Mechanisms of action of proteinase-activated receptor agonists on human platelets.

1. 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)]. 2. 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. 3. Aggregation inhibitors, including prostacyclin (PGI(2)), 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. 4. Thrombin was the most potent platelet agonist, followed by PAR1AP, TRAP and PAR4AP. 5. The aggregatory potencies of PAR-APs were not modified by the aminopeptidase inhibitor, amastatin. 6. Subthreshold concentrations of PAR1AP potentiated the effects of PAR4AP to stimulate maximal aggregation. 7. Both PGI(2) and GSNO reduced PAR agonist-induced aggregation and diminished GPIIb/IIIa up-regulation. 8. PAR agonist-induced aggregation was aspirin-insensitive indicating a minor role for TXA(2). 9. 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. 10. PAR4AP-induced aggregation (but not PAR1AP-induced aggregation) was entirely ADP-dependent (abolished by apyrase) and resistant to phenanthroline (MMP-2-independent). 11. 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.