Mechanisms of action of proteinase‐activated receptor agonists on human platelets

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Abstract
We studied the activation of human platelets by thrombin and proteinase activated receptor (PAR)-activating peptides (PAR-APs) [SFLLRNPNDKYEPF-amide (TRAP), TFLLR-amide (PAR1AP) and AYPGKF-amide (PAR4AP)]. PAR agonist-induced platelet aggregation, glycoprotein (GP) Ib and GPIIb/IIIa surface expression and ADP release were measured by light aggregometry, flow cytometry and chemiluminescence. Aggregation inhibitors, including prostacyclin (PGI2), nitric oxide-releasing agent (S-nitroso-glutathione, GSNO), aspirin, apyrase, and phenanthroline were used to study the susceptibility of PAR agonist-induced aggregation to pharmacological inhibition. Thrombin was the most potent platelet agonist, followed by PAR1AP, TRAP and PAR4AP. The aggregatory potencies of PAR-APs were not modified by the aminopeptidase inhibitor, amastatin. Subthreshold concentrations of PAR1AP potentiated the effects of PAR4AP to stimulate maximal aggregation. Both PGI2 and GSNO reduced PAR agonist-induced aggregation and diminished GPIIb/IIIa up-regulation. PAR agonist-induced aggregation was aspirin-insensitive indicating a minor role for TXA2. In contrast, phenanthroline and apyrase significantly enhanced the anti-aggregatory effects of aspirin against thrombin-, PAR1AP- and TRAP-induced aggregation suggesting the involvement of ADP- and MMP-2-dependent pathways. PAR4AP-induced aggregation (but not PAR1AP-induced aggregation) was entirely ADP-dependent (abolished by apyrase) and resistant to phenanthroline (MMP-2-independent). Thus, the mechanisms of PAR1 and 4-induced platelet aggregation are distinct and depend differentially on their ability to interact with pathways of aggregation, along with the subsequent activation of GPIIb/IIIa receptors.