P2Y12 Receptor Antagonists: Do They Really Inhibit Shear-Mediated Platelet Activation within MCS?

Abstract

Purpose Shear-mediated platelet activation (SMPA) by supra-physiological blood flow found within MCS is a main driver and contributor of device-related thrombosis. Thus, antiplatelet drugs targeting the main pathways of platelet activation are routinely prescribed. We have shown, that aspirin and GP IIbIIIa blockers have limited ability in mitigating SMPA, and do NOT protect platelets from integrin shedding and microparticle (MP) generation. In contrast, ticagrelor, a direct P2Y12 purinergic receptor antagonist, drastically inhibits platelet prothrombinase activity induced by high shear. The aim of the study was to define the role of the P2Y12 pathway in SMPA and evaluate the effect of P2Y12 antagonists on platelet procoagulant activity and MP generation induced by high shear stress. Methods The following P2Y12 antagonists were used: clopidogrel active metabolite (10 µM), cangrelor (1 µM) and ticagrelor (1 µM). After 10 min incubation with the agent, human platelets were exposed to continuous high shear stress (70 dyn/cm2, 10 min) in a hemodynamic shearing device. Platelet procoagulant activity was assessed using the annexin V binding assay, detecting procoagulant surface exposure, and platelet activity state (PAS) assay measuring thrombin generation. MPs were quantified by flow cytometry as CD41+/AnV+ particles distinguished from platelets by their FS/SS. Results Ticagrelor and cangrelor showed similar inhibitory effect, consistently decreasing both platelet procoagulant activity and microparticle generation induced by shear stress. The number of AnV+ platelets, AnV+ MPs, and PAS levels were decreased by 26%, 27%, and 30%, respectively, as compared with non-treated controls subjected to shear. Clopidogrel active metabolite, known for its low affinity to the targeted receptor, reduced PAS by 15%, while annexin V binding was diminished in more than 10 times for platelets and 2 times for MPs, indicating a significant reduction of platelet procoagulant surface exposure but limited inhibition of thrombin generation. Conclusion P2Y12 purinergic receptor antagonists inhibit shear-mediated platelet procoagulant surface formation and microparticle generation, both contributors in overall platelet prothrombotic activity induced by high shear stress within MCS. Our results suggest that P2Y12 pathway is involved in the regulation of SMPA.