Phosphorylation of Focal Adhesion Vasodilator‐Stimulated Phosphoprotein at Ser157 in Intact Human Platelets Correlates with Fibrinogen Receptor Inhibition

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Abstract
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 αIIbβ3) 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.