VWF + ristocetin–induced platelet aggregation is independent of cGK activation. (A) VWF-induced platelet aggregation. Washed platelets were stirred in an aggregometer at 1000 rpm and stimulated with 15 μg/mL VWF and 1 mg/mL ristocetin, 7.5 μg/mL VWF and 1 mg/mL ristocetin, or 15 μg/mL VWF alone as indicated. (B) Inhibition of VWF-induced platelet aggregation by cGMP analogs. Washed platelets were preincubated for 1 minute with 8-pCPT-cGMP or Rp-8-pCPT-cGMPS (200 μM both) then placed in an aggregometer and stimulated with 15 μg/mL VWF and 1 mg/mL ristocetin. (C) Stirring-induced VASP phosphorylation. Washed platelets (3 × 10⁸/mL) were incubated at 37°C in a turbidometric platelet aggregometer at 0 rpm or 1000 rpm, and stimulated with 7.5 μg/mL VWF and 1 mg/mL ristocetin (lane P-VASP 1) or 15 μg/mL and 1 mg/mL ristocetin (lane P-VASP 2). After the times indicated, SDS-stop solution was directly added to the tubes and platelet lysates were analyzed by Western blotting for VASP phosphorylation. Shown are representatives of 3 different experiments.