Phospholipase C-γ2 via p38 and ERK1/2 MAP kinase mediates diperoxovanadate-asparagine induced human platelet aggregation and sCD40L release.

Objective: Redox imbalance either inside platelets or in their immediate surroundings prove detrimental to their physiologic functions during haemostasis. This study was therefore aimed to assess the effect of peroxide radicals on platelet functions and underlying signalling mechanisms using asparagineconjugated diperoxovanadate (DPV-Asn). Methods: Platelet aggregation, ATP secretion, TxB(2) release, intra-platelet calcium mobilization, protein tyrosine phosphorylation, GPIIbIIIa activation by PAC1 labelling and sCD40L release (enzyme-linked immunosorbent assay) was monitored using various concentrations of DPV-Asn. Cell viability was assessed by Annexin V labelling, MTT assay, LDH leakage and mitochondrial membrane potential by JC-1. Results: Platelet aggregation induced by DPV-Asn was chiefly regulated by dense granule secretion, thromboxane A(2) (TxA(2)) generation, intra-platelet [Ca2+] influx, GPIIbIIIa activation and sCD40L release, which were significantly reduced in presence of U73122 (PLC inhibitor), aspirin (COX), SB203580 (p38 inhibitor), and PD98059 (ERK inhibitor). This was further corroborated by enhanced tyrosine phosphorylation of numerous platelet proteins including PLC-gamma 2, which apparently played a central role in transducing peroxide signals to regulate [Ca2+] influx and phosphorylation of p38 and ERK1/2 MAP kinase. Discussion: Peroxide radicals critically regulate the thrombo-inflammatory functions of platelets via the PLC gamma 2-p38-ERK1/2-TxA(2) pathway, which closely resembles the clinical scenario of various pathologies like hyperglycemia and atherosclerosis during which oxidative stress disrupts platelet functions.