CARD12: Shear Stress Renders Redistribution of P2Y12 and PAR-1 Receptors from Platelets to Microparticles: A Cue to Limited Effectiveness of Antiplatelet Therapy in MCS

Background: Platelet exposure to hypershear stress generated by mechanical circulatory support (MCS) results in platelet dysfunction, thrombosis, and bleeding coagulopathy. Antiplatelet agents, targeting ADP-evoked P2Y12 and thrombin-evoked PAR1 platelet receptors, have limited effectiveness reducing MCS thrombosis, rather favoring bleeding. Shear-mediated platelet dysfunction is associated with the downregulation of platelet adhesion receptors, decreased aggregation response, and generation of platelet-derived microparticles (PDMPs). We tested the hypothesis that MCS shear stress alters the surface expression of agonist receptors P2Y12 and PAR1 on platelets and PDMPs, thus affecting platelet aggregation response induced by ADP, collagen, and TRAP-6. Methods: Human platelets were exposed to shear stress (30-70 dynes/cm2, 10’) or sonication; PDMPs were isolated via differential centrifugation. P2Y12, PAR1, & αIIbβ3 expression on platelets and PDMPs was visualized by immunostaining and multi-colored flow cytometry. Platelets were distinguished from PDMPs by forward/side scatter vs. standard fluorescent nanobeads SPHEROTM. Platelet aggregation in plasma induced by ADP, collagen, and TRAP-6 was assessed by optical aggregometry. Results: Platelet exposure to increasing shear stress and sonication resulted in a decrease in P2Y12+ and PAR1+ platelet populations (Fig. 1A&B). The baseline surface density of P2Y12 on platelets was very low and slightly increased after 70 dyne/cm2 shear (Fig. 1C). While also low, PAR1 density on platelets tended to decrease with increasing shear stress (Fig. 1D). Shear stress induced generation of P2Y12+ and PAR1+ PDMPs, accounting for 3.5±0.6% and 4.3±0.5% after 70 dyne/cm2 shear, respectively. Remarkably, P2Y12 and PAR1 surface density on PDMPs was between 7 and 10-fold higher than on platelets (Fig. 1C&D). When added to intact platelets, PDMPs significantly decreased platelet aggregation in plasma induced by ADP, collagen, and TRAP-6. Conclusions: Elevated shear stress promotes generation of PDMPs carrying increased amounts of P2Y12 and PAR1 on their surface; the surface density of these receptors on sheared platelets was also affected. Sheared PDMPs reduced platelet aggregation likely acting as competitive inhibitors imposing steric difficulties for platelets to aggregate and thus favoring MCS bleeding phenotype and limiting the effectiveness of antiplatelet agents.