VWF- or low-dose thrombin-induced p38 phosphorylation is PKG dependent. (A) Human platelets were preincubated without or with a PKG inhibitor, Rp-pCPT-cGMPS (200 μM), at 22°C for 5 minutes, and then stimulated with botrocetin in the presence or absence of VWF at 37°C for 1 minute. (B) Human platelets were treated with or without the PKG inhibitor Rp-Br-PET-cGMPS (100 μM) and then stimulated with or without a low-dose thrombin (0.05 μ/mL) at 37°C for 2 minutes (the order of lanes from the same blot was rearranged for better presentation). (C) Platelets from wild-type mice (WT) or PKG I knockout mice (KO) were incubated with buffer or with botrocetin (5 μg/mL) in the presence or absence of VWF (20 μg/mL) at 37°C for 0.5 minute. Platelets from panels A, B, and C were then solubilized and immunoblotted with an antibody specific for phosphorylated forms of p38 (P-p38) or with an antibody against phosphorylation-independent epitopes of p38 (p38). Note that VWF or thrombin-induced p38 phosphorylation is inhibited by PKG inhibitors and in PKG knockout platelets.