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 αIIbβ3, 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 αIIbβ3 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.