THE EFFECTS OF ASPIRIN, CARPROFEN, DERACOXIB, AND MELOXICAM ON HEMOSTASIS AND SYSTEMIC PROSTAGLANDINS IN DOGS A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by SHAUNA LEANNE BLOIS In partial fufilment of requirements for the degree of Doctor of Veterinary Science August 2008 © Shauna Leanne Blois, 2008 Library and Bibliotheque et 1*1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-42554-1 Our file Notre reference ISBN: 978-0-494-42554-1 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library permettant a la Bibliotheque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par Plntemet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans loan, distribute and sell theses le monde, a des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, electronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. 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Canada ABSTRACT THE EFFECTS OF ASPIRIN, CARPROFEN, DERACOXIB, AND MELOXICAM ON HEMOSTASIS AND SYSTEMIC PROSTAGLANDINS IN DOGS Shauna Leanne Blois Advisor: University of Guelph 2008 Doctor D.G. Allen Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used in veterinary medicine to provide analgesic and anti-inflammatory benefits to patients. The adverse effects associated with NSAID use are believed to be largely due to inhibition of the enzyme cyclooxygenase (COX)-l. As such, COX-2-selective NSAIDs were developed in attempt to limit the development of NSAID-associated adverse effects. Recent reports in the human medical literature have suggested an increased incidence of thromboembolic events associated with the use of COX-2 selective NSAIDs. There is speculation that COX-2 selective NSAIDs may lead to an imbalance in prostaglandin levels, with a relative increase in thromboxane versus prostacyclin. Thromboxane promotes platelet aggregation and vasoconstriction, while prostacyclin counteracts these effects. This study examined the effects of NSAIDs on hemostasis and cardiovascular prostaglandin levels in healthy dogs. Ten dogs were given four NSAIDs and one placebo in a cross-over design at dosages consistent with current therapeutic recommendations. The NSAIDs administered included aspirin, carprofen, deracoxib, and meloxicam. Parameters measured before and after 7 days of NSAID administration included platelet optical aggregometry, platelet function analysis (using the PFA-100), and plasma thromboxane and prostacyclin levels. Administration of NSAIDs did not cause a significant effect on platelet function measured by the PFA-100. Platelet aggregation induced by 50 ^im of adenosine diphosphate (ADP) mildly decreased after deracoxib administration. Deracoxib did not affect platelet function measured by other aggregation studies and the PFA-100. Aspirin, carprofen, and meloxicam did not affect platelet function. Plasma thromboxane levels decreased after aspirin administration compared to after deracoxib administration, while NSAID administration did not affect plasma prostacyclin levels. This study showed that treatment with COX-2 selective NSAIDs in healthy dogs did not result in platelet dysfunction or an imbalance in plasma thromboxane and prostacyclin levels. Administration of aspirin, carprofen, deracoxib, and meloxicam had minimal impact on platelet function in healthy dogs. Further evaluation of COX-2 selective inhibitors should be performed, especially in patients prone to thromboembolic events. ACKNOWLEDGEMENTS Many thanks to my DVSc. advisory committee, Drs. Dana Allen, Peter Conlon, and Darren Wood for their support throughout my research project. My committee helped provide the initial idea for the project and were always ready to offer valuable advice to make the project grow in the right direction. Special thanks to Dr. Dana Allen for being a fantastic advisor both in and outside of the clinic during my residency. Thank you to Drs. Marilyn Dunn (Universite de Montreal) and Maureen Barry for agreeing to be a part of my DVSc. examination committee. I would also like to thank the Ontario Veterinary Pet Trust Foundation for their generous financial support of this project. The technical expertise of Barb Jefferson was invaluable throughout the duration of the project. Many thanks to Michelle Ross for taking the time to teach us the necessary techniques to perform platelet aggregometry. Thank you to Gabrielle Monteith and Dr. William Sears for their assistance with the statistical analysis. A special thanks to the staff (and the dogs) at the Central Animal Facility, University of Guelph, for facilitating the sample collection and drug administration for this project. DECLARATION OF WORK PERFORMED I declare that with the exception of the items below, all work reported in this thesis was performed by me. Complete blood cell count, serum biochemical profile, urinalysis, one stage prothrombin time, activated partial thromboplastin time, and fibrinogen levels were analysed by the technicians in the Animal Health Laboratory, University of Guelph, Guelph, Ontario. The animal care was provided by the Central Animal Facility, University of Guelph, Guelph, Ontario. Technicians, and animal care attendants performed restraint and blood collection, and administered nonsteroidal anti-inflammatory drugs. The pharmacy at the Ontario Veterinary College, University of Guelph, Guelph, Ontario, provided the nonsteroidal anti-inflammatory drugs at the requested dosages. Statistical randomization of the project was performed with the assistance of William Sears, Population Medicine, Ontario Veterinary College, University of Guelph, Guelph, Ontario. Statistical analysis was performed by Gabrielle Monteith, Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario. 11 TABLE OF CONTENTS Page Acknowledgements i Declaration of Work Performed ii Table of Contents iii List of Figures vi Glossary viii CHAPTER 1: LITERATURE REVIEW 1.0 INTRODUCTION 1 1.1 PRIMARY HEMOSTASIS: FORMATION OF THE PLATELET PLUG 3 Megakaryocytes 3 Platelet Structure and Function 4 . Platelet Adhesion, Activation, and Aggregation 6 Disorders of Primary Hemostasis 8 Summary 10 1.2 SECONDARY HEMOSTASIS: STABILIZING THE PLATELET PLUG 10 The Coagulation Cascade 10 The Fibrinolytic System 13 Disorders of Secondary Hemostasis 14 Summary 15 1.3 MEASURES OF PRIMARY HEMOSTASIS 16 Sample Collection 16 Platelet Number and Morphology 17 Buccal Mucosal Bleeding Time 17 Von Willebrand Factor Analysis 18 Platelet Aggregometry 18 Platelet Function Analyzer 20 Other Measurements of Primary Hemostasis 21 Summary 21 1.4 MEASURES OF SECONDARY HEMOSTASIS & FIBRINOLYSIS 22 Partial Thromboplastin and Prothrombin Times 22 Other Measurements of Secondary Hemostasis 23 Fibrin Degradation Products and D-dimers 24 Global Tests of Hemostasis 25 Measurements of Endogenous Anticoagulants 25 Summary 26 1.5 THE ARACHIDONIC ACID PATHWAY 26 Summary 27 m 1.6 THE PHARMACOLOGY OF NSAIDS 29 COX-2 Selective Inhibitors 30 Adverse effects of NSAIDs 31 Summary 32 1.7 EFFECTS OF NSAIDS ON THE CARDIOVASCULAR SYSTEM 33 COX-2 Selective Inhibitors and Cardiovascular Outcomes in Humans 33 Effects of NSAIDs on the Canine Cardiovascular System 35 Summary 36 References 38 CHAPTER 2: RESARCH PROJECT 51 2.0 PRIMARY AND SECONDARY HEMOSTASIS 51 Introduction 51 Materials and Methods 52 Animals 52 Study Design 53 Blood Collection 54 Platelet Count, Hematocrit, and Leukocyte count 55 Analysis of Hemostasis 55 Platelet Function Analysis as Measured by the PFA-100™ 55 Platelet Aggregation 56 Plasma Thromboxane Levels 59 Plasma Prostacyclin Levels 61 One-stage PT, Activated PTT, and Fibrinogen Concentration . 61 Statistical Analysis 61 Results 63 Platelet Count, Hematocrit, and Leukocyte Count 63 Platelet Function Analysis as Measured by the PFA-100™ and Platelet Aggregometry 63 Plasma PGI2 and TBX2 Levels 69 Measures of Secondary Hemostasis 71 Discussion 74 Limitations and Future Areas of Study 82 Conclusions 83 References 85 CHAPTER 3: CONCLUSIONS 91 Summary 91 4.0 APPENDICES 93 Appendix la: Circulating platelet count 93 Appendix lb: Circulating hematocrit 93 lv Appendix 1c: Circulating white blood cell count 93 Appendix Id: Closure time as measured by the PFA-100™