-09-01

    Epidermal stem cells play a central role in tissue homeostasis, wound repair, tumor initiation, and gene therapy. A major impediment to the purification and molecular characterization of epidermal stem cells is the lack of a quantitative assay for cells capable of long-term repopulation in vivo, such as exists for hematopoietic cells. The tremendous strides made in the characterization and purification of hematopoietic stem cells have been critically dependent on the availability of competitive transplantation assays, because these assays permit the accurate quantitation of long-term repopulating cells in vivo. We have developed an analogous functional assay for epidermal stem cells, and have measured the frequency of functional epidermal stem cells in interfollicular epidermis. These studies indicate that cells capable of long-term reconstitution of a squamous epithelium reside in the interfollicular epidermis. We find that the frequency of these long-term repopulating cells is 1 in 35,000 total epidermal cells, or in the order of 1 in 104 basal epidermal cells, similar to that of hematopoietic stem cells in the bone marrow, and much lower than previously estimated in epidermis. Furthermore, these studies establish a novel functional assay that can be used to validate immunophenotypic markers and enrichment strategies for epidermal stem cells, and to quantify epidermal stem cells in various keratinocyte populations. Thus further studies using this type of assay for epidermis should aid in the progress of cutaneous stem cell-targeted gene therapy, and in more basic studies of epidermal stem cell regulation and differentiation.

  2. A novel assay revealed that ribonucleotide reductase is functionally important for interstrand DNA crosslink repair.

    PubMed

    Fujii, Naoaki; Evison, Benjamin J; Actis, Marcelo L; Inoue, Akira

    2015-11-01

    Cells have evolved complex biochemical pathways for DNA interstrand crosslink (ICL) removal. Despite the chemotherapeutic importance of ICL repair, there have been few attempts to identify which mechanistic DNA repair inhibitor actually inhibits ICL repair. To identify such compounds, a new and robust ICL repair assay was developed using a novel plasmid that contains synthetic ICLs between a CMV promoter region that drives transcription and a luciferase reporter gene, and an SV40 origin of replication and the large T antigen (LgT) gene that enables self-replication in mammalian cells. In a screen against compounds that are classified as inhibitors of DNA repair or synthesis, the reporter generation was exquisitely sensitive to ribonucleotide reductase (RNR) inhibitors such as gemcitabine and clofarabine, but not to inhibitors of PARP, ATR, ATM, Chk1, and others. The effect was observed also by siRNA downregulation of RNR. Moreover, the reporter generation was also particularly sensitive to 3-AP, a non-nucleoside RNR inhibitor, but not significantly sensitive to DNA replication stressors, suggesting that the involvement of RNR in ICL repair is independent of incorporation of a nucleotide RNR inhibitor into DNA to induce replication stress. The reporter generation from a modified version of the plasmid that lacks the LgT-SV40ori motif was also adversely affected by RNR inhibitors, further indicating a role for RNR in ICL repair that is independent of DNA replication. Intriguingly, unhooking of cisplatin-ICL from nuclear DNA was significantly inhibited by low doses of gemcitabine, suggesting an unidentified functional role for RNR in the process of ICL unhooking. The assay approach could identify other molecules essential for ICLR in quantitative and flexible manner. PMID:26462050

  3. Quantitative analysis of platelets aggregates in 3D by digital holographic microscopy

    PubMed Central

    Boudejltia, Karim Zouaoui; Ribeiro de Sousa, Daniel; Uzureau, Pierrick; Yourassowsky, Catherine; Perez-Morga, David; Courbebaisse, Guy; Chopard, Bastien; Dubois, Frank

    2015-01-01

    Platelet spreading and retraction play a pivotal role in the platelet plugging and the thrombus formation. In routine laboratory, platelet function tests include exhaustive information about the role of the different receptors present at the platelet surface without information on the 3D structure of platelet aggregates. In this work, we develop, a method in Digital Holographic Microscopy (DHM) to characterize the platelet and aggregate 3D shapes using the quantitative phase contrast imaging. This novel method is suited to the study of platelets physiology in clinical practice as well as the development of new drugs. PMID:26417523

  4. A protocol for heterologous expression and functional assay for mouse pheromone receptors.

    PubMed

    Dey, Sandeepa; Zhan, Senmiao; Matsunami, Hiroaki

    2013-01-01

    Innate social behaviors like intermale aggression, fear, and mating rituals are important for survival and propagation of a species. In mice, these behaviors have been implicated to be mediated by peptide pheromones that are sensed by a class of G protein-coupled receptors, vomeronasal receptor type 2 (V2Rs), expressed in the pheromone-detecting vomeronasal organ (VNO) (Chamero et al., Nature 450:899-902, 2007; Haga et al., Nature 466:118-122, 2010; Kimoto et al., Curr Biol 17:1879-1884, 2007; Leinders-Zufall et al., Nat Neurosci 12:1551-1558, 2009; Papes et al., Cell 141:692-703, 2010). Matching V2Rs with their cognate ligands is required to understand what receptors the biologically relevant pheromones are acting on. However, this goal has been greatly limited by the unavailability of appropriate heterologous tools commonly used to carry out receptor deorphanization, due to the fact that this family of receptors fails to traffic to the surface of heterologous cells. We have demonstrated that calreticulin, a housekeeping chaperone commonly expressed in most eukaryotic cells, is sparsely expressed in the vomeronasal sensory neurons (VSNs). Stable knock down of calreticulin in a HEK293T derived cell line (R24 cells) allows us to functionally express V2Rs on the surface of heterologous cells. In this chapter we describe protocols for maintenance and expansion of the R24 cell line and functional assays for V2Rs using these cells. PMID:24014358

  5. Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA).

    PubMed

    Miller, Daniel E; Patel, Zubin H; Lu, Xiaoming; Lynch, Arthur T; Weirauch, Matthew T; Kottyan, Leah C

    2016-01-01

    Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype. PMID:27585267

  6. Variable Effect of P2Y12 Inhibition on Platelet Thrombus Volume in Flowing Blood

    PubMed Central

    Mendolicchio, G. L.; Zavalloni, D.; Bacci, M.; Corrada, E.; Marconi, M.; Lodigiani, C.; Presbitero, P.; Rota, L.; Ruggeri, Z. M.

    2010-01-01

    Background and objectives Patients treated by percutaneous coronary intervention (PCI) receive aspirin and P2Y12 ADP receptor inhibitors to reduce thrombotic complications. The choice of methodology for monitoring the effects of treatment and assessing its efficacy is still a topic of debate. We evaluated how decreased P2Y12 function influences platelet aggregate (thrombus) size measured ex vivo. Methods and Results We used confocal videomicroscopy to measure in real time the volume of platelet thrombi forming upon blood perfusion over fibrillar collagen type I at the wall shear rate of 1,500 s−1. The average volume was significantly smaller in 31 patients receiving aspirin and clopidogrel (19) or ticlopidine (12) than 21 controls, but individual values were above the lower limit of the normal distribution, albeit mostly within the lower quartile, in 61.3% of cases. Disaggregation of platelet thrombi at later perfusion times occurred frequently in the patients. Vasodilator-stimulated phosphoprotein (VASP) phosphorylation, reflecting P2Y12 inhibition, was also decreased in the patient group and only 22.6% of individual values were above the lower normal limit. We found no correlation between thrombus volume formed onto collagen fibrils and level of P2Y12 inhibition, suggesting that additional and individually variable factors can influence the inhibitory effect of treatment on platelet function. Conclusions Measuring platelet thrombus formation in flowing blood reflects the consequences of anti-platelet therapy in a manner that is not proportional to P2Y12 inhibition. Combining the results of the two assays may improve the assessment of thrombotic risk. PMID:21083646

  7. Role of platelets in neuroinflammation: a wide-angle perspective

    PubMed Central

    2010-01-01

    Objectives This review summarizes recent developments in platelet biology relevant to neuroinflammatory disorders. Multiple sclerosis (MS) is taken as the "Poster Child" of these disorders but the implications are wide. The role of platelets in inflammation is well appreciated in the cardiovascular and cancer research communities but appears to be relatively neglected in neurological research. Organization After a brief introduction to platelets, topics covered include the matrix metalloproteinases, platelet chemokines, cytokines and growth factors, the recent finding of platelet PPAR receptors and Toll-like receptors, complement, bioactive lipids, and other agents/functions likely to be relevant in neuroinflammatory diseases. Each section cites literature linking the topic to areas of active research in MS or other disorders, including especially Alzheimer's disease. Conclusion The final section summarizes evidence of platelet involvement in MS. The general conclusion is that platelets may be key players in MS and related disorders, and warrant more attention in neurological research. PMID:20128908

  8. Aspirin decreases platelet uptake on Dacron vascular grafts in baboons

    SciTech Connect

    Mackey, W.C.; Connolly, R.J.; Callow, A.D.; Keough, E.M.; Ramberg-Laskaris, K.; McCullough, J.L.; O'Donnell, T.F. Jr.; Melaragno, A.; Valeri, C.R.; Weiblen, B.

    1984-07-01

    The influence of a single dose of aspirin (5.4-7.4 mg/kg) on platelet uptake on 4-mm Dacron interposition grafts was studied in a baboon model using gamma camera scanning for 111-Indium labeled platelets. In vitro assessment of platelet function after aspirin administration revealed that in the baboon, as in the human, aspirin abolished arachidonic acid-induced platelet aggregation, prolonged the lag time between exposure to collagen and aggregation, and decreased plasma thromboxane B2 levels. Aspirin also prolonged the template bleeding time. Scans for 111-Indium labeled platelets revealed that pretreatment with a single dose of aspirin decreased platelet uptake on 4-mm Dacron carotid interposition grafts. This decrease in platelet uptake was associated with a significant improvement in 2-hour graft patency and with a trend toward improved 2-week patency.

  9. Isotopic rubidium ion efflux assay for the functional characterization of nicotinic acetylcholine receptors on clonal cell lines

    SciTech Connect

    Lukas, R.J.; Cullen, M.J.

    1988-11-15

    An isotopic rubidium ion efflux assay has been developed for the functional characterization of nicotinic acetylcholine receptors on cultured neurons. This assay first involves the intracellular sequestration of isotopic potassium ion analog by the ouabain-sensitive action of a sodium-potassium ATPase. Subsequently, the release of isotopic rubidium ion through nicotinic acetylcholine receptor-coupled monovalent cation channels is activated by application of nicotinic agonists. Specificity of receptor-mediated efflux is demonstrated by its sensitivity to blockade by nicotinic, but not muscarinic, antagonists. The time course of agonist-mediated efflux, within the temporal limitations of the assay, indicates a slow inactivation of receptor function on prolonged exposure to agonist. Dose-response profiles (i) have characteristic shapes for different nicotinic agonists, (ii) are described by three operationally defined parameters, and (iii) reflect different affinities of agonists for binding sites that control receptor activation and functional inhibition. The rubidium ion efflux assay provides fewer hazards but greater sensitivity and resolution than isotopic sodium or rubidium ion influx assays for functional nicotinic receptors.

  10. Comparative analysis of three functional predictive assays in lymphocytes of patients with breast and gynaecological cancer treated by radiotherapy

    PubMed Central

    Padjas, Anna; Kedzierawski, Piotr; Florek, Agnieszka; Kukolowicz, Pawel; Kuszewski, Tomasz; Góźdz, Stanislaw; Lankoff, Anna; Wojcik, Andrzej

    2012-01-01

    Purpose There is a need to develop predictive tests that would allow identifying cancer patients with a high risk of developing side effects to radiotherapy. We compared the predictive value of three functional assays: the G0 aberration assay, the G2 aberration assay and the alkaline comet assay in lymphocytes of breast cancer and gynaecological cancer patients. Material and methods Peripheral blood was collected from 35 patients with breast cancer and 34 patients with gynaecological cancer before the onset of therapy. Chromosomal aberrations were scored in lymphocytes irradiated in the G0 or G2 phase of the cell cycle. DNA repair kinetics was performed with the alkaline comet assay following irradiation of unstimulated lymphocytes. The results were compared with the severity of early and late side effects to radiotherapy. Results No correlation was observed between the results of the assays and the severity of side effects. Moreover, each assay identified different patients as radiosensitive. Conclusions There is no simple correlation between the in vitro sensitivity of lymphocytes and the risk of developing early and late side effects. PMID:23378851

  11. Expanding the repertoire of secretory peptides controlling root development with comparative genome analysis and functional assays

    PubMed Central

    Ghorbani, Sarieh; Lin, Yao-Cheng; Parizot, Boris; Fernandez, Ana; Njo, Maria Fransiska; Van de Peer, Yves; Beeckman, Tom; Hilson, Pierre

    2015-01-01

    Plant genomes encode numerous small secretory peptides (SSPs) whose functions have yet to be explored. Based on structural features that characterize SSP families known to take part in postembryonic development, this comparative genome analysis resulted in the identification of genes coding for oligopeptides potentially involved in cell-to-cell communication. Because genome annotation based on short sequence homology is difficult, the criteria for the de novo identification and aggregation of conserved SSP sequences were first benchmarked across five reference plant species. The resulting gene families were then extended to 32 genome sequences, including major crops. The global phylogenetic pattern common to the functionally characterized SSP families suggests that their apparition and expansion coincide with that of the land plants. The SSP families can be searched online for members, sequences and consensus (http://bioinformatics.psb.ugent.be/webtools/PlantSSP/). Looking for putative regulators of root development, Arabidopsis thaliana SSP genes were further selected through transcriptome meta-analysis based on their expression at specific stages and in specific cell types in the course of the lateral root formation. As an additional indication that formerly uncharacterized SSPs may control development, this study showed that root growth and branching were altered by the application of synthetic peptides matching conserved SSP motifs, sometimes in very specific ways. The strategy used in the study, combining comparative genomics, transcriptome meta-analysis and peptide functional assays in planta, pinpoints factors potentially involved in non-cell-autonomous regulatory mechanisms. A similar approach can be implemented in different species for the study of a wide range of developmental programmes. PMID:26195730

  12. Decreased mean platelet volume in panic disorder

    PubMed Central

    Göğçegöz Gül, Işıl; Eryılmaz, Gül; Özten, Eylem; Hızlı Sayar, Gökben

    2014-01-01

    Aim The relationship between psychological stress and platelet activation has been widely studied. It is well known that platelets may reflect certain biochemical changes that occur in the brain when different mental conditions occur. Platelet 5-hydroxytryptamine (5-HT) is also extensively studied in psychiatry. The mean platelet volume (MPV), the accurate measure of platelet size, has been considered a marker and determinant of platelet function. The aim of the present study was to search for any probable difference in the MPV of subjects with panic disorder (PD). Methods A total of 37 drug-free subjects, aged 18 to 65 years, diagnosed with PD, with or without agoraphobia, according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth edition (DSM-IV) criteria and 45 healthy control subjects were included in the study. Platelet count and MPV were measured and recorded for each subject. Results There were no statistically significant differences between groups in terms of female/male ratio, age, or body mass index between the PD group and control group (P=0.91, P=0.82, and P=0.93, respectively). The MPV was found to be significantly lower in the PD group compared with the control group (8.8±0.9 fL vs 9.2±0.8 fL; P=0.02). All the participants had MPV values in the standard range of 6.9–10.8 fL. Conclusion We concluded that abnormalities of the 5-HT1A receptor function in the central nervous system of subjects with a diagnosis of PD are also mirrored in as an alteration in platelet activity. Measurements of platelet activity may be used as a tool for neuropsychiatric and psychopharmacological research and for studying how certain mental diseases and medications affect the central nervous system. PMID:25214790

  13. Platelet-rich plasma (PRP) applied during total knee arthroplasty☆

    PubMed Central

    Guerreiro, João Paulo Fernandes; Danieli, Marcus Vinicius; Queiroz, Alexandre Oliveira; Deffune, Elenice; Ferreira, Rosana Rossi

    2015-01-01

    Objective To evaluate the efficacy of platelet-rich plasma regarding healing, pain and hemostasis after total knee arthroplasty, by means of a blinded randomized controlled and blinded clinical study. Methods Forty patients who were going to undergo implantation of a total knee prosthesis were selected and randomized. In 20 of these patients, platelet-rich plasma was applied before the joint capsule was closed. The hemoglobin (mg/dL) and hematocrit (%) levels were assayed before the operation and 24 and 48 h afterwards. The Womac questionnaire and a verbal pain scale were applied and knee range of motion measurements were made up to the second postoperative month. The statistical analysis compared the results with the aim of determining whether there were any differences between the groups at each of the evaluation times. Results The hemoglobin (mg/dL) and hematocrit (%) measurements made before the operation and 24 and 48 h afterwards did not show any significant differences between the groups (p > 0.05). The Womac questionnaire and the range of motion measured before the operation and up to the first two months also did not show any statistical differences between the groups (p > 0.05). The pain evaluation using the verbal scale showed that there was an advantage for the group that received platelet-rich plasma, 24 h, 48 h, one week, three weeks and two months after the operation (p < 0.05). Conclusions In the manner in which the platelet-rich plasma was used, it was not shown to be effective for reducing bleeding or improving knee function after arthroplasty, in comparison with the controls. There was an advantage on the postoperative verbal pain scale. PMID:26229915

  14. Fluorescence Adherence Inhibition Assay: A Novel Functional Assessment of Blocking Virus Attachment by Vaccine-Induced Antibodies.

    PubMed

    Asati, Atul; Kachurina, Olga; Karol, Alex; Dhir, Vipra; Nguyen, Michael; Parkhill, Robert; Kouiavskaia, Diana; Chumakov, Konstantin; Warren, William; Kachurin, Anatoly

    2016-01-01

    Neutralizing antibodies induced by vaccination or natural infection play a critically important role in protection against the viral diseases. In general, neutralization of the viral infection occurs via two major pathways: pre- and post-attachment modes, the first being the most important for such infections as influenza and polio, the latter being significant for filoviruses. Neutralizing capacity of antibodies is typically evaluated by virus neutralization assays that assess reduction of viral infectivity to the target cells in the presence of functional antibodies. Plaque reduction neutralization test, microneutralization and immunofluorescent assays are often used as gold standard virus neutralization assays. However, these methods are associated with several important prerequisites such as use of live virus requiring safety precautions, tedious evaluation procedure and long assessment time. Hence, there is a need for a robust, inexpensive high throughput functional assay that can be performed rapidly using inactivated virus, without extensive safety precautions. Herein, we report a novel high throughput Fluorescence Adherence Inhibition assay (fADI) using inactivated virus labeled with fluorescent secondary antibodies virus and Vero cells or erythrocytes as targets. It requires only few hours to assess pre-attachment neutralizing capacity of donor sera. fADI assay was tested successfully on donors immunized with polio, yellow fever and influenza vaccines. To further simplify and improve the throughput of the assay, we have developed a mathematical approach for calculating the 50% titers from a single sample dilution, without the need to analyze multi-point titration curves. Assessment of pre- and post-vaccination human sera from subjects immunized with IPOL®, YF-VAX® and 2013-2014 Fluzone® vaccines demonstrated high efficiency of the assay. The results correlated very well with microneutralization assay performed independently by the FDA Center of

  15. Fluorescence Adherence Inhibition Assay: A Novel Functional Assessment of Blocking Virus Attachment by Vaccine-Induced Antibodies

    PubMed Central

    Asati, Atul; Kachurina, Olga; Karol, Alex; Dhir, Vipra; Nguyen, Michael; Parkhill, Robert; Kouiavskaia, Diana; Chumakov, Konstantin; Warren, William; Kachurin, Anatoly

    2016-01-01

    Neutralizing antibodies induced by vaccination or natural infection play a critically important role in protection against the viral diseases. In general, neutralization of the viral infection occurs via two major pathways: pre- and post-attachment modes, the first being the most important for such infections as influenza and polio, the latter being significant for filoviruses. Neutralizing capacity of antibodies is typically evaluated by virus neutralization assays that assess reduction of viral infectivity to the target cells in the presence of functional antibodies. Plaque reduction neutralization test, microneutralization and immunofluorescent assays are often used as gold standard virus neutralization assays. However, these methods are associated with several important prerequisites such as use of live virus requiring safety precautions, tedious evaluation procedure and long assessment time. Hence, there is a need for a robust, inexpensive high throughput functional assay that can be performed rapidly using inactivated virus, without extensive safety precautions. Herein, we report a novel high throughput Fluorescence Adherence Inhibition assay (fADI) using inactivated virus labeled with fluorescent secondary antibodies virus and Vero cells or erythrocytes as targets. It requires only few hours to assess pre-attachment neutralizing capacity of donor sera. fADI assay was tested successfully on donors immunized with polio, yellow fever and influenza vaccines. To further simplify and improve the throughput of the assay, we have developed a mathematical approach for calculating the 50% titers from a single sample dilution, without the need to analyze multi-point titration curves. Assessment of pre- and post-vaccination human sera from subjects immunized with IPOL®, YF-VAX® and 2013–2014 Fluzone® vaccines demonstrated high efficiency of the assay. The results correlated very well with microneutralization assay performed independently by the FDA Center of

  16. Effect of Platelet-Rich Plasma (PRP) versus Autologous Whole Blood on Pain and Function Improvement in Tennis Elbow: A Randomized Clinical Trial

    PubMed Central

    Raeissadat, Seyed Ahmad; Sedighipour, Leyla; Rayegani, Seyed Mansoor; Bahrami, Mohammad Hasan; Bayat, Masume; Rahimi, Rosa

    2014-01-01

    Background. Autologous whole blood and platelet-rich plasma (PRP) have been both suggested to treat chronic tennis elbow. The aim of the present study was to compare the effects of PRP versus autologous whole blood local injection in chronic tennis elbow. Methods. Forty patients with tennis elbow were randomly divided into 2 groups. Group 1 was treated with a single injection of 2 mL of autologous PRP and group 2 with 2 mL of autologous blood. Tennis elbow strap, stretching, and strengthening exercises were administered for both groups during a 2-month followup. Pain and functional improvements were assessed using visual analog scale (VAS), modified Mayo Clinic performance index for the elbow, and pressure pain threshold (PPT) at 0, 4, and 8 weeks. Results. All pain and functional variables including VAS, PPT, and Mayo scores improved significantly in both groups 4 weeks after injection. No statistically significant difference was noted between groups regarding pain scores in 4-week follow-up examination (P > 0.05). At 8-week reevaluations, VAS and Mayo scores improved only in PRP group (P < 0.05). Conclusion. PRP and autologous whole blood injections are both effective to treat chronic lateral epicondylitis. PRP might be slightly superior in 8-week followup. However, further studies are suggested to get definite conclusion. PMID:24579044

  17. Breakdown of paraendothelial barrier function during Marburg virus infection is associated with early tyrosine phosphorylation of platelet endothelial cell adhesion molecule-1.

    PubMed

    Böckeler, Michael; Ströher, Ute; Seebach, Jochen; Afanasieva, Tatiana; Suttorp, Norbert; Feldmann, Heinz; Schnittler, Hans-Joachim

    2007-11-15

    Marburg virus (MARV) infection often causes fulminant shock due to pathologic immune responses and alterations of the vascular system. Cytokines released from virus-infected monocytes/macrophages provoke endothelial activation and vascular hyperpermeability and contribute to the development of shock. Tyrosine phosphorylation of cell-junction proteins is important for the regulation of paraendothelial barrier function. We showed that mediators released from MARV-infected monocytes/macrophages, as well as recombinant tumor necrosis factor (TNF)- alpha /H2O2 and interferon (IFN)- gamma , caused tyrosine phosphorylation of platelet endothelial cell adhesion molecule-1 (PECAM-1) but not of the vascular endothelial (VE) cadherin/catenin complex proteins. Tyrosine phosphorylation of PECAM-1 was associated with delayed opening of interendothelial junctions. Interestingly, we observed an early increase in water permeability in response to TNF- alpha /H2O2 that was not due to an opening of the interendothelial junctions. These data indicate 2 distinct mechanisms for the TNF- alpha /H2O2-mediated decrease in endothelial barrier function involving tyrosine phosphorylation of PECAM-1 but not requiring tyrosine phosphorylation of VE-cadherin or catenin proteins. PMID:17940969

  18. Effect of local administration of platelet-derived growth factor B on functional recovery of peripheral nerve regeneration: A sciatic nerve transection model

    PubMed Central

    Golzadeh, Atefeh; Mohammadi, Rahim

    2016-01-01

    Background: Effects of platelet-derived growth factor B (PDGF-B) on peripheral nerve regeneration was studied using a rat sciatic nerve transection model. Materials and Methods: Forty-five male, white Wistar rats were divided into three experimental groups (n = 15), randomly: Normal control group (NC), silicon group (SIL), and PDGF-B treated group (SIL/PDGF). In NC group, left sciatic nerve was exposed through a gluteal muscle incision and after homeostasis muscle was sutured. In the SIL group, the left sciatic nerve was exposed in the same way and transected proximal to tibio-peroneal bifurcation leaving a 10-mm gap. Proximal and distal stumps were each inserted into a silicone conduit and filled with 10 μL phosphate buffered solution. In SIL/PDGF group, the silicon conduit was filled with 10 μL PDGF-B (0.5 ng/mL). Each group was subdivided into three subgroups of five and were studied in 4, 8, 12 weeks after surgery. Results: Behavioral testing, sciatic nerve functional study, gastrocnemius muscle mass, and histomorphometric studies showed earlier regeneration of axons in SIL/PDGF than in SIL group (P < 0.05). Conclusion: Local administration of PDGF-B combined with silicon grafting could accelerate functional recovery and may have clinical implications for the surgical management of patients after facial nerve transection. PMID:27274342

  19. Attenuated Reactive Gliosis and Enhanced Functional Recovery Following Spinal Cord Injury in Null Mutant Mice of Platelet-Activating Factor Receptor.

    PubMed

    Wang, Yuanyi; Gao, Zhongwen; Zhang, Yiping; Feng, Shi-Qing; Liu, Yulong; Shields, Lisa B E; Zhao, Ying-Zheng; Zhu, Qingsan; Gozal, David; Shields, Christopher B; Cai, Jun

    2016-07-01

    Platelet-activating factor (PAF) is a unique phosphoglycerine that mediates the biological functions of both immune and nervous systems. Excessive PAF plays an important role in neural injury via its specific receptor (PAFR). In this study, we hypothesized that PAF signaling activates reactive gliosis after spinal cord injury (SCI), and blocking the PAF pathway would modify the glia scar formation and promote functional recovery. PAF microinjected into the normal wild-type spinal cord induced a dose-dependent activation of microglia and astrocytes. In the SCI mice, PAFR null mutant mice showed a better functional recovery in grip and rotarod performances than wild-type mice. Although both microglia and astrocytes were activated after SCI in wild-type and PAFR null mutant mice, expressions of IL-6, vimentin, nestin, and GFAP were not significantly elevated in PAFR null mutants. Disruption of PAF signaling inhibited the expressions of proteoglycan CS56 and neurocan (CSPG3). Intriguingly, compared to the wild-type SCI mice, less axonal retraction/dieback at 7 dpi but more NFH-labeled axons at 28 dpi was found in the area adjacent to the epicenter in PAFR null mutant SCI mice. Moreover, treatment with PAFR antagonist Ginkgolide B (GB) at the chronic phase rather than acute phase enhanced the functional recovery in the wild-type SCI mice. These findings suggest that PAF signaling participates in reactive gliosis after SCI, and blocking of this signaling enhances functional recovery and to some extent may promote axon regrowth. PMID:26084439

  20. Improving platelet transfusion safety: biomedical and technical considerations.

    PubMed

    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

    2016-03-01

    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

  1. 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

  2. 2-Arachidonoylglycerol enhances platelet formation from human megakaryoblasts

    PubMed Central

    Gasperi, Valeria; Avigliano, Luciana; Evangelista, Daniela; Oddi, Sergio; Chiurchiù, Valerio; Lanuti, Mirko; Maccarrone, Mauro; Valeria Catani, Maria

    2014-01-01

    Platelets modulate vascular system integrity, and their loss is critical in haematological pathologies and after chemotherapy. Therefore, identification of molecules enhancing platelet production would be useful to counteract thrombocytopenia. We have previously shown that 2-arachidonoylglycerol (2-AG) acts as a true agonist of platelets, as well as it commits erythroid precursors toward the megakaryocytic lineage. Against this background, we sought to further interrogate the role of 2-AG in megakaryocyte/platelet physiology by investigating terminal differentiation, and subsequent thrombopoiesis. To this end, we used MEG-01 cells, a human megakaryoblastic cell line able to produce in vitro platelet-like particles. 2-AG increased the number of cells showing ruffled surface and enhanced surface expression of specific megakaryocyte/platelet surface antigens, typical hallmarks of terminal megakaryocytic differentiation and platelet production. Changes in cytoskeleton modeling also occurred in differentiated megakaryocytes and blebbing platelets. 2-AG acted by binding to CB1 and CB2 receptors, because specific antagonists reverted its effect. Platelets were split off from megakaryocytes and were functional: they contained the platelet-specific surface markers CD61 and CD49, whose levels increased following stimulation with a natural agonist like collagen. Given the importance of 2-AG for driving megakaryopoiesis and thrombopoiesis, not surprisingly we found that its hydrolytic enzymes were tightly controlled by classical inducers of megakaryocyte differentiation. In conclusion 2-AG, by triggering megakaryocyte maturation and platelet release, may have clinical efficacy to counteract thrombocytopenia-related diseases. PMID:25427281

  3. Platelets enhance neutrophil transendothelial migration

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Platelets are increasingly recognized as important mediators of inflammation in addition to thrombosis. While platelets have been shown to promote neutrophil (PMN) adhesion to endothelium in various inflammatory models, it is unclear whether platelets enhance neutrophil transmigration across inflame...

  4. Sickle cell disease biochip: a functional red blood cell adhesion assay for monitoring sickle cell disease.

    PubMed

    Alapan, Yunus; Kim, Ceonne; Adhikari, Anima; Gray, Kayla E; Gurkan-Cavusoglu, Evren; Little, Jane A; Gurkan, Umut A

    2016-07-01

    Sickle cell disease (SCD) afflicts millions of people worldwide and is associated with considerable morbidity and mortality. Chronic and acute vaso-occlusion are the clinical hallmarks of SCD and can result in pain crisis, widespread organ damage, and early movtality. Even though the molecular underpinnings of SCD were identified more than 60 years ago, there are no molecular or biophysical markers of disease severity that are feasibly measured in the clinic. Abnormal cellular adhesion to vascular endothelium is at the root of vaso-occlusion. However, cellular adhesion is not currently evaluated clinically. Here, we present a clinically applicable microfluidic device (SCD biochip) that allows serial quantitative evaluation of red blood cell (RBC) adhesion to endothelium-associated protein-immobilized microchannels, in a closed and preprocessing-free system. With the SCD biochip, we have analyzed blood samples from more than 100 subjects and have shown associations between the measured RBC adhesion to endothelium-associated proteins (fibronectin and laminin) and individual RBC characteristics, including hemoglobin content, fetal hemoglobin concentration, plasma lactate dehydrogenase level, and reticulocyte count. The SCD biochip is a functional adhesion assay, reflecting quantitative evaluation of RBC adhesion, which could be used at baseline, during crises, relative to various long-term complications, and before and after therapeutic interventions. PMID:27063958

  5. Sickle cell disease biochip: a functional red blood cell adhesion assay for monitoring sickle cell disease

    PubMed Central

    ALAPAN, YUNUS; KIM, CEONNE; ADHIKARI, ANIMA; GRAY, KAYLA E.; GURKAN-CAVUSOGLU, EVREN; LITTLE, JANE A.; GURKAN, UMUT A.

    2016-01-01

    Sickle cell disease (SCD) afflicts millions of people worldwide and is associated with considerable morbidity and mortality. Chronic and acute vaso-occlusion are the clinical hallmarks of SCD and can result in pain crisis, widespread organ damage, and early movtality. Even though the molecular underpinnings of SCD were identified more than 60 years ago, there are no molecular or biophysical markers of disease severity that are feasibly measured in the clinic. Abnormal cellular adhesion to vascular endothelium is at the root of vaso-occlusion. However, cellular adhesion is not currently evaluated clinically. Here, we present a clinically applicable microfluidic device (SCD biochip) that allows serial quantitative evaluation of red blood cell (RBC) adhesion to endothelium-associated protein-immobilized microchannels, in a closed and preprocessing-free system. With the SCD biochip, we have analyzed blood samples from more than 100 subjects and have shown associations between the measured RBC adhesion to endothelium-associated proteins (fibronectin and laminin) and individual RBC characteristics, including hemoglobin content, fetal hemoglobin concentration, plasma lactate dehydrogenase level, and reticulocyte count. The SCD biochip is a functional adhesion assay, reflecting quantitative evaluation of RBC adhesion, which could be used at baseline, during crises, relative to various long-term complications, and before and after therapeutic interventions. PMID:27063958

  6. High-throughput functional screening using a homemade dual-glow luciferase assay.