Vasodilator-stimulated phosphoprotein (VASP) is phosphorylated on Ser¹⁵⁷ by protein kinase C-dependent and -independent mechanisms in thrombin-stimulated human platelets

VASP (vasodilator-stimulated phosphoprotein) is an actin- and profilin-binding protein that is expressed in platelets at high levels and plays a major role in negatively regulating secretory and adhesive events in these cells. VASP is a major substrate for cAMP- and cGMP-regulated protein kinases and it has been shown to be directly phosphorylated on Ser¹⁵⁷ by PKC (protein kinase C). In the present paper, we show that, in human platelets, VASP is phosphorylated by PKC on Ser¹⁵⁷, but not Ser²³⁹, in response to phorbol ester stimulation, in a manner blocked by the PKC inhibitor BIM I (bisindolylmaleimide I). In response to thrombin, VASP was also phosphorylated on Ser¹⁵⁷, but this response was only partially inhibited by BIM I, indicating PKC-dependent and -independent pathways to VASP phosphorylation by thrombin. Using inhibitors, we have ruled out the possibility that the PKC-independent pathway acts through guanylate cyclase generation of cGMP, or through a phosphoinositide 3-kinase-dependent kinase. Inhibition of Rho kinase, however, substantially reduced Ser¹⁵⁷ VASP phosphorylation, and its effects were additive with BIM I. This implicates Rho kinase and PKC as the major kinases that phosphorylate VASP Ser¹⁵⁷ in response to thrombin in platelets.