Fibrin polymerization is not essential for platelet spreading or GPVI binding. (A) Fibrinogen was coated onto glass coverslips and converted into fibrin by treatment with thrombin. GPRP (5 μM) was used to inhibit polymerization of fibrin monomers. Hirudin was used to neutralize any residual thrombin before washing and blocking. Platelets (2 × 107/mL) were allowed to spread, followed by actin staining with Alexa-488 phalloidin. Scale bar, 5 μm. The results are representative of 3 experiments. (B) Fibrinogen was coated onto the wells of Nunc Maxisorp plates and converted into fibrin by treatment with thrombin. GPRP (5 μM) was used to inhibit polymerization of fibrin monomers. The ectodomain of GPVI was incubated, and following washing, adherent GPVI was detected with a HRP-conjugated antibody. The results are shown as mean ± SEM of 3 experiments performed in duplicate. *P < .05; **P < .01.
