von Willebrand factor activates endothelial nitric oxide synthase in blood platelets by a glycoprotein Ib-dependent mechanism

Abstract

Background: The molecular regulation of endothelial nitric oxide synthase (eNOS) in blood platelets and the signalling events induced by platelet-derived NO are poorly defined. In particular, the ability of von Willebrand factor (VWF) to stimulate cyclic guanosine monophosphate (cGMP) formation in platelets has produced conflicting data. Objectives: To determine the mechanisms leading to eNOS activation and clarify the downstream signaling pathways activated by platelet-derived NO in response to VWF. Methods: We used three independent markers of NOsignaling, [3H] L-citrulline production, cGMP accrual and immunoblotting of vasodilator-stimulated phosphoprotein (VASP) to examine the NO signaling cascade in response to VWF. Results: VWF increased NO synthesis and bioavailability, as evidenced by increased [3H] L-citrulline production and cGMP accrual, respectively. VWF-induced eNOS activation was GPIb-IX-dependent and independent of integrin αIIbβ3. cGMP formation in response to VWF required Ca2+ mobilization, Src family kinases, phosphatidylinositol 3-kinase and phospholipase C, but not protein kinase C. This suggests that a cross-talk between the signaling mechanisms regulates platelet activation and NO synthesis. VWF-induced cGMP accrual was completely blocked by apyrase and indomethacin, demonstrating an essential role for platelet-derived ADP and thromboxane A2 (TxA2). Elevated cGMP levels led to increased VASP phosphorylation at serine239 that was both protein kinase G (PKG)- and protein kinase A (PKA)-dependent. Conclusions: We demonstrate that VWF activates eNOS through a specific Ca2+-dependent GPIb receptor-signaling cascade that relies on the generation of platelet-derived ADP and TxA2. Furthermore, we provide the first evidence to suggest that platelet derived-NO/cGMP activates PKA in addition to PKG. © 2006 International Society on Thrombosis and Haemostasis.