Summary. Adenosine diphosphate (ADP) is an important platelet agonist and ADP released from platelet dense granules amplifies responses to other agonists. There are three known subtypes of ADP receptor on platelets: P2X 1 , P2Y 1 and P 2T receptors. Sustained ADP-induced aggregation requires co-activation of P2Y 1 and P 2T receptors. AR-C69931MX, a selective P 2T receptor antagonist and novel antithrombotic agent, was studied to characterize further the function of the P 2T receptor. The roles of the P2Y 1 receptor and thromboxane A 2 were assessed using the selective P2Y 1 antagonist A2P5P and aspirin respectively. Aggregation was measured by whole blood single-platelet counting and platelet-rich plasma turbidimetry, using hirudin anticoagulation. Dense granule release was estimated using [14 C]-5-hydroxytryptamine (HT)-labelled platelets. Ca 21 mobilization, P-selectin expression, Annexin V binding and microparticle formation were determined by flow cytometry. P 2T receptor activation amplified ADPinduced aggregation initiated by the P2Y 1 receptor, as well as amplifying aggregation, secretion and procoagulant responses induced by other agonists, including U46619, thrombin receptor-activating peptide (TRAP) and collagen, independent of thromboxane A 2 synthesis, which played a more peripheral role. P 2T receptor activation sustained elevated cytosolic Ca 21 induced by other pathways. These studies indicate that the P 2T receptor plays a central role in amplifying platelet responses and demonstrate the clinical potential of P 2T receptor antagonists.
Receptors for prostanoids on platelets include the EP3 receptor for which the natural agonist is the inflammatory mediator prostaglandin E(2) (PGE(2)) produced in atherosclerotic plaques. EP3 is implicated in atherothrombosis and an EP3 antagonist might provide atherosclerotic lesion-specific antithrombotic therapy. DG-041 (2,3-dichlorothiophene-5-sulfonic acid, 3-[1-(2,4-dichlorobenzyl)-5-fluoro-3-methyl-1H-indol-7-yl]acryloylamide) is a direct-acting EP3 antagonist currently being evaluated in Phase 2 clinical trials. We have examined the contributions of EP3 to platelet function using the selective EP3 agonist sulprostone and also PGE(2), and determined the effects of DG-041 on these. Studies were in human platelet-rich plasma or whole blood and included aggregometry and flow cytometry. Sulprostone enhanced aggregation induced by primary agonists including collagen, TRAP, platelet activating factor, U46619, serotonin and adenosine diphosphate, and enhanced P-selectin expression and platelet-leukocyte conjugate formation. It inhibited adenylate cyclase (measured by vasodilator-stimulated phosphoprotein phosphorylation) and enhanced Ca(2+) mobilization. It potentiated platelet function even in the presence of aspirin and/or AR-C69931 (a P2Y(12) antagonist). DG-041 antagonized the effects of sulprostone on platelet function. The effect of PGE(2) on platelet aggregation depended on the nature of the agonist and the concentration of PGE(2) used as a consequence of both pro-aggregatory effects via EP3 and anti-aggregatory effects via other receptors. DG-041 potentiated the protective effects of PGE(2) on platelet aggregation by inhibiting the pro-aggregatory effect via EP3 stimulation. DG-041 remained effective in the presence of a P2Y(12) antagonist and aspirin. DG-041 warrants continued investigation as a potential agent for the treatment of atherothrombosis without inducing unwanted bleeding risk.
There is great interest in assessing the efficacy of treatment with clopidogrel and aspirin in patients with cardiovascular disease using procedures that can be used in a remote setting. Here we have established methods to assess the effects of clopidogrel and aspirin on platelets based on measurements of platelet P-selectin. Platelets were stimulated in whole blood by adding the combination of adenosine diphosphate and the TXA(2) mimetic U46619 (ADP/U4, designed to assess P2Y(12) inhibition) or the combination of arachidonic acid and epinephrine (AA/Epi, designed to assess COX-1 inhibition). The stimulated samples were then fixed using a fixative solution that provides stability for at least 9 days, and sent to a central laboratory for analysis of P-selectin by flow cytometry. Measurements were performed in blood from healthy volunteers and patients with cardiovascular disease. The inhibitory effects of clopidogrel and aspirin were assessed ex vivo and the effects of the direct acting P2Y(12) antagonist cangrelor and aspirin were assessed in vitro. Measurements of platelet aggregation were also performed for comparison. In healthy volunteers clopidogrel ex vivo and cangrelor in vitro markedly inhibited P-selectin expression induced by ADP/U4. Aspirin did not inhibit and did not interfere with the effects of clopidogrel or cangrelor using this test. There was very little overlap of results obtained in the absence and presence of clopidogrel or cangrelor. In contrast, over half of 42 patients with cardiovascular disease did not respond well to clopidogrel treatment, although cangrelor was still effective. Aspirin markedly inhibited P-selectin expression induced by AA/Epi. Clopidogrel had much less effect and did not interfere with the effects of aspirin. There was no overlap of results obtained in the absence and presence of aspirin. Aspirin provided near-complete inhibition in 29 of 30 patients with cardiovascular disease. Aggregometry measurements agreed well with the P-selectin data obtained ex vivo following both clopidogrel and aspirin treatment. It is concluded that measurements of P-selectin performed on fixed blood samples following platelet stimulation in whole blood in a remote setting can be used effectively to monitor the effects of clopidogrel and aspirin.
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 were related to their flavanol content. There was also inhibition of monocyte and neutrophil activation, but here it was concluded that cocoa constituents other than flavanols may contribute to the inhibition that was observed. It can be concluded that cocoa flavanols, their metabolites and possibly other cocoa constituents can modulate the activity of platelets and leukocytes in vitro and ex vivo. The research suggests that the consumption of certain cocoa products may provide a dietary approach to maintaining or improving cardiovascular health.
Summary. Background: Mild platelet function disorders (PFDs) are complex and difficult to diagnose. The current gold standard test, light transmission aggregometry (LTA), including lumi-aggregometry, is time and labour intensive and blood samples must be processed within a limited time after venepuncture. Furthermore, many subjects with suspected PFDs do not show a platelet abnormality on LTA. Objective: To assess the diagnostic potential of an easy-to-use remote platelet function test (RPFT) as a diagnostic pre-test for suspected PFDs. Methods: A remote platelet function test was compared with lumi-aggregometry in participants recruited to the Genotyping and Phenotyping of Platelets Study (GAPP, ISRCTN 77951167). For the RPFT, whole blood was stimulated with platelet agonists, stabilized with PAMFix and returned to the central laboratory for analysis of P-selectin and CD63 by flow cytometry. Results: For the 61 study participants (42 index cases and 19 relatives) there was a good agreement between lumi-aggregometry and the RPFT, with diagnosis being concordant in 84% of cases (j = 0.668, P < 0.0001). According to both tests, 29 participants were identified to have a deficiency in platelet function and 22 participants appeared normal. There were four participants where lumi-aggregometry revealed a defect but the RPFT did not, and six participants where the RPFT detected an abnormal platelet response that was not identified by lumi-aggregometry. Conclusion: This study suggests that the RPFT could be an easy-to-use pretest to select which participants with bleeding disorders would benefit from extensive platelet phenotyping. Further development and evaluation of the test are warranted in a wider population of patients with excessive bleeding and could provide informative screening tests for PFDs.
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