VWF-induced phosphorylation of p38 in human platelets. (A,B) Washed platelets (5 × 10⁸/mL, 200 μL) were incubated for 0.5, 1, 2, or 5 minutes in a platelet aggregometer stirring at 1000 rpm with buffer, ristocetin alone (0.6 mg/mL), or with ristocetin and VWF (20 μg/mL). (A) The platelets were then solubilized and immunoblotted with a rabbit antibody specifically recognizing phosphorylated form of p38 (P-p38) or with a rabbit anti-p38 antibody to indicate comparable loading levels (p38). (B) Solubilized platelets also were immunoblotted with a rabbit antibody specifically recognizing the phosphorylated form of ERK (P-ERK) or with a rabbit anti-ERK2 antibody to indicate comparable loading levels (ERK). (C) Washed platelets (5 × 10⁸/mL, 200 μL) were incubated for 0.5 or 5 minutes in a platelet aggregometer (stirring at 1000 rpm) with buffer (ctrl), 1 mg/mL ristocetin (risto), ristocetin and VWF (20 μg/mL). The platelets were then solubilized and immunoblotted with a rabbit antibody specifically recognizing phosphorylated forms of p38 (P-p38) or immunoblotted with a rabbit anti-p38 antibody to indicate comparable loading levels (p38). (D) Immunoblotting results as in experiments described in panel C were scanned and quantitated using NIH Image software. Results shown are from 4 experiments (mean ± SD). (E) Platelets were preincubated with a mouse IgG (40 μg/mL), an anti-GPIbα monoclonal antibody, SZ2 (40 μg/mL), or RGDS (1 mM) at 22°C for 5 minutes, and then stimulated with VWF and ristocetin (VWF). (F) Platelets were preincubated with a mouse IgG (20 μg/mL) or anti-human integrin α_IIbβ₃ monoclonal antibodies SZ21 or 2G12 (20 μg/mL), or RGDS (1 mM) at 22°C for 5 minutes, and then incubated without or with thrombin (0.05 μ/mL). (G) Platelets were preincubated with buffer (control) or RGDS (1 mM) at 22°C for 5 minutes and then incubated with or without thrombin (0.05 μ/mL). Immunoblotting results were scanned and quantitated using NIH Image software. Results shown are from 3 experiments (mean ± SD).