Glycoprotein shedding increases coagulation factor binding to platelets and enhances the formation of thrombin and fibrin. (A) Washed platelets were preincubated with vehicle, GW280264X (GW; 5 µM, 15 minutes), or 5G6 Fab fragment, which are known to block ADAM17-mediated shedding of GPIbα (10 µg/mL, 15 minutes). After 60 minutes of stimulation (37°C) with ionomycin, ABT-737, or CRP-XL plus SFLLRN, flow cytometry was used to measure platelet binding of VWF with fluorescent anti-VWF mAb (in the presence of ristocetin) (Ai), OG488-prothrombin (Aii), OG488-factor Xa (Aiii), or AF488-factor Va (Aiv). Binding of prothrombin, factor Xa, and factor Va was evaluated for the PS-exposing platelet population, identified with AF647-annexin A5. Data are mean ± SD, n = 3-6. (B) Platelet thrombi were formed on collagen and posttreated with SFLLRN for 60 minutes at 37°C in the presence of vehicle or GW (5 µM). After incubation, the thrombi were stained with OG488-prothrombin or were incubated with recalcified plasma supplemented with fluorogenic substrate Z-Gly-Gly-Arg-AMC to measure thrombin generation. Representative fluorescence images (Bi) and quantification of prothrombin fluorescence (Bii). (Biii) Time course (under stasis) of generation of thrombin from the surface of platelet thrombi formed in the presence or absence of GW. Data are mean ± SD, n = 3. (C) Kinetics of fibrin formation at a layer of adhered platelets on collagen, posttreated with ionomycin for 10 minutes, and inducing maximal shedding, in the presence of vehicle or GW. Fibrin formation was then allowed by perfusion of recalcified normal plasma at 250 s⁻¹, while recording fluorescence images. Shown are times to fibrin formation and representative time traces of fibrin formation (expressed as SAC). Data are mean ± SD, n = 6. *P < .05.