A critical role of thrombin/PAR-1 in ADP-induced platelet secretion and the second wave of aggregation

Abstract

The stable or second wave of platelet aggregation often observed in ADP-stimulated platelet-rich plasma (PRP) with an artificially lowered extracellular calcium level has been attributed to enhanced thromboxane A2 (TXA2 ) generation and inhibition of ectonucleotidase activity. However, the role of thrombin in ADP-induced platelet secretion and the second wave of aggregation is unknown. We employed aggregometry, flow cytometry, immunoblotting and ELISA to determine whether and how thrombin participates in ADP-induced platelet secretion and the second wave of aggregation. ADP induces a phosphoinositide 3-kinase (PI3K) pathway-dependent thrombin generation, presumably resulting from the cleavage of αII b β3 -associated prothrombin. Generated thrombin subsequently activates protease-activated receptor-1 (PAR-1) and mediates dense granule secretion and the second wave of platelet aggregation in ADP-stimulated citrated PRP. Thus, ADP-induced dense granule secretion and the second wave of platelet aggregation in PRP were similarly and non-additively blocked by thrombin inhibitor hirudin, PAR-1 antagonist SCH-79797 or PI3K inhibitor wortmannin. Moreover, ADP stimulation caused the dissociation of prothrombin from αII b β3 and an increased plasma thrombin level; both were prevented by wortmannin. Furthermore, the wortmannin-inhibited second wave of platelet aggregation by ADP was restored by a subaggregation concentration of PAR-1 activating peptide SFLLRN. Blocking TXA2 production with indomethacin or restoring extracellular calcium to physiological concentration did not influence this thrombin/PAR-1 dependence. A PI3K-dependent thrombin generation and the resultant PAR-1 activation serve as an indispensable mechanism to relay the platelet activation process induced by ADP.