ANGIOTENSIN-(1-7)/MAS AXIS COMPENSATES ABSENT BRADYKININ IN BDKRB2-/- AND KLKB1-/- MICE TO REGULATE THROMBOSIS RISK by CHAO FANG Submitted in partial fulfillment of requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. Alvin Schmaier Department of Pathology CASE WESTERN RESERVE UNIVERSITY January, 2014 Case Western Reserve University School of Graduate Studies We hereby approve the thesis/dissertation of Chao Fang Candidate for the Doctor of Philosophy degree* George R. Dubyak (Committee Chair) Alvin H. Schmaier Jonathan S. Stamler Eugene Podrez Clive D. Hamlin Date 10/02/2013 *We also certify that written approval has been obtained for any proprietary material contained therein. Table of Contents List of Tables .................................................................................................................................... 3 List of Figures ................................................................................................................................... 3 Acknowledgement ............................................................................................................................ 6 Contribution of Co-authors ............................................................................................................... 7 List of Abbreviations ......................................................................................................................... 8 Abstract ......................................................................................................................................... 10 Chapter 1: Renin Angiotensin System (RAS) and thrombosis ............................................... 13 Plasma Kallikrein-Kinin (KKS) and Renin-Angiotensin Systems (RAS) .................................... 14 KKS and contact activation .................................................................................................... 14 RAS and hypertension modulation ........................................................................................ 15 Interaction between KKS and RAS ............................................................................................ 17 Influence of KKS, RAS and Mas receptor on thrombosis risk ................................................... 19 The antithrombotic nature of KKS .......................................................................................... 19 The prothrombotic nature of RAS .......................................................................................... 19 Mas receptor in thrombosis .................................................................................................... 20 Platelet spreading and GPVI activation ..................................................................................... 21 Signaling during platelet activation ........................................................................................ 21 Platelet spreading .................................................................................................................. 24 Integrin-mediated platelet spreading ...................................................................................... 25 Nitric oxide and prostacyclin as platelet inhibitors ..................................................................... 26 1. Nitric oxide: ........................................................................................................................ 28 2. Prostacyclin: ....................................................................................................................... 28 Overall hypothesis for the research ........................................................................................... 29 Chapter 2: Angiotensin-(1-7)/Mas Decreases Thrombosis in Bdkrb2-/- Mice by Increasing NO and Prostacyclin to Reduce Platelet Spreading and GPVI Activation ............................. 31 INTRODUCTION ........................................................................................................................ 32 RESULTS ................................................................................................................................... 34 Characterization of angiotensin-(1-7) and Mas of the renin angiotensin system in Bdkrb2-/- mice. ....................................................................................................................................... 34 Influence of Mas on thrombosis protection in Bdkrb2-/- mice. ................................................ 38 Platelet function of Bdkrb2-/- mice. ......................................................................................... 41 Bdkrb2-/- platelet spreading .................................................................................................... 43 GPVI activation in Bdkrb2-/- platelets ..................................................................................... 47 1 DISCUSSION ............................................................................................................................. 54 Chapter 3: Angiotensin-(1-7)/Mas Protects Klkb1-/- Mice from Thrombosis by Increased Prostacyclin to Reduce Vascular Tissue Factor through Elevated Sirt1 ............................... 60 INTRODUCTION ........................................................................................................................ 61 RESULTS ................................................................................................................................... 63 Characterization of Klkb1-/- mice ............................................................................................ 63 Thrombosis protection is not just defective contact activation in Klkb1-/- mice ...................... 64 Role of Mas receptor in thrombosis protection in Klkb1-/- mice .............................................. 67 Thrombosis protection is mediated by elevated plasma PGI2 and vascular Sirt1 ................. 70 DISCUSSION ............................................................................................................................. 76 Chapter 4: Methods and Materials ............................................................................................. 82 Methods for Chapter 2 (bradykinin B2 receptor deficient mice)................................................. 83 Methods for Chapter 3 (prekallikrein deficient mice) ................................................................. 93 Chapter 5: Summary and Future Directions ........................................................................... 101 Mechanisms for the compensatory effect of Angiotensin/Mas axis on Bradykinin/B2R signaling ................................................................................................................................................. 102 Mechanisms for the beneficial effect of PGI2 .......................................................................... 103 Mechanisms for reduced GPVI activation and integrin dependent spreading defect in Bdkrb2-/- mice. ...................................................................................................................... 104 Molecular mechanisms by which PGI2 maintain vascular homeostasis. ............................ 105 Conclusions .............................................................................................................................. 105 Literature Cited ........................................................................................................................... 106 2 List of Tables Table I Primers for Real-time PCR in Bdkrb2-/- mice 86 Table II Primers for Real-time PCR in Klkb1-/- mice 98 List of Figures Figure 1 Kallikrein Kinin System (KKS) and Contact activation 15 Figure 2 Kallikrein Kinin System (KKS), Renin Angiotensin System 18 (RAS) and their interactions Figure 3 Platelet activation by GPVI through the immune receptor in 24 platelets Figure 4 Nitric oxide and prostacyclin as platelet inhibitors 27 Figure 5 Influences of angiotensin receptor blocker (ARB) on the 35 Bdkrb2-/- mice Figure 6 Angiotensin-(1-7) level in Bdkrb2-/- mice 36 Figure 7 Measurement of Ang-(1-7) formation by PRCP or ACE2 in 37 kidney from Bdkrb2+/+ and Bdkrb2-/- mice Figure 8 Mas receptor expression in Bdkrb2-/- mice 38 Figure 9 Proposed mechanism by which elevation of angiotensin II 39 (AngII) leads to thromboprotection in Bdkrb2-/- mice Figure 10 The influence of the receptor Mas on thrombosis risk in 40 Bdkrb2-/- mice Figure 11 cGMP and cAMP levels in Bdkrb2-/- platelet 42 Figure 12 ADP induced fibrinogen binding in Bdkrb2-/- platelets 42 3 Figure 13 α- and γ- thrombin induced platelet activation in Bdkrb2-/- 43 platelets Figure 14 Static adhesion to collagen with washed platelets 44 Figure 15 Spreading on collagen in Bdkrb2-/- platelets 45 Figure 16 Platelet spreading on GFOGER 46 Figure 17 Bdkrb2-/- platelet spreading on fibrinogen and CRP 46 Figure 18 Incubation of washed Bdkrb2-/- platelets reverses their 47 spreading defect Figure 19 Collagen-related peptide (CRP) and convulxin (CVX) activation 47 of Bdkrb2-/- platelets Figure 20 GPVI level in Bdkrb2-/- platelets 48 Figure 21 Influence of Mas antagonist A-779 on GPVI activation in 49 Bdkrb2-/- platelets Figure 22 Influence of NO on GPVI activation in Bdkrb2-/- platelets 50 Figure 23 Influence of prostacyclin on GPVI activation in Bdkrb2-/- 51 platelets Figure 24 Ligation-dependent Syk phosphorylation in Bdkrb2-/- platelets 52 Figure 25 Bone marrow transplantation