Endothelial nitric oxide synthase plays a minor role in inhibition of arterial thrombus formation

Summary

Endothelial NO synthase (eNOS) expressed in the vascular en-dothelium or formed within platelets was postulated to inhibit platelet activation and aggregation. We have assessed the role of eNOS in platelet aggregation in vitro and in vivo by comparison of WT and eNOS-/- mice. Aggregometer studies revealed that collagen over a concentration range of 0.36–10 µg aggregated WT and eNOS-/- platelets to the same extent (10 µg: WT 86.7±4.7%, eNOS-/- 91±12%, n=6). Collagen treatment did not result in a significant increase in cGMP formation and VASP phosphorylation. Thrombin-induced P-selectin surface expression was unchanged in eNOS-/- platelets. In line with these findings no eNOS protein was detectable within the platelets of WT mice. In vivo, bleeding time after tail tip resection tended to be shorter in eNOS/- mice (WT: 116±35 s; eNOS-/- 109±37 s, n.s). Similarly, time to occlusion of the A.carotis after focal induction of thrombosis was 501±76 s (WT) and 457±95 s (eNOS-/-) (n.s.). These data demonstrate that eNOS-deficiency minimally affects platelet aggregation and is not associated with accelerated arterial thrombosis in vivo. Thus, in the mouse endothelial NO synthase does not play a major role in the autocrine modulation of platelet function and in thrombosis of conduit vessels in vivo.

Keywords

Platelets - nitric oxide - platelet physiology - transgenic animals - gene targeting / knock-out

• References

• 1 Lüscher TF, Noll G. The pathogenesis of cardiovascular disease: role of the endothelium as a target and mediator. Atherosclerosis 1995; 118: S81-S90
• 2 Mombouli JV, Vanhoutte PM. Endothelial dysfunction: from physiology to therapy. J Mol Cell Cardiol 1999; 31: 61-74
• 3 Freedman JE, Loscalzo J, Benoit SE. et al Decreased platelet inhibition by nitric oxide in two brothers with a history of arterial thrombosis. J Clin Invest 1996; 97: 979-87
• 4 Michelson AD, Benoit SE, Furman MI. et al Effects of nitric oxide/EDRF on platelet surface glycoproteins. Am J Physiol 1996; 270: H1640-H1648
• 5 Walter U, Geiger J, Haffner C. et al Platelet-vessel wall interactions, focal adhesions, and the mechanism of action of endothelial factors. Agents Actions Suppl 1995; 45: 255-68
• 6 Horstrup K, Jablonka B, Honig-Liedl P. et al Phosphorylation of focal adhesion vasodilator-stimulated phosphoprotein at Ser157 in intact human platelets correlates with fibrinogen receptor inhibition. Eur J Biochem 1994; 225: 21-7
• 7 Wade ML, Fitzpatrick FA. Nitric oxide modulates the activity of the hemoproteins prostaglandin I2 synthase and thromboxane A2 synthase. Arch Biochem Biophys 1997; 347: 174-80
• 8 Wang GR, Zhu Y, Halushka PV. et al Mechanism of platelet inhibition by nitric oxide: in vivo phosphorylation of thromboxane receptor by cyclic GMP-dependent protein kinase. Proc Natl Acad Sci U S A 1998; 95: 4888-93
• 9 de Graaf JC, Banga JD, Moncada S. et al Nitric oxide functions as an inhibitor of platelet adhesion under flow conditions. Circulation 1992; 85: 2284-90
• 10 Mehta JL, Chen LY, Kone BC. et al Identification of constitutive and inducible forms of nitric oxide synthase in human platelets. J Lab Clin Med 1995; 125: 370-7
• 11 Radomski MW, Palmer RM, Moncada S. An L-arginine/ nitric oxide pathway present in human platelets regulates aggregation. Proc Natl Acad Sci U S A 1990; 87: 5193-7
• 12 Chen LY, Mehta JL. Further evidence of the presence of constitutive and inducible nitric oxide synthase isoforms in human platelets. J Cardiovasc Pharmacol 1996; 27: 154-8
• 13 Radomski MW, Rees DD, Dutra A. et al S-nitrosoglutathione inhibits platelet activation in vitro and in vivo. Br J Pharmacol 1992; 107: 745-9
• 14 Pawloski JR, Swaminathan RV, Stamler JS. Cellfree and erythrocytic S-nitrosohemoglobin inhibits human platelet aggregation. Circulation 1998; 97: 263-7
• 15 Freedman JE, Sauter R, Battinelli EM. et al Deficient platelet-derived nitric oxide and enhanced hemostasis in mice lacking the NOSIII gene. Circ Res 1999; 84: 1416-21
• 16 Massberg S, Sausbier M, Klatt P. et al Increased adhesion and aggregation of platelets lacking cyclic guanosine 3',5'-monophosphate kinase I. J Exp Med 1999; 189: 1255-64
• 17 Gödecke A, Decking UK, Ding Z. et al Coronary hemodynamics in endothelial NO synthase knockout mice. Circ Res 1998; 82: 186-94
• 18 Schwarz UR, Geiger J, Walter U. et al Flow cytometry analysis of intracellular VASP phosphorylation for the assessment of activating and inhibitory signal transduction pathways in human platelets--definition and detection of ticlopidine/clopidogrel effects. Thromb Haemost 1999; 82: 1145-52
• 19 Smolenski A, Bachmann C, Reinhard K. et al Analysis and regulation of vasodilator-stimulated phosphoprotein serine 239 phosphorylation in vitro and in intact cells using a phosphospecific monoclonal antibody. J Biol Chem 1998; 273: 20029-35
• 20 Kurz KD, Main BW, Sandusky GE. Rat model of arterial thrombosis induced by ferric chloride. Thromb Res 1990; 60: 269-80
• 21 Murohara T, Parkinson SJ, Waldman SA. et al Inhibition of nitric oxide biosynthesis promotes P-selec selectin expression in platelets. Role of protein kinase C. Arterioscler Thromb Vasc Biol 1995; 15: 2068-75
• 22 Gries A, Bode C, Peter K. et al Inhaled nitric oxide inhibits human platelet aggregation, P-selectin expression, and fibrinogen binding in vitro and in vivo. Circulation 1998; 97: 1481-7
• 23 Lamping KG, Faraci FM. Role of sex differences and effects of endothelial NO synthase deficiency in responses of carotid arteries to serotonin. Arterioscler Thromb Vasc Biol 2001; 21: 523-8
• 24 Freedman JE, Loscalzo J, Barnard MR. et al Nitric oxide released from activated platelets inhibits platelet recruitment. J Clin Invest 1997; 100: 350-6
• 25 Huang PL, Huang Z, Mashimo H. et al Hypertension in mice lacking the gene for endothelial nitric oxide synthase. Nature 1995; 377: 239-42
• 26 Feelisch M, Rassaf T, Mnaimneh S. et al Concomitant S-, N-, and heme-nitros(yl)ation in biological tissues and fluids: implications for the fate of NO in vivo. FASEB J 2002; 16: 1775-85
• 27 Murata T, Ushikubi F, Matsuoka T. et al Altered pain perception and inflammatory response in mice lacking prostacyclin receptor. Nature 1997; 388: 678-82
• 28 Koszalka P, Özüyaman B, Huo Y. et al Targeted disruption of cd73/Ecto-5'-nucleotidase alters thromboregulation and augments vascular inflammatory response. Circ Res 2004; 95: 814-21
• 29 Zweier JL, Wang P, Kuppusamy P. Direct measurement of nitric oxide generation in the ischemic heart using electron paramagnetic resonance spectroscopy. J Biol Chem 1995; 270: 304-7
• 30 Li Z, Xi X, Gu M. et al A stimulatory role for cGMP-dependent protein kinase in platelet activation. Cell 2003; 112: 77-86
• 31 Randriamboavonjy V, Schrader J, Busse R. et al Insulin induces the release of vasodilator compounds from platelets by a nitric oxide-G kinase-VAMP-3-dependent pathway. J Exp Med 2004; 199: 347-56