Arf6 KO platelets have enhanced spreading but normal static adhesion. (A) Quantification of static platelet adhesion on Fg- and BSA-coated surface. Washed calcein-labeled platelets (2.5 × 108/mL) from WT and KO mouse were incubated on Fg- or BSA-coated surfaces for 30 minutes at 37°C. The number of adherent platelets was measured using a plate reader with excitation/emission at 485/538 nm, and referenced to a standard curve. Each bar represents platelets from a single mouse. Statistical analysis was performed using Student t test. (B) Quantification of platelet surface area when spread on Fg-coated surfaces. Washed platelets from WT and KO mice (2.0 × 107/mL) were supplemented with 1 mM Ca2+ and then incubated on Fg-coated surfaces for the indicated times. They were fixed with 4% paraformaldehyde, and DIC images were taken using Nikon Eclipse E600 microscope (Nikon) with a 100X/1.40 NA, DIC H oil objective lens (Nikon) with a Zeiss camera (AxioCam MR). Images were processed with Zen 2011 (blue edition; Zeiss) and quantified by Image J (v1.47; National Institutes of Health). (C-D) Representative DIC images of WT (C) and KO (D) platelets spread at the 120 minutes point. The data are representative of at least 3 independent experiments.
