Effects of NO and selected cyclic nucleotide analogues on VASP Ser157 and VASP Ser239 phosphorylation. Aspirin-treated and gel-filtered human platelets were preincubated for 15 minutes in the absence or presence of a PKG activator (0.1 mM 8-Br-PET-cGMP) or PKA activator (0.2 mM SP-5,6-DCL-cBIMPS) or for 30 minutes with the PKG antagonist (0.5 mM Rp-8-pCPT-cGMPS). The platelets were then stimulated with thrombin (0.01 U/mL) alone or with NO (1.7 × 10^-10 mol NO/mL/min) for 0.5 (A-B) or 1 minute (C) in the presence of CP/CPK. Shape change was monitored by light transmission, and aliquots were withdrawn for measurement of VASP phosphorylation using monoclonal anti-PSer157-VASP5C6 and anti-P-Ser239 antibodies as described in “Materials and methods.” The 5C6 antibody recognizes phosphorylated VASP-Ser157 preferentially phosphorylated by PKA, whereas the VASP-16C2 antibody recognizes phosphorylated VASP Ser239 preferentially phosphorylated by PKG. The results are from a single experiment representative of 3.