Loyola University Chicago Loyola eCommons Dissertations Theses and Dissertations 1996 Comparative Pharmacologic Studies on Synthetic and Recombinant Inhibitors of Thrombin Demetra D. Callas Loyola University Chicago Follow this and additional works at: https://ecommons.luc.edu/luc_diss Part of the Pharmacology Commons Recommended Citation Callas, Demetra D., "Comparative Pharmacologic Studies on Synthetic and Recombinant Inhibitors of Thrombin" (1996). Dissertations. 3406. https://ecommons.luc.edu/luc_diss/3406 This Dissertation is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Dissertations by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 1996 Demetra D. Callas LOYOLA UNIVERSITY CHICAGO COMPARATIVE PHARMACOLOGIC STUDIES ON SYNTHETIC AND RECOMBINANT INHIBITORS OF THROMBIN VOLUME I (CHAPTERS I, II, III, IV) A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL IN CANDIDACY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS BY DEMETRA D. CALLAS CHICAGO, ILLINOIS MAY 1996 LOYOLA UNIVERSITY MEDICAL CENTER LIBRARY Copyright by Demetra D. Callas, 1996 All rights reserved. ii ACKNOWLEDGEMENTS I would like to take this opportunity to thank all of those who helped me and supported me throughout my graduate studies. I will always be grateful to my mentor, Dr. Jawed Fareed, for his guidance and support during the past six years. Dr. Fareed has not been only a source of scientific knowledge for me, but has also taught me many other aspects that are important and contribute to the survival of a scientist in our days and for these I will always be thankful. I thank the members of my dissertation committee, Dr. Sandor Bajusz, Dr. Edward W. Bermes Jr., Dr. Joseph R. Davis, Dr. Yale Nemerson and Dr. Loek Van de Kar, for their contributions, suggestions and critical evaluation of various parts of this dissertation and for their efforts in evaluating overall this body of work. I also thank Dr. Umberto Cornelli for his critical and insightful comments on this manuscript. I express my sincere appreciation to my colleagues at the Hemostasis Research Laboratories for teaching me many of the techniques used in these studies: Peter Bacher, Mike Koza, Debra Hoppensteadt, Dr. Ahmad Ahsan and Dr. Omer Iqbal. I also appreciate the assistance received by Dr. Tom Herndon, Scott Asselmeyer and Dr. Kaiding Fu. I thank our secretary, Ms. Elayne Grzeda, for her help and for always being cheerful and encouraging. I thank my classmate, colleague and friend Walter Jeske for his suggestions and his attentive ears, since the beginning of our graduate careers. iii I thank Dr. Jeanine Walenga for her insights, comments, suggestions and support with many of the projects and in manuscript preparation. I thank Peter Molnar and Dan Murphy for their invaluable assistance with the computer-related work and slide preparation. I thank Ms. Debra Arendziak for expediting and facilitating some of my work in the department of Pathology. I am thankful to Dr. Volker Eschenfelder, Dr. John Fenton II, Dr. Bill Drohan and Dr. Richard Schwarz for their generous contributions and suggestions. Last, but not least, I am most thankful to Dr. Klaus Breddin, Ms. Dietrich and Dr. Wolfram Raake for teaching me, guiding me and helping me establish the rat laser-induced thrombosis model, with utmost patience and sincere interest. Most of all, I would like to acknowledge and thank my parents and my brothers for their infinite love, moral support, trust and confidence they have in me and for never failing to make me smile through difficult times. IV DEDICATION To my parents TABLE OF CONTENTS ACKNOWLEDGEMENTS .................................. m LIST OF FIGURES ...................................... xvii LIST OF TABLES ...................................... xxii LIST OF STANDARD ABBREVIATIONS ...................... xxviii LIST OF NON-STANDARD ABBREVIATIONS . xxxii VOLUME I Chapter I. PURPOSE . 1 II. REVIEW OF LITERATURE .............................. 8 A. Overview of Hemostasis . 8 B. Role of Serine Proteases in the Regulation of Coagulation, Fibrinolytic, Kallikrein and Complement Pathways. .................... 12 1. Biochemical Characteristics of Serine Proteases . 17 2. Role of Serine Proteases and Cofactors in the Hemostatic and Hemorrhagic Processes . 20 a. High Molecular Weight Kininogen (HMWK) ............. 22 b. Prekallikrein ................................. 23 c. Factor XII . 23 d. Factor XI ................................... 24 e. Factor IX ................................... 25 f. Factor VIII/von Willebrand Factor Complex . 26 g. Tissue Factor (TF) ............................. 27 h. Factor VII . 28 i. Factor X . 29 J. Factor XIII .................................. 30 k. Plasmin . 30 1. Tissue Type Plasminogen Activator (tPA) ............... 31 m. Urokinase Type Plasminogen Activator (uPA) ............ 32 3. Regulation of Serine Proteases by Serpins . 32 v a. Antithrombin . 33 b. Heparin Cofactor II ............................. 33 c. Tissue Factor Pathway Inhibitor (TFPI) ................ 35 d. Protein C . 36 e. Protein S ................................... 37 f. Factor V . 37 g. a 2Plasmin Inhibitor ............................. 38 h. Plasminogen Activator Inhibitors ..................... 38 C. Thrombin: Structure, Function, Physiologic, Pathologic and Pharmacologic Mechanisms . 39 1. Biology of Thrombin Formation and Structure of the Thrombin Molecule .................................... 39 2. Molecular Variants of Thrombin ....................... 43 3. Thrombin Standardization ........................... 43 4. Physiologic Regulation of Thrombin Formation .............. 44 5. Cellular Interactions of Thrombin . 45 6. Thrombin Receptors ............................... 45 D. Pharmacologic Control of Thrombin's Action .................. 46 1. Inhibition of Thrombin Receptors ....................... 47 2. Inhibition of Cellular Processes Mediated by Thrombin ......... 47 3. Inhibition of Thrombin Generation ...................... 48 4. Inhibition of Formed Thrombin ........................ 53 E. Thrombin Inhibitors .................................. 55 1. Inhibitors of Plasma Origin . 55 2. Synthetic Inhibitors ............................... 56 a. Compounds Restricted to the Specificity Pocket of the Catalytic Domain of Thrombin . 57 b. Compounds Extending Unidirectionally Beyond the Specificity Pocket of the Catalytic Site of Thrombin .................... 58 Vl c. Compounds Extending Bidirectionally Beyond the Specificity Pocket of the Catalytic Domain of Thrombin ................... 59 3. Peptide Inhibitors . 61 a. Aldehyde Derivatives . 62 b. Chloromethyl Ketone Derivatives .................... 64 c. Nitrile Derivatives ............................. 65 d. Boronic acid Derivatives . 66 e. Other Peptide Analogues ......................... 68 4. Recombinant Thrombin Inhibitors ....................... 69 a. Aptamers . 69 b. Hirudin and its Variants .......................... 69 5. Thrombin Inhibitors not Directed Against its Catalytic Site ....... 72 6. Chimeric Thrombin Inhibitors ......................... 73 7. Thrombin Directed Antibodies . 75 F. Clinical Applications of Thrombin Inhibitors ................... 76 1. Antithrombin . 77 2. Argatroban .................................... 77 3. Efegatran . 79 4. Inogatran ...................................... 80 5. Napsagatran .................................... 80 6. Hirudin ....................................... 80 7. Hirulog . 85 G. Drug Interactions . 86 H. Synopsis ......................................... 92 III. MATERIALS AND METHODS ............................ 93 A. Synthesis and Characterization of Serine Protease Inhibitors .......... 93 Vll B. Analytical Profile of Peptide Thrombin Inhibitors ................ 96 1. D-Phe-Pro-Arg-H, D-MePhe-Pro-Arg-H and Boc-D-Phe-Pro-Arg-H Purity Determination . 97 2. Ac-(D)Phe-Pro-boroArg-OH Purity Determination ............ 98 C. Analytical Profile of the Unfractionated Heparin ................. 98 D. Biochemical Assays . 99 1. Thrombin Titration Assay ........................... 99 2. Thrombin Time (TT) in a Fibrinogen Based System .......... 101 3. Antithrombin Assays ............................. 102 a. Preparation of Normal Human Plasma (NHP) ............ 103 b. Antithrombin Amidolytic Assay with NHP .............. 103 c. Preparation of Normal Rabbit Plasma (NRP) . 104 d. Antithrombin Amidolytic Assay with NRP . 105 4. Anti-Xa Assays ................................. 105 a. Anti-Xa Amidolytic Assay with NHP ................. 106 b. Anti-Xa Amidolytic Assay with NRP ................. 106 5. Inhibitory Actions of Various Thrombin Inhibitors on Specific Serine Proteases . 107 a. Inhibition of Chymotrypsin . 108 b. Inhibition of Trypsin ........................... 108 c. Inhibition of Activated Protein C (APC) . 109 d. Inhibition of Glandular Kallikrein . 109 e. Inhibition of Tissue Plasminogen Activator (tPA) ......... 109 f. Inhibition of Urokinase . 110 g. Inhibition of Plasmin ........................... 110 6. Inhibition of Plasminogenolysis . 110 a. Pure Plasminogen System . 111 b. NHP System ................................ 112 7. Inhibition of Extrinsic Activation Systems in Fibrinogen Deficient Human Plasma by Thrombin Inhibitors . 113 a. Inhibition of Extrinsic Thrombin