Impact of C-terminal amidation on platelet activation by PAR1 and PAR4 activating peptides: Communication from the ISTH SSC Subcommittee on Platelets in Health and Disease.

Introduction: Platelet protease-activated receptors (PAR)1 or PAR4 can be specifically activated with thrombin receptor-activating peptides, available with either amide (-NH2), or free-acid (-OH) in their C-terminus.

Aim: To assess differences in potency on platelet activation and aggregation induced by PAR1 and PAR4-activating peptides (AP) with either C-terminal amide or free-acid groups.

Method: Platelet aggregation was evaluated using light transmission aggregometry (LTA) in platelet-rich plasma (PRP) and washed platelets. Platelet activation markers CD62P (P-selectin), CD63, and PAC-1 binding were evaluated by flow cytometry in blood anticoagulated with citrate or hirudin and in washed platelets. Platelet responses were recorded following stimulation with PAR1-AP (SFLLRN) or PAR4-AP (AYPGKF), in either their free-acid or amidated forms. EC50 values were calculated from concentration-response curves.

Results: Amidated peptides displayed consistently higher potency than their free-acid counterparts. Median EC50 values for PAR1-AP-NH2 were 2.6-12.6 times lower than for PAR1-AP-OH, and median EC50 values for PAR4-AP-NH2 were 4.7-10 times lower than for PAR4-AP-OH. PAR4-AP-OH showed low potency and did not reach maximal platelet activation for several activation markers.

Conclusion: The amidated forms of either PAR1-AP and PAR4-AP agonists were significantly more potent, compared to their free-acid counterparts. Results obtained with one form should not be assumed to apply to the other, as structural differences can substantially affect biological activity. Thus, the SSC recommends that the complete peptide structure is reported in studies using these agonists.