Arf6 KO platelets have enhanced platelet clot retraction without noticeable defect on myosin light-chain phosphorylation (MLC-P), actin polymerization, or Rac1/RhoA activation. (A) Washed platelets from WT and KO mice (3 × 10⁸/mL) were supplemented with 0.5 mg/mL human Fg and 1 mM Ca²⁺. Clot retraction was initiated with thrombin (0.1 U/mL), and images were taken at the indicated times (i) and quantified (ii). Clot size in panel A was measured using Image J v1.48 and normalized to clot size at time 0 (clot volume %). The data are representative of at least 5 independent experiments. (B) KO mice showed no defect in MLC-P upon thrombin stimulation. Washed platelets from WT and KO mice were prepared at 4 × 10⁸/mL in HEPES Tyrode buffer (pH 7.4) and kept resting or stimulated with thrombin (0.1 U/mL) for the indicated times. The reaction was stopped by addition of sodium dodecyl sulfate–polyacrylamide gel electrophoresis sample buffer containing both protease inhibitor and phosphatase inhibitor cocktails. The lysates were probed for MLC-P by western blotting. β-Actin was used as a loading control. The blots shown are representative of at least 3 independent experiments. (C) Arf6 deletion did not affect thrombin-induced F-actin formation. Resting and thrombin-stimulated platelets (2 × 10⁷) were fixed and permeabilized with Triton X-100 in the presence of tetramethylrhodamine (TRITC)-phalloidin. The bound TRITC-phalloidin was solubilized and measured using a microplate spectrofluorimeter. The data are representative of 2 independent experiments. (D-E) KO platelets showed no defect in Rac1/RhoA activation. Washed platelets (5 × 10⁸/mL) from WT and KO mice were kept resting or stimulated with thrombin (0.1 U/mL) for the indicated times. The reactions were stopped by addition of 2× GTPases-pulldown lysis buffer containing a protease inhibitor cocktail. Rac1-GTP/RhoA-GTP were recovered as described in “Methods.” (Di and Ei) Western blotting of the pellets (Rac1-GTP or RhoA-GTP) and the supernatant (total Rac1 or total RhoA). (Dii and Eii) Quantification of Rac1-GTP/total Rac1 and RhoA-GTP/total RhoA. Quantification was performed using ImageQuantTL, and the ratio of Rac1-GTP/total Rac1 or RhoA-GTP/total RhoA was plotted in a bar graph. Blots shown are representative of at least 2 independent experiments.