VWF-induced LIMK1 phosphorylation and LIMK1-dependent cofilin phosphorylation. (A-C) Immunoblots of washed human platelets, stimulated with VWF (10 μg/mL) in the absence or presence of ristocetin (0.25 mg/mL) in a platelet aggregometer at 37°C for 6 minutes with: (A and B) an antibody recognizing phosphorylated Thr^508/505(T^508/505) in LIMK and an anti-LIMK1 antibody (loading control); and (C) an antibody specific for Ser³ (S³ )-phosphorylated cofilin and an anticofilin antibody (loading control). In (B), platelets were preincubated with a function-blocking anti-GPIbα antibody, AK2 (10 μg/mL), or 10 µg/mL of control IgG for 6 minutes and then stimulated with VWF and ristocetin. (D) Immunoblots of WT and LIMK1^−/− mouse platelets stimulated with VWF (10 µg/mL) in the presence of botrocetin (2 µg/mL) with an antibody against Ser³ -phosphorylated cofilin or an anticofilin antibody.