Thrombin-induced platelet PAR4 activation: role of glycoprotein Ib and ADP

Thrombin activates human platelets via the cleavage of two protease-activated G-protein coupled receptors (PARs), PAR1 and PAR4 that respond to low and high concentrations of thrombin, respectively. The aim of the present study was to examine the relative contributions of GPIbalpha and ADP receptors in response to thrombin-induced PAR1 and PAR4 stimulation. Platelet responses (aggregation, secretion and calcium mobilization) elicited by low thrombin concentrations were impaired when thrombin interaction with GPIbalpha was blocked. In contrast, blockade of thrombin interaction with GPIbalpha had no effect when PAR4-coupled responses were specifically elicited by high thrombin concentrations in the presence of PAR1 antagonists or after PAR1 desensitization. These results confirmed that unlike PAR1, PAR4 does not require GPIbalpha as a cofactor for thrombin-mediated activation. Both apyrase and selective antagonists of P2Y1 and P2Y12 inhibited PAR1-coupled responses but did not modify PAR4-coupled responses, indicating that in contrast to PAR1, PAR4 signals are not reinforced by ADP secretion and binding to the platelets. These results provide the direct evidence that, in human platelets, GPIbalpha and ADP act in synergy to amplify PAR1 coupled responses while PAR4 is activated independently of GPIbalpha and ADP.