ALLOSTERIC REGULATION OF PROTHROMBIN ACTIVATION BY FACTOR Va MAHESHEEMA ALI Bachelor of Science in Botany, Microbiology, Chemistry Osmania University, India April 2000 Master of Science in Organic Chemistry Osmania University, India April 2002 Submitted in partial fulfillment of requirements for the degree DOCTOR OF PHILOSOPHY IN CLINICAL AND BIOANALYTICALCHEMISTRY at the CLEVELAND STATE UNIVERITY May 2016 We hereby approve this dissertation for Mahesheema Ali Candidate for the Doctor of Philosophy in Clinical-Bioanalytical Chemistry Degree for the Department of Chemistry and the CLEVELAND STATE UNIVERSITY College of Graduate Studies ________________________________________ Dissertation Chairperson, Dr. Michael Kalafatis Department of CHEMISTRY ______________________ Date ________________________________________ Dissertation Committee Member, Dr. Edward F. Plow Department of MOLECULAR CARDIOLOGY Cleveland Clinic Foundation ______________________ Date ________________________________________ Dr. Anton A. Komar Department of BIOLOGY ______________________ Date ________________________________________ Dr. David J. Anderson Department of CHEMISTRY ______________________ Date ________________________________________ Dr. Crystal M. Weyman Department of BIOLOGY ______________________ Date Date of Defense: April 22nd, 2016 DEDICATION I dedicate this thesis to my loving family. My beloved husband, Mir Ali, has shown unwavering support and encouragement during past five years of my doctoral journey. I am grateful for his love and continuous support, which gave me the strength and courage to pursue my dream and to make it come true. I would like to give special thanks to my wonderful children--Maaz, Maryum, and Idris--for their support and patience throughout. I would like to thank my parents, Mrs. Kaneez Fathima and Mr.Yousuf Ali for all that I have become today, for the constant support in my academic career and personal life. They believed in me and encouraged me to strive in my life. I would like to thank my mother-in-law, Mrs.Moida Bano, and father-in-law Mr. Masood Ali, for all the encouragement of our family. Special thanks also goes to my sisters Mahjabeen, Afreen Fathima, Arshia Fathima and my brothers Kamran Mahmood and Rizwan Mahmood. They were always there when I needed them. The love of family is life’s greatest blessing. I love you all very much. ACKNOWLEDGEMENTS I would like to express my sincere gratitude and appreciation to my advisor, Dr. Michael Kalafatis, a great educator and a wonderful mentor. His invaluable advice and insightful comments have shaped my career for the future. He is patient and kind, and I thank him for the rewarding experience in his research lab. I wish to thank the members of my dissertation committee for their valuable time, collegial guidance and support to help improve my research. I thank Dr. Edward F. Plow, Dr. David J. Anderson, Dr. Crystal M. Weyman, and Dr. Anton A. Komar. I would like to thank Dr. Mary McDonald for reading this text for grammatical and syntactical issues. I especially would like to thank, the department secretaries, Richelle Emery and Michele Jones, for all their administrative help to make this achievement possible. I would like to thank Dr. Jamila Hirbawi for being a wonderful mentor and friend throughout and providing me with great support to be able to accomplish my work. I would like to thank Dr. Joseph Wiencek for his tremendous support. I appreciate his help and support. I would like to greatly acknowledge, Gregory M. Guzzo, research technologist, who has helped me tremendously in my day-to-day activities. I thank you Greg, for being there for me and supporting me as a wonderful friend. I also wish to acknowledge, Katie Turner and Jasmine Manouchehri for being wonderful friends and supporting me. They will never know how much their support meant to me. I would like to thank all my colleagues from Cleveland State University, especially those who have helped me along the way. I would like to thank the entire faculty who have taught and mentored me from Chemistry Department at Cleveland State University. Finally, I thank one and all. Thank you. ALLOSTERIC REGULATION OF PROTHROMBIN ACTIVATION BY FACTOR Va MAHESHEEMA ALI ABSTRACT Upon vascular injury, the proteolytic conversion of prothrombin to thrombin occurs in the presence of prothrombinase. Prothrombinase is an enzymatic complex between factor Va (fVa) and factor Xa (fXa) assembled on a membrane surface in the presence of divalent metal ions. Blood coagulation factor V (FV) is a multi-domain protein (A1-A2-B-A3-C1-C2) with no procoagulant activity and circulates in blood as a procofactor. Our investigation demonstrates that fV is activated by thrombin in a kinetically preferred order, by a first cleavage at Arg709, followed by cleavage at Arg1545 to ultimately generate the active cofactor species, fVa. The cofactor binds to membrane surfaces and effectively serves as receptor for membrane-bound fXa. Although membrane-bound fXa alone is capable of activating prothrombin through initial cleavage at Arg271, followed by the cleavage at Arg320 (Prethrombin-2 Pathway); the rate of activation is not physiologically compatible with survival. However, incorporation of fVa into prothrombinase results in a 300,000-fold increase in the catalytic efficiency of fXa for thrombin generation with the order of cleavages reversed, with initial cleavage at Arg320 followed by Arg271 (Meizothrombin Pathway), resulting in physiologically relevant rates of thrombin formation at the place of vascular injury. We have shown that fXa interacts with prothrombin through amino acid region 478-482 in a fVa-dependent manner. We further demonstrate that basic amino acids from exosite I of prothrombin are involved in recognizing fXa within prothrombinase also in a fVa-dependent manner. Finally, we characterized the allosteric vi interactions of these amino acid residues within prothrombin required for optimum prothrombin activation rates and optimal thrombin function. Our data suggest that amino acids Leu480 and Gln481 allosterically interact with basic amino acids from exosite I on prothrombin, thus modulating the enzymatic activity of fXa within prothrombinase. Our results also provide a logical explanation for the deleterious physiological consequences of natural mutations in proexosite I (Arg382→Cys) and (Arg388→Hys) and demonstrate that patients harboring these mutations are impaired in their ability to form a fibrin clot and, thus, are prone to severe bleeding. Overall our data underline the crucial physiological importance of fVa for thrombin generation and clot formation. vii TABLE OF CONTENTS ABSTRACT……………………………………………………………………………………...vi LIST OF TABLES…………………………………………………………………………….....xi LIST OF FIGURES………………………………………………………………......................xii CHAPTER I INTRODUCTION………………………………………………………………….1 1.1 Cardiovascular Disease and Associated Risk………………………………………1 1.2 Hemostasis…………………………………………………………………………2 1.3 Primary Hemostasis………………………………………………………………..3 1.4 The Coagulation Cascade: Secondary Hemostasis………………………………...4 1.5 Blood coagulation fV……………………………………………………………....9 1.6 Activation of fV………………………………………………………………….11 1.7 fV Inactivation…………………………………………………………………...13 1.8 Formation of the Prothrombinase Complex……………………………………...13 1.9 Interaction of fVa with Phospholipid Membranes……………………………….16 1.10 Interaction of fVa with fXa………………………………………………………18 1.11 Interaction of fVa with Prothrombin……………………………………………..18 1.12 Thrombin…………………………………………………………………………18 1.13 Clinical Significance……………………………………………………………..20 1.14 References………………………………………………………………………..22 CHAPTER II THE MOLECULAR MECHANISM CORRELATING THROMBIN CLEAVAGE AND ACTIVATION OF FACTOR V……………………………………………33 viii 2.1 Abstract………………………………………………………………………...33 2.2 Introduction…………………………………………………………………….35 2.3 Experimental Procedures……………………………………………………....37 2.4 Results…………………………………………………………………………44 2.5 Discussion……………………………………………………………………..62 2.6 References……………………………………………………………………..65 CHAPTER III ALLOSTERIC INTERACTIONS REGULATING PROTHROMBIN ACTIVATION AND THROMBIN ACTIVITY………………………………………………..71 3.1 Abstract………………………………………………………………………71 3.2 Introduction………………………………………………………………….73 3.3 Experimental Procedures…………………………………………………….76 3.4 Results………………………………………………………………………..84 3.5 Discussion…………………………………………………………………...110 3.6 References……………………………………………………………………114 CHAPTER IV OVERALL CONCLUSION……………………………………………………122 ix 4.1 Conclusion……………………………………………………………….122 4.2 References………………………………………………………………126 x LIST OF TABLES Table 2.1 Functional Properties of Recombinant fV Molecules……………………………………….57 Table 3.1 Molecular Weight of Prothrombin Fragments ………………………………………………91 Table 3.2 Kinetic Constants of Wild-Type and Selected rIIa Mutant Molecules toward S-2238…………………………………………………………………………………………..101 Table 3.3 Kinetic Constants of Plasma FII and rFII Mutant Molecules Activation by Prothrombinase…………………………………………………………………………………103 xi LIST OF FIGURES Figure 1.1 The Coagulation Cascade……………………………………………………………...8 Figure 1.2 Schematic of fV………………………………………………………………………10 Figure 1.3 Schematic of Human fV……………………………………………………………...12 Figure 1.4 Prothrombin Activation………………………………………………………………15 Figure 1.5 Prothrombinase Complex…………………………………………………………….17 Figure 2.1 Recombinant Mutants Generated to Explicate the Role of Individual Cleavage Site..……………………………………………………………………………………………...46 Figure 2.2 SDS-PAGE Analysis of Purified Recombinant Proteins (3µg/lane) before (0 min) and after Activation with Thrombin