Abstract
This study probed the differential utilization of P2Y1 and P2Y12 receptors in mobilizing CD62P (P-selectin) from intracellular granules following activation of human platelets with adenosine 5′-diphosphate (ADP, 100 µmol·L−1) Platelet-rich plasma (PRP) was prepared from the blood of adult humans. CD62P was measured by flow cytometry following activation of PRP with ADP in the absence and presence of the selective antagonists of P2Y1 and P2Y12 receptors, MRS2500 and PSB0739 (both 0.155–10 µmol·L−1), respectively. Effects of the test agents on ADP-activated, CD62P-dependent formation of neutrophil:platelet (NP) aggregates were also measured by flow cytometry, while phosphatidylinositol 3-kinase (PI3K) activity was measured according to Akt1 phosphorylation in platelet lysates. Treatment with MRS2500 or PSB0739 at 10 µmol·L−1 almost completely attenuated (94.6% and 86% inhibition, respectively) ADP-activated expression of CD62P and also inhibited NP aggregate formation. To probe the mechanisms involved in P2Y1/P2Y12 receptor-mediated expression of CD62P, PRP was pre-treated with U73122 (phospholipase C (PLC) inhibitor), 2-aminoethoxy-diphenyl borate (2-APB, inositol triphosphate receptor antagonist), calmidazolium chloride (calmodulin inhibitor), or wortmannin (PI3K inhibitor). U73122, 2-APB, and wortmannin caused almost complete inhibition of ADP-activated expression of CD62P, while calmidazolium chloride caused statistically significant, partial inhibition. PSB0739, but not MRS2500, caused potent inhibition of PI3K-mediated phosphorylation of Akt1. Optimal mobilization of CD62P by ADP-stimulated platelets is critically dependent on the co-activation of platelet P2Y1 and P2Y12 receptors. P2Y12 receptor activation is the key event in activation of PI3K, while activation of the P2Y1 receptor appears to create a high cytosolic Ca2+ environment conducive to optimum PI3K activity.
Highlights
Platelets are well recognized as being key players in orchestrating inflammatory responses, those involving neutrophils, driving activities such as heterotypic neutrophil:platelet (NP)aggregation, adhesion to vascular endothelium, extravasation, and formation of neutrophil extracellular traps (NETs) [1,2,3,4,5,6,7]
Our findings demonstrate that the mobilization of CD62P from α-granules following exposure of human platelets to adenosine -diphosphate (ADP) in vitro is markedly attenuated by blockade of either P2Y1 or P2Y12 receptors, consistent with a critical requirement for receptor co-activation
In an extension of these studies, Leon et al reported that pharmacologic blockade of either P2Y1 or P2Y12 receptors resulted in almost complete inhibition of upregulation of CD62P expression by ADP-activated human platelets [23], as noted in the current study
Summary
Aggregation, adhesion to vascular endothelium, extravasation, and formation of neutrophil extracellular traps (NETs) [1,2,3,4,5,6,7] In this setting, mobilization of the adhesion molecule, CD62P (P-selectin), located in platelet α-granules, is a major trigger of NP interactions via binding to its counter ligand, P-selectin glycoprotein ligand-1 (PSGL-1), constitutively expressed on neutrophils and other types of leukocyte [4,5,7]. Platelet activation per se, as well as platelet-driven neutrophil activation, are potentially protective against microbial and viral pathogens [8,9,10], if excessive, these events increase the potential risk of intravascular occlusion [1,7,11,12,13,14] This contention is supported by findings that NP aggregates, NETs, as well as NET-derived constituents, are major components of intravascular thrombi [11,12,13,14]. TXA2 interacts with the prostanoid TP receptor, while ADP activates two major platelet-activating purinergic receptors viz. P2Y1 and P2Y12 [16], functioning both as a primary activator of platelets as well as a cofactor, augmenting responses to other primary activators
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