Browsing by keyword "Receptors, Purinergic P2Y12"

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    • Agonist and antagonist effects of diadenosine tetraphosphate, a platelet dense granule constituent, on platelet P2Y1, P2Y12 and P2X1 receptors
      Chang, Hung; Yanachkov, Ivan B.; Michelson, Alan D.; Li, YouFu; Barnard, Marc R.; Wright, George E.; Frelinger, Andrew L. III (2009-12-01)
      INTRODUCTION: Diadenosine 5',5'''-P(1),P(4)- tetraphosphate (Ap(4)A) is stored in platelet dense granules, but its effects on platelet function are not well understood. METHODS AND RESULTS: We examined the effects of Ap(4)A on platelet purinergic receptors P2Y(1), P2Y(12) and P2X(1). Flow cytometry was used to measure the effects of Ap(4)A in the presence or absence of ADP on: a) P2Y(12)-mediated decrease in intraplatelet phosphorylated vasodilator stimulated phosphoprotein (VASP), b) P2Y(1)-mediated increase in platelet cytosolic Ca(2+), and c) P2X(1)-mediated intraplatelet entry of extracellular Ca(2+). ADP-stimulated platelet shape change (P2Y(1)-mediated) and aggregation (P2Y(1)- and P2Y(12)-mediated) were measured optically. Ap(4)A inhibited 3 microM ADP-induced: a) platelet aggregation (IC(50) 9.8+/-2.8 microM), b) P2Y(1)-mediated shape change, c) P2Y(1)-mediated increase in platelet cytosolic Ca(2+) (IC(50) 40.8+/-12.3 microM), and d) P2Y(12)-mediated decrease in VASP phosphorylation (IC(50)>250 microM). In the absence of added ADP, Ap(4)A had agonist effects on platelet P2X(1) and P2Y(12), but not P2Y(1), receptors. CONCLUSION: Ap(4)A, a constituent of platelet dense granules, is a) an antagonist of platelet P2Y(1) and P2Y(12) receptors, where it inhibits the effects of ADP, and b) an agonist of platelet P2X(1) and P2Y(12) receptors.

    • Hematocrit alters VerifyNow P2Y12 assay results independently of intrinsic platelet reactivity and clopidogrel responsiveness
      Kakouros, Nikolaos; Kickler, T S.; Laws, K M.; Rade, Jeffrey J. (2013-10-01)
      BACKGROUND: The VerifyNow P2Y12 assay assesses the adequacy of clopidogrel therapy by measuring ADP-induced platelet activation in whole blood. Low hematocrit is associated with high clopidogrel on-treatment platelet reactivity (HTPR) defined by this assay. OBJECTIVES: To characterize the effect of hematocrit on VerifyNow values and determine if it is due to hematocrit-dependent changes in intrinsic platelet reactivity or an in vitro assay phenomenon. PATIENTS/METHODS: Adenosine diphosphate-induced platelet activation was measured using the VerifyNow P2Y12 assay, whole blood impedance and light transmission platelet aggregometry (LTA) before and after clopidogrel loading in 113 patients undergoing elective cardiac catheterization. Iso-TRAP-induced platelet activation was additionally measured using the VerifyNow device. Multivariate modeling employing clinical and laboratory variables was used to investigate the association between hematocrit and VerifyNow values. RESULTS: VerifyNow P2Y12 reaction units (PRU) and iso-TRAP Base units before and after clopidogrel loading, but not their relative change, exhibited strong negative correlation with hematocrit (P < /= 0.0005 for both). While hematocrit remained a strong predictor of post-clopidogrel PRU (P = 0.001) in multivariate modeling, it was independent of post-clopidogrel ADP-induced platelet reactivity as measured by LTA (P = 0.001). Correcting for the effects of hematocrit resulted in a 15-39% reduction in the prevalence of HTPR defined by thresholds of 208-236 PRU. CONCLUSIONS: The effect of hematocrit on VerifyNow PRU values is an in vitro phenomenon that is independent of intrinsic change in ADP-induced platelet reactivity and clopidogrel responsiveness. Correcting for hematocrit when using this assay may more accurately identify patients with HTPR that may benefit from alternative antiplatelet therapy.

    • Platelet antistaphylococcal responses occur through P2X1 and P2Y12 receptor-induced activation and kinocidin release
      Trier, Darin A.; Gank, Kimberly D.; Kupferwasser, Deborah; Young, Nannette Y; French, William J.; Michelson, Alan D.; Kupferwasser, Leon I.; Xiong, Yan Q.; Bayer, Arnold S.; Yeaman, Michael R. (2008-12-01)
      Platelets (PLTs) act in antimicrobial host defense by releasing PLT microbicidal proteins (PMPs) or PLT kinocidins (PKs). Receptors mediating staphylocidal efficacy and PMP or PK release versus isogenic PMP-susceptible (ISP479C) and -resistant (ISP479R) Staphylococcus aureus strains were examined in vitro. Isolated PLTs were incubated with ISP479C or ISP479R (PLT/S. aureus ratio range, 1:1 to 10,000:1) in the presence or absence of a panel of PLT inhibitors, including P2X and P2Y receptor antagonists of increasingly narrow specificity, and PLT adhesion receptors (CD41, CD42b, and CD62P). PLT-to-S. aureus exposure ratios of > or = 10:1 yielded significant reductions in the viability of both strains. Results from reversed-phase high-performance liquid chromatography indicated that staphylocidal PLT releasates contained PMPs and PKs. At ratios below 10:1, the PLT antistaphylococcal efficacy relative to the intrinsic S. aureus PMP-susceptible or -resistant phenotype diminished. Apyrase (an agent of ADP degradation), suramin (a general P2 receptor antagonist), pyridoxal 5'-phosphonucleotide derivative (a specific P2X(1) antagonist), and cangrelor (a specific P2Y(12) antagonist) mitigated the PLT staphylocidal response against both strains, correlating with reduced levels of PMP and PK release. Specific inhibition occurred in the presence and absence of homologous plasma. The antagonism of the thromboxane A(2), cyclooxygenase-1/cyclooxygenase-2, or phospholipase C pathway or the hindrance of surface adhesion receptors failed to impede PLT anti-S. aureus responses. These results suggest a multifactorial PLT anti-S. aureus response mechanism involving (i) a PLT-to-S. aureus ratio sufficient for activation; (ii) the ensuing degranulation of PMPs, PKs, ADP, and/or ATP; (iii) the activation of P2X(1)/P2Y(12) receptors on adjacent PLTs; and (iv) the recursive amplification of PMP and PK release from these PLTs.

    • The active metabolite of prasugrel inhibits adenosine diphosphate- and collagen-stimulated platelet procoagulant activities
      Frelinger, Andrew L. III; Jakubowski, Joseph A.; Li, YouFu; Barnard, Marc R.; Linden, Matthew Dean; Tarnow, Inge; Fox, Marsha L.; Sugidachi, Atsuhiro; Winters, Kenneth J.; Furman, Mark I.; et al. (2008-02-01)
      BACKGROUND: Prasugrel is a novel antiplatelet prodrug of the same thienopyridine class as clopidogrel and ticlopidine. Metabolism of prasugrel generates the active metabolite R-138727, an antagonist of the platelet P2Y(12) adenosine diphosphate (ADP) receptor, leading to inhibition of ADP-mediated platelet activation and aggregation. ADP also enhances the platelet response to collagen, and these two agonists contribute to the generation of platelet procoagulant activity. We therefore examined whether R-138727 inhibits ADP- and collagen-triggered platelet procoagulant activities. METHODS AND RESULTS: As shown by whole blood flow cytometry, R-138727 inhibited surface phosphatidylserine expression on ADP plus collagen-stimulated platelets and tissue factor (TF) expression on ADP-, collagen-, and ADP plus collagen-stimulated monocyte-platelet aggregates. R-138727 reduced monocyte-platelet aggregate formation, thereby further inhibiting TF expression. ADP, collagen, and ADP plus collagen accelerated the kinetics of thrombin generation in recalcified whole blood and R-138727 significantly inhibited this acceleration. Clot strength in a modified thromboelastograph system was also inhibited by R-138727 (IC50 0.7 +/- 0.1 microM). CONCLUSIONS: In addition to its previously known inhibitory effects on platelet activation and aggregation, the active metabolite of prasugrel, R-138727, inhibits platelet procoagulant activity in whole blood (as determined by phosphatidylserine expression on platelets and TF expression on monocyte-platelet aggregates), resulting in the functional consequences of delayed thrombin generation and impaired clot development.