114 research outputs found
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P2Y12 Receptor Blockade Augments Glycoprotein IIbâIIIa Antagonist Inhibition of Platelet Activation, Aggregation, and Procoagulant Activity
Background: New antiplatelet agents that provide greater, more consistent inhibition of the platelet ADP receptor P2Y12 may be used in combination with glycoprotein (GP) IIbâIIIa antagonists, but their combined effect on platelet function and procoagulant activity is not well studied. Therefore, the objective of this study was to evaluate the independent and complementary effects of P2Y12 and GPIIbâIIIa inhibition on platelet function and procoagulant activity. Methods and Results: Healthy donor blood was treated with the active metabolite of prasugrel (Râ138727 5 ÎŒmol/L), GPIIbâIIIa antagonists (abciximab 3 ÎŒg/mL or eptifibatide 0.9 ÎŒg/mL), and combinations thereof, exposed to physiologically relevant agonists (collagen and ADP) and then evaluated for markers of platelet activation and procoagulant activity. Significant interactions between Râ138727 and GPIIbâIIIa antagonists were observed. Râ138727 and the GPIIbâIIIa antagonists had additive inhibitory effects on collagenâstimulated platelet aggregation and on the collagen plus ADPâstimulated level of activated platelet surface GPIIbâIIIa. Râ138727 and abciximab each inhibited collagen plus ADPâstimulated platelet phosphatidylserine expression and prothrombin cleavage, and the combination produced greater inhibition than achieved with abciximab alone. In contrast, eptifibatide did not inhibit, but instead enhanced, collagen plus ADPâstimulated prothrombin cleavage. Addition of Râ138727 reduced prothrombin cleavage in eptifibatideâtreated samples, suggesting a novel mechanism for potential benefit from combined prasugrel and eptifibatide treatment. Conclusions: The complementary effects of abciximab and Râ138727 on platelet activation, aggregation, and procoagulant activity suggest their combined use may, to a greater degree than with either agent alone, reduce thrombus formation in vivo
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Antiplatelet Activity, P2Y1 and P2Y12 Inhibition, and Metabolism in Plasma of Stereoisomers of Diadenosine 5âČ,5âČâł-P1,P4-dithio-P2,P3-chloromethylenetetraphosphate
Background: Diadenosine tetraphosphate (Ap4A), a constituent of platelet dense granules, and its P1,P4-dithio and/or P2,P3-chloromethylene analogs, inhibit adenosine diphosphate (ADP)-induced platelet aggregation. We recently reported that these compounds antagonize both platelet ADP receptors, P2Y1 and P2Y12. The most active of those analogs, diadenosine 5âČ,5âłâł-P1,P4-dithio-P2,P3-chloromethylenetetraphosphate, (compound 1), exists as a mixture of 4 stereoisomers. Objective: To separate the stereoisomers of compound 1 and determine their effects on platelet aggregation, platelet P2Y1 and P2Y12 receptor antagonism, and their metabolism in human plasma. Methods: We separated the 4 diastereomers of compound 1 by preparative reversed-phase chromatography, and studied their effect on ADP-induced platelet aggregation, P2Y1-mediated changes in cytosolic Ca2+, P2Y12-mediated changes in VASP phosphorylation, and metabolism in human plasma. Results: The inhibition of ADP-induced human platelet aggregation and human platelet P2Y12 receptor, and stability in human plasma strongly depended on the stereo-configuration of the chiral P1- and P4-phosphorothioate groups, the SPSP diastereomer being the most potent inhibitor and completely resistant to degradation in plasma, and the RPRP diastereomer being the least potent inhibitor and with the lowest plasma stability. The inhibitory activity of SPRP diastereomers depended on the configuration of the pseudo-asymmetric carbon of the P2,P3-chloromethylene group, one of the configurations being significantly more active than the other. Their plasma stability did not differ significantly, being intermediate to that of the SPSP and the RPRP diastereomers. Conclusions: The presently-described stereoisomers have utility for structural, mechanistic, and drug development studies of dual antagonists of platelet P2Y1 and P2Y12 receptors
Consensus recommendations on flow cytometry for the assessment of inherited and acquired disorders of platelet number and function : communication from the ISTH SSC Subcommittee on Platelet Physiology
Flow cytometry is increasingly used in the study of platelets in inherited and acquired disorders of platelet number and function. However, wide variation exists in specific reagents, methods, and equipment used, making interpretation and comparison of results difficult. The goal of the present study was to provide expert consensus guidance on the use of flow cytometry for the evaluation of platelet disorders. A modified RAND/UCLA survey method was used to obtain a consensus among 11 experts from 10 countries across four continents, on the appropriateness of statements relating to clinical utility, pre-analytical variables, instrument and reagent standardization, methods, reporting, and quality control for platelet flow cytometry. Feedback from the initial survey revealed that uncertainty was sometimes due to lack of expertise with a particular test condition rather than unavailable or ambiguous data. To address this, the RAND method was modified to allow experts to self-identify statements for which they could not provide expert input. There was uniform agreement among experts in the areas of instrument and reagent standardization, methods, reporting, and quality control and this agreement is used to suggest best practices in these areas. However, 25.9% and 50% of statements related to pre-analytical variables and clinical utility, respectively, were rated as uncertain. Thus, while citrate is the preferred anticoagulant for many flow cytometric platelet tests, expert opinions differed on the acceptability of other anticoagulants, particularly heparin. Lack of expert consensus on the clinical utility of many flow cytometric platelet tests indicates the need for rigorous multicenter clinical outcome studies
Platelet activation in cystic fibrosis
Cystic fibrosis (CF) is caused by a mutation of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). We examined platelet function in CF patients because lung inflammation is part of this disease and platelets contribute to inflammation. CF patients had increased circulating leukocyte-platelet aggregates and increased platelet responsiveness to agonists compared with healthy controls. CF plasma caused activation of normal and CF platelets; however, activation was greater in CF platelets. Furthermore, washed CF platelets also showed increased reactivity to agonists. CF platelet hyperreactivity was incompletely inhibited by prostaglandin E(1) (PGE(1)). As demonstrated by Western blotting and reverse-transcriptase-polymerase chain reaction (RT-PCR), there was neither CFTR nor CFTR-specific mRNA in normal platelets. There were abnormalities in the fatty acid composition of membrane fractions of CF platelets. In summary, CF patients have an increase in circulating activated platelets and platelet reactivity, as determined by monocyte-platelet aggregation, neutrophil-platelet aggregation, and platelet surface P-selectin. This increased platelet activation in CF is the result of both a plasma factor(s) and an intrinsic platelet mechanism via cyclic adenosine monophosphate (cAMP)/adenylate cyclase, but not via platelet CFTR. Our findings may account, at least in part, for the beneficial effects of ibuprofen in CF
Variability of Individual Platelet Reactivity Over Time in Patients Treated With Clopidogrel Insights From the ELEVATEâTIMI 56 Trial
AbstractBackgroundThe degree of antiplatelet response to clopidogrel has been associated with clinical outcomes. Studies have investigated whether adjustment of antiplatelet therapies based on a single platelet function test is beneficial.ObjectivesThe aim of the study was to test the stability of platelet reactivity measurements over time among patients treated with standard and double doses of clopidogrel.MethodsThe ELEVATEâTIMI 56 (Escalating Clopidogrel by Involving a Genetic StrategyâThrombolysis In Myocardial Infarction 56) investigators genotyped 333 patients with coronary artery disease and randomized them to various clopidogrel regimens. Patients with at least 2 platelet function results on the same maintenance dose of clopidogrel (75 mg or 150 mg) were analyzed. Platelet aggregation was measured using P2Y12 reaction units (PRU).ResultsIn total, the mean platelet reactivity and the total number of nonresponders (PRU â„230) with clopidogrel did not change between 2 periods for the 75-mg (22.4% vs. 21.9%; p = 0.86) and 150-mg doses of clopidogrel (11.5% vs. 11.5%; p = 1.00). In contrast, when evaluating each patient individually, 15.7% of patients taking clopidogrel 75 mg and 11.4% of patients taking 150 mg had a change in their responder status when tested at 2 different time points (p < 0.001). Despite being treated with the same dose of clopidogrel, >40% of patients had a change in PRU >40 on serial sampling, which approximates the average PRU difference caused by increasing the clopidogrel dose from 75 mg to 150 mg.ConclusionsMeasurements of platelet reactivity vary over time in a significant proportion of patients. Thus, treatment adjustment according to platelet function testing at a single time point might not be sufficient for guiding antiplatelet therapy in clinical or research settings. (Escalating Clopidogrel by Involving a Genetic StrategyâThrombolysis In Myocardial Infarction 56 [ELEVATEâTIMI 56]; NCT01235351
The Human Endogenous Circadian System Causes Greatest Platelet Activation during the Biological Morning Independent of Behaviors
Platelets are involved in the thromboses that are central to myocardial infarctions and ischemic strokes. Such adverse cardiovascular events have day/night patterns with peaks in the morning (~9 AM), potentially related to endogenous circadian clock control of platelet activation. The objective was to test if the human endogenous circadian system influences (1) platelet function and (2) platelet response to standardized behavioral stressors. We also aimed to compare the magnitude of any effects on platelet function caused by the circadian system with that caused by varied standardized behavioral stressors, including mental arithmetic, passive postural tilt and mild cycling exercise.We studied 12 healthy adults (6 female) who lived in individual laboratory suites in dim light for 240 h, with all behaviors scheduled on a 20-h recurring cycle to permit assessment of endogenous circadian function independent from environmental and behavioral effects including the sleep/wake cycle. Circadian phase was assessed from core body temperature. There were highly significant endogenous circadian rhythms in platelet surface activated glycoprotein (GP) IIb-IIIa, GPIb and P-selectin (6-17% peak-trough amplitudes; p †0.01). These circadian peaks occurred at a circadian phase corresponding to 8-9 AM. Platelet count, ATP release, aggregability, and plasma epinephrine also had significant circadian rhythms but with later peaks (corresponding to 3-8 PM). The circadian effects on the platelet activation markers were always larger than that of any of the three behavioral stressors.These data demonstrate robust effects of the endogenous circadian system on platelet activation in humans--independent of the sleep/wake cycle, other behavioral influences and the environment. The 9 AM timing of the circadian peaks of the three platelet surface markers, including platelet surface activated GPIIb-IIIa, the final common pathway of platelet aggregation, suggests that endogenous circadian influences on platelet function could contribute to the morning peak in adverse cardiovascular events as seen in many epidemiological studies
Using flow cytometry to monitor glycoprotein IIb-IIIa activation
Platelet-to-platelet aggregation is critical to the formation of hemostatic thrombi which limit bleeding following vascular injury and also contributes to obstructive thrombi in acute myocardial infarction, stroke, or other thrombotic diseases. Platelet aggregation is mediated by platelet surface glycoprotein (GP) IIb-IIIa (integrin αIIbÎČ3, CD41/61) on adjacent platelets. Upon platelet activation by adenosine diphosphate (ADP), thrombin, or other platelet agonists, GPIIb-IIIa undergoes conformational changes from a ârestingâ bent conformation to an âactivatedâ extended conformation. In GPIIb-IIIaâs activated conformation, a binding site is exposed which interacts with the arginine-glycine-aspartic acid (RGD) residues in the fibrinogen alpha chain, permitting fibrinogen binding and cross-bridging of adjacent activated platelets. Consequently, changes in the state of GPIIb-IIIa activation closely correlate with fibrinogen binding and the degree of platelet-platelet aggregation. In contrast to radiolabeled ligand methods used for bulk receptor-binding studies, flow cytometry allows the rapid analysis of fibrinogen receptor expression on single cells, thereby enabling analysis of the kinetics of GPIIb-IIIa activation and differences between platelets in their expression of activated GPIIb-IIIa. The present review will consider the use of flow cytometry to monitor GPIIb-IIIa activation and its application in clinical and research settings
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