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

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While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. 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™ 94

Appendix le: Maximal platelet aggregation with 1 urn of PAF 94

Appendix If: Maximal platelet aggregation with 0.5 urn of PAF 94

Appendix lg: Maximal platelet aggregation with 100 urn of ADP 95

Appendix lh: Maximal platelet aggregation with 50 urn of ADP 95

Appendix li: Rate of platelet aggregation with 1 urn of PAF 96

Appendix lj: Rate of platelet aggregation with 0.5 urn of PAF 96

Appendix Ik: Rate of platelet aggregation with 100 urn of ADP 96

Appendix 11: Rate of platelet aggregation with 50 urn of ADP 97

Appendix lm: Prostacyclin (6-keto prostaglandin Fla) levels 97

Appendix In: Thromboxane B2 levels 97

Appendix lo: Ratio of thromboxane B2 to prostacyclin 98

Appendix lp: Prothrombin time 98

Appendix lq: Partial, thromboplastin time 99

Appendix lr: Fibrinogen levels 99 LIST OF FIGURES

Page

CHAPTER 1: LITERATURE REVIEW

Figure 1.1: Diagram of a normal platelet. 5

Figure 1.2: Scanning electron micrograph of a platelet thrombus. 8

Figure 1.3: The coagulation cascade. 12

Figure 1.4: Production and actions of prostaglandins and thromboxane. 28

CHAPTER 2: RESEARCH PROJECT

Figure 2.1: Example of a platelet aggregometry curve. 59

Figure 2.2: Platelet count (mean ± SD) before and after 7 days of NSAID

administration in 10 healthy dogs. 63

Figure 2.3: Closure time (mean ± SD) as measured by the PFA-100™ before

and after 7 days of NSAID administration in 10 healthy dogs. 64

Figure 2.4: Percent aggregation (mean ± SD) induced by 1 [im PAF before

and after 7 days of NSAID administration in 10 healthy dogs. 65

Figure 2.5: Percent aggregation (mean ± SD) induced by 0.5 ^im PAF before

and after 7 days of NSAID administration in 10 healthy dogs. 65

Figure 2.6: Percent aggregation (mean ± SD) induced by 100 pirn ADP before

and after 7 days of NSAID administration in 10 healthy dogs. 66

Figure 2.7: Percent aggregation (mean ± SD) induced by 50 [i.m ADP before

and after 7 days of NSAID administration in 10 healthy dogs. 66

Figure 2.8: Rate of aggregation (mean ± SD) induced by 1 [im PAF before

and after 7 days of NSAID administration in 10 healthy dogs. 67

VI Figure 2.9: Rate of aggregation (mean ± SD) induced by 0.5 \xm PAF before

and after 7 days of NSAID administration in 10 healthy dogs. 67

Figure 2.10: Rate of aggregation (mean ± SD) induced by 100 urn ADP before

and after 7 days of NSAID administration in 10 healthy dogs. 68

Figure 2.11: Rate of aggregation (mean ± SD) induced by 50 \im ADP before

and after 7 days of NSAID administration in 10 healthy dogs. 68

Figure 2.12: Prostacyclin (6-keto prostaglandin FXa) levels (mean ± SD) before

and after 7 days of NSAID administration in 10 healthy dogs. 69

Figure 2.13: Thromboxane B2 levels (mean ± SD) before and after 7 days

of NSAID administration in 10 healthy dogs. 70

Figure 2.14: Ratio of thromboxane to prostacyclin (mean ± SD) before and

after 7 days of NSAID administration in 10 healthy dogs. 70

Figure 2.15: Prothrombin time (mean ± SD) before and after 7 days of NSAID

administration in 10 healthy dogs. 72

Figure 2.16: Partial thromboplastin time (mean ± SD) before and after 7 days

of NSAID administration in 10 healthy dogs. 72

Figure 2.17: Fibrinogen level (mean ± SD) before and after 7 days of NSAID

administration in 10 healthy dogs. 73

vii GLOSSARY

6-keto PG Fla 6-keto prostaglandin Fla (prostacyclin metabolite)

AA Arachidonic acid

AchE Acetylcholinesterase

ACT Activated clotting time

ADP Adenosine diphosphate

ADP-CTs Closure times measured by the Platelet Function Analyzer-100 with an adenosine diphosphate cartridge

ANOVA Analysis of variance

ASA Acetylsalicylic acid

AT Antithrombin

BMBT Buccal mucosal bleeding time

CaCl2 Calcium chloride cAMP Cyclic adenosine monophosphate

CBC Complete blood count cGMP Cyclic guano sine monophosphate

Col-ADP Collagen-adenosine diphosphate cartridges used in the Platelet Function Analy2er-100

Col-Epi Collagen-epinephrine cartridges used in the Platelet Function Analyzer-100

COX Cyclooxygenase

DAG Diacylglycerol

DIC Disseminated intravascular coagulation

EDTA Ethylene diamine tetraacetic acid

EIA Enzyme immunoassay kit

vin EPI-CTs Closure times measured by the Platelet Function Analyzer-100 with an epinephrine cartridge

FDP Fibrin/fibrinogen degradation products

HMWK High molecular weight kininogen

IMHA Immune mediated hemolytic anemia

IMT Immune mediated thrombocytopenia

IP3 Inositol triphosphate

LOX Lipoxygenase

PGI2 Prostacyclin

PIVKA Proteins induced by vitamin K antagonism/absence

MPV Mean platelet volume

NO Nitric oxide

NOS Nitric oxide synthase

NSAID Nonsteroidal anti-inflammatory drug

OCS Open canilicular system

PAF Platelet actiating factor

PAI Plasminogen activator inhibitor

PC Protein C

PE Phosphatidyl enthanolaminc

PG Prostaglandin

PI Phosphoinositol

PK Prekallekrein

PPP Platelet-poor plasma

PRP Platelet-rich plasma

PS Phosphatidylserine

ix PSAIg Platelet surface-associated immunoglobulin

PT One-stage prothrombin time

PTC Phosphatidylcholine

PTT Activated partial thromboplastin time

SP Sphingosine

TBS Tris-buffered saline

TEG Thromboelastography

TF Tissue factor

TFPI Tissue factor pathway inhibitor t-PA Tissue plasminogen activator

TT Thrombin time

TXA, Thromboxane A2

TXB2 Thromboxane B2

\iL Microliter (10"6 liter) u-PA Uroplasminogen activator vWd von Willebrand disease vWf von Willebrand factor vWf:Ag von Willebrand factor antigen CHAPTER 1; LITERATURE REVIEW

1.0 INTRODUCTION

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been widely used to provide short-term and long-term analgesia, and anti-inflammatory benefits to human and veterinary patients. By inhibiting the key enzyme cyclooxygenase (COX) in the arachidonic acid (AA) pathway, NSAIDs produce both analgesic and toxic effects.1"4 The toxic effects of NSAIDs are thought to result from blocking the COX-1 enzyme in this pathway.3"5 As such, COX-2 selective NSAIDs have been developed in an attempt to minimize the adverse effects associated with these drugs.3'4'"'7

Recently, there have been reports of an increased incidence of myocardial infarction and other adverse cardiovascular events associated with the use of COX-2 selective NSAIDs in humans.8'11 No such reports have been made in veterinary species. The increased incidence of adverse cardiovascular events associated with COX-2 inhibitors may be attributed to the induction of an imbalance in two important prostaglandins, thromboxane

(TXAj) and prostacyclin (PGI2). Thromboxane induces vascular constriction and platelet aggregation in circulation, while PGL inhibits platelet aggregation and induces vasodilation.12,

13 COX-2 inhibitors may lead to decreased levels of PGI2, leaving the actions of TXA2 unopposed, and potentially contributing to a prothrombotic environment in circulation.14'16

Many conditions that affect dogs can alter the natural hemostatic balance and lead to a prothrombotic state. Dogs with sepsis, malignancy, protein-losing nephropathies, heartworm infestation, parvoviral infection, thrombocytosis, hyperadrenocorticism, and immune-mediated hemolytic anemia have been reported to be at increased risk for developing thromboembolic complications.17"28 There is currently little known about the use

1 of NSAIDs in these patients and whether the use of COX-2 selective inhibitors increases the risk of vascular obstruction in dogs.

One previous study examining the effects of NSAIDs on canine coagulation used subjects with osteoarthritis, and relied on owner compliance for medication administration.

Deracoxib therapy resulted in thromboelastograms suggestive of hypercoagulability in some patients, but did not alter platelet aggregometry signifkandy.29 The effect of inflammation due to osteoarthritis on the hemostatic system is unknown, and may have altered results of this study. Also, the study used client-owned dogs and as such, owner compliance with

NSAID administration may not have been complete, leading to possible alteration of the results.

The objective of the current study was to examine the effects of enteral administration of aspirin, carprofen, deracoxib, and meloxicam at therapeutic dosages on primary and secondary hemostasis, as well as on thromboxane and prostacyclin levels, of healthy dogs housed in a research facility, These NSAIDs were chosen for the study based on their widespread use in canine medicine for analgesic benefits. The null hypothesis was that NSAID use would not alter parameters of primary and secondary hemostasis, or diromboxane and prostacyclin levels, in healthy dogs.

The first part of the literature review serves as a summary of the primary and secondary hemostatic systems, and the fibrinolytic system. Defects of the coagulation system leading to hypo- and hypercoagulation are discussed, as are common methods for evaluating hemostasis. The literature review continues with an overview of the pharmacology of NSAIDs. The use of NSAIDs in human and veterinary medicine is discussed, with particular emphasis placed on the effects of NSAIDs on the cardiovascular system.

2 1.1 PRIMARY HEMOSTASIS: FORMATION OF THE PLATELET PLUG

Platelets are the body's first line of defense when injury to the vasculature occurs.

They function to minimize blood loss by adhering to die subendothelium of the vessel and aggregating. After an insult to the vasculature, the blood vessel constricts immediately.

Platelet adhesion occurs within seconds, while platelet aggregation may take up to several minutes. The interaction between endothelium and platelets to achieve this first response to . a vessel defect is known as primary hemostasis. Although often described in a stepwise manner, the events of primary and secondary hemostasis normally occur simultaneously.

Platelets also have important roles in inflammation and wound healing, as well as releasing substances that modulate the activity of other blood cells and blood vessels.

Megakaryocytes

Platelets in circulation originate from megakaryocytes in the bone marrow and, to a lesser extent, from the liver and spleen.30 Megakaryocytes arise from multipotential progenitor cells, colony-forming units generating granulocytes, erythroblasts, macrophages and megakaryocyte (CFU-GEMM) cells, which also serve as the progenitors for die other hematopoietic cells in the bone marrow.31, "2 Megakaryocytopoiesis is stimulated by interleukin (IL)-3, IL-6, IL-11, stem cell factor, thrombopoietin (TPO), and other growth factors.30'32"35 Thrombopoietin stimulates the burst-forming unit megakaryocyte (BFU-MK) progenitor cells to proliferate and differentiate.30,31

Megakaryocyte progenitors express CD34 antigen throughout their maturation in the bone marrow, but become CD34 negative as they acquire megakaryocyte-specific receptors such as glycoprotein (GP) Ilb-IIIa."' ' At the final stages of megakaryocyte maturation within the bone marrow, the megakaryocyte nucleus becomes increasingly lobulated and the

3 cytoplasm contains many azurophilic granules. Mature megakaryocytes have an extensive microtubule and microfilament network, which reorganize to form pseudopodia.37,38 The megakaryocyte pseudopodia then extend into the bone marrow sinuses, fragmenting to produce individual platelets that are released into systemic circulation. Megakaryocytes present in the lung, spleen, and liver may also contribute to platelet production. The exact mechanism controlling final platelet production is unknown, but it is thought that TPO is the primary growth factor involved.30'32

Platelet Structure & Function

Circulating canine platelets are small (< 3x7 [Am), anucleate, cytoplasmic fragments with a normal circulating life span of 5-7 days.26,30'39 The platelet membrane consists of an outer glycocalyx, a plasma membrane, and an invaginated open canalicular system (OCS).

Many extracellular domains are contained within the glycocalyx, including the integrins and the leucine-rich glycoproteins used in cell signaling.40

Platelet integrins are involved in platelet adhesion and aggregation during primary hemostasis, and consist of an a and a |3 subunit. The combination of a and (3 subunit types determines the type of receptor formed on the platelet surface, The GPIIb-IHa integrin is the most common surface receptor found on platelets and megakaryocytes, and is also found within the OCS. It has a high affinity for von Willebrand factor (vWf), fibronectin and vitronectin. Glycoprotein Ilb-IIIa is involved in platelet shape change, granule secretion, and reorganization of membrane phospholipids associated with platelet aggregation and clot retraction.40,41 Another common platelet surface glycoprotein is the GPIb-IX-V complex, part of the leucine-rich glycoprotein family. The GPIb-IX-V complex is the site of vWf and

4 thrombin binding, and appears to play a role in platelet cytoskeleton rearrangement.40' 42

Other important platelet receptors include the thrombin receptor and P-selectin.40

The platelet cytoskeleton is comprised of contractile actin filaments, which along with platelet myosin and intracellular calcium concentration changes, allow for platelet shape change in response to agonist binding.40 The platelet microtubule system and intermediate filaments comprise the remainder of the platelet cytoskeleton.40

Figure 1.1 — Diagram of a normal platelet, showing receptors linked to G-proteins, phospholipid

metabolism, and platelet granules. ADP - adenosine diphosphate; ATP - adenosine triphosphate;

PAF - platelet activating factor; TXA2 - thromboxane A2; IP3 - inositol triphosphate; vWF - von

Willebrand factor. Used with written permission from Brooks M. and Catalfamo J.L., Platelet

Dysfunction. In Bonagura J.D. (ed): Kirk's Current Veterinary Therapy XIII. Philadelphia, W.B.

Saunders. © Elsevier (2000).

The platelet cytoplasm contains three types of intracellular granules: alpha granules, dense granules, and glycogen granules. Alpha granules comprise the largest population within the platelet, and contain coagulation factors, platelet-specific proteins, growth factors,

5 and GPs. Dense granules store adenosine triphosphate (ATP), guanosine diphosphate

(GDP), serotonin, magnesium, and calcium. Membranes of the alpha and dense granules each contain glycoproteins important in platelet activation, such as GPIIb-IIIa and GPIb.40

Glycogen granules, free in platelet cytoplasm, are the primary energy source for cell metabolism.40

Platelet Adhesion, Activation, and Aggregation

In normal circulation, platelets do not interact with vascular endothelium or other circulating blood cells.43 However, platelets can become activated by vascular endothelium defects or by platelet agonists, such as platelet-activating factor (PAF), ADP, or collagen, present in the circulation.44,45

Endothelial defects created by vascular injury expose circulating platelets to subendothelial collagen fibrils, which act as potent platelet agonists under high shear flow stress.43 Platelet adhesion to endothelium is mediated primarily by the interactions between the GPIb/IX/V complex and vWf. Platelet adhesion continues as platelet receptors adhere to proteins in die connective tissue of collagen. Platelets spread along the vascular defect, covering the surface with a single layer. Platelet adhesion can occur in a similar fashion after exposure to platelet agonists.45

After contact with the endothelium, or stimulation by platelet agonists, platelets become activated. Activated platelets undergo a series of morphological and functional changes. As platelet agonists bind to their specific receptors on the platelet surface, intracellular calcium levels increase via extracellular calcium influx and release from dense granules. Calcium mediates phospholipid metabolism, and also initiates protein kinase and other downstream protein phosphorylation reactions.46"48 Intracellular cyclic adenosine

6 monophosphate (cAMP) levels decline in response to the elevated intracellular calcium levels, indirectly enhancing platelet aggregation. Platelet GPIIb-IIIa becomes activated, acting as a binding site for adhesive proteins.47

Platelet membrane phospholipids are involved in platelet activation. At rest, negatively charged phospholipids (phosphatidylserine — PS; phosphatidyl enthanolamine —

PE) are found primarily on the inner leaflet of the phospholipid bilayer while neutral phospholipids (phosphatidylcholine - PC; sphingosine - SP) are found on the outer leaflet,411'

4S Once platelets are activated, several changes within the phospholipid arrangement occur: phosphoinositol (PFj turnover proceeds more rapidly to produce vast quantities of inositol triphosphate (IP3) and 1,2-diacylglycerol (DAG); arachidonic acid (AA) is liberated; and negatively charged PS translocates to the outer leaflet of the phospholipid bilayer. Release of

AA from the platelet phospholipid membrane allows for metabolism of prostaglandin intermediates, and production of thromboxane (TX) A2 through the activity of cyclooxygenase (COX)-l.47

Throughout platelet activation, platelets develop long, cytoplasm-filled pseudopodia.

The platelets spread along the defect while intracellular granules fuse with the OCS and

secrete their contents. Secretion of granule contents further enhances the hemostatic response. Adenosine diphosphate, serotonin, and epinephrine secreted from dense granules of activated platelets serve as agonists to recruit more platelets. Additionally, activated

platelets and endothelial cells synthesize new agonists, such as PAF and TXA,, leading to

further platelet recruitment and aggregation.30'40,45

There is wide variation among species in regards to platelet structure and function.45

While canine platelets are often used as models for human medicine, there are many differences between the platelet morphology and function of the two species and caution

7 must be used when interpreting platelet studies across species. Canine platelets have fewer glycoproteins in the plasma membrane, and have a circulating lifespan that is several days

(approximately 3 days) shorter than human platelets.49 Clotting times in dogs and other domestic animals are shorter than in humans. Additionally, aggregation studies have shown canine and human platelets respond differently to platelet agonists.45' M''[

Figure 1.2 - Scanning electron micrograph of a platelet thrombus. Activated platelets have

undergone a shape change, are adherent to the underlying matrix, and have formed

interplatelet bonds. Used with written permission from Brooks M.B., Catalfamo J.L.,

Platelets Disorders and von Willebrand Disease. In Ettinger S.J., Feldman E.C. (eds):

Textbook of Veterinary Internal Medicine, 6th edition. Philadelphia, WB Saunders. © Elsevier

(2005).

Disorders of Primary Hemostasis

Disorders of primary hemostasis can be caused by decreased platelet production, platelet sequestration, increased platelet consumption, excessive platelet loss, platelet

dysfunction, and platelet destruction. Vascular or platelet abnormalities can result in

petechiation, bleeding at multiple sites, hemorrhage of the mucous membranes, and prolonged bleeding from vascular injury.

8 Thrombocytopenia in veterinary patients can result from primary or secondary immune-mediated mechanisms, or can be drug-related. Primary immune-mediated thrombocytopenia (IMT) results from accelerated platelet destruction by the mononuclear- phagocyte system due to the presence of antiplatelet antibodies. Thrombocytopenia can occur secondary to other immune system disorders, such as immune-mediated hemolytic anemia (IMHA), neoplasia, infection, or drug therapy.52"54 Greyhounds and Cavalier King

Charles Spaniels (CKCS) often have incidental mild thrombocytopenia, and an increased mean platelet volume is seen in CKCS.55"5'

Platelet dysfunction can occur secondary to hepatic disease, uremia, infection, or neoplasia and may contribute to bleeding tendencies in IMT dogs.53, 58"61 Drug-related platelet dysfunction has been reported in dogs in association with COX-1 inhibitors, certain antibiotics, and synthetic colloids.12, 62, a Von Willebrand disease, due to a quantitative or qualitative lack of vWf, is the most common canine heritable disease leading to impairment of platelet function.64'65 Platelet dysfunction has been associated in dogs with mitral valve disease and subaortic stenosis.66'67 Thrombocytopenia and platelet dysfunction can each lead to clinical bleeding, depending on the severity of the defect. Thrombocytopenia is unlikely to induce hemorrhage unless the platelet count is <20,000-50,000/uL.68

Hyperaggregable platelets have been observed in association with malignancy, nephrotic syndrome, and heartworm infection in dogs.18'20 Erythropoietin therapy results in increased platelet production and reactivity in dogs.65 Enhanced platelet activation occurs in humans with diabetes mellitus and asthma, but this has not been reported in veterinary medicine.70"72 Platelet hyperfunction may contribute to inappropriate thrombosis.

Thrombocytosis may result from a primary bone disorder or occur secondary to a systemic disease or physiological cause. Essential thrombocythemia is a rare

9 myeloproliferative disorder characterized by megakaryocyte hyperplasia with morphological abnormalities, and circulating thrombocytosis.22"24 Dogs with essential thrombocytosis can have platelet hyperaggregability.24' 25 Reactive thrombocytosis can occur secondary to immune-mediated disease, infection, neoplasia, trauma, iron deficiency, or stress.

Thrombocytosis can increase the risk for thromboembolic disease.25

Summary

Exposure to subendothelial collagen and the release of a platelet agonist in the circulation allow for platelet changes that facilitate initial adhesion and subsequent platelet aggregation. Many simultaneous reactions involving functional and morphological changes of the platelet are involved in initiating primary hemostasis. The platelet plug that is formed provides the scaffolding on which the secondary hemostatic system can act. Quantitative or qualitative defects in platelet function can lead to hemorrhage, while platelet hyperactivity and dirombocytosis place a patient at risk of developing thromboembolic disease.

1.2 SECONDARY HEMOSTASIS: STABILIZING THE PLATELET PLUG

As platelets become activated and aggregate, a platelet clot is formed.

Phosphatidylserine exposure on the outer leaflet of the activated platelet is important in inducing secondary hemostasis.46 Tissue factor, released during vascular endothelial damage, concurrendy stimulates secondary hemostasis.

The Coagulation Cascade

The secondary hemostatic pathway results in formation of a cross-linked fibrin plug.

Intrinsic, extrinsic, and common pathways of this coagulation cascade have been identified in

10 vitro (Figure 1.3). However, the extrinsic pathway appears most important for coagulation in vivo. Additionally, there is interaction of coagulation factors between the intrinsic and extrinsic branches of the cascade in vivo. Coagulation factors circulate as inactive zymogens until activation.30

The extrinsic pathway of the coagulation cascade begins with release of tissue factor

(TF) from damaged tissue or activated endothelium, monocytes, or macrophages. Tissue factor forms a complex with factor VII, which is activated upon exposure to calcium. The

TF-VIIa complex ('a' denotes activated coagulation factor) then activates Factor X (common pathway) and Factor IX (intrinsic pathway) after exposure to calcium and phospholipids.

The activity of the extrinsic branch of the cascade is considered the major stimulus for secondary hemostasis.30

Stimulation of the coagulation cascade can also be induced by activation of factor

XII by contact with a negatively charged surface in the intrinsic pathway. Factor Xlla, prekallekrein (PK), high molecular weight kininogen (HMWK), and Factor XI become closely associated, resulting in the production of kallekrein and more Factor Xlla. Factor

Xlla activates Factor XI, which leads to activation of Factor Xa in the presence of calcium, phospholipid, and Factor Villa.30

The extrinsic and intrinsic pathways lead to activation of Factor Xa, the beginning of the common pathway. Factors Xa and Va combine with calcium to form a prothrombinase complex. This complex cleaves prothrombin to thrombin (Factor Ha), which subsequently cleaves fibrinogen to form fibrin monomers. Factor XHIa forms an insoluble, cross-linked fibrin clot in the presence of calcium. Thrombin also acts on other parts of the coagulation cascade, including the activation of Factors XIII, V, and VIII and protein C.30

11 While the traditional "cascade" model of coagulation correlates with laboratory, or in vitro, evaluation of coagulation, the cell-based model of coagulation correlates more closely with in vivo hemostasis. It is now known that coagulation is initiated on tissue factor-bearing cell surfaces. Coagulation factors and vWf act on the surface of activated platelets, propagating hemostasis in vivo. 73 This model of hemostasis better reflects the cellular control of hemostasis and allows a more thorough understanding of in vivo hemostasis.

Figure 1.3 — The coagulation cascade. The intrinsic and extrinsic pathways begin with the

interaction of Factor XII with a negatively charged surface and by the release of tissue factor,

respectively, and lead to the. start of the common pathway. The eventual result is formation

of a cross-linked fibrin clot. Used with written permission and modified from Carr A.P.,

Inherited Coagulopathies, in Ettinger S.J., Feldman E.G. (eds): Textbook of Veterinary Internal

Medicine, 6th edition. Philadelphia, WB Saunders. © Elsevier (2005).

12 The fibrinolytic system

As the primary and secondary hemostatic systems are activated in the body, inhibitors of hemostasis are also activated to limit excessive clotting. Normal vascular endothelium has antithrombotic properties, but endothelial cells undergo changes upon activation by injury, platelet agonists, inflammatory mediators, and substances released from platelets such as AA and ADP.4' Two major endothelial mediators, prostacyclin (PGL) and nitric oxide (NO), have antithrombotic properties. Endothelial cells synthesize prostacyclin, which induces vasodilation and inhibits platelet adhesion and aggregation. Prostacyclin synthesis is upregulated upon activation of endothelial cells, increasing the activation of platelet membrane-bound adenylate cyclase and cAMP production to attenuate platelet response. Nitric oxide, synthesized by endothelial cell nitric oxide synthase (NOS), has similar effects to PGI2 since it mediates endothelial relaxation and inhibits platelet aggregation by increasing platelet cyclic guanosine monophosphate (cGMP) levels.43, u

Bradykinin appears to be important in the induction of PG12 and NO from endothelial cells.74 Endothelial antithrombotic properties also include heparin-like substances such as vascular wall heparan sulfate proteoglycans. Vascular wall enzymes, such as ADPase, degrade platelet agonists.43 Endothelial cells express thrombomodulin, which activates the

anticoagulant protein C when bound to thrombin.30

Antimrombin (AT; formerly AT 111) is the primary anticoagulant in the body, and

acts in synergy with endogenous heparan .sulfate or exogenous heparin to inactivate

thrombin. Circulating thrombin binds with the AT-heparin complex, and the resulting

complex (thrombin-AT; TAT) and is cleared from circulation by the liver. Tissue factor pathway inhibitor (TFPI) binds to the TF-VIIa complex and inhibits further activation of

13 Factor X. Other inhibitors of secondary hemostasis include proteins C and S, which inactivate Factors V and VIII.30'75

After formation of a fibrin clot, the fibrinolytic system helps dissolve the clot to

maintain a patent vascular system. The actions of this system are normally confined to the

site of the fibrin clot. Tissue plasminogen activator (t-PA) is released from activated

endothelial cells to cleave plasminogen, forming plasmin. Uroplasminogen activator (u-PA) activates plasmin outside of the blood vessels, Plasmin degrades fibrin, fibrinogen, vWf, and

Factors Va and Villa. Protein C promotes the activity of plasmin, while plasminogen

activator inhibitor (PAI) inhibits it. Circulating a2-antiplasmin binds to and inactivates

plasmin to regulate its actions.30,76

Disorders of Secondary Hemostasis

Disorders of secondary hemostasis can be related to a lack of one or more

coagulation factors, due to inhibition of coagulation factors, or resulting from excessive

fibrinolysis. Coagulation factor dysfunction can be associated with hematomas, localized

bleeding, bleeding into muscles and joints, and delayed onset of bleeding (after the initial

platelet plug fails to become stabilized). Some inherited factor deficiencies are not clinically

detectable. Increased activity of coagulation factors, low levels of circulating anticoagulants,

or impairment of the fibrinolytic system can result in thromboembolic complications.30

Inherited coagulopathies include Hemophilia A, B, and C due to lack of Factors

VIII, IX, and XI, respectively. The severity of bleeding resulting from Hemophilia A or B is

variable. Hemophilia A and B have been described in dogs and cats, and are heritable

conditions.64 Hemophilia C has been described in Kerr)' Blue Terriers, with a tendency for

mild post-traumatic or post-operative bleeding in affected dogs.77 Factor XII deficiency

14 (Hageman trait) can cause a prolongation of partial thromboplastin time, but is generally not associated with clinical bleeding. Hageman trait is an inherited disorder in cats and dogs.78

Vitamin K deficiency results in inactivation of the vitamin-K dependent coagulation proteins, Factors II, VII, IX, X, and Proteins G and S.30 Anticoagulant rodenticides antagonize vitamin K, resulting in alteration of the coagulation cascade and bleeding.

Vitamin K deficiency can also result from malnutrition, decreased synthesis in the intestinal tract, and decreased absorption by the biliary system. Severe liver disease (i.e., reduction of liver mass by at least 75%) can result in abnormalities, of coagulation tests but rarely results in clinical bleeding alone. Acquired inhibitors of coagulation are uncommon in veterinary medicine but can result from immunoglobulin production secondary to immune-mediated diseases, liver disease, drug therapy, blood product transfusion reactions, or disseminated intravascular coagulation (DIC). Acquired inhibitors of coagulation can inhibit activity of one or more coagulation factors and can result in alteration of hemostatic laboratory tests, thrombosis, or hemorrhage.'8"80

Dogs with sepsis have been found to have abnormally low levels of circulating anticoagulants, possibly increasing their risk of hypercoagulability and thrombosis.17

Hypercoagulability has also been identified in dogs with nephrotic syndrome and parvoviral enteritis.19, 21 Dogs with hyperadrenocorticism have an increased incidence of thromboembolic events, possibly due to increased levels of circulating coagulation factors or decreased circulating antithrombin.26

Summary

The hemostatic system has evolved to minimize blood loss after a vascular insult.

Through the coordinated actions of platelets, coagulation factors, and other blood

15 components, fibrin clots are formed to seal defects in the vasculature. The extrinsic pathway of the coagulation cascade is likely the most important stimulant of secondary hemostasis.

The coagulation cascade culminates with formation of an insoluble fibrin plug. The fibrinolytic system provides balance to the hemostatic system by ensuring that the fibrin clot is formed at the appropriate site and does not become too extensive. Alterations of the coagulation cascade, fibrinolytic system, or endogenous anticoagulant mechanisms can lead to complications ranging from clinical bleeding to thromboembolic disease.

1.3 MEASURES OF PRIMARY HEMOSTASIS

Sample Collection

Proper venipuncture technique to collect blood for hemostatic testing is essential for obtaining accurate results. Venous occlusion prior to venipuncture should be brief, as prolonged occlusion can lead to activation of the hemostatic system. A sharp needle, with a wide lumen, attached to a syringe is used for blood collection. The vacutainer system is not recommended, as it may induce contact activation or lead to imprecise filling or mixing of

blood in collection tubes. The first few drops of the blood sample should not be used for hemostatic testing, as the blood may be contaminated with tissue thromboplastin and thus

may lead to premature activation of the coagulation cascade in vitro.

Depending on the test being performed, the blood can then be placed in a citrate or

EDTA collection tube. Blood should be placed into the tube carefully, flowing along the wall of the tube into die sample container. Citrated tubes contain 3,2 or 3.8% sodium

citrate, and a ratio of 9 parts blood to 1 part citrate is achieved when tubes are filled

properly. '' " Inversion of the tube is performed several times to ensure that the blood

mixes evenly with the citrate. Centrifugation to obtain citrated plasma must be performed

16 within 2 hours of collection, and it is recommended that centrifugation be performed immediately after collection.81 It is important to use species-specific reagents for coagulation testing, where applicable.83 Citrated plasma is used for many of the tests evaluating the coagulation cascade. Platelet count and morphology is usually performed using EDTA blood.81

Platelet Number and Morphology

Primary hemostasis can be assessed by evaluating platelet number and function.

Platelet number is routinely assessed on a complete blood count (CBC), with normal canine platelet levels ranging from 117-418 X 109/L.a Mean platelet volume (MPV) is often reported as part of a CBC and reflects the average platelet size of the sample. Canine MPV values can range from 7-14 fL.a Platelet number, morphology, and degree of clumping can be assessed by a direct blood smear evaluation.

Buccal Mucosal Bleeding Time

In vivo primary hemostasis can be assessed in dogs using the buccal mucosal bleeding time (BMBT). The BMBT is often used as a preliminary screening test to evaluate platelet function, and is performed by measuring the time to bleeding cessation after making a small, standardized incision in the buccal mucosa. Normal BMBT in dogs is 1-4 minutes.60, 84

Prolonged BMBTs have been observed with thrombocytopenia, uremia, and von Willebrand disease (vWd), and after aspirin therapy.6"'85 However, there can be high variability when measuring BMBT in dogs and its utility in predicting likelihood of hemorrhage in humans without a history of excessive bleeding is poor.86,87 a Animal Health Laboratory (AHL), University of Guelph, Guelph, Ontario. -

17 Von WiUebrand Factor Analysis

Von WiUebrand factor, essential for platelet adhesion, is deficient in vWd. Plasma levels of von WiUebrand factor antigen (vWf:Ag) can be measured using an electroimmunoassay. Von WiUebrand factor multimer analysis can * be performed by electrophoresis, to determine quaUtative deficiencies in vWf. Functional vWf assays are also available, and include an assay that assesses the ability of vWf to bind to coUagen in vitro.65'm

Platelet Aggregometry

Platelet function is commonly measured in vitro using aggregometry, which is considered to be the gold standard for platelet function analysis.89 Optical platelet aggregation studies are performed using platelet-rich plasma (PRP) and the change in optical density of die platelet suspension is measured. Platelets in the sample change from their discoid, resting state to a more rounded form with pseudopodia when a platelet agonist is added. Initially, this shape change results in decreased light transmission through the sample. However, as the platelets aggregate, a large increase in Ught transmission occurs.

Both the rate and the extent of change in Ught transmission are recorded. Light transmission through the sample is proportional to die amount of platelet aggregation.90,91

Whole blood aggregation is measured using impedance or electrical aggregometry.

Current is passed between two electrodes immersed in the sample. As platelets aggregate around the electrodes they increase the electrical impedance between the electrodes, The rate and extent of electrical impedance between the electrodes is recorded.91'93

Platelet aggregation is performed at a constant temperature (usually 37°C) and the sample is continuously agitated. Agonists commonly used for canine platelet aggregation studies in veterinary medicine include ADP, coUagen, arachidonic acid, serotonin, PAF, and

18 thrombin.51,00 Common agonists used in human in vitro platelet aggregation studies include

ADP, collagen, epinephrine, thrombin, and ristocetin.94

Platelet aggregation is variable among species. Canine and human platelets show similar aggregation responses to ADP, but aggregation in response to collagen produces a longer lag time in canine versus human platelets. Additionally, human platelets are more responsive to epinephrine, AA, and ristocetin agonists than are canine platelets.95 The variation in platelet response to agonists among species may be attributable to structural differences (e.g., presence of an open canalicular system), type and number of agonist receptors present on the platelet membrane, and the ability of the platelet to synthesize and

50 51 95 96 respond to TXA2. ' ' ' Platelet aggregometry is subject to variability among dogs, and this variability may be at least partially attributable to breed, since dogs of varying breeds may have differing platelet responses to arachidonic acid and variable TXA, sensitivity.90,97'98 In addition, canine platelets that are less responsive to AA stimulation also appear to be less sensitive to ADP and collagen than the platelets of other species.8

Use of PRP permits standardization of platelet concentration in the samples.90

Preparation of PRP leads to a more artificial environment as the other cellular components of blood are removed and the procedure may lead to activation of the platelets. Impedance aggregometry avoids the preparation of PRP, and unlike optical aggregometry, allows analysis of lipemic samples, A whole blood sample maintains the interaction of platelets with other components of blood.93 However, impedance aggregometry is less precise and has lower reproducibility than optical aggregometry.92 Neither optical nor impedance aggregometry is suitable for measurement of platelet function in thrombocytopenic patients.9' Another disadvantage of platelet aggregation studies is that they do not simulate

19 the hemodynamic environment of platelet-endothelial interaction at the site of vascular injury.

Platelet Function Analyzer

The Platelet Function Analyzer (PFA)-100 (Dade Behring, Newark, DE, USA) is a point-of care test that evaluates platelet adhesion and aggregation in an environment that simulates high shear blood flow at an injured blood vessel wall. The primary indication for use of the PFA-100 is the detection of intrinsic platelet defects and vWd." Coagulopathies do not influence the results of the PFA-100.100

The PFA-100 uses citrated blood placed in a sample well in a test cartridge that contains a collagen membrane coated with a platelet agonist (ADP or epinephrine). The sample is incubated at 37°C, and the blood sample is aspirated through a central 150 (.tm aperture under high shear conditions. Platelets are stimulated to adhere and aggregate under the high shear conditions and exposure to the agonist. Time to achieve closure of the aperture with a platelet plug (closure time, or CT) is measured, and is indicative of platelet function.101

The ADP and epinephrine PFA-100 cartridges have been tested in dogs. While closure times with the ADP cartridges are highly reproducible in dogs, the epinephrine cartridges have a wider reference interval with poor reproducibility, and are therefore not as useful." Epinephrine is a primary platelet agonist in humans, but results of previous optical aggregometry studies have shown it to be a poor agonist for canine platelet aggregation.99'101

Anemia is known to be associated with increased bleeding in humans and laboratory animals, which may be due to the change in viscosity of the circulating blood, platelet dysfunction, or decreased platelet-endothelial wall contact.102,103 As hematocrit in the citrated

20 blood samples decreases to <30%, the readings obtained by the PFA-100 become progressively prolonged.99' 10° Thus, clinical utility of the PFA-100 is currentiy limited for anemic patients.

Prolonged closure times have been reported in dogs with thrombocytopenia, thrombopathia, and vWd.99'100 Aspirin-induced thrombopathia in humans is characterized by a prolonged epinephrine closure time (EPI-CT), but a normal ADP closure time (ADP-CT).

The EPI-CT was markedly prolonged in dogs two hours after an intravenous injection of aspirin. The post-treatment ADP-CT was mildly prolonged after treatment with the same dose, but did not exceed the reference interval in all dogs.100

Other Measurements of Primary Hemostasis

Flow cytometry can be used to detect specific platelet markers including platelet membrane proteins, glycoprotein receptors, and activation markers. Markers for PS and P- selectin have been identified on activated canine platelets using flow cytometry. Detection of platelet-leukocyte aggregates and presence of platelet microparticles by flow cytometry have also been used to identify platelet activation.104 Detection of platelet surface-associated immunoglobulin (PSAIg) with flow cytometry has been used in the diagnosis of IMT.105' m

While the test has good sensitivity, it lacks specificity. Increased PSAIg is associated with

IMT, but cannot differentiate between primary and secondary IMT.106,107

Summary

Platelet function can be measured in many ways, and most tests require specialized equipment. Platelet aggregometry is considered the gold standard in platelet function testing. The PFA-100 is thought to provide testing conditions that mimic the in vivo

21 environment. Choosing the most appropriate tests depends on available equipment and goals of the study.

1.4 MEASURES OF SECONDARY HEMOSTASIS & FIBRINOLYSIS

Partial Thromboplastin and Prothrombin Times

The partial thromboplastin time (PTT; activated PIT) is commonly used to assess

the intrinsic and common coagulation pathways, measuring the combined activities of prekallektein, HMWK, and Factors XII, XI, IX, VIII, X, V, II, and I. The PTT is measured by adding an excess of procoagulant phospholipids (which do not contain tissue factor) and

a contact activator to citrated plasma at 37°C. The citrate in the sample chelates calcium, and

the reaction generally does not proceed beyond the activation of Factor XI. After incubation for a period of time, calcium chloride (CaClj) is added to the sample, allowing for the eventual formation of fibrin monomers and a fibrin clot. The PTT is measured from the

time of addition of the CaCl, to the formation of the clot, and the clot may be detected

optically or electrochemically.'5"'81

The prothrombin time (PT; one-stage PT) is used to evaluate the extrinsic and

common coagulation pathways, assessing the combined activities of Factors I, II, V, VII,

and X. The PT is performed by adding a calcium-thromboplastin reagent, which contains an

excess of phospholipid and tissue factor to citrated plasma at 37°C. Activation of the

extrinsic and common pathway proceeds to the point of fibrin clot formation. The time

from addition of the reagent to clot formation is the PT.81

Both the PT and PTT detect in ntro deficiencies of coagulation factors. These tests

are referred to as group tests as they test the function of a group of factors rather than

individual factors. A factor must be decreased to 20-25% of normal before the PT or PTT

22 becomes prolonged. Therefore these tests are not sensitive enough to detect mild factor deficiencies. Qualitative factor abnormalities and circulating inhibitors of factors can also lead to prolongation of the PT or PTT. A PT or PTT prolonged >20-25% above the reference interval is considered abnormal.30,81 While some authors consider a shortened PT or PTT indicative of hypercoagulability, it is not a consistent finding in reported cases of thrombosis.21, 28, 78, 8I Newer point-of-care coagulation analyzers (e.g., the SCA 2000

Veterinary Coagulation Analyzer, Synbiotics, San Diego, California) allow more convenient and immediate measurements of PT and PTT.108

Other Measurements of Secondary Hemostasis

Specific clotting factor protein analysis can be performed to determine the level of activity of a particular factor. Factor analysis is usually performed after detection of a prolonged PT/PTT. If a prolonged PT/PTT corrects when normal plasma is added to the patient's plasma, a factor deficiency is likely. If the PT/PTT fails to correct, presence of a circulating anticoagulant is likely. Factor analysis can be assessed by measuring the ability of the patient's plasma to correct clotting of plasma products known to be deficient in a particular factor. Chromatogenic assays can be used to assess the ability of a factor to lead to enzymatic cleavage of a substrate. Factor activity is proportional to the degree of colour change in chromatogenic factor assays.81,109

The vitamin K-dependent coagulation factors (Factors II, VII, IX, and X) are activated in the liver via a vitamin K-mediated carboxylation reaction. Proteins induced by vitamin K antagonism/absence (PIVKA) are incompletely carboxylated vitamin In­ dependent coagulation factors (Factors II, VII, IX, and X), and are elevated during periods

23 of vitamin K deficiency or antagonism. Chromatography and immunoassays can be used to measure PIVKA.110

Thrombin time (TT; thrombin clotting time - TCT) measures the time required for conversion of fibrinogen to fibrin. In this test, a thrombin reagent is added to citrated plasma at 37°C, and the time to fibrin clot formation is measured. Prolongation of TT can occur due to quantitative or qualitative defects in fibrinogen, or can result from circulating substances that inhibit the action of thrombin on fibrinogen.81

Fibrin Degradation Products and D-dimers

Plasmin en2ymatically degrades fibrin and fibrinogen at specific sites, producing fibrin/ fibrinogen degradation products (FDPs). Activity of the fibrinolytic system can be assessed by measuring FDP concentration using serum or citrated plasma latex agglutination immunoassays.30, U1 Increased concentration of FDPs is suggestive of excessive fibrinolysis, but the assay is not specific for cross-linked fibrin.111

D-dimer assays are specific for the lysis of cross-linked fibrin. Citrated plasma is used for the detection of large fragments of plasmin-digested fibrin, which contain the D- dimer antigen. Increased D-dimer levels indicate both excessive coagulation and fibrinolysis, such as that observed in disseminated intravascular coagulation.30' n2'113 Both the FDP and

D-dimer immunoassays use substrates coated with human antibodies (to FDPs and D- dimers, respectively). Specific reactivity to canine antigen is not known for these assays, but some cross-reaction is present.30

24 Global Tests of Hemostasis

The activated clotting time (ACT) is used as a point-of-care whole blood coagulation screening test.114 The ACT uses a chemical contact activator such as diatomaceous earth in a tube to activate the intrinsic coagulation pathway. The ACT is measured as the time from blood contacting the activator to the formation of a clot.81 A prolonged ACT may be the result of an abnormality in the intrinsic or common pathway, presence of coagulation inhibitors, or heparin therapy. Thrombocytopenia may prolong the ACT due to the lack of phospholipid availability.81, "5 The ACT is considered less sensitive for detection of intrinsic pathway defects than the partial thromboplastin time, and can produce variable results with poor repeatability.81

Thromboelastog'raphy (TEG) records the entire course of coagulation, measuring die interaction between components of the primary and secondary hemostatic systems.

Thromboelastography measures the viscoelastic properties of blood by allowing formation of a blood clot in a low-shear environment and testing its strength. The strength of the clot is measured over time in a tracing. Several parameters of clot formation are measured by

TEG, including strength of the clot, stability of the clot, and time needed for clot formation.81

Measurements of Endogenous Anticoagulants

Antithrombin (AT; AT III) measurement can be performed using chromatogenic, or functional assay techniques. To'' measure AT activity, a heparin-containing reagent is added to plasma. Excess thrombin or Factor Xa is then added to the assay, and increased AT activity results in decreased activity of thrombin or Factor Xa, leading to less colour change

25 in the assay. Abnormally low AT activity may increase risk of inappropriate thrombus formation.17,81

Protein C (PC) immunological assays measure concentration of PC antigen, or functional assays can be used to measure PC activity. Measurements of other coagulation inhibitors such as cx2-macroglobulin and oc-antitrypsin can be performed. Decreased coagulation inhibitor activity may predispose a patient to thrombosis.81

Summary

Tests of secondary hemostasis can be used to assess imbalances in the coagulation cascade. Hypocoagulable states can result from deficient or dysfunctional coagulation factors, or from the presence of excess anticoagulants. Hypercoagulable states can be due to increased availability of components of the secondary hemostatic system such as coagulation factors or fibrinogen, or deficiencies of anticoagulants. D-dimer and FDP levels can be evaluated when there is clinical suspicion of DIC. Global tests examine the interaction of several components of the hemostatic system, and can identify hyper- or hypocoagulable states. While most tests require sample submission to a reference laboratory, more point-of- care tests are being developed and validated for use in veterinary medicine as a rapid and convenient measure of hemostasis.

1.5 THE ARACHIDONIC ACID PATHWAY

Prostaglandins are formed mainly from the precursor arachidonic acid (AA). After mobilization from phospholipids, AA undergoes sequential biotransformation into prostaglandin (PG) G2 and PGH2 via the cyclooxygenase (COX) and hydroperoxidase

(HOX) activities of PGG2/H2 synthase. PGH2 is subsequently converted to a variety of

26 eicosanoids, depending on die cell type, including PGE2, PGD2, PGF2a, PGI2, and

3 thromboxane (TX)A2.''

Cyclooxygenase is the key regulatory enzyme of this pathway, catalyzing the conversion of arachidonic acid to PGG2 and PGH2. Two genes encoding for PGG2/H2 synthase, widely known as COX-1 and -2, have been identified in humans and veterinary species. A third isoenzyme, COX-3, has recently been identified in the canine brain and appears similar to COX-1. Cyclooxygenase-3 appears to play an important role in mediating inflammation and fever, and is thought to be the target of acetominophen in the brain116

Recendy, COX-3 expression has been identified in canine osteosarcoma cells.117 Both COX-

1 and COX-2 catalyze the conversion of AA to PGG2 and, subsequently, PGH2. The presence of local prostanoid synthases determines the type of prostanoids produced in a given tissue (Figure 1.4).4

Prostanoids are lipid mediators involved in diverse functions, including coagulation of blood, bone metabolism, nerve growth, blood vessel tone, and immune responses. While production of prostaglandins is widespread throughout the body, they serve as autocrine or paracrine mediators to signal changes within the immediate environment.2

Summary

Cyclooxygenase-1 and -2 are key enzymes in the production of prostanoids, These prostanoids have widely varying functions in the body. Inhibition of COX-1 and -2 is thought to be responsible for both the therapeutic and adverse effects of NSAIDs.

27 i U « U U i( H« l Membrane phospholipids " jj " " )] ]> "

PhospholipaseAj Divers* physical, chemical. Inflammatory, and mitogonie stimuli

• Arachidonic • Coxibs acid 1 Prostaglandin G2 Prostaglandin G2 Prostaglandin G/H Prostaglandin G/H synthase 1 synthase 2 (cyclooxygenase-1) (cyclooxygenase-2) Prostaglandin H2 Prostaglandin H2 I i Tissue-specific isomerases

Prostanoids Prostacyclin Thromboxane A2 Prostaglandin D2 Prostaglandin E2 Prostaglandin Fa

Receptors IP TP«'TFJ> DP,, DP, j EP„EP„EPa,EP4 FP"'IPl

Platelets, Brain, kidney, Uterus, airways, Endothelium, vascular smooth- Mast cells, vascular smooth- vascular smooth- kidney, muscle cells, brain, muscle cells, muscle cells, platelets, brain macrophages, airways kidney platelets eye

Figure 1.4 - Production and Actions of Prostaglandins and Thromboxane.

Arachidonic acid is liberated from membrane phospholipids by phospholipase A2, which is

activated by diverse stimuli. Arachidonic acid is converted to the unstable intermediate

prostaglandin H2, then converted by tisstie-specific isomerases to multiple prostanoids. These

bioactive lipids activate specific cell-membrane receptors of the superfamily of G-protein-

coupled receptors. IP - prostacyclin receptor, TP - thromboxane receptor, DP -prostaglandin D2

receptor, EP - prostaglandin E2 receptor, FP - prostaglandin F2ct receptor.

Used with written permission from FitzGerald, GA and Patrono, C. The coxibs, selective

inhibitors of cyclooxygenase-2. N. Engl J. Med 2001; 345(6): 433-442. © Massachusetts Medical

Society (2001). All rights reserved.

28 1.6 PHARMACOLOGY OF NSAIDS

Arachidonic acid undergoes a hairpin conformation change to interact with the active site on the COX enzyme. Traditional NSAIDs act as competitive inhibitors, blocking the interaction of arachidonic acid and COX, and impairing the production of prostaglandins.3 Inhibition of prostaglandin is thought to be responsible for both the therapeutic and adverse effects of NSAIDs.4,6

Two isoenzymes of COX, COX-1 and COX-2, have been identified in humans and veterinary species. COX-1 is considered a constitutive form of the COX enzyme, present under basal conditions in many cells, and responsible for such functions as gastric cytoprotection, regulation of renal blood flow, and normal platelet activity. In contrast,

COX-2 is thought of as an inducible form of the COX enzyme, present in very low levels in normal tissue but rapidly induced in inflammatory conditions.118' 1" COX-1 mRNA and protein has been identified in canine tissues including the gastrointestinal tract, spleen, liver, kidney, lung, ovary, and cerebral cortex. COX-2 mRNA has also been identified in these tissues, but at a much lower level than COX-1 mRNA. However, expression of COX-2 protein was not identified in these tissues, suggesting the role of COX-2 as an enzyme inducible by inflammation.119 The generalization of COX-1 as die constitutive isoenzyme and COX-2 as the inducible isoenzyme is likely oversimplified, as there is evidence of considerable overlap between the two domains.14,12°

Dual inhibitors of the arachidonic acid pathway have recently been introduced to veterinary medicine. Tepoxalin (Zubrin™) has recently been introduced as a COX and lipoxygenase (LOX) inhibitor, having inhibitory effects on COX-1, COX-2, and 5-LOX.'2'

By inhibiting both the COX and LOX enzymes, tepoxalin's antiinflammatory benefits are believed to be greater than COX inhibitors alone.121,122

29 COX-2 selective inhibitors

Traditional NSAIDs non-selectively blocked both the COX-1 and COX-2 isoforms.

Blocking COX-1 production of constitutive prostaglandins is thought to result in the adverse effects associated with NSAID use, which lead to the development of COX-2 selective inhibitors. The first generation of human COX-2 selective inhibitors included rofecoxib (Vioxx™) and celecoxib (Celebrex™). More recent COX-2 selective inhibitors introduced to the human market include lumiracoxib, etoricoxib, and valdecoxib and its pro­ drug parecoxib.7

Clinical trials in veterinary medicine have identified several COX-2 selecdve (COX-1 sparing) inhibitors. In dogs, carprofen (Rimadyl™) and deracoxib (Deramaxx™) have demonstrated COX-1 sparing activity on target tissues such as gastric mucosa. Etodolac also acts as COX-1 sparing, but its effects on COX-2 are variable depending on the target tissue.123 Firocoxib (Previcox™) is reported to have little impact (0 to 3%) on COX-1 activity at serum levels necessary to provide peak inhibition of COX-2.119,124 In vitro studies have determined ketoprofen, aspirin, and etodolac to be COX-1 selective in dogs, while COX-2 selective NSAIDs include piroxicam, meloxicam, celecoxib, and carprofen.125"128 In vivo trials characterize meloxicam as COX-2 selective, while aspirin has COX-1 selective activity.129

Nonsteroidal anti-inflammatories currently licensed for use in dogs in Canada include carprofen, deracoxib, ketoprofen, and meloxicam.130

Various techniques have been used to describe COX-1 :COX-2 selectivity ratios in veterinary species. There is evidence of species variation in the activity in the expression of

COX, and caution must be used when extrapolating results of studies across species lines.

Comparing activity of NSAIDs in canine cell lines versus cell lines of other species

30 demonstrated this variability.128 There is evidence that while etodolac is COX-1 selective in dogs, it is COX-2 selective in humans.128'131

Adverse effects of NSAIDs

Adverse effects associated with NSAID therapy in dogs include gastrointestinal irritation, renal and hepatic toxicity, alteration of hemostasis, and decreased fertility.132, 133

The incidence of NSAID associated adverse effects in veterinary species is unknown, but it has been suggested that dogs and cats are more sensitive to NSAID toxicosis than humans.134 Dogs tolerate NSAIDs better than cats, which are susceptible to adverse effects of NSAIDs due to their lack of glucuronyl transferase. Increased susceptibility to NSAID adverse effects in dogs compared to humans may be related to a number of factors including extensive enterohepatic recirculation, higher absorption rates across the gastrointestinal tract, and a longer drug half-life in dogs.135

One of the most common adverse effects related to veterinary NSAID use is gastrointestinal toxicosis.135 The gastrointestinal toxicity of NSAIDs in humans and veterinary species appears to be related to the inhibition of prostaglandin synthesis, as well as due to direct injury to the gastrointestinal tract mucosa.4 As gastrointestinal mucosal protection and other homeostatic mechanisms are largely mediated by COX-1, COX-2- selective NSAIDs were developed in attempt to limit the development of NSAID-associated adverse effects.2,7

Several large clinical trials have addressed the efficacy and associated risk of COX-2 selective inhibitors in humans. Improved analgesic effects and reduced gastrointestinal toxicity of COX-2 selective inhibitors have been reported in these trials.8'136"138 . Similarly,

COX-2 selective inhibitors have been shown to spate protective gastric prostaglandins in

31 dogs, and are thought to result in decreased gastrointestinal toxicity and fewer other adverse effects in veterinary species.12'' Chronic therapy with carprofen or meloxicam showed a decreased incidence of gastric lesions in dogs compared to therapy with ketoprofen, etodolac, or flunixin.139 Long-term carprofen therapy appears to be well tolerated.140

Reported rates of gastrointestinal adverse effects are low in dogs treated with firocoxib.141,142

Cases of gastrointestinal perforation secondary to deracoxib or meloxicam therapy have been reported, but are often associated with excessive doses or use of the medication in close temporal association with other NSAIDs or corticosteroids.143, 144 However, a recent study reported the constitutive expression of COX-2 in the canine gastroduodenal mucosa, suggesting a possible role of COX-2 in the protection of the upper gastrointestinal tract.140

Constitutive expression of COX-2 is important for normal renal development, and may help maintain normal renal blood flow.7'146 Therefore, treatment with COX-2 selective inhibitors may induce renal dysplasia in developing animals and may predispose to renal injury, especially in low-volume states. Other potential adverse effects of COX-2 inhibitors include delayed fracture healing, impairment of reproduction, and retarded organ development in neonates.146

Summary

Nonsteroidal anti-inflammatory drugs vary in their inhibition of the COX enzymes.

Cyclooxygenase-2 selective inhibitors are thought to reduce toxicity, especially gastrointestinal ulceration, associated with NSAID use.

32 1.7 EFFECTS OF NSAIDS ON THE CARDIOVASCULAR SYSTEM

COX-2 selective inhibitors and cardiovascular outcomes in humans

No prospective trials identifying potential cardiovascular risk associated with use of

COX-2 inhibitors have yet been performed. However, some clinical trials of COX-2 inhibitors examining rate of gastrointestinal adverse effects have reported adverse cardiovascular effects in humans. The Vioxx Gastrointestinal Outcome Research study

(VIGOR) identified an increased risk of myocardial infarction in patients with rheumatoid arthritis administered rofecoxib compared to those taking naproxen (0.4% versus 0.1%, 95% confidence interval, 0.1% to 0.6%).8 The Celecoxib Long-term Arthritis Safety study

(CLASS) examined use of celecoxib in patients with osteoarthritis or rheumatoid arthritis, compared to use of ibuprofen or diclofenac. In contrast to VIGOR, CLASS did not identify any significant increase in risk of myocardial infarction or other serious cardiovascular events associated with celecoxib use. In a trial of patients with colonic adenomas, celecoxib use was associated with a dose-related increase in the incidence of death from cardiovascular causes, myocardial infarction, stroke, or heart failure.147

Observational studies examining NSAID use and adverse cardiovascular outcomes also offer contradictory data. A study of NSAID use in the Tennessee Medicaid program showed that users of high-dose rofecoxib were 1.70-fold more likely than non-users to have serious coronary heart disease, and the rate was increased to 1.93-fold among new users.

Low-dose rofecoxib users or users of other NSAIDs did not experience this increased risk of adverse cardiovascular effects.9 An analysis of acute myocardial infarction rates in elderly patients administered NSAIDs in Ontario examined the use of COX-2 selective inhibitors, naproxen, and other NSAIDs. This study did not show a significant difference in acute myocardial infarction among new users of celecoxib, rofecoxib, naproxen, and non-

33 naproxen NSAIDs. However, use of valdecoxib after coronary bypass surgery resulted in an increase in adverse cardiovascular outcomes, compared to a placebo.10,11

Several case control studies suggest that the differences observed in VIGOR could be explained by a cardioprotective effect of naproxen, most likely as due to antithrombotic properties. In these studies, the relative risk of myocardial infarction was lower in patients taking naproxen than,in those taking other NSAIDs, or in non-users of NSAIDs.149"151

Controversy surrounds the proposal that naproxen is cardioprotective, with some studies showing a similar rate of myocardial- infarction among naproxen users compared to non- users of NSAIDs.9' I52' 1:>3 No prospective studies examining the potential cardioprotective role of naproxen have been performed.

Concern regarding the cardiovascular safety of COX-2 selective inhibitors, and their possible prothrombotic effects, may be attributed to a possible imbalance of thrombotic and anti-thrombotic factors in the blood. Non-selective NSAIDs block both TXA2, released via

COX-1 activity in platelets, and PGI2, a product of endothelial COX-2 activity.

Thromboxane acts to increase platelet aggregation in the blood and to promote vasoconstriction.12 The actions of prostacyclin, inhibition of platelet aggregation and function and vasodilation, oppose diose of TXA2,'" COX-2 selective inhibition may result in

14 impaired antithrombotic activity of PGI,, allowing unopposed thrombotic effects of TXA2. "

16 Therefore, one potential cause of the increase in adverse cardiovascular events associated with COX-2 selective inhibitors may be due to a potential prothrombotic mechanism.

Additionally, preliminary research has suggested that upregulation of COX-2 may play a key role in cardioprotection and may increase vascular protective responses to injury.154,155

Research examining a possible hemostatic imbalance induced by selective COX-2 inhibition has produced variable results. A study of healthy humans showed no effects on

34 systemic prostacyclin or thromboxane formation after administration of aspirin or rofecoxib.

Platelet function, measured by expression of CD62P, was also not affected.156 However, decreased prostacyclin levels were reported in response to rofecoxib and celecoxib in other

157,158 studies. Meloxicam administration caused significant reduction in serum TXB2 levels in humans, with a minor increase in closure time measured by PFA-100 but no reduction in platelet aggregation.'59

Adverse cardiovascular outcomes may be related to specific COX-2 inhibitors, such as rofecoxib, suggesting the effect may be due to the drugs' intrinsic chemical properties.

Rofecoxib use was found more likely to be associated with serious coronary heart disease when compared to celecoxib.160 Rofecoxib has been shown to act as a potent prooxidant in humans, increasing susceptibility of cell membrane lipids to oxidative damage. This property of rofecoxib may lead to instability of atherosclerotic plaques and increased risk of thrombus formation, which is not shared by other COX-2 inhibitors such as celecoxib and valdecoxib. 6

While data are inconclusive in implicating COX-2 inhibitors with increased cardiovascular risk, rofecoxib has been withdrawn from the global market.162 Drug authorities have issued warnings about similar events during the use of other coxibs, and a large study involving cancer patients was discontinued due to concerns regarding an increased number of cardiovascular effects associated with celecoxib.'63'165

Effect of NSAIDs on the canine cardiovascular system

Unlike humans, thromboembolic disease leading to cardiovascular disease is infrequently identified in dogs. There have been no reports associating the use of COX-2 selective drugs (e.g., meloxicam, deracoxib, carprofen) with thromboembolic disease in dogs.

35 Aspirin (acetylsalicylic acid; ASA) has long been used for its antithrombotic benefits in veterinary medicine. Aspirin causes irreversible acetylation of COX-1 in platelets, preventing TXA2 production and inducing antithrombotic effects for the platelet's lifespan

(5-7 days). Ultra-low dose aspirin therapy (0.5 mg/kg body weight q 12 hours) is more effective in decreasing in vivo platelet aggregation in dogs than higher doses due to differential effects on the platelet and the endothelial cell.166

Few studies have examined the role of COX-2 inhibition in canine hemostasis. A study investigating the effect of carprofen on hemostatic variables concluded that the drug had minor, but clinically irrelevant, effects on platelet aggregation.167 Carprofen was shown

168 to have no effect on serum TXB2 levels in healthy dogs. The effects of various NSAIDs on platelet function, hemostasis, and prostaglandin production was examined in dogs with osteoarthritis. The study identified decreased platelet aggregation and clot strength with carprofen and aspirin therapy, while meloxicam minimally inhibited platelet aggregation.

Deracoxib therapy was shown to increase clot strength on some parameters measured by thromboelastography, but platelet aggregation was not affected. Serum PGI2 and TXB2 levels, as well as platelet TXB2 levels, were not affected by therapy with any NSAIDs used in

29 the study. Whole blood TXB2 levels are reported to decrease after administration of tepoxalin, a dual COX and LOX inhibitor, but not after meloxicam administration.121

Summary

The use of COX-2 selective inhibitors has been linked to adverse cardiovascular events in humans, but this phenomenon has not been reported in veterinary medicine. The increase in prothrombotic events in people may be linked to inhibition of prostacyclin synthesis, leaving the actions of thromboxane unopposed. One study examining COX-1

36 and -2 selective NSAID use in dogs did not show significant alteration of prostacyclin or thromboxane levels associated with NSAID treatment. However, this study examined effects of NSAIDs on coagulation in dogs with mild to moderate osteoarthritis.29 The effects of inflammation secondary to osteoarthritis may have confounded the results of that study.

Despite the widespread use of NSAIDs in veterinary medicine, there is limited information available regarding the effects of NSAIDs on canine coagulation. Controlled, blinded, randomi2ed trials examining commonly used NSAIDs at therapeutic dosages on canine coagulation are lacking. This study examines the effects of four commonly used

NSAIDs (aspirin, carprofen, deracoxib, and meloxicam) on canine platelet function and serum prostacyclin and thromboxane levels. Normal dogs were used as the study subjects to help avoid potential confounding factors of illness on coagulation. The aim of this study was to provide further information on the effect of NSAIDs currentiy used in canine practice on primary and secondary hemostasis.

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120. Jones CJ, Budsberg SC. Physiologic characteristics and clinical importance of the cyclooxygenase isoforms in dogs and cats. J Am Vet Med Assoc 2000;217(5):721-729.

121. Agnello K, Reynolds L, Budsberg S. In vivo effects of tepoxalin, an inhibitor of cyclooxygenase and lipoxygenase, on prostanoid and leukotriene production in dogs with chronic osteoarthritis. Am J I"VR

122. Curry SL, Cogar SM, Cook JL. Nonsteroidal antiinflammatory drugs: a review. / Am Anim Hosp Assoc 2005;41 (5) :298-309.

123. Sessions JK, Reynolds LR, Budsberg SC. In vivo effects of carprofen, deracoxib, and etodolac on prostanoid production in blood, gastric mucosa, and synovial fluid in dogs with chronic osteoarthritis. Am J Vet Res 2005;66(5):812-817.

124. Meade EA, Smith WL, DeWitt DL. Differential inhibition of prostaglandin endoperoxide synthase (cyclooxygenase) isozymes by aspirin and other non-steroidal anti­ inflammatory drugs. / Biol Cbem 1993;268(9):6610-6614.

125. Streppa HK, Jones CJ, Budsberg SC. Cyclooxygenase selectivity of nonsteroidal anti­ inflammatory drugs in canine blood. Am] Vet Res 2002;63(l):91-94.

126. Kay-Mugford P, Benn SJ, LaMarre J, et al. In vitro effects of nonsteroidal anti­ inflammatory drugs on cyclooxygenase activity in dogs. Am] Vet Res 2000;61(7):802-810.

127. Brideau C, Van Staden C, Chan CC. In vitro effects of cyclooxygenase inhibitors in whole blood of horses, dogs, and cats. Am J Vet Res 2001 ;62(11):1755-1760.

128. Ricketts AP, Lundy KM, Seibel SB. Evaluation of selective inhibition of canine cyclooxygenase 1 and 2 by carprofen and other nonsteroidal anti-inflammatory drugs. Am J Vet Res 1998;59(11):1441-1446.

129. Jones CJ, Streppa HK, Harmon BG, et al. In vivo effects of meloxicam and aspirin on blood, gastric mucosal, and synovial fluid prostanoid synthesis in dogs. Am J Vet Res 2002;63(11) :1527-1531.

130. North American Compendiums. Compendium of Veterinary Products. 10th ed. Hensall, Ontario: North American Compendiums, 2007.

131. Cryer B, Feldman M. Cyclooxygenase-1 and cyclooxygenase-2 selectivity of widely used nonsteroidal anti-inflammatory drugs. Am J Med \998;104(5):413-421.

132. Lees P, Landoni MF, Giraudel J, et al. Pharmacodynamics and pharmacokinetics of nonsteroidal anti-inflammatory drugs in species of veterinary interest. / Vet Pharmacol Ther 2004;27(6):479-490.

46 133. Lascelles BD, McFarland JM, Swann H. Guidelines for safe and effective use of NSAIDsin dogs. VetTber20Q5;6(3):237-251.

134. Mathews KA. Nonsteroidal anti-inflammatory analgesics. Indications and contraindications for pain management in dogs and cats. Vet Clin North Am Small Anim Pract 2000;30(4):783-804.

135. Jones RD, Baynes RE, Nimitz CT. Nonsteroidal anti-inflammatory drug toxicosis in dogs and cats: 240 cases (1989-1990). ] Am Vet Med Assoc 1992;201(3):475-477.

136. Farkouh ME, Kirshner H, Harrington RA, et al. Comparison of lumiracoxib with naproxen, and ibuprofen in the Therapeutic Arthritis Research and Gastrointestinal Event Trial (TARGET), cardiovascular outcomes: randomised controlled trial. Lancet 2004;364(9435):675-684.

137. Silverstein FE, Faich G, Goldstein JL, et al. Gastrointestinal toxicity with celecoxib vs nonsteroidal anti-inflammatory drugs for osteoarthritis and rheumatoid arthritis: the CLASS study: A randomized controlled trial. Celecoxib Long-term Arthritis Safety Study. JAMA 2000;284(10):1247-1255.

138. Lanas A, Hunt R. Prevention of anti-inflammatory drug-induced gastrointestinal damage: benefits and risks of therapeutic strategies. Ann Med 2006;3S(6):415-428.

139. Luna SP, Basilio AC, Steagall PV, et al. Evaluation of adverse effects of long-term oral administration of carprofen, etodolac, flunixin meglumine, ketoprofen, and meloxicam in dogs. Am J Vet Res 2007;68(3):258-264.

140. Raekallio MR, Hielm-Bjorkman AK, Kejonen J, et al. Evaluation of adverse effects of long-term orally administered carprofen in dogs. / Am Vet Med Assoc 2006;228(6):876-880.

141. Ryan WG, Moldave K, Carithers D. Clinical effectiveness and safety of a new NSAID, firocoxib: a 1,000 dog study. Vet Tber 2006;7(2)\l 19-126.

142. Steagall PV, Mantovani FB, Ferreira TH, et al. Evaluation of the adverse effects of oral firocoxib in healthy dogs. / Vet Pharmacol Ther 2007;30(3):218-223.

143. Lascelles BD, Blikslager AT, Fox SM, et al. Gastrointestinal tract perforation in dogs treated with a selective cyclooxygenase-2 inhibitor: 29 cases (2002-2003). ] Am Vet Med Assoc 2005;227(7):11'12-1117.

144. Enberg TB, Braun LD, Kuzma AB. Gastrointestinal perforation in five dogs associated with die administration of meloxicam. jr/ECC2006;16(l):34-43.

145. Wooten JG, Blikslager AT, Ryan KA, et al. Cyclooxygenase expression and prostanoid production in pyloric and duodenal mucosae in dogs after administration of nonsteroidal anti-inflammatory drugs. Am] Vet Res 2008;69(4):457-464.

146. Bergh MS, Budsberg SC. The coxib NSAIDs: potential clinical and pharmacologic importance in veterinary medicine. J Vet Intern Med 2005;! 9(5) :633-643.

47 147. Solomon SD, McMurray JJ, Pfeffer MA, et al. Cardiovascular risk associated with celecoxib in a clinical trial for colorectal adenoma prevention. N Engl J Med 2005;352(11):1071-1080.

148. Mamdani M, Juurlink DN, Lee DS, et al. Cyclo-oxygenase-2 inhibitors versus non­ selective non-steroidal anti-inflammatory drugs and congestive heart failure outcomes in elderly patients: a population-based cohort study. Lancet 2004;363(9423):1751-1756.

149. Rahme E, Pilote L, LeLorier J. Association between naproxen use and protection against acute myocardial infarction. Arch Intern Mft/2002;162(10):1111-1115.

150. Watson DJ, Rhodes T, Cai B, et al. Lower risk of thromboembolic cardiovascular events with naproxen among patients with rheumatoid arthritis. Arch Intern Med 2002;162(10):1105-1110.

151. Solomon DH, Glynn RJ, Levin R, et al. Nonsteroidal anti-inflammatory drug use and acute myocardialinfarction.^4r^7«^r«M^2002;162(10):1099-1104.

152. Hippisley-Cox J, Coupland C. Risk of myocardial infarction in patients taking cyclo- oxygenase-2 inhibitors or convcndonal non-steroidal anti-inflammatory drugs: population based nested case-control analysis. BMJ 2005;330(7504):1310-1316.

153. Johnsen SP, Larsson H, Tarone RE, et al. Risk of hospitalization for myocardial infarction among users of rofecoxib, celecoxib, and other NSAIDs: a population-based case- control study. Arch Intern Med 2005;! 65(9):978-984.

154. Shinmura K, Tang XL, Wang Y, et al. Cyclooxygenase-2 mediates the cardioprotective effects of the late phase of ischemic preconditioning in conscious rabbits. Proc Natl Acad Sri U SA 2000;97(18):10197-10202.

155. Howard PA, Delafontaine P. Nonsteroidal anti-Inflammatory drugs and cardiovascular risk. J Am Coll Cardiol 2004;43(4);5V)-525.

156. Weber AA, Heim HK, Schumacher M, et al. Effects of selective cyclooxygenase isoform inhibition on systemic prostacyclin synthesis and on platelet function at rest and after exercise in healthy volunteers. Platelets 2007;18(5):379-385.

157. Catella-Lawson F, McAdam B, Morrison B, et al. Effects of specific inhibition of cyclooxygenase-2 on sodium balance, hemodynamics, and vasoactive eicosanoids. JPET 1999;289(2):735-741.

158. McAdam B, Catella-Lawson F, Mardini I, et al. Systemic biosynthesis of prostacyclin by cyclooxygenase (COX)-2: the human pharmacology of a selective inhibitor of COX-2. Proc NatlAcadSciUSA 1999;96(l):272-277.

159. de Meijer A, Vollaard H, de Metz M, et al. Meloxicam, 15 mg/day, spares platelet function in healthy volunteers. Clin Pharmacol Ther 1999;66(4):425-430.

48 160. Graham DJ, Campen D, Hui R, et al. Risk of acute myocardial infarction and sudden cardiac death in patients treated with cyclo-oxygenase 2 selective and non-selective non­ steroidal anti-inflammatory drugs: nested case-control study. L#»«#2005;365(9458):475-481.

161. Mason RP, Walter MF, McNulty HP, et al. Rofecoxib increases susceptibility of human LDL and membrane lipids to oxidative damage: a mechanism of cardiotoxicity. / Cardiovasc Pharmacol 2006;47 Suppl 1:S7-14.

162. US Food and Drug Administration. FDA Issues Public Health Advisory on Vioxx as its Manufacturer Voluntarily Withdraws the Product. Available at: http://www.fda.gov/ bbs/topics/news/2004/NEW01122.html. Accessed August 10, 2007.

163. US Food and Drug Administration. COX-2 Selective (includes Bextta, Celebrex, and Vioxx) and Non-Selective Non-Steroida] Anti-Inflammatory Drugs (NSAIDs). Available at: http://www.fda.gOv/cder/drug/infopage/COX2/defaiilt.htm#COX2. Accessed August 10, 2007.

164. US. Food and Drug Administration. FDA Alert for Practitioners on Celecoxib (marketed as Celebrex). Available at: http://www.fda.gov/cder/drug/infopage/celebrex/ celebrex-hcp.htm. Accessed 08/10, 2007.

165. US Department of Health and Human Services. NIH Halts Use of COX-2 Inhibitor in Large Cancer Prevention Trial. Available at: http://www.nih.gov/news/pr/dec2004/od- 17.htm. Accessed August 10, 2007.

166. Rackear D, Feldman B, Farver T, et al. The effect of three different dosages of acetylsalicylic acid on canine platelet aggregation. JAAHA 1988;12:201-238.

167. Hickford FH, Barr SC, FJrb HN. Bffect of carprofcn on hemostatic variables in dogs. Am] Vet Res 2001;62(10):1642-1646.

168. McKellar QA, Pearson T, Bogan J A, et al. Pharmacokinetics, tolerance, and serum thromboxane inhibition of carprofen in the dog./ SmAnim Pract 1990;31:443-448.

49 CHAPTER 2: RESEARCH PROJECT

2.0 PRIMARY AND SECONDARY HEMOSTASIS

Introduction

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used in veterinary

medicine to provide analgesic and anti-inflammatory benefits to patients. The most

commonly reported adverse effects associated with NSAID use in dogs and cats include vomiting and diarrhea secondary to gastrointestinal irritation.1,2 Many of NSAIDs' adverse

effects, including gastrointestinal irritation, are believed to be largely due to inhibidon of

cyclooxygenase (COX)-l. As such, COX-2-selective NSAIDs were developed in attempt to limit the development of NSAID-associated adverse effects.3,4

Recent reports in the human medical literature have suggested an increased incidence

of myocardial infarction and other adverse cardiovascular events associated with the use of

COX-2 selective NSAIDs in humans.3"8 This increased incidence of thromboembolic events

may result from a relative increase in thromboxane (TXA^ levels compared to prostacyclin

(PGI^ levels. Unlike humans, thromboembolic events leading to cardiovascular disease are

infrequently identified in dogs.10 There have been no reports associating the use of COX-2

selective drugs (e.g., meloxicam, deracoxib, carprofen) with diromboembolic disease in dogs.

Dogs appear to be more resistant to diromboembolic complications than humans.

However, certain pathologic conditions are known to be associated with hypercoagulability

in dogs. Increased numbers and activity of platelets may lead to thromboembolism.

Systemic conditions such as malignancy, hyperadrenocorticism, and systemic inflammatory

conditions may also lead to thromboembolic events due to an increase in the activity of

coagulation factors involved with secondary hemostasis."'31

50 Studies evaluating the effects of NSAIDs on primary and secondary hemostasis in veterinary species are limited. No studies have reported the effects of therapeutic dosages of

NSAIDs on hemostatic function of healthy dogs. The aim of this study was to provide further information on the effect of commonly used NSAIDs in canine practice on the primary and secondary hemostatic system. Specifically, the effects of these NSAIDs would be evaluated based on platelet function tests, thromboxane and prostacyclin levels, and measures of secondary hemostasis (prothrombin and partial thromboplastin times, and fibrinogen levels). The null hypotheses of this study included:

i.. Aspirin, carprofcn, deracoxib, and mcloxicam used at anti-inflammatory

levels would not affect platelet function as measured by optical

aggregometry.

ii. Aspirin, carprofen, deracoxib, and meloxicam used at anti-inflammatory

levels would not affect platelet function as measured by optical the platelet

function analyzer (PFA)-100.

iii. Aspirin, carprofen, deracoxib, and meloxicam used at anti-inflammatory

levels would not affect free plasma thromboxane levels,

iv. Aspirin, carprofen, deracoxib, and meloxicam used at anti-inflammatory

levels would not affect free plasma prostacyclin levels.

Materials and Methods

Animals — This study was designed in accordance with the standards of the

Canadian Council on Animal Care and die Ontario Animals for Research Act, and was approved by the University of Guelph Animal Care Committee. Ten healdiy mature hound- cross dogs (5 males and 5 females) that were part of a research colony were used in the

51 study. The male dogs were sexually intact, while the females were spayed. Dpgs weighed between 23.8 and 32.3 kg (mean 27.3). All dogs had received routine immunization against canine distemper virus, parvovirus, adenovirus types 1 and 2, parainfluenza virus, rabies virus, and Bordatella bronchiseptka'within 12 months of initiating the study. All dogs had been prophylactically dewormed with fenbendazole within 2 months of initiating the study. The dogs were free of apparent clinical disease in the 2 months prior to and during the study, and had not received any medication in the 4 weeks prior to commencement of the study.

All dogs were confirmed to be healthy on the basis of physical examination, complete blood count, serum biochemistry profile, and urinalysis conducted 2 weeks prior to onset of the study. Platelet count and closure time measured using a platelet-function analyzer (PFA-100) were used to assess primary hemostasis and were within reference intervals in all dogs. One-stage prothrombin time (PT), activated partial thromboplastin time (PTT), and fibrinogen concentration results were within reference intervals, indicating normal secondary hemostatic abilities of all dogs.

Dogs were housed in indoor kennels, fed a standard commercially available kibble formulated for dogs, and allowed leash walks outside in a restricted area.

Study Design - Ten dogs were orally administered four NSAIDs and one placebo in a cross-oyer design at dosages consistent with current therapeutic recommendations."2"2'

The dogs were administered aspirinb (10 mg/kg PO, q 12 hours), carprofenc (4.4 mg/kg PO, q 24 hours), deracoxibd (2 mg/kg PO, q 24 hours), and meloxicanf (0.1 mg/kg PO, q 24

Acetylsalicylic acid, Pharmasciencc Inc., Montreal, Quebec. c Rimadyl, Pfizer Animal Health Canada, Kirkland, Quebec. Deramaxx, Novartis Animal Health Canada Inc., Mississauga, Ontario. 6 Metacam, Boehringer Ingelheim Canada Ltd., Burlington, Ontario.

52 hours). The chosen doses of each NSAID represent what is commonly used in clinical practice for anti-inflammatory therapy. The placebo consisted of an empty gelatin capsule.

Each drug and the placebo were administered for 7 days, with a 21-day washout period between subsequent treatments. The 21-day washout period was chosen to allow for at least

14 elimination half-lives for each drug to elapse and for at least 2 new platelet generations to form. The elimination half-life of deracoxib increases with dose but is approximately 3 hours for doses up to 8 mg/kg in dogs. Elimination half-lives for carprofen and meloxicam in dogs are 13-18 hours and 12-36 hours, respectively.23'25 Aspirin has an elimination half-life of 4.5-8.5 hours in dogs, but irreversibly binds to platelets rendering the bound platelet ineffective for the duration of its life (up to 7 days in dogs).2S'27

The study was performed in a crossover design so that each dog received each medication in a randomized order. The drugs were given with food, with the exception of

the NSAIDs administered on day 7 to avoid lipemia in the blood samples collected on that

day.

A Latin Square design was used to determine the random treatment order for each

dog to control for carryover and period effects.28 The columns and treatment periods in the

Latin Square were randomly shuffled using three random number generators.29,30

Blood collection was performed on day 0 prior to initiating each treatment and on

day 7 of each treatment. Sedation was not used nor was it required for blood collection

from any of die dogs. Blood was collected approximately 2-3 hours after NSAID

administration to correspond to peak serum NSAID concentration for most of the NSAIDs

used in the study. The peak serum concentrations after aspirin, carprofen, and deracoxib

range from 0.5 to 3 hours, while the peak serum concentration after meloxicam

administration is up to 7.5 hours.23"25'31 Blood was collected for the tests listed below. Food

53 was withheld for a minimum of 12 hours prior to blood sample collection for hemostatic

testing. The investigators were blinded to the treatments administered to each dog. The

study was conducted over a 4-month period.

Blood collection — Venous blood samples were collected from the jugular vein

using a 20-gauge, thin walled butterfly catheter needle. The first 2 mL of blood was

collected in a 3 mL syringe and immediately transferred to an ethylenediamine tetraacetic

acid (EDTA) plastic tube. The butterfly catheter was then attached to a 35 mL syringe

containing 3.5 mL of 3.2% sodium citrate and the remainder of the blood sample was

obtained and gently mixed. Approximately 35 mL of blood was collected each sampling day.

All dogs were acclimated to blood collection procedures, allowing rapid and efficient

collection of samples.

Platelet count, hematocrit, and leukocyte count - Approximately 2 mL of

EDTA whole blood was used to quantify platelet number, hematocrit, and leukocyte

number using an automated hematologic analyzer/

Analysis of hemostasis - Primary hemostasis was evaluated in each dog by the

measure of closure time using a platelet function analyzer, and platelet aggregation.

Secondary hemostatic evaluation included PT, PTT, and concentration of fibrinogen (factor

I)-

Adiva 120, Bayer Inc., Toronto, Ontario.

54 Platelet function analysis as measured by the PFA-100 ^ - Platelet function was performed using the platelet function analyzer (PFA)-100™,g as described elsewhere.32 The

PFA-100 has previously been evaluated for use in dogs.33' 34 Briefly, a sample reservoir cartridge containing a membrane coated with collagen plus adenosine-5'-diphosphate (ADP) was warmed to 22°C. The cartridge was placed into the analyzer, and 800 p.L of citrated whole blood was transferred by use of an adjustable piston micropipetterh into a sample reservoir. The cartridge and sample were further warmed to 37°C within the analyzer prior to assessing platelet function. The sample was aspirated from the reservoir under constant vacuum through a capillary and a microscopic aperture (147 \xm). The membrane coating stimulates platelet aggregation, and a clot forms. The time required to occlude the aperture is reported as closure time (CT), Analysis was performed within 1 hour of sample collection.

The analysis was repeated on 2 samples due to error reports (flow obstruction) by the analyzer. The maximum CT measured by the PFA-100 is 300 sec. Longer CTs are reported as >300 sec.

Platelet aggregation - An aliquot of citrated whole blood was centrifuged (80 X g for 15 minutes). The supernatant of platelet-rich plasma (PRP) was removed using a polyethylene pipette1 and transferred to polystyrene tubes' that were sealed with caps. When an insufficient quantity of PRP was harvested, the sample was centrifuged a second time and the supernatant was again collected. The tubes were mixed with gentle rotation and allowed to sit for 10 minutes undisturbed. The remaining blood was centrifuged (3000 X g for 15

g PFA-100, Dade-Behring, Mississauga, Ontario. 1 Research Pipette, Eppendorf, Mississauga, Ontario. 1 Transfer pipette, Fisher Scientific, Pittsburg, Pennsylvania. J Centrifuge Tubes, Fisher Scientific, Pittsburg, Pennsylvania.

55 minutes) and the supernatant of platelet-poor plasma (PPP) was- removed using a polyethylene pipette and transferred to polystyrene tubes that were sealed with caps.

As previously described, a standard manual platelet counting method was used to determine

the platelet concentration of each sample of PRP.35 A 20-(JiL aliquot of PRP was added to a

commercial dilution system for platelet determinations'1 to achieve a final dilution 1:100. The

solution was gently mixed and allowed to sit undisturbed for 10 minutes. The two counting

chambers on a hemocytometer1 were filled with the diluted PRP solution, and the

hemocytometer was placed in a humidified chamber for 10 minutes. Using light

microscopy, the chambers were viewed at 500X magnification, and a platelet count was

obtained from the 25 main squares of each of the two counting chambers. If the counts

from the two chambers had >5% discrepancy, the counts were repeated. The mean value

was determined for the counted chambers, and the value multiplied by 1000 to provide an

estimate of die platelet count of the PRP.

An appropriate volume of autologous PPP was mixed with the PRP aliquot in test

tubes to yield PRP with a final platelet count of 200 x 109/L. The diluted PRP was gendy

mixed by tube rotation, sealed with a plastic cap, and allowed to sit undisturbed for 30

minutes.

Two platelet aggregation agonists, ADPm and platelet activating factor (PAF)n were

used for aggregometry. The agonists were prepared and used within 2 hours of initiation of

aggregometry. Each agonist was used at a high concentration (that which induced maximal

platelet aggregation), and a low concentration that less consistendy induced platelet

Unopette microcollection system, Becton-Dickinson, Franklin Lakes, New jersey. Bright-line hemocytometer, American Optical, Buffalo, New York. m F.quine muscle adenosine 5'-diphosphate, Sigma-Aldrich Co., St. Louis, Missouri n Synthetic PAF-16, EMD Biosciences, San Diego, California.

56 aggregation and was half of the high concentration. Two concentrations of ADP were used,

100 and 50 \im. A stock solution of 10 mM ADP was diluted with pH 7.4 Tris-buffered

saline (TBS) to achieve the desired concentrations. Platelet activating factor was used at concentrations of 1 and 0.5 p,m. The PAF was dissolved in ethanol at a concentration of 10

mM and diluted with pH 7.4 TBS to achieve the desired concentrations.

Platelet aggregation was performed within 4 hours of sample collection, and was measured optically using a dual channel aggregometer.0 A 250 ^L aliquot of PPP was transferred, using an adjustable piston micropipetter, to a disposable, siliconi2ed glass

cuvette in one channel of the aggregometer. A 225 \xL aliquot of autologous PRP was

transferred, using an adjustable piston micropipetter, to a disposable, siliconized glass

cuvette containing a small magnetic stir bar in the other channel of the aggregometer. The

samples were allowed to warm to 37°C in the aggregometer, and were continuously stirred at

900 rounds/minute to allow for adequate platelet distribution. Maximal transmission was

determined by measuring transmission of light through the PPP, and determined to be a

baseline transmission of 100%. Minimum transmission was determined by measuring light

transmission through the stirred PRP, and determined to be a baseline transmission of 0%.

A 25 piL aliquot of agonist was added to the cuvette containing PRP to induce

aggregation. Continuous stirring allowed adequate distribution of the agonist through the

PRP. During incubation, aggregation was recorded as the increase in light transmission

allowed through the PRP over time. Samples were recorded continuously until aggregation

was complete, at which time light transmission would reach a plateau. In cases of partial

aggregation, recording was continued until disaggregation was complete and light

transmission reached 0%.

0 Chnoro-log 440VS, Chrono-log Corporation, Haverton, Pennsylvania

57 Each sample was run in duplicate at two ADP and two PAF concentrations, giving 4 aggregation curves (run in duplicate) for each dog. If the aggregation curves recorded appeared markedly different, the sample was repeated in duplicate. Aggregation was evaluated using two variables; maximal aggregation and initial aggregation velocity. Maximal aggregation was measured as the total percentage increase of light transmission. Initial aggregation velocity was measured as the percentage increase in aggregation per minute. In samples where disaggregation occurred, time to disaggregation was measured.

1 minute

Figure 2.1 - Example of a platelet aggregometry curve. Percent aggregation is shown on the Y-axis and time is shown on the X-axis. Rate of aggregation is measured as the percent change in aggregation over the first minute of recording. Maximal extent of aggregation is measured as a percentage increase from the baseline until the aggregation response reaches a plateau.

Plasma Thromboxane Levels - Thromboxane B2 levels were measured using a

P competitive enzyme immunoassay kit (EIA). Thromboxane B2 is a stable metabolite of

36 TXA2, which is rapidly hydrolysed after synthesis. The kit uses a TXB2 acetylcholinesterase

(AchE) tracer in a constant concentration, while the concentration of free TXB2 in the

p Thromboxane B2 EIA kit, Cayman Chemical Company, Ann Arbor, Michigan

58 assayed sample is variable. The tracer and the free TXB2 compete for a limited number of

TXB2-specific rabbit antiserum binding sites in the assay wells. The amount of tracer that binds to the rabbit antiserum is inversely proportional to the concentration of free TXB, in the sample.

Ninety-six well EIA plates were used to perform the assays. The sample wells in the plate were coated with mouse monoclonal anti-rabbit antiserum binding sites. The plate included wells for an 8-point standard curve and wells to assay plasma samples to determine concentration of TXB2. Serial dilutions of the commercially prepared TXB2 included with the assay kits were performed to achieve standard curve points of 1000, 500, 250, 125, 62.5,

31.3, 15.6, and 7.8 pg/mL. Plasma samples were assayed in duplicate by adding 50 |xL of plasma to a well. Fifty \iL of TXB2 AchE tracer and 50 |xL TXB2 EIA antiserum (included with the assay kit) were then added to each sample well.

Each EIA kit was covered with a plastic film and allowed to incubate at room temperature for 18 hours. The plate was then washed 5 times with wash buffer to remove

unbound reagents. An AchE substrate reagent was added to the plate wells in aliquots of

200 fj,l and the plate was incubated on an orbital shaker in the dark for 30 minutes. The product of the incubation was a noticeable yellow colour, and the intensity of this reaction was determined spectrophotometrically at 412 nm by a computerized assay plate reader.1*

The intensity of colour change was proportional to the amount of TXB2 AchE tracer, and

inversely proportional to the amount of free TXB2, bound (Figure 2.2).

Data analysis was performed using a computer spreadsheet service provided by the

assay kit manufacturers. Samples with a coefficient of variation (CV) greater than 10%

between duplicates were assayed again in duplicate. A control subject was assayed in

q SpectraMax Miniplate reader, Biotek Instruments, Winooski, Vermont

59 duplicate on each assay plate. According to the manufacturer's data, cross reactivity of TXB2 with other eicosanoids is less than 1%. The detection limit of the assay was 11 pg/mL.

Inter-assay coefficient of variation for the TXB2 assays was 9.3%, and intra-assay coefficient of variation ranged from 2.4-9.0%. The TXB2 assay kit has been used in previous canine studies.37"41

Plasma Prostacyclin Levels - Prostacyclin levels were measured by using a

r competitive EIA kit to assay levels of the PGI2 analogue 6-keto prostaglandin (PG) Flct.

The kit follows the same principles as described for the TXB2 assay kit, with few technical differences. The eight-point standard curve for this kit included concentrations of 500, 250,

125, 62.5, 31.3, 15.6, 7.8, and 3.9 pg/mL. In this assay, 100 ^L of plasma was added to each sample well and each plate was initially incubated at 4°C for 18 hours. After reconstitution with the AchE substrate, the plates were then incubated on an orbital shaker in the dark for

40 minutes.

Spectrophotometric readings and data analysis were performed as with the TXB2 kits. According to manufacturer's data, cross reactivity of 6-keto-PG Fla with other eicosanoids is less than 1%. The detection limit of the assay was 11 pg/mL. Inter-assay

coefficient of variation for the PGI2 assays was 5.0%, intra-assay coefficient of variation ranged from 1-6.2%. The PGL assay kit has been used in a previous canine study.40

r 6-keto-prostacyclin Fla EIA kit, Cayman Chemical Company, Ann Arbor, Michigan

60 One-stage PT, activated PTT, and fibrinogen concentration - A 1 mL aliquot

of PPP was used to measure one-stage PT, activated PTT, and fibrinogen concentration.

Testing was performed by laboratory personnel at the Animal Health Laboratory, Ontario

Veterinary College, Guelph, ON, Canada, using an Amelung KC 4 delta analyzer (Trinity

Biotech, Ireland).

Statistical analysis — An analysis of variance (ANOVA), including treatment

carryover and period/effect as factors in the model, was run on the differences (pre-

treatment versus post-treatment) for the parameters of interest. If the overall treatment

effect was significant, a post hoc Tukey test was used to determine if there was a greater

change for that parameter between treatments.. A multivariate t adjustment was made to

compare within an individual treatment back to the baseline. The level of significance was

set at p s 0.05. All statistical analysis was performed using statistical software. *

61 Results

Platelet count, hematocrit, white blood cell count - No significant differences were detected in platelet count (Figure 2.2), hematocrit, or white blood cell count before or after treatments (Appendices la, lb, lc).

300

< o K, 200

*•> Pre c I Post 3 8 1 U too 10 &

aspirin carprofen deracoxib meloxlcam placebo

Figure 2.2 — Platelet count (mean ± SD) before and after 7 days of NSAID administration in 10 healthy dogs. The dashed line represents the lower limit of the laboratory reference interval for normal canine platelet count (117 x 109/L); upper limit of reference interval was 418 x 109/L.

Platelet function analysis as measured by the PFA-100 and platelet aggregometry - There was no significant difference in platelet function measured by the

PFA-100 before and after administration of aspirin, carprofen, or deracoxib. While the adenosine diphosphate closure time (ADP-CT) measured by the PFA-100 was mildly increased after meloxicam administration, this difference was not significant (p=0.07; Figure

2.3; Appendix Id). Prolonged ADP-CTs were measured in 2 dogs: dog 3 had a prolonged

62 ADP-CT (100 sec) after deracoxib administration and dog 7 had a prolonged ADP-CT (119 sec) after meloxicam administration. No dog had an ADP-CT below the reference interval.

Figure 2.3 - Closure time (mean ± SD) as measured by the PFA-100 before and after 7 days of

NSAID administration in 10 healthy dogs. The dashed lines represent the published normal reference interval of 52-98 sec.

Maximal platelet aggregation was not significantly affected by NSAID treatment when induced by 1 or 0.5 |xm of PAF (Figures 2.4 & 2.5; Appendices le & £). Maximal platelet aggregation induced by 50 |im ADP was significantly decreased from 65.2 to 52.7% after deracoxib administration (p=0.03; Figure 2.7; Appendix lh). Carprofen administration resulted in a mild decrease in maximal platelet aggregation induced by 50 and 100 [Am of

ADP, but these changes were not significant (p=0.059 and p=0.065, respectively). Rate of aggregation measured by percent change in aggregation over 1 minute was not significantiy affected by NSAID administration after aggregation was induced by PAF or ADP (Figures

2.8-2.11; Appendices li-11).

63 Figure 2.4 - Percent aggregation (mean ± SD) induced by 1 p,m PAF before and after 7 days of

NSAID administration in 10 healthy dogs.

Figure 2.5 - Percent aggregation (mean ± SD) induced by 0.5 firm PAF before and after 7 days of

NSAID administration in 10 healthy dogs.

64 Figure 2.6 - Percent aggregation (mean ± SD) induced by 100 ^m ADP before and after 7 days of

NSAID administration in 10 healthy dogs.

Figure 2.7 - Percent aggregation (mean ± SD) induced by 50 ^m ADP before and after 7 days of

NSAID administration in 10 healthy dogs.

* Signifkandy (psO.05) decreased mean post-treatment value when compared to the mean pre- treatment value.

65 Figure 2.8 - Rate of aggregation (mean ± SD) induced by 1 ^m PAF before and after 7 days of

NSAID administration in 10 healthy dogs.

Figure 2.9 — Rate of aggregation (mean ± SD) induced by 0.5 (xm PAF before and after 7 days of

NSAID administration in 10 healthy dogs.

66 Figure 2.10 — Rate of aggregation (mean + SD) induced by 100 [xm ADP before and after 7 days of

NSAID administration in 10 healthy dogs.

Figure 2.11 — Rate of aggregation (mean ± SD) induced by 50 ^.m ADP before and after 7 days of

NSAID administration in 10 healthy dogs.

67 Plasma prostacyclin and thromboxane levels - No significant differences were found in PGI2 levels after NSAID treatment (Figure 2.12, Appendix lm). Aspirin treatment decreased TXB2 levels, but this difference was not significant (p=0.07). Thromboxane B2 levels were significantly higher (p = 0.05) after deracoxib treatment compared to after aspirin treatment (Figure 2.13, Appendix In). The ratio of TXB2 to PGI2 was decreased after aspirin administration, although this difference was not significant (p=0.06). The ratio of

TXB2 to PGI2 was not affected by administration of any other NSAID (Figure 2.14,

Appendix lo).

300

E 200 s '".'.1 Pre I • Post

o IOO

aspirin carprofen deracoxib meloxicam placebo

Figure 2.12 - Prostacyclin (6-keto prostaglandin Fia) levels (mean ± SD) before and after 7 days of

NSAID administration in 10 healthy dogs. The dashed line represents the lower limit of detection of the assay (11 pg/mL).

68 Figure 2.13 - Thromboxane B2 levels (mean ± SD) before and after 7 days of NSAID administration in 10 healthy dogs. The dashed line represents the lower limit of detection of the assay (11 pg/mL).

Figure 2.14 - Ratio of thromboxane to prostacyclin (mean ± SD) before and after 7 days of NSAID administration in 10 healthy dogs.

69 Measures of secondary hemostasis - Secondary hemostasis was measured using prothrombin time (PT) and partial thromboplastin time (FIT). NSAID treatment did not have a significant effect on PT or PTT (Figures 2.15 & 2.16; Appendices lp & lq). Six dogs had mildly prolonged PT values (range: 15.1 to 16.7 sec) and 4 dogs had mildly shortened

PT values (range: 6.4 to 8.7 sec) prior to treatment. Five dogs had mildly prolonged PT values (one each after aspirin, deracoxib, and placebo, and 2 each after carprofen administration; range 15.2 to 19.3 sec) and 2 dogs had mildly shortened PT values (one each after aspirin and placebo administration; range 8.2 to 8.7 sec) after treatment. Three dogs had mildly prolonged PTT values prior to treatment (range 23.3 to 24.4 seconds). Four dogs had mildly prolonged PTT values (one each after deracoxib and meloxicam, and 2 each after carprofen administration; range 23.1 to 26.5 sec) after treatment. These mildly altered values for PT and PTT were not clinically or statistically significant. Fibrinogen level was significandy decreased after meloxicam treatment (p = 0.03) but remained within the normal reference interval (Figure 2.17; Appendix It). One dog had an elevated fibrinogen level (3.6 g/L; reference interval: 0.9-2.3 g/L) after placebo treatment.

70 20

<~, 15 J

« E *3 ;Pre .£10 XI (Post E 2 1 a. 5

aspirin carprofen deracoxlb meloxlcam placebo

Figure 2.15 - Prothrombin time (mean ± SD) before and after 7 days of NSAID administration in

10 healthy dogs. Dashed lines indicate the laboratory reference interval (9-15 seconds).

Figure 2.16 - Partial thromboplastin time (mean ± SD) before and after 7 days of NSAID administration in 10 healthy dogs. Laboratory reference interval is 15-23.5 seconds.

71 Figure 2.17 - Fibrinogen level (mean ±.SD) before and after 7 days of NSAID administration in 10 healthy dogs. Dashed lines indicated the laboratory reference interval (0.9-2.3 g/L).

72 Discussion

The purpose of this study was to examine tine effects of commonly used nonsteroidal anti-inflammatory drugs (NSAIDs) on primary and secondary hemostasis in healthy dogs.

Recent reports from the human medical literature have suggested that treatment with some

COX-2 selective NSAIDs may be associated with thromboembolic complications such as myocardial infarction.5"8 There is speculation that COX-2 selective NSAIDs may lead to an

9 43, imbalance in systemic prostaglandin levels, with a relative increase in TXA2 versus PGI2. '

44 Specifically, this study examined the effects of oral administration of therapeutic doses of aspirin, carprofen, deracoxib, and meloxicam on platelet function (measured with the Platelet

Function Analyzer [PFAJ-100 and optical aggregometry), prothrombin time (PT), partial thromboplastin time (PTT), fibrinogen levels, and systemic levels of thromboxane (TX) B2 and prostacyclin (PGI2) in healthy dogs. To the authors' knowledge, the present study is the first to measure the effects of NSAIDs on canine platelet function with optical aggregometry and the PFA-100.

Complete blood count (CBC) parameters (i.e., platelet count, hematocrit, and white blood cell count) and measures of secondary hemostasis (PT, PTT, and fibrinogen) remained within the normal reference intervals after NSAID administration in this study. Inhibition of COX by NSAIDs is not expected to alter the CBC or secondary hemostasis in veterinary patients or humans.

Analysis of platelet function by the PFA-100 and by optical aggregometry in this study showed minimal effects of NSAID administration on platelet function. The adenosine diphosphate closure time (ADP-CT) measured by the PFA-100 was not significantly affected by administration of any of the NSAIDs. Deracoxib administration mildly but significandy decreased maximal platelet aggregation induced by 50 ^m of ADP from a mean of 65.2% to

73 a mean of 52.7% (p-0.03). Aspirin, carprofen, or meloxicam administration did not significantly affect maximum extent of aggregation induced by ADP. Maximum extent of platelet aggregation induced by platelet activating factor (PAF) was not affected by administration of any of the NSAIDs. Rate of platelet aggregation was not affected by any of the NSAIDs in this study.

Plasma PGI2 levels were not significantly affected by NSAID administration in this study. Thromboxane B2 levels were significantly reduced after aspirin administration versus deracoxib administration (p=0.05), but were not affected by administration of carprofen or meloxicam.

As a primarily COX-1 antagonist, aspirin was expected to decrease platelet function in this study. There are conflicting results in the literature with respect to platelet function testing after administration of aspirin. Nielsen et al (2007) showed that approximately 7-8 mg/kg/day of aspirin administered orally to healthy dogs for four days significantly decreased platelet aggregation responses detected with whole blood aggregometry induced by ADP and prolonged EPI-CT, but not ADP-CT.43 In another study, aspirin decreased platelet aggregation responses to ADP and collagen at a dose of 4 mg/kg every 12 hours for seven days.40 Intravenous administration of 20 mg/kg of aspirin given once to dogs resulted in increased ADP-CT and EPI-CT measured by the PFA-100.34 Aspirin given at doses as low as 0.5 mg/kg orally every 12 hours decreased platelet aggregation in response to arachidonic acid (AA).26 However, other studies have shown a lack of effect of COX-1 inhibitors on platelet aggregation. Aspirin at a dose of 3.5 mg/kg every 12 hours for seven treatments did not alter platelet aggregation in response to collagen.46 Similarly, the COX-1 inhibitor phenylbutazone failed to alter platelet aggregation in response to ADP but decreased aggregation induced by arachidonic acid.47

74 It is unknown why aspirin at 10 m/kg administered orally every 12 hours failed to produce platelet inhibition in the present study. Aspirin has a dose-related effect on primary hemostasis and platelet function. Aspirin administration at and-thrombotic doses is reportedly sufficient to block platelet COX generation, leading to decreased TXA2 synthesis.

As platelets are anucleate, acetylation is irreversible and the platelet is unable to produce additional COX.48 However, ultra-low doses of aspirin may allow nucleated endothelial cells

49 to recover and continue production of PGI2. Doses exceeding 0.5 mg/kg administered over a period of several days may not result in platelet inhibition for several reasons. After oral administration, aspirin is rapidly hydrolyzed to form salicylate.50 Build-up of salicylate can reverse the anti-aggregatory effects of aspirin.26,51 Additionally, higher doses of aspirin

26 may lead to increased inhibition of endothelial PGI2 production. While PGI2 levels were not affected by aspirin administration in this study, a build-up of salicylate may have prevented aspirin from affecting platelet function tests. While aspirin blocks platelet COX generation, other platelet agonists (such as PAF, ADP, collagen, and epinephrine) can still induce platelet aggregation.52 The concentrations of PAF and ADP used in this study may have been sufficient to overcome the inhibitory action of aspirin, allowing platelet aggregation to proceed. Lower concentrations of ADP and PAF may have resulted in platelet dysfunction measured by aggregometry. It is unknown how die concentrations of

ADP and PAF used in this or other studies compare to the normal intravascular environment during primary hemostasis. Breed and inter-individual variations in platelet function among dogs have been documented35,53'54 and may account for the variability in the effects of aspirin on platelet function reported in this and other studies.

Up to 45% of the human population is resistant to the antiplatelet effects of aspirin.

Additionally, the optimal dose of aspirin that produces antiplatelet effects in humans is

75 55 60 variable. ' Aspirin blocks platelet TXA2 production and can attenuate full activation of platelets by other pathways and agonists. The mechanism by which some people are aspirin resistant is unknown, but it is thought that these patients are able to activate platelets by pathways independent of the TXA2 pathway, Platelets from humans with aspirin resistance have increased sensitivity to ADP, and patients with aspirin resistance have been shown to have higher plasma ADP levels.'1''62 Hypersensitivity to other platelet agonists such as ADP could explain the mechanism responsible for aspirin resistance.

As expected, TXB2 levels after aspirin therapy were significantly lower than those after deracoxib therapy. However, TXB2 levels after aspirin administration were not significantly different than baseline TXB2 levels, This suggests that oral administration of anti-inflammatory doses of aspirin may have little impact on circulating free TXB2 levels.

Similarly, Brainard et al (2007) found no change in free scrum TXB2 levels in dogs with osteoarthritis after treatment with 5 mg/kg of aspirin given orally every 12 hours. However,

40 a marked decline in platelet-produced TXB2 was identified in these dogs. This decline in platelet-produced TXB2 correlated with a mild and marked decrease in maximal platelet aggregation induced by ADP and collagen, respectively.40 These studies show that aspirin administration does not alter serum levels of free plasma TXB2 in dogs, but instead impairs the capacity of platelets to produce TXB2 when whole blood is allowed to clot, and the platelet-produced TXB2 may be the most appropriate TXB2 measurement that correlates with platelet function impairment caused by aspirin administration.

The lack of effect on maximal platelet aggregation after aspirin therapy may have important therapeutic implications, as aspirin is often used for anti-thrombotic effects in veterinary patients at an ultra-low dose (0.5 mg/kg/day), while anti-inflammatory doses used for aspirin range from 10 mg/kg/day to 35 mg/kg every 8 hours.63 Canine platelets are

76 known to be less sensitive to certain agonists (such as AA, ADP, and collagen) resulting from variations in platelet surface membranes, compared to humans.10' 53' 64"67 Further investigation into the effects of aspirin on platelet function and TXB2 levels at various aspirin doses is warranted to determine the significance of the findings reported in the current study and to investigate the suitability of aspirin as an antithrombotic in dogs.

In vitro and in vivo studies show that carprofen is preferentially a COX-2 selective inhibitor.37' 41'-68 This .study showed that carprofen had no significant effect on platelet function as measured by aggregometry or by the PFA-100.' Similar to a previous study,40

TXB2 and PGI2 levels were unaffected by administration of carprofen.

The effect of carprofen on platelet function has been variable in previous studies.

Some studies have demonstrated that carprofen decreased platelet aggregation induced by

ADP and collagen as well as decreased clot strength measured by thromboelastography

(TEG). 4CI'69 However, a study of short-term carprofen administration (for four days) in dogs with traumatic fractures showed no evidence of decreased platelet aggregation induced by

ADP and collagen.70 Carprofen administration had no effect on ADP-CT or EPI-CT measured by the PFA-100 in a previous study.71 One study reports prolongation of skin bleeding time with long-term carprofen use,72 which is contradictory to other reports of carprofen having no significant effects on BMBT.69'70

The lack of effect. on platelet function after carprofen administration was an expected finding. As a COX-2 selective inhibitor, carprofen is likely to have minimal impact on COX-1 in platelets and therefore no effect on platelet function, However, caution should still be used when considering the use of carprofen in patients with a primary hemostatic disorder or with concurrent anticoagulant therapy given the results of previous studies reporting an impairment of platelet function associated with carprofen use. While

77 carprofen is considered a COX-2 selective inhibitor, it likely has some COX-1 inhibition and may result in platelet function impairment. Further investigation regarding the effects of carprofen on platelet function is needed. The lack of effect of carprofen on free TXB2 and

PGI2 is not supportive of this COX-2 selective inhibitor producing an imbalance in circulating levels of these two prostaglandins.

Deracoxib administration resulted in a mild decrease in the extent of maximal aggregation induced by 50 |xm of ADP, but aggregometry induced by other agonists and

ADP-CT measured by the PFA-100 were not affected. This finding is contradictory to a previous study that found deracoxib therapy in dogs with osteoarthritis increased clot strength measured by TEG, suggestive of hypercoagulability, but did not alter platelet aggregometry induced by ADP or collagen.40 Consistent with a previous study,40 deracoxib did not alter TXB2 or PGI2 levels in the current study. The mild decrease in platelet aggregation produced by deracoxib administration reported by this study is unlikely to be mediated by a decrease in platelet TXB, production as this drug is highly COX-2 selective and did not change free plasma TXB2 levels. However, platelet-produced TXB2 levels were not measured and a subtle impairment of platelet COX-1 activity therefore cannot be ruled out. While deracoxib is considered the most selective COX-2 inhibitor tested in this study,37 it may still cause enough COX-1 inhibition to affect platelet function as evidenced by the mild decrease in aggregation induced by 50 [xm of ADP. No clinical signs of excessive bleeding (e.g., hematoma formation after venipuncture) after administration of either drug were noted in any of the dogs in the study. The clinical significance of the mildly reduced platelet aggregation induced by ADP after deracoxib administration is likely low but warrants further investigation.

78 Similar to carprofen and deracoxib, meloxicam is also considered to be a COX-2 selective NSAID. As expected, the platelet aggregometry results and the results from PFA-

100 analysis do not support a change in platelet function associated with meloxicam administration. Additionally, meloxicam administration did not alter TXB2 or PGI2 levels in healthy dogs. Intravenous or subcutaneous administration of meloxicam did not affect

ADP-CT or EPI-CT measured by the PFA-100, BMBT, or intraoperative bleeding score in previous studies.73"76 Similarly, platelet function is not affected by meloxicam therapy in humans.77,78 Because meloxicam administration did not appear to alter platelet function in healthy dogs in this and previous studies, it may be an appropriate choice for perioperative use. Whether meloxicam use in dogs with primary hemostatic defects or with concurrent administration of anticoagulant drugs is appropriate remains uncertain and requires further study. Like carprofen and deracoxib, meloxicam did not alter free plasma TXB2 or PGI2 levels and there is no evidence that these drugs create an imbalance in these systemic prostaglandins. However, the effect of these drugs on dogs with predisposition to hypercoagulability requires further investigation.

Compared to other studies of optical aggregometry assessment of platelet function, this study used higher concentrations of ADP to induce aggregation. Concentrations of

ADP typically used to induce platelet aggregation in platelet rich plasma range from 10 to 25

|iM,79'80 compared to the concentrations of 50 and 100 fiM of ADP used in this study. It is possible that the higher agonist concentrations used resulted in over-stimulation of the platelets and masked subtle platelet dysfunction. However, at the beginning of the study, both PAF and ADP were titrated to determine the lowest concentration of agonist that would result in maximal aggregation responses as measured by the optical aggregometry.

This concentration was used as the "high" agonist concentration, and therefore while it is

79 possible that the platelets were over-stimulated in this study it is considered unlikely.

Variability among breeds of dogs (hound crosses were used in this study) and inter- individual variability may account for the higher concentration of ADP required to induce platelet aggregation in this study.

Recendy, Brainard et al (2007) examined the effects of aspirin (5 mg/kg PO q 12 hours), carprofen (4 mg/kg PO q 24 hours), deracoxib (2 mg/kg PO q 24 hours), and meloxicam (0.1 mg/kg PO q 24 hours) on canine hemostasis.40 Each NSAID was given for

10 days with at least a 14-day washout period between NSAIDs. Platelet aggregation was performed using ADP and collagen agonists. Platelet aggregation induced by ADP was significantly decreased after treatment with aspirin, carprofen, and meloxicam. Aspirin also significandy decreased platelet aggregation induced by collagen. Thromboelastography showed an increase in one measure of hypercoagulability after deracoxib administration, while carprofen resulted in several parameters being consistent with hypocoagulation.40

The results presented by Brainard et al (2007) have several limitations. Platelet aggregation was not consistently performed in all subjects in the study leading to a small sample number (four to six dogs per drug tested). The study examined the effects of

NSAIDs on hemostasis in dogs with osteoarthritis. Systemic inflammatory conditions such, as sepsis and parvoviral enteritis are known to cause hypercoagulability attributable to upregulation of pro-inflammatory cytokines in dogs."' 1S Degenerative and inflammatory joint diseases in humans have been shown to activate the coagulation and fibrinolytic pathways to various degrees.81 Osteoarthritis is an inflammatory disease and its effects on the coagulation system and pro-inflammatory cytokine profile in dogs is unknown, but may have influenced results in the study by Brainard et al (2007). Additionally, drug administration

80 relied on owner compliance, which may result in variability of drug doses or dosing schedules.40

Graff et al (2007) demonstrated that COX-2 selective inhibitor therapy in healthy human volunteers did not alter maximal platelet aggregation as measured by platelet aggregometry, nor were platelet activation markers detected by flow cytometry affected. *

However, administration of COX-2 selective inhibitors resulted in a significant decrease in

PGI2 levels while TXB2 levels were unchanged. Additionally, COX-2 selective inhibitors caused increased TXB2 generation during platelet aggregation. This study showed that

COX-2 selective inhibitors may favour a relative increase in TXB2 levels compared to PGI2 levels, leading to a proaggreatory environment and the possibility of diromboembolic events.82 The study by Graff et al (2007) used NSAIDs that are considered to have a much higher COX-2 selectivity than die COX-2 selective NSAIDs approved for use in dogs.4'37,41,

68,83 ^e reiatjve imbalance in TXB2 and PGI2 levels reported by that study may be due to this higher COX-2 selectivity. In contrast to these data, selective COX-2 inhibition in another human study did not affect plasma TXB2 or PGI2 levels, but did lead to an increase

84 in urinary TXB2 excretion. Dogs are not as predisposed to development of myocardial infarctions and other thromboembolic complications compared to humans,10 suggesting that there may be other factors aside from selective COX inhibition involved in reports suggesting an increased rate of myocardial infarctions associated with COX-2 selective inhibitors in humans.

The effects of other NSAIDs on canine hemostatic function have been examined,

Therapy with ketoprofen, primarily a COX-1 selective NSAID, resulted in prolongation of

EPI-CT using the PFA-100 and decreased whole blood platelet aggregation induced by collagen.71, 85 Ketoprofen administration did not prolong BMBT in canine studies.75, 85

81 Tepoxalin, a dual COX and lipoxygenase (LOX) inhibitor, has both COX-1 and COX-2

activity in vivo. Tepoxalin has been shown to decrease TXB2 levels in dogs, but no studies have documented its effects on platelet function.86 As tepoxalin inhibits both COX-1 and

COX-2, it is expected to result in decreased platelet function.

2.4 LIMITATIONS AND FUTURE AREAS OF STUDY

A limiting factor of this study was the small number of dogs used. While aspirin administration was associated with lower levels of plasma TXB2, this result failed to achieve statistical significance. Using 10 dogs, the power for this measurement was limited (57%).

Increasing the number of subjects-in the study to 15 would result in a power of 89%. The increase in power may have resulted in a statistically significant reduction in TXB2 after aspirin administration. Increasing the number of study subjects may have also increased the power of the study sufficiently to result in statistically significant reduction in platelet aggregation induced by ADP after carprofen administration. To ensure a more comprehensive evaluation of platelet function, future studies should incorporate the use of

Col-Epi cartridges in PFA-100 studies, as well as other platelet agonists such as collagen and different agonist concentrations when employing platelet aggregometry. Markers of platelet activity, such as P-selectin detected by flow cytometry, may aid in platelet function evaluation in future studies. Other measures of hemostasis, including TEG, should also be considered for future studies. Measuring platelet-produced TXB2 levels may be more accurate in determining the impact of NSAIDs on platelet COX function and should be performed along with measuring free TXB2 and PGI2 in future studies in this area.

Additionally, measurement of serum drug concentrations would be helpful to determine that

82 each NSAID achieved therapeutic concentrations in the patient when platelet function and levels of TXB2 and PGI2 are being evaluated.

The results of this study have several important clinical implications that warrant further investigation. The failure of aspirin given orally at 10 mg/kg every 12 hours to inhibit platelet function raises questions regarding its utility as an antithrombotic agent.

Further study of platelet function and various doses of aspirin is needed. The COX-2 specific inhibitors carprofen, deracoxib, and meloxicam had minimal effect on platelet function in this study. Based on the results from this and previous studies, meloxicam may be an appropriate NSAID choice in the perioperative period given its lack of platelet inhibition. Study of NSAIDs in the perioperative period using assessment such as intraoperative bleeding score concurrently with platelet function evaluation, is needed.

Additionally, investigation into the use of these NSAIDs in dogs with underlying primary hemostatic disorders or with concurrent anticoagulant therapy is recommended to fully evaluate the safety of these drugs. There was no evidence of a prothrombotic environment created by administration of carprofen, deracoxib, and meloxicam to healthy dogs. The effects of NSAIDs on platelet function and levels of TXB2 and PGI2 in dogs with underlying disorders that predispose to hypercoagulability is needed to fully evaluate the safety of these drugs.

Conclusions

The results of the present study refuted some of the initial hypotheses. Aspirin, a

COX-1 inhibitor, did not result in a decrease in platelet function in this study. Therapy with

COX-2 selective NSAIDs did not consistently affect platelet function as measured by PFA-

100 and platelet aggregometry. Deracoxib administration produced conflicting platelet

83 aggregation data, resulting in decreased maximal aggregation induced by one agonist but not affecting aggregation induced by the other agonists. No significant effect on platelet function was detected with PFA-100 for any of the NSAIDs administered. Aspirin, carprofen, deracoxib, or meloxicam did not significantly change TXB2 or PGI2 levels compared to baseline levels. Use of COX-2 selective inhibitors did not result in a detectable prothrombotic state nor did COX-2 selective inhibitors produce a consistent impairment of platelet function in healthy dogs in this study.

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90 CHAPTER 3: CONCLUSIONS

This study demonstrated that administration of commonly used nonsteroidal antiinflammatory drugs (NSAIDs) had minimal effect on platelet function as measured by optical aggregometry and by the platelet function analyzer (PFA)-100 in healthy dogs. Of the NSAIDs used, deracoxib was the only drug that resulted in a mild defect platelet function as measured by platelet aggregometry induced by 50 \xxn of adenosine diphosphate

(ADP). Aspirin, carprofen, and meloxicam did not significantly alter platelet function.

While platelet dysfunction was not identified by the use of optical aggregometry or the PFA-

100, the possibility of platelet dysfunction undetected by these tests cannot be excluded.

Thromboxane (TX) B2 and prostacyclin (PGI^ levels were not altered by NSAID administration. There was no evidence of cyclooxygenase (COX)-2 selective inhibitors creating a prothrombotic environment in healthy dogs.

Summary

The major points derived from this research project include:

1. Administration of oral therapeutic doses of aspirin, carprofen, and meloxicam for

seven days did not alter platelet function in healthy dogs.

2. Administration of oral therapeutic doses of deracoxib for seven days resulted in a

mild decrease in the extent of maximal aggregation induced by 50 \im of ADP in

healthy dogs. However, no other measures of platelet function were affected by

deracoxib administration and therefore this effect is likely not clinically relevant.

3. The lack of effect of oral therapeutic doses of aspirin (10 mg/kg every 12 hours) for

seven days in healthy dogs was unexpected. Further evaluation of the effects of

91 various doses of aspirin on platelet function is warranted and may include studies

using epinephrine cartridges in the PFA-100, different agonists and agonist

concentrations for platelet aggregometry studies, and thromboelastography (TEG)

with platelet mapping to assess the effects of platelet antagonists.

4. Compared to baseline values, thromboxane B2 levels were not significantly altered by

administration of aspirin, deracoxib, carprofen or meloxicam for seven days in

healthy dogs. Thromboxane B2 levels were significandy lower after aspirin

administration only when compared to levels after deracoxib administration for

seven days in healthy dogs.

5. Prostacyclin levels were not altered by administration of aspirin, carprofen,

deracoxib, or meloxicam for seven days in healthy dogs.

6. Measures of secondary hemostasis (prothrombin time, PT; partial thromboplastin

time, PTT; fibrinogen) were not affected by administration of aspirin, carprofen,

deracoxib, or meloxicam for seven days in healthy dogs.

7. Evaluation of the effects of NSAIDs on primary and secondary hemostasis in

unhealthy dogs is warranted.

92 4.0 APPENDICES

Appendix la: Circulating platelet count (x 109/L)

Pre-treatment Post-treatment

DoS aspirin carprofen deracoxib mdoxicatn placebo aspirin carprofen deracoxib meloxicam placebo 1 247 250 235 • 271 227 240 226 235 195 218 2 353 346 335 358 351 319 300 304 330 340 3 184 194 201 199 228 190 198 213 217 184 4 3.39 315 383 316 322 308 301 279 292 291 5 223 284 240 225 247 200 279 261 225 251 6 188. 208 213 206 • 195 . 218 183 .206. .206 203 7 , 160 150 156 160 175 192 182 151 200 179 8 • ' 219 212 214 ' 214 201 202 242 . 159 •• 196. '200 9 -.284'•• • :271 290 246 170 278' • ,293 • '."242. 269 • 283 10 ": 216: •• • 285 281 250 .254 •287' 276 : 266- 309 268

Appendix lb: Circulating hematocrit (L/L)

Pre-treatment Post-treatment Dog aspirin carprofen deracoxib meloxicam placebo aspirin carprofen deracoxib meloxicam placebo 1 0.50 0.50 0.45 0.48 0,52 0,49 0.48 •0.47 0.50 0.50 2 0.51 0.54 0.54 0.54 0.52 0.51 0.51 0.52 0,51 •0.54 3 0.48 0.48 0.50 0.49 0,53 0.47 0.51 0.49 0.48 0.50 4 0.53 0;53 0.52 0,52 0.56. 0.52 0.54 ' 0.56 0.52 . ,0.50 5 0.S3 0.50 ' ' 0,52 0.51 0.49' • 0.51 - .0,49 ". 0.5$;. :"•••'0.51' 0,54 6 0.44 0.46 0.46 0.45 0.45 0.47- 0.47- •. 0.44 0.45 0.44 7 . 0.50 •' 0.52 0.53 0.51- 0.52.- • 0.51- ' 0,54' .'''.'•••.0.56' : 0.49 0.53 8 0.48 :* * 0.51 0.49 • 0,50 0.48 ' 0.45 0.45 0.49 • 0,49 0.49 9 0.44 0.44: 0.51 0.47 0.55 ' 0.47 0,47 0.45 0.47 0,47 10 0.47 0.48 0.48 0.48 0.48 0.49 0.49 6.53 0.49 0.50

Appendix lc: Circulating white blood cell count (x 109/L)

Pre-treatment Post-treatment Dog aspirin carprofen deracoxib meloxicam placebo aspirin carprofen deracoxib meloxicam placebo 1 6.9 7.2 7.0 6.6 7.3 7.1 6.5 . 7.7 6.7 7.5 2 8.1 7.9 8.1 8;5 9.2 7.8 7.9 7.2' 7.6 12.0 3 11.9 8,4 8.2 8.6 8.1 9.6 6,9 7.8 1 7.8. . 4.9 4 7.6 8.6 9.4 7.5 9.1 11.0 7,6 ' " ,8.8 n 7.2 5 4.5 ' ' 4.8 5.4 5.4 4.0 4.3- 5.5 . 6.8 5.4 4.3 6 8.2 9.1 10.2 .8.6 10.5 • 9.7 9.1 ' 7,1 8.6 8.9 7 • 6,8 • 7,9 8.8 . 6,5 6.8 '7.6 . 8.1 • '8.3' 8.3 6.1 8 6.8 '6.5 S.7 6.6 8.3 • 6.2 6.1 6.0 9.8 6.8 9 ' 6.7 6,7 7.8 7.5 7.1 6.8 7,8 7.1 7.7 7.3 10 7,7 6.2 7.1 6,9 6.8 7.6 7.4 8.1 7.4 8.6

93 Appendix Id: Closure time as measured by the PFA-100

Pre-treatment Post-treatment Dog aspirin carprofen deracoxib n-iclnxiomi placebo aspirin carprofen deracoxib meloxicam placebo 1 91 82 76 66 63 69 . 73 62- 63 73 2 54 58 . 58 56 69 54. 55 -1 . •• 60: • • 97 51 3 58 .. :. 76 75 ' 73 ..." '03- - 72- ' - - 97' ' .' .76 85 4 66 .'. 68 •• .. - 87 62 '>!' -•••;. 60: •• ".• '67' ''•- \'M: 60 82

5 • • 72 ' • •y"-6& • 67 '71 • "'•%• ••• -n- ••" - .- 91. - •••••; 70- ••; 66 82 . 77. 6 •78 ' ' '. •-' 77 : 82 71-- • - 88' : 66 '79 86 93 7 • - •• 85 '•' -• ' '55 ' 79 63 57 62. 65 " ' •' 74' 75 8 .62 65 57 59 58 65 61. 87' 68 69 9 74 75 74 72 76 83 84 73 63. 73 10 55 75 60 57 55 96 83 63 76 54

Note: Values outside of the published reference range (52-98 sec) are highlighted by darker background shading.

Appendix le: Maximal platelet aggregation with 1 |xm of PAF

Pre-treatment Post-treatment Dog aspirin carprofen deracoxib meloxicam placebo aspirin carprofen deracoxib meloxicam placebo 1 68.1 69.4 70.0 63.8 58.8 63.1 . 68.8 '69.4 63,8 63.1 2 64,4 77.5 63.8 79,4 31 ;9 • 71.9 41.9- 73.1 71,9 80.0 3 57.5 63.8 67,5 65.6 66.9 65,0 60.6 76,9 65.6 65.6 4 80.0 53.1 58.8 64.4 63.8 70.6 74.4 61.3 54.4 60,6 5 73.8 65.0 78.8 73.8 75.6 81,3 55.0 80.6 71.9 65.6 6 84.4 ' 64.4 70.6 73.1 73i8 76.9 75.0 • 56,9 68.1 55.0 7 60.0 60,6 . 61.3 74.4 60.6 71.8 58.8 •59..4- 66,3 63.8 8 67J5- 68,1 61.3 65,6 66.3 ,66,9 71,3 60.0 66.9 79.4 9 64,4 -• '•'- 58.1 69.4 61,3 65.6 78.8 57-;5-. ; 64:4 -60.0 ' 64.4 10 74.4- , 75.6 • 67.5 69.4 61.3 • 70,6, 68.8 ' ¥": '60'D; 68.1. 64.4

Appendix If: Maximal platelet aggregation with 0.5 [Am of PAF

Pre-treatment Post-treatment Dog aspirin carprofen deracoxib meloxicam placebo aspirin carprofen deracoxib meloxicam placebo 1 55.0 66.3 60.0 61,9 65,0 66,3 81,3 87.5 67,5 65.0 2 63.8 86.9 55.6 21.9 65.6 73,1 66,3 76.9 63.1 65.6 3 58.1 61.9 65.0 78.8 63.8 69.4 66.9 81.3 76.9 63,8 4 '71.9 55.0 58.1 65.0 50.0 65.6 65.0 58.8 77.5 50.0 5 69.4 57.5 74.4 76.9 70.0 79.4 54.4 73.8 75.0 "0.0 6 72.5 55.0 68.1 76.3 71.3 78,8 71.3 . 65.0. 64.4 71.3 7 64.4 62.5 71.9 56.9 62.5 78.8 59.4 60.0 63.1 62.5 8 • 71,9- -':-.--75.6'- ' 61,3 66,3, • -• =70.4, .•/5&3 • -. 78.1' •;--.;>v60.'6" .-"-; 67.5 7U.U : ! 9 6%'S' ' 55;0' V -76.3. .53*8-'- .; ••:'•&,(>••• . \70;6'' '*'.- ':7S#"'. ;::f::?U& ••r.'"-*MV- .,80.0 10 1AA 78.1 68.8 61,9 68.1' ' 67;5; 68.8' •'- .' 61;9 ., .'69.4 ,'68.1

94 Appendix lg: Maximal platelet aggregation with 100 (Am of ADP

Pre-treatment Post-treatment Dog aspirin carprofen deracoxib meloxicarn placebo aspirin carprofen deracoxib meloxicarn placebo 1 54.4 57.5 67.5 47.5 55.6 40,6 21.9 63.1 75.0 43.8 2 66.3 62.5 62.5 60.6 61.3 65,6 43.1 45.0 61.9 55.0 3 51.9 62.5 60.0 53.8 63.8 63.1 56,3 56.3 31.3 65.0 4 62.5 65.0 56.9 60.0 54.4 75.6 69,4 41,9 68,1 53.1 5 59.4 6S.8 51.3 59.4 57,5 55,0 70.6 75.8 73,1 76,9 6 52.5 75.0 70.6 58.8 69.4 72.5 ,59.4 513 . 68.1 75.6 7 56.3 813 70.0 33.8 60.6 • 47.5 ' 63.1 • 61.3 ! 61.3 ,. 58.8 8 -. 71-? 75.6 •"".68.8' 46.3 . 7SJ 36,3 ••-.• -:'72.'5' ,/,v'"56.9 : 58.1 58.8 9 ..-.,.71.3' ... '(St9 ' . 65.6 74,4 68,1 :-"7&a. . . - -H4-.; -..'.. ,81.3: ;'\55:0. 66.9 10 ' 67.5'' •••'-' '60:0 66,9 - • -71,3 63.8" 70.&- !-';'. ' 40.6 ' • 56:3 66.9 66.3

Appendix In: Maximal platelet aggregation with 50 \im of ADP

Pre-treatment Post-treatment Dog aspirin carprofen deracoxib "icloxicam placebo aspirin carprofen deracoxib meloxicarn placebo 1 60.0 65.0 65.6 31.3 70.6 44.4 66.9 67.5 35,0 61.9 2 50.6 78,1 69,4 75.0 42,5 58.1 44.4 60.0 55.0 60.6 3 54.4 48.1 48.8 55.0 65,0 38.8 42.5 51.3 . 60,6 54.4 4 55.6 57.5 68.8 61.3 55X) • 56.9 45.0 53.1 33.8 31.9 5 65.0 70.0 83.8 73.1 57.5 75,0 21.9 •59.4 58.1 . 68.1 6 64.4 53.1 61.3 68,1 71,9 65.6*. • 66,3 57.5. 53.1 19.4 7 . 40.0 "' •' 54.4 66.9 •58.1 56,3 69,4 51.9 • ,'.;;.:,. .35:6 ".. 33.8 ' 53.1' : 8 .69.4 ••,. 58.8' •• 58,8 . 68.1 71;3 . 52.5' .•- 68,8 „ ; .. ' '44,4 " ;" = -. 58.1. 68.1

9 ..60.6 51.9 •• 70.6 61.9 ' 784- 68.8 '•••'. 40.0 '••••-.• 50:0- •f • 56.3. •'-' 57.5 10 62.5 71.9 58.1 66.9 73,1 43.8 54,4 •' 48.1 66.9' • 67.5

95 Appendix li: Rate of platelet aggregation with 1 |im of PAF

Pre-treatment Post-treatment Dog aspirin carprofen deracoxib meloxicam placebo aspirin carprofen deracoxib meloxicam placebo 1 9.8 17.9 16.3 12.9 13.1 13.1 13.3 18,1 13,5- 12.9 2 19.1 14.0 15.8 18.5 6.9 19.1 15,0 •14.1, 13.5. 14.8 3 ' 17.6' - • • it.s-- 14.4 16.3 14.6 • "14.4 13.8 ' • 14.9 •J.' '•: 14.9 : 16.0 4 • 17.3. '• 10,1 •• 10.3 15.9 • 11.5 '12.5 , 14.6- . 12.9 1.1.6 •••••12.5 5 173'' 13.1. 22,1 16.6 • 19.1 . 22.6 13,5 . / 15.6' .17.4. . 19.0 6 17,8 12.9- -• 15.9 17.6 16.9 ' 19.1 20.0 15.1 . 14.5 " 6.5 7 . 11.0 13.9 13.0 12.6 14.0. 14.3 •11,5 12.4 11,6 16.0 8 13.3 ' 15.3 13.5 13.9 16.4 15.0 14.5 10.3 17.3 15.3 9 .16.0 15,1 13.9 14.4 18.1 •17.8 14.6 16.9 • 13.5 15.9 10 15.6 17.6 15.6 15.3 14.5 15.4 17.5 13.8 15.0 15.4

Appendix lj: Rate of platelet aggregation with 0.5 \im of PAF

Pre-treatment Post-treatment

DoS aspirin carprofen deracoxib meloxicam placebo aspirin carprofen deracoxib meloxicam placebo 1 9.1 • 19.5 n.6 12,0 11,5 14.6 16.1 ' .'19.4 19,1 13,9 2 • -11.5 • 18.0 15.0 10.4 • ' 12,5/' " 11.5 '•- •'15-3 '/•.- 16.1 12,5 13.6 3 • • 13,1 • 10.3 •15.5 16.5 16.1 - 14.4 14.1 18.1 • • 16.4 13.6 4 ; 17.8 11.5 - '- 13.5 15.5 ' i'i,4.' :12'.6! '13.3 15.'8" ' ' 12.8 14.9 5 16.9 13.5 21.3 18.8 19.4 20.0 ' 12.5 ' 16.4 17.4 16.5 6 ' 16.0 13.5 16.5 18.5 16,4 22.9 19.5 14.8 15.0 7.6 7 14.0 14.5 12.9 9.9 14,3 15.8 13.9 12.8 12.1 17.5 8 14.8 16.6 14.6 14.6 15.4 14.3' 14.4 10.9 17.5 16.1 9 15.3 13.4 15.9 13.4 23.3 17.5" 15.0 17,6' 13.3 16.8 10 • 15,1 19.3 15.5 17,0 17.5 15.1 17.8 14.9 14.9 16.4

Appendix Ik: Rate of platelet aggregation with 100 (xm of ADP

Pre-treatment Post-treatment

DoS aspirin carprofen deracoxib meloxicam placebo aspirin carprofen deracoxib meloxicam placebo 1 7.1 16,5 12.5 13.4 11.8 11.0 15.0 12.9 10.3 10.3 2 13.5 14.5 13,4 12.6 8.8 13.5 12,6 ' 13.0 • 8,4 14.0 3 - 10.5 1S.4 10.1 12.8 12.0 10.9 11.6 14.6 14.5 12.6 4 17.9 11.3 12.9 12.1 8.5 12.4 12.0 12.6 8.3 10.0 5 12.6 ' 10.1 16.6 11.5 13.3 16.0 8.4 12,0 12.8 13.9 6 13.3 12.3 14.9 15.5 13.5 13,0 16.6 12.1 13.3 5.0 7 8.4 10.8 9.8 10.4 9.5 12.8 8.5 10,5 8.0 11.6 8 • 15;5- • 13.8 8.9 12,3 12.6 13.8 12.8 7.9 15.5' 13.5 9 '.' 14.4- . ' '11.5 .13.4 ' ' 10.8 • 45,9. "'16".5'' ': ."12.3 :'- .. 12.3 . 11.5 12.5

10 13,0 .15.5 11.(5 11.4 '"" »#•'. ; 11.0- •'.". -»J3.a. v.'\ 'U£\ .;•:•'. J2;5" ll.l

96 Appendix 11: Rate of platelet aggregation with 50 \im of ADP

Pre-treatment Post-treatment

DoS aspirin carprofen deracoxib meloxicam placebo aspirin carprofen deracoxib meloxicam placebo

1 6,8- 446 • 15.3 • ••• 8,5 9.3 • .10.3 • •• 13.5 •••• .13.0 :...133 .. 10.5 2 ; 12.3 ' 13.4- ' 13.5 14.8 . 6,9 ' ii 1 ' ' 111 ' ' '. ' 143" ' 8.3' 11.6 3 10,9 .12.9 ' 9.6 12.5 11.6 9.4 10.3 13,3 12.8 11.6 4 12.1 11.4 19.1 14.5 10.9 11.5 11.4 12.6 9.1 10.5 5 12.5 11.4 17.6 11.8' 12.4 ' 19.3 8,3 11.1 12.5 14.4 6 13.1 10.4 10.9 13.8 14.5 ' 14.6 16,0 13.4 13,6 5.4 7 8.0 •9.1 11.0 9.9 11.0 11.9 8.5 7.6 7.0 13.0 8 • 15.0 . 11.6 8.3 .12.6 13.9 1.1,1 12,0 7.5: 13,6 .12.4 9 13.9 10.4 13.3 10.9 .18,0.. • 14.5" . 10,8 .*... 12.4 ••. n.i 13.3 10 j 11.4 J 4.4 11.5 11.5 14.3 11.4 12.8 11.0 11.6 12.6

Appendix lm: Prostacyclin (pg/ml)

Pre-treatment Post-treatment Dog aspirin carprofen deracoxib meloMCiun nlacebo aspirin carprofen deracoxib meloxicam placebo 1 243.7 156.6 249.5 204.8 113.5 132.5 159.2 269.5 213.9 111.0 2 120.0 112.0 112,3 224.5 229.2 133.5 62.8 214.0 166.2 237.7 3 145.9 225.1 231.0 82.4 175.8 187.1 150.4 221.4 182.2 177.2 4 77.9 98.6 225.1 174.3 128,4 109.8 216.2 114.8 156.3 159.5 5 139.9 209.9 202.5 142.0 209.1 320.0 143.0 266.6 82.9 160.6 6 185.8 173.0 83.6 214.9 107.3 152.7 111.0 86,2 291.4 188.5 7 183.7 . , 154.0 128,0 120.4 148.0 237.3 157.8' ." . 223.5 249.6 . 118.5 8 122.4 ' - 166.9 207.5 - 88.3 174.8' 154,7" , •' &A' - ''• 217.7.; ' ' '$3-5 160.0 9 121.4 • 109.8- -104.8 - 206,3 - -ms- •<\m\ v-r-pm-.•••"••V2WJ0T ' V:l'8.&4/ , 118.4 ,! 10 AD0:\ /' wU 237.9 ' 128,3 144.'4 'toKr ".""t894 *"-" -&y 302.1 228.1

Appendix In: Thromboxane B2 (pg/ml)

Pre-treatment Post-treatment Dog aspirin carprofen deracoxib meloxicam placebo aspirin carprofen deracoxib meloxicam placebo 1 460.8 425.0 425.0 173,6 460.8 201.0 538.0 470,2 143.5 302.6 2 486.6 197.2 75.2 271.5 425.0 227.0 555.7 306.6 377.1 152.3 3 105.6 220.0 277.6 310.3 14.8 5.2 281.2 291.8 171.0 382.7 4 11.6 113.7 204,4 ' ' 98.2 37,4 . 66.1 246.3 298.7 173.1 193.7 5 - 297.9 . .75.6 . 257.5 346.3 81.6. 2664 105,6 342.7 ' .145.2 27,6 6 418.6' 252.4 235.5 136.1 1'98.2: 289.2, 7,7.7 135.5, ' ,28.9 178,2 7 162.9- ' -446.0- ; 171,6- -' 186.8 224.9 - 1932 605,0* .'• • ;365.1 69.7 214.0 8 264.0 •87.1 406.0 291.0 •; :M.\ 89.4- •'... 82,0 :•.- / 322.7 ' ,11.7 258.1 9 85.2 . 208.2 109,9 12.1 • 342.1 29.5 4.6 . ,'.'• '690.7 .138:8 104.1 10 , ..728.8 39,2 289.7 72,0 .438.6 35S.6 ' 214.1 82::0 •'.. 78.4' 393.6

97 Appendix lo: Ratio of Thromboxane B2 to Prostacyclin

Pre-treatment Post-treatment Dog aspirin carprofen deracoxib meloxicam placebo aspirin carprofen deracoxib meloxicam placebo 1 1 9 n -* 1." "h 4.1 1.5 3.4 l/7 or -> - 2 4.1 1 *! i)~ I 2 19 1." 1.0 14 2 3 0.6 3 D.~ 10 1 2 3 8 0.1 00 19 1.3 0.9 22 4 0.1 1.2 ".9 0.6 0.3 0.0 1.1 2.6 1.1 1.2 5 2.1 0.4 1 3 2 4 0 4 0.8 0." 1.3 1.8 0.2 6 2 3 1.5 28 U 6 J.8 19 0.7 1.6 0.1 0.9 7 ()') 2.9 r" ill I 6 1 5 0 8 3 8 16 03 IS 8 22 0.5 2.H 5.$ 1.0 0.6 0 8 1.5 0.1 1.6 9 o- ll> 1 0 u I 3B 0 2 0 0 25 0.7 09 10 4 9 0 2 I 2 ii 0 3 0 3 4 1.1 l"f 03 1-

Appendix lp: Prothrombin time (seconds)

Pre-treatment Post-treatment DOR aspirin carprofen deracoxib meloxicam placebo aspirin carprofen deracoxib meloxicam placebo 1 11.7 . 12.3 11.2 12.0 14,9 11.4 9.1 11.1 13.3 2 122.. ;. is.?. 13.8 •14.0 ' ' 14.2 X&iTT- 11.6 3 •-UA- -'£'•. •••r'H.D' ;*""• til ' 11.3. / 10.3- •'• •' 9.2; "•; 8.0* •. 11.4' '•• 'MA. : • 12.4 4 • * 13.2 . 13.8 11.6 13.4 9,6 11.0 13.3 5 ; ' . "ri.3 12.9 12.4 13,0 11.7 15.0 12.4 14.2 6 • 12.8 12,4 11,4 13,6 11.9 12,4 12,9 11.4 7 12.1' 11.8 11.2 13.1' 12.4 12;2 13.3 8 11.7 10.8 12.3 12.5 10.7 11.0 10.7 11.9 12.2 9 11.4 11,6 11.7 10,8 11.2 11,1 11.0 10.4 •Hi 10 12.8 10.7 12,8 11.8 11,8 12,1. , . 9.3 11.2 12.8

Note: Values outside of the laboratory reference interval (9-15 sec) are highlighted by darker background shading.

98 Appendix lq: Partial thromboplastin time (seconds)

Pre-treatment Post-treatment Dog aspirin carprofcn dcracoxib meloxicam placebo aspirin carprofcn dcracoxib meloxicam placebo 1 .19.3 ' 23.0' 19.6 19.7 20.3" 19.7 20.3 22.0 21.0 19.0 2 17.3 16.0 17.4 17.6 21.3 17.7 17,3 16.8. 19.1 17.7 3 20.0 18.5 IHI 19.S 19.2 20.7 22.3 18,9 20.3 19.3 4 19.4 18.4 19.4 18.6 18.7 20.0 20.9 .19.4 21.3 5 19.5 19.1 20.6 19.5 19.8 17.8 19,6 17.3 19.5 19.5 6 18.9 . 19.1 19.2 18.6 18.6 19,1 18.8 17.3 18.6 17.2 7 18.6 18.3 18.7 21.3 19.7 21.7 19.1 17.9 22.3 19.7 8 18.7 2o;o 19.1 19.8 18.8 18.4 , , 1,8.4 •19;5 22.3 9 15.9' ' . 19.7 21.9 18.5 16.8 19.3 ..".-; is.?.; •; ;- 18.1. 19.1 10 18.4 1

Note: Values outside of the laboratory reference interval (15-23.5 sec) are highlighted by darker background shading.

Appendix lr: Fibrinogen levels (g/L)

Pre-treatment Post-treatment Dog aspirin cai-profcn dcracoxib meioxicam placebo aspirin carprofcn dcracoxib meloxicam placebo 1 1.2 1,3 1.2 ' 1.4 1.2 1.2 1.2 1.4 1.2 1.1 2 1.8 1.7 1.9 1.7 2.1 1.8 1.7 1,7 1.5 2.1 3 1.2 1.2 1.6 1.2 0.9 • 1.3 1.2 .. 1.0 :• '.".i,o. jj{|H8|fl|yBfp| ; 4 ' 2.0' 1.5 1.9 1.4 1.5 1.6' • . 13 ''"•'•>•£&. -< "• i-2' ...... ,13 5 1.4. ' 1.2' 1.3 1.5 ' .1:7 1.3 • - -' 1.5 '' v.y-.i^ p::-'.. ,'i;s • • '1,5 6 '1.4 1.4 1.3 ..'1.5' • 1.5 -.- 1.4. r-- 1.4. ' 1.3 7 • 1.4' 1.3 1.2 1.4 . 2.0 ' 1.3 1.3- 1.1 1.2 1.7 8 ' • 1.3 '' 1.2 ' -1.3 1,3 1.3' 1.2 1.5 - 1.8 1.3 1.2 9 1.6 1.4 1.5 1.5 1.3 1.3 1.3 1.5 1.5 1.2 10 1.2 1.6 1.4 1.8 1.3 1.2 1.2 1.4 1.4 1.4

Note; Values outside of the published reference range (0.9-2.3 g/L) are highlighted by darker background shading.

99