SYNERGISTIC PHOSPHORYLATION OF THE FOCAL ADHESION-ASSOCIATED VASODILATOR-STIMULATED PHOSPHOPROTEIN IN INTACT HUMAN PLATELETS IN RESPONSE TOCGMP-ELEVATING AND CAMP-ELEVATING PLATELET INHIBITORS

The mechanism underlying the synergistic inhibition of platelet activa tion by cGMP- and cAMP-elevating vasodilators was investigated using w ashed human platelets and platelet-rich plasma. With both types of hum an platelet preparations, low concentrations of sodium nitroprusside i ncreased the cAMP-elevating potency of low concentrations of prostagla ndin E(1) (PG-E(1)). Using threshold concentrations of both sodium nit roprusside and PG-E(1), the NO-donor potentiated the effect of PGE(1) with respect to the phosphorylation of the focal adhesion-associated v asodilator-stimulated phosphoprotein (VASP) at serine(157). In contras t, threshold concentrations of cell-membrane permeant selective activa tors of the platelet cGMP-dependent protein kinase or the cAMP-depende nt protein kinase had only additive effects on VASP serine(157) phosph orylation in washed human platelets. The data demonstrate that low int racellular levels of cGMP effectively inhibit type III cGMP-inhibited phosphodiesterase in human platelets despite the high levels of cGMP-d ependent 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 seri ne(157) phosphorylation, whereas the supra-additive effects observed w ith the combination of sodium nitroprusside and PG-E(1) are due to cGM P-mediated inhibition of type III phosphodiesterase. VASP phosphorylat ion at serine(157) may be an important component underlying the synerg istic inhibition of human platelets by cGMP- and cAMP-elevating agents .