cGMP analogs or a nitric oxide donor-induced phosphorylation of p38 in platelets. (A) Washed human platelets (200 μL) were incubated under stirring in the platelet aggregometer with 2 μL water (control) or PKG activators 8-bromo-cGMP (100 μM), 8-pCPT-cGMP (100 μM), or glyco-SNAP1 (100 μM) at 37°C for 0.5 minute. Platelets were then solubilized and analyzed by immunoblotting with a rabbit antibody recognizing phosphorylated form of p38 to detect p38 phosphorylation (P-p38) and with a rabbit anti-p38 antibody to indicate comparable loading levels (p38). (B) Platelets were preincubated without (control) or with DMSO, a PKG inhibitor, KT5823 (5 μM), or PKA inhibitors, KT5720 (5 μM) or H89 (50 μM), at 22°C for 5 minutes and then stimulated with 8-pCPT-cGMP (100 μM) for 0.5 minute. These platelets were solubilized and analyzed by immunoblotting with a rabbit antibody recognizing phosphorylated form of p38 (P-p38) and with a rabbit anti-p38 antibody to indicate comparable loading levels (p38). (C,D) Washed human platelets were treated with 8-bromo-cGMP for 0.5 minute or 5 minutes and then immunoblotted with P-p38 antibody and with p38 antibody as described in panel A. Immunoblotting results were scanned and quantitated using NIH Image software. Quantitative results from 3 experiments (mean ± SD) are shown in panel C, and a representative experiment is shown in panel D.