F-actin/G-actin ratio and F actin content

The ratio of filamentous actin (F-actin) to monomeric actin (G-actin) was determined by immunoblots. Washed platelets (0.5 x 10⁸/ml) were activated with collagen (10 μg/ml) ot thrombin (0.05 unit/ml) and lysed with an equal volume of 2 × lysis buffer containing 100 mM Tris-HCl (pH 7.4), 10 mM EGTA, 2 mM MgCl₂ and 2% Triton X 100 containing protease and phosphatase inhibitor cocktail (Thermo scientific) and kept on ice for 15 minutes. Lysates were centrifuged at 100,000 g for 1 hour to separate Triton-X-100-soluble G-actin from Triton-X-100-insoluble F-actin. Supernatant containing G-actin was collected, and the F-actin pellets were washed twice in cold lysis buffer and solubilized in 8 M urea. The supernatants and the pellets were reconstituted to the same initial volume. Fractions were proportionally loaded onto SDS-polyacrylamide gels, electrophoresed and transferred onto PVDF membrane for probing with an anti-actin antibody. Densitometric quantification of the western blot was used to determine the F-actin versus G-actin content. The F-actin to G-actin ratio of resting platelets was considered as 1. FITC-phalloidin binding to platelets was also used to quantify F-actin content as previously described [22]. Resting and activated platelets were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and stained with 10 μM FITC-phalloidin for 1 hour. FITC fluorescence was monitored by flow cytometry.