Integrins and other adhesion receptors are essential components for outside-in and inside-out signaling through the cell membrane. The platelet glycoprotein IIb-IIIa (also known as fibrinogen receptor or integrin alpha IIb beta 3) is activated by platelet agonists, inhibited by cyclic-nucleotide-elevating agents, and is involved in the activation of protein tyrosine kinases including the 125-kDa focal adhesion kinase (pp125FAK). However, the molecular details of glycoprotein IIb-IIIa regulation are not well understood. Here we report that in ADP-activated human platelets cAMP- and cGMP-dependent protein-kinase-mediated phosphorylation of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) at Ser157 correlates well with glycoprotein IIb-IIIa inhibition. Human platelets contain similar concentrations of glycoprotein IIb-IIIa complexes (fibrinogen binding sites) and VASP. Using gel-filtered platelets, cAMP-elevating agents [e.g. prostaglandin E1 and the forskolin analog 6-(3-dimethylaminopropionyl)forskolin (NKH 477)] caused VASP Ser157 phosphorylation and inhibited glycoprotein IIb-IIIa activation up to 70-100%. NO-generating, cGMP-elevating agents [e.g. 3-morpholinosydnonimine hydrochloride (SIN1) and sodium nitroprusside] stimulated VASP Ser157 phosphorylation and inhibited glycoprotein IIb-IIIa activation up to a maximal extent of 30-50%. The effects of cAMP- and cGMP-elevating agents on VASP phosphorylation and fibrinogen binding were reversible and could be mimicked by membrane-permeant selective activators of platelet cAMP- or cGMP-dependent protein kinase, respectively. Using threshold concentrations, the nitrovasodilator SIN 1 potentiated the effects of the forskolin analog NKH 477 with respect to inhibition of platelet aggregation, VASP phosphorylation and glycoprotein IIb-IIIa inhibition. It is proposed that the inhibition of glycoprotein IIb-IIIa induced by cyclic nucleotide involves cAMP-and cGMP-dependent protein-kinase-mediated VASP phosphorylation at Ser157.