Synergistic phosphorylation of the focal adhesion-associated vasodilator-stimulated phosphoprotein in intact human platelets in response to cGMP- and cAMP-elevating platelet inhibitors

Abstract

The mechanism underlying the synergistic inhibition of platelet activation by cGMP- and cAMP-elevating vasodilators was investigated using washed human platelets and platelet-rich plasma. With both types of human platelet preparations, low concentrations of sodium nitroprusside increased the cAMP-elevating potency of low concentrations of prostaglandin E 1 (PG-E 1). Using threshold concentrations of both sodium nitroprusside and PG-E 1, the NO-donor potentiated the effect of PG-e 1 with respect to the phosphorylation of the focal adhesion-associated vasodilator-stimulated phosphoprotein (VASP) at serine 157. In contrast, threshold concentrations of cell-membrane permeant selective activators of the platelet cGMP-dependent protein kinase or the cAMP-dependent protein kinase had only additive effects on VASP serine 157 phosphorylation in washed human platelets. The data demonstrate that low intracellular levels of cGMP effectively inhibit type III cGMP-inhibited phosphodiesterase in human platelets despite the high levels of cGMP-dependent protein kinase present in this cell type. This study provides the first evidence that the simultaneous activation of both cGMP-and cAMP-dependent protein kinase results in additive effects on VASP serine 157 phosphorylation, whereas the supra-additive effects observed with the combination of sodium nitroprusside and PG-E 1 are due to cGMP-mediated inhibition of type III phosphodiesterase. VASP phosphorylation at serine 157 may be an important component underlying the synergistic inhibition of human platelets by cGMP-and cAMP-elevating agents.