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To cite this article: Naseem KM, Riba R. Unresolved roles of platelet nitric oxide synthase. J Thromb Haemost 2008; 6: 10-9.Summary. Endothelial-derived nitric oxide (NO) is a key regulator of platelet function, inhibiting both adhesion to the extracellular matrix and aggregation at sites of vascular injury. Platelets also have the capacity to synthesize and release bioactive NO, which is thought to make a significant contribution to the vascular pool of NO. The regulation of platelet NO production is poorly understood and studies examining the physiological role of platelet-derived NO have produced contradictory and controversial findings. In the present article, we discuss the current understanding of the biochemical and molecular regulation of platelet NO synthesis and outline the potential physiological and clinical significance of this molecule.

To cite this article: Riba R, Oberprieler NG, Roberts W, Naseem KM. von Willebrand factor activates endothelial nitric oxide synthase in blood platelets by a glycoprotein Ib-dependent mechanism. J Thromb Haemost 2006; 4: 2636-44.Summary. 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 NO signaling, [ 3 H] 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 [ 3 H] L-citrulline production and cGMP accrual, respectively. VWFinduced eNOS activation was GPIb-IX-dependent and independent of integrin a IIb b 3 . cGMP formation in response to VWF required Ca 2+ 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 A 2 (TxA 2 ). Elevated cGMP levels led to increased VASP phosphorylation at serine 239 that was both protein kinase G (PKG)-and protein kinase A (PKA)-dependent. Conclusions: We demonstrate that VWF activates eNOS through a specific Ca 2+ -dependent GPIb receptor-signaling cascade that relies on the generation of platelet-derived ADP and TxA 2 . Furthermore, we provide the first evidence to suggest that platelet derived-NO/cGMP activates PKA in addition to PKG.

The molecular regulation of nitric oxide synthase (NOS) in blood platelets is an uncharacterised area of platelet biology. We investigated the mechanism of collagen-stimulated NO synthesis in platelets. Our aim was to identify the key collagen receptor and downstream signalling mechanisms linking collagen to NOS activation. Collagen and the GpVI-specific platelet activator collagen-related peptide (CRP-XL) stimulated NO synthesis, as evidenced by increased [(3)H]L-citrulline production, and cyclic GMP (cGMP) formation. After platelet activation by collagen and CRP-XL was normalised, we found no differences in NOS activation or cGMP formation in response to these agonists. Blocking the interaction of collagen with integrin alpha(2)beta(1), a second collagen receptor, failed to affect NOS activation by collagen. These data indicate that collagen-induced NO synthesis is linked to GpVI activation. cGMP formation in response to collagen and CRP-XL required increased intracellular Ca(2+), Src family kinases, phosphatidylinositol 3-kinase (PI3-K) and protein kinase C. By comparison, Gp VI-independent cGMP formation induced by thrombin was Src kinase-dependent, but was independent of PI3-K and PKC. Thus the mechanisms of collagen- and CRP-XL-induced NOS activation were identical, but distinct from that of thrombin. Platelet activation in response to collagen leads to secretion of adenosine diphosphate (ADP) and thromboxane A(2) (TxA(2)). Our results demonstrate that collagen-stimulated cGMP synthesis was enhanced significantly by platelet-derived ADP and TxA(2). These results reveal that collagen stimulates platelet NOS activation through a specific Ca(2+)-dependent GpVI receptor signalling cascade, and demonstrate that collagen-induced cGMP accrual requires the release of secondary platelet agonists.

To cite this article: Roberts W, Riba R, Homer-Vanniasinkam S, Farndale RW, Naseem KM. Nitric oxide specifically inhibits integrin-mediated platelet adhesion and spreading on collagen. J Thromb Haemost 2008; 6: 2175-85.Summary. Background: Nitric oxide (NO) inhibits platelet adhesion to collagen, although the precise molecular mechanisms underlying this process are unclear. Objectives: Collagenmediated adhesion is a multifaceted event requiring multiple receptors and platelet-derived soluble agonists. We investigated the influence of NO on these processes. Results: S-nitrosoglutathione (GSNO) induced a concentration-dependent inhibition of platelet adhesion to immobilized collagen. Maximal adhesion to collagen required platelet-derived ADP and TxA 2 . GSNO-mediated inhibition was lost in the presence of apyrase and indomethacin, suggesting that NO reduced the availability of, or signaling by, ADP and TxA 2 . Exogenous ADP, but not the TxA 2 analogue U46619, reversed the inhibitory actions of GSNO on adhesion. Under adhesive conditions NO inhibited dense granule secretion but did not influence TxA 2 generation. These data indicated that NO may block signaling by TxA 2 required for dense granule secretion, thereby reducing the availability of ADP. Indeed, we found TxA 2 -mediated activation of PKC was required to drive dense granule secretion, a pathway that was inhibited by NO. Because our data demonstrated that NO only inhibited the activation-dependent component of adhesion, we investigated the effects of NO on individual collagen receptors. GSNO inhibited platelet adhesion and spreading on a 2 b 1 specific peptide ligand GFOGER. In contrast, GSNO did not inhibit GPVI-mediated adhesion to collagen, or adhesion to the GPVI specific ligand, collagen related peptide (CRP). Conclusions: NO targets activationdependent adhesion mediated by a 2 b 1 , possibly by reducing bioavailability of platelet-derived ADP, but has no effect on activation-independent adhesion mediated by GPVI. Thus, NO regulates platelet spreading and stable adhesion to collagen.