Complement factor C4a does not activate protease‐activated receptor 1 (PAR1) or PAR4 on human platelets

Background Protease-activated receptor (PAR) 1 and PAR4 are key thrombin signal mediators for human platelet activation and aggregation in response to vascular injury. They are primarily activated by thrombin cleavage of the N-terminus to expose a tethered ligand. In addition to the canonical activation by thrombin, a growing panel of proteases can also elicit PAR1- or PAR4-mediated signal transduction. Recently, complement factor C4a was reported as the first endogenous agonist for both PAR1 and PAR4. Further, it is the first endogenous nontethered ligand that activates PAR1 and PAR4. These studies were conducted with human microvascular cells; the impact of C4a on platelet PARs is unknown. Objectives The goal of this study was to interrogate PAR1 and PAR4 activation by C4a on human platelets. Methods Platelet-rich plasma (PRP) was isolated from healthy donors. PRP was stimulated with C4a, and the platelet aggregation was measured. Human embryonic kidney (HEK) 293 Flp-In T-rex cells were used to further test if C4a stimulation can initiate PAR1- or PAR4-mediated Gαq signaling, which was measured by intracellular calcium mobilization. Results C4a failed to elicit platelet aggregation via PAR1- or PAR4-mediated manner. In addition, no PAR1- or PAR4-mediated calcium mobilization was observed upon C4a stimulation on HEK293 cells. Conclusions Complement factor C4a does not activate PAR1 or PAR4 on human platelets. These data show that PAR1 and PAR4 activation by C4a on microvascular cells likely requires a cofactor, which reinforces the concept of cell type-specific regulation of protease signaling.