Parstatin Mediates Platelet Activation That Is Unaffected By PAR Antagonism

Thrombin cleaves PAR-1 at amino acids arg⁴¹-ser⁴² to yield the canonical tethered ligand as well as a soluble peptide, parstatin, which has been reported to have divergent physiological functions. In animal models, this peptide demonstrates anti-angiogenic, anti-inflammatory, and cardioprotective properties. In ex vivo studies, parstatin was also demonstrated to affect platelet aggregation and enhance GPIIb/IIIa-mediated adhesion, suggesting this cleaved peptide may participate in pathological thrombosis. With recent approval of a first-in-class PAR-1 antagonist as an antiplatelet agent, it is clinically imperative to fully appreciate the physiologic mechanisms through which thrombin-activation of PAR-1 contributes to platelet aggregation and thrombosis. As such, the effects of PAR antagonism in modulating parstatin-mediated platelet activation requires evaluation.

In this study, we characterized parstatin-mediated effects on platelets and investigated the potential involvement of platelet thrombin receptor (PAR-1, PAR-4)-associated signaling in this phenomenon. Light-transmission aggregometry was used to measure aggregation response in washed platelet preparations, and flow cytometry was used to assess expression of protein markers indicative of platelet activation. Consistent with previous reports, we demonstrated parstatin induces P-Selectin surface expression (degranulation), GPIIb/IIIa activation (PAC-1 binding), and aggregation independent of thrombin receptor cleavage (n=10, healthy donors). Interestingly, platelet shape change was not observed following parstatin treatment, even in the presence of PAR-1 activating peptide (PAR-1-AP, SFLLRN), suggesting parstatin-mediated activation does not signal through G_12/13-dependent mechanisms, and may override canonical G_12/13-associated PAR-1 signaling. Pretreatment with G_q-selective PAR-1 antagonist, ML161 (3 µM), or PAR-4-selective antagonist, ML354 (500 nM) did not inhibit parstatin-mediated platelet activation. These findings are consistent with previous reports suggesting this peptide may signal through a G_i-dependent mechanism. Platelet PAR receptors couple to Gα_q and Gα_12/13, but direct coupling to Gα_i is controversial; therefore, parstatin-mediated activation may occur through a signaling cascade unrelated to canonical PAR-associated mechanisms.