PHOSPHORYLATION OF FOCAL ADHESION VASODILATOR-STIMULATED PHOSPHOPROTEIN AT SER157 IN INTACT HUMAN PLATELETS CORRELATES WITH FIBRINOGEN RECEPTOR INHIBITION

Integrins and other adhesion receptors are essential components for ou tside-in and inside-out signaling through the cell membrane. The plate let glycoprotein IIb-IIIa (also known as fibrinogen receptor or integr in 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 kinas e (ppl25(FAK)). However, the molecular details of glycoprotein IIb-III a regulation are not well understood. Here we report that in ADP-activ ated human platelets cAMP- and cGMP-dependent protein-kinase-mediated phosphorylation of the focal adhesion vasodilator-stimulated phosphopr otein (VASP) at Ser157 correlates well with glycoprotein IIb-IIIa inhi bition. Human platelets contain similar concentrations of glycoprotein IIb-IIIa complexes (fibrinogen binding sites) and VASP. Using gel-fil tered platelets, cAMP-elevating agents [e.g. prostaglandin E, and the forskolin analog 6-(3-dimethylaminopropionyl)forskolin (NKH 477)] caus ed VASP Ser157 phosphorylation and inhibited glycoprotein IIb-IIIa act ivation up to 70-100%. NO-generating, cGMP-elevating agents [e.g. 3-mo rpholinosydnonimine hydrochloride (SIN1) and sodium nitroprusside] sti mulated VASP Ser157 phosphorylation and inhibited glycoprotein IIb-III a activation up to a maximal extent of 30-50%. The effects of cAMP- an d 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, respect ively. Using threshold concentrations, the nitrovasodilator SIN 1 pote ntiated the effects of the forskolin analog NKH 477 with respect to in hibition of platelet aggregation, VASP phosphorylation and glycoprotei n IIb-IIIa inhibition. It is proposed that the inhitition of glycoprot ein IIb-IIIa induced by cyclic nucleotide involves cAMP-and cGMP-depen dent protein-kinase-mediated VASP phosphorylation at Ser157.