Divergent effects of C4a, C4adesArg, and thrombin on platelet aggregation and phosphorylation of ERK and Akt in human endothelial cells.

Abstract

Prior studies have established C4a as an untethered ligand for protease-activated receptors (PAR)1 and PAR4, which can increase ERK phosphorylation and [Ca2+]i influx in human endothelial cells (ECs). C4adesArg is a stable metabolite produced from C4a through cleavage of an arginine at the carboxyl terminus by plasma carboxypeptidases B/N. PAR1 and PAR4 are typical receptors for thrombin and transduce cellular responses to the serine protease generated by the activation of coagulation pathways. Here, we aim to address whether C4adesArg can induce the same effects as C4a through PAR1 and PAR4, and whether C4a and C4adesArg can activate the same downstream signaling effectors as thrombin through PAR1 and PAR4. We demonstrated that C4adesArg induces ERK phosphorylation and [Ca2+]i influx with the reduced efficacy as compared to C4a in human ECs. Distinct from C4a, C4adesArg-induced ERK phosphorylation was only inhibited by the PAR4 antagonist tcY-NH2, indicating that C4adesArg-mediated ERK phosphorylation is PAR4-dependent. Both C4a and C4adesArg at a concentration of 3 M failed to induce platelet aggregation. Moreover, both C4a and C4adesArg induce significant Akt phosphorylation, whereas thrombin causes Akt dephosphorylation in human ECs. Our study revealed that the absence of the C-terminal arginine in C4a decreases its efficacy and changes its preference for receptor of ERK and Akt activations in human ECs, suggesting that the C-terminal arginine of C4a might govern its binding specificity and/or affinity to PAR1 and/or PAR4. Unlike thrombin, both C4a and C4adesArg fail to induce platelet aggregation at supraphysiological concentrations. Contrary to thrombin, both C4a and C4adesArg induce significant Akt phosphorylation, indicating a unique role of C4a and C4adesArg in inflammation and coagulation through their association with PAR1 and/or PAR4.