Platelet adhesion and activation at the vascular wall are the initial steps leading to arterial thrombosis and vascular occlusion. Prostacyclin and nitric oxide inhibit platelet adhesion, acting via cyclic adenosine monophosphate (cAMP)-and cyclic guanosine monophosphate (cGMP)-dependent protein kinases. A major downstream target for both cAMP-and cGMPdependent protein kinases is the vasodilator-stimulated phosphoprotein (VASP). To test the significance of VASP for the regulation of platelet adhesion in vivo, we studied platelet-vessel wall interactions using VASP-deficient (VASP ؊/؊ ) mice. Under physiologic conditions, platelet adhesion to endothelial cells was significantly enhanced in VASP null mutants when compared with wild-type mice (P < .05). Platelet recruitment in VASP null mice involved P-selectin and the fibrinogen receptor glycoprotein IIb-IIIa (GPIIb-IIIa). Under pathophysiologic conditions, the loss of VASP increased platelet adhesion to the postischemic intestinal microvasculature, to the atherosclerotic endothelium of ApoE-deficient mice, and to the subendothelial matrix following endothelial denudation (P < .05 vs wild type). Importantly, platelet adhesion in VASP null mutants was unresponsive to nitric oxide. These data show for the first time in vivo that VASP is involved in down-regulation of platelet adhesion to the vascular wall under both physiologic and pathophysiologic conditions. ( IntroductionThe adhesion of platelets to the vascular wall is central to the pathogenesis of atherogenesis and arterial thrombosis. 1,2 Nitric oxide (NO) and prostacyclin are of major importance for the regulation of platelet-vessel wall interactions. They activate soluble guanylyl cyclase and adenylate cyclase, respectively, initiating a subsequent rise in platelet cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP). [3][4][5] Cyclic GMPdependent protein kinase I (cGKI) and cAMP-dependent protein kinase (cAK) are thought to be the major downstream targets of the NO/cGMP and prostacyclin/cAMP signaling cascades in platelets. 3,6,7 A common substrate of both cAK and cGK is the vasodilatorstimulated phosphoprotein (VASP). [8][9][10] VASP was isolated initially from human platelets but it is also expressed in a wide variety of other cells and tissues. 11 VASP is the founding member of a family of proline-rich proteins designated the Ena/VASP protein family, which comprises VASP, Drosophila Enabled (Ena), a substrate of the Abelson tyrosine kinase (Abl), the mammalian Ena homolog Mena, and the Ena-VASP-like protein Evl. 12-14 These proteins share highly homologous N-terminal and C-terminal domains (Ena-VASP homology domains 1 and 2, designated EVH1 and EVH2) and proline-rich central domains. 12-14 VASP has been found to be associated with focal adhesions, stress fibers, cell-cell contacts, and highly dynamic membrane regions in platelets, smooth muscle cells, endothelial cells, and fibroblasts. 11,15 VASP directly binds to profilin, zyxin, and to the focal adhesion and cell-cell cont...

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Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are established regulators of actin-based motility, platelet aggregation, and growth cone guidance. However, the molecular mechanisms involved essentially remain elusive. Here we report on a novel mechanism of VASP action, namely the regulation of tensile strength, contractility, and rigidity of the actin cytoskeleton. Compared to wild-type cells fibroblasts derived from VASP-deficient mice have thicker and more stable actin stress fibres. Furthermore focal adhesions are enlarged, myosin light chain phosphorylation is increased, and the rigidity of the filament-supported plasma membrane is elevated about three- to fourfold, as is evident from atomic force microscopy. Moreover, fibronectin-coated beads adhere stronger to the surface of VASP-deficient cells. The resistance of these beads to mechanical displacement by laser tweezers is dramatically increased in an F-actin-dependent mode. Cytoskeletal stabilization coincides with slower cell adhesion and detachment, while overall adhesion is increased. Interestingly, many of these effects observed in VASP (-/-) cells are recapitulated in VASP-overexpressing cells, hinting towards a balanced stoichiometry necessary for appropriate VASP function. Taken together, our results suggest that VASP regulates surface protrusion formation and cell adhesion through modulation of the mechanical properties of the actin cytoskeleton.