Fibrin stimulates spreading in a GPVI-dependent manner. Fibrinogen was coated onto glass coverslips and converted into fibrin by treatment with thrombin. Hirudin was used to neutralize any residual thrombin before washing and blocking. (A) Alexa-488 fibrinogen was used to visualize fibrin formation. Platelets (2 × 107/mL) were allowed to spread on coated coverslips, followed by actin staining with Alexa-568 phalloidin. Scale bar, 5 μm. (B) Platelets (2 × 107/mL) were allowed to spread on nonfluorescent fibrinogen or fibrin-coated coverslips, followed by actin staining with Alexa-488 phalloidin. Where shown, platelets were preincubated with dasatinib (10 μM) or hirudin (5 U/mL). Scale bar, 5 μm. (C) WT or GPVI KO platelets were allowed to spread on fibrinogen or fibrin-coated coverslips, followed by actin staining with Alexa-488 phalloidin. Scale bar, 5 μm. The results are shown as mean ± standard error of the mean (SEM) of 3 experiments. **P < .01; ***P < .001.
