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

Abstract

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 asparagine-conjugated diperoxovanadate (DPV-Asn).

Methods

Platelet aggregation, ATP secretion, TxB₂ 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 A₂ (TxA₂) 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-γ2, 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 PLCγ2-p38-ERK1/2-TxA₂ pathway, which closely resembles the clinical scenario of various pathologies like hyperglycemia and atherosclerosis during which oxidative stress disrupts platelet functions.

Keywords:

• Reactive oxygen species
• Diperoxovanadate-asparagine
• Platelet aggregation
• Phospholipase C-γ2
• Thromboxane A₂
• Soluble CD40L

Acknowledgements

The authors are grateful to Dr T. Ramasarma (Department of Biochemistry, Indian Institute of Sciences, Bangalore, India) for his excellent guidance regarding understanding of peroxovanadate chemistry. The excellent technical help of Mr A.L. Vishwakarma and Mrs M. Chaturvedi for the Flow Cytometry study, Sophisticated Analytical Instrument Facility (SAIF); CSIR-Central Drug Research Institute, Lucknow, is duly acknowledged.