EFFECTS OF NITRITE AND NITROXYL ON HUMAN VASCULAR AND PLATELET FUNCTION Rustem Dautov MBBS (Hons), FRACP Department of Medicine, Faculty of Health Sciences University of Adelaide & Department of Cardiology, The Queen Elizabeth Hospital South Australia May 2014 A thesis submitted to the University of Adelaide as the requirement for the degree of Doctor of Philosophy Dedicated to my parents, my love Inara and children Zilya, Latipha and Temir ii Table of Contents Abstract!.............................................................................................................!x! Declaration!.......................................................................................................!xii! Publications, presentations and awards related to the work conducted towards this thesis!.........................................................................................................!xiii! Acknowledgements!...........................................................................................!xv! List of abbreviations!.......................................................................................!xvii! Chapter 1:! Introduction!.................................................................................!1! 1.1 Nitric oxide physiology!........................................................................................!2! 1.1.1 Introduction!.........................................................................................................!2! 1.1.2 Nitric oxide effects on vascular tone!...................................................................!2! 1.1.3 Nitric oxide production!.......................................................................................!4! 1.1.4 Nitric oxide diffusion!..........................................................................................!7! 1.1.5 Nitric oxide consumption!....................................................................................!7! 1.1.6 Molecular bases of NO action, including alternatives to direct activation of soluble guanylate cyclase!.............................................................................................!9! 1.1.7 Nitric oxide bioreactivity!.....................................................................................!9! 1.2 Nitric oxide pathophysiology: “endothelial dysfunction”!..................................!11! 1.2.1 Normal endothelial function!..............................................................................!11! 1.2.2 Endothelial dysfunction!.....................................................................................!12! 1.2.3 Clinical measurements of endothelial function!.................................................!13! 1.2.3.1 Invasive intra-coronary studies!................................................................................!14! 1.2.3.2 Assessment of endothelial function in peripheral vascular beds!..............................!15! 1.2.3.2.1!Venous!occlusion!plethysmography!................................................................!15! 1.2.3.2.2!Flow>mediated!vasodilatation!.........................................................................!16! 1.2.3.2.3!Pulse!wave!analysis!..........................................................................................!16! 1.2.3.2.4!Peripheral!arterial!tonometry!..........................................................................!17! 1.2.4 Biochemical assessment of endothelial function!...............................................!17! 1.2.5 Does arginine supplementation reverse endothelial dysfunction?!.....................!19! 1.2.6 Mechanism of endothelial dysfunction!..............................................................!19! iii 1.2.6.1 NO half-life and oxidative stress!.............................................................................!21! 1.2.6.2 Impaired nitric oxide synthesis: L-arginine and BH4 availability!............................!22! 1.2.6.3 Enhanced activity and expression of arginase!..........................................................!22! 1.2.6.4 Dysregulation of dimethylarginine dimethylaminohydrolase!..................................!23! 1.2.6.5 Reduced sensitivity to NO!.......................................................................................!23! 1.3 Nitric oxide resistance!.......................................................................................!23! 1.3.1 Introduction!.......................................................................................................!23! 1.3.2 Relationship between NO resistance and endothelial function!..........................!25! 1.3.3 Platelet function and NO resistance!...................................................................!26! 1.3.4 Epidemiology of NO resistance!.........................................................................!27! 1.3.5 Mechanisms of NO resistance!...........................................................................!29! 1.3.5.1 Nitric oxide bioavailability!......................................................................................!30! 1.3.5.2 Integrity of the NO/cGMP signaling pathway!.........................................................!31! 1.3.5.3 cGMP-independent effects of NO!............................................................................!33! 1.3.6 Prognostic value of NO resistance!.....................................................................!34! 1.3.7 Amelioration of NO resistance!..........................................................................!37! 1.3.7.1 Ascorbic acid!...........................................................................................................!37! 1.3.7.2 ACE inhibitors!.........................................................................................................!37! 1.3.7.3 Perhexiline!...............................................................................................................!38! 1.3.7.4 Glycaemic control!....................................................................................................!39! 1.3.7.5 Statins!......................................................................................................................!40! 1.3.8 Influence of thioredoxin-interacting protein (TXNIP) on NO signaling: implications regarding NO resistance and its amelioration!........................................!41! 1.4 Platelets: anatomy, physiology and pathophysiology!........................................!43! 1.4.1 Normal platelet function and anatomy!..............................................................!43! 1.4.2 Platelet adhesion!................................................................................................!45! 1.4.2.1 Von Willebrand Factor (vWf)!..................................................................................!45! 1.4.2.2 GP Ib-IX-V receptor complex!..................................................................................!45! 1.4.3 Platelet activation!..............................................................................................!46! 1.4.3.1 Platelet activation via ADP receptors!......................................................................!48! 1.4.3.2 Platelet activation via thrombin receptors!................................................................!48! 1.4.3.3 Platelet activation via thromboxane A2 receptors!....................................................!48! 1.4.4 Platelet aggregation!...........................................................................................!49! 1.4.5 Characterization of platelet aggregation!............................................................!49! 1.4.5.1 Platelet aggregometry!..............................................................................................!50! iv 1.4.5.1.1!Optical!platelet!aggregometry!.........................................................................!50! 1.4.5.1.2!Impedance!platelet!aggregometry!...................................................................!51! 1.4.5.1.3!The!rapid!platelet!function!analyzer!................................................................!51! 1.4.5.1.4!Shear>dependent!assay:!PFA>100!....................................................................!52! 1.4.5.2 Measures of platelet activation!................................................................................!52! 1.4.5.2.1!Soluble!platelet!activation!markers!.................................................................!52! 1.4.5.2.2!Flow!cytometry!................................................................................................!53! 1.5 Nitrate-Nitrite-Nitric oxide pathway!.................................................................!53! 1.5.1 Nitrogen living cycle!.........................................................................................!53! 1.5.2 Sources of nitric oxide!.......................................................................................!55! 1.5.3 Sources of nitrate and nitrite!..............................................................................!56! 1.5.4 Bioconversion of nitrate in the digestive tract!...................................................!57! 1.5.5 Nitrite bioactivation and hypoxic potentiation in humans!.................................!58! 1.5.5.1 Role of haemoglobin!................................................................................................!59! 1.5.5.2 Role of myoglobin!...................................................................................................!60!