GPVI-mediated thrombus stabilization of shear-induced platelet aggregates in a microfluidic stenosis

Supraphysiological shear rates (>2000 s^-1) amplify von Willebrand factor unfurling and increase platelet activation and adhesion. These elevated shear rates and shear rate gradients also play a role in shear-induced platelet aggregation (SIPA). The primary objective of this study is to investigate the contributions of major binding receptors to platelet deposition and SIPA in a stenotic model. Microfluidic channels with stenotic contractions ranging from 0% to 75% are fabricated and coated with human type I collagen at 100 μg/mL. Fresh human blood is reconstituted to 40% hematocrit and treated to stain platelets. Platelet receptors α_IIbβ₃, GPIb, or GPVI are blocked with inhibitory antibodies or proteins to reduce platelet function under flow at 500, 1000, 5000, or 10,000 s^-1 over 5 min of perfusion. Additional validation experiments are performed by dual-blocking receptors and performing coagulability testing by rotational thromboelastometry. Control samples exhibit SIPA correlating to increasing shear rate and increasing stenotic contraction. Inhibition of α_IIbβ₃ or GPIb receptors causes a nearly total reduction in platelet adhesion and a loss of aggregation at >1000 s^-1. GPVI inhibition does not notably reduce platelet adhesion at 500 or 1000 s^-1 but affects microthrombus stability at 5000-10,000 s^-1 following aggregation formation in 50%-75% stenotic channels. Inhibition of von Willebrand factor-binding receptors completely blocks adhesion and aggregation at shear rates >1000 s^-1. Inhibition of GPVI reduces platelet adhesion at 5000-10,000 s^-1 but renders thrombi susceptible to fragmentation. This study yields further insight into mechanisms regulating rapid growth and stabilization of arterial thrombi at supraphysiological shear rates.