Anti-GPVI Fab reveals distinct roles for GPVI signaling in the first platelet layer and subsequent layers during microfluidic clotting on collagen with or without tissue factor.

The collagen receptor glycoprotein VI (GPVI) drives strong platelet activation, however its role at later stages of clotting remains less clear. Controlled timing of addition of anti-human GPVI Fab (clone E12) with microfluidic venous whole blood flow over collagen (± lipidated tissue factor, TF) produced distinct effects on platelets, fibrin, P-selectin exposure, and phosphatidylserine (PS) exposure. On collagen alone, Fab present initially potently reduced platelet deposition on collagen, while Fab added 90 s after initial platelet deposition, stopped subsequent platelet accumulation (despite the absence of fibrin). With thrombin generation via TF, Fab added at either t = 0 or 90 s had no effect on platelet deposition. However, Fab added initially, but not at 90-s, blocked fibrin formation. Gly-Pro-Arg-Pro ablated fibrin formation without effect on platelet accumulation (regardless of Fab added at t = 0 or 90 s), indicating thrombin signaling can suffice over GPVI signaling. Still, Fab moderately reduced P-selectin exposure with thrombin present and fibrin absent. On collagen/TF, Fab present initially ablated PS exposure, but had no effect when added 30 to 90-s later. The thrombin generated via PS exposure had an important role in driving platelet deposition in the presence of Fab, since inhibition of PS via annexin V binding in the presence of Fab significantly inhibited platelet deposition. We conclude GPVI signaling in the first platelet layer on collagen dictates thrombin and fibrin production, but the role of GPVI at subsequent times after formation of the first monolayer is obscured by thrombin-induced signaling.