Animal Models Of Thrombosis Research Articles

Tissue-type plasminogen activator: a historical perspective and personal account

Over the past two decades tissue-type plasminogen activator (t-PA), the main physiological plasminogen activator, has been developed as a fibrin-specific thrombolytic agent for the treatment of various thromboembolic diseases. Milestones in this development include: first purification of human t-PA from uterine tissue, elucidation of the interactions regulating physiological fibrinolysis, thus providing a molecular basis for the concept of fibrin-specific plasminogen activation, first animal models of thrombosis and pilot studies in patients supporting the therapeutic potential of t-PA, cloning and expression of recombinant t-PA providing sufficient amounts for large scale clinical use, and demonstration of its therapeutic benefit in large multicenter clinical trials, mainly in patients with acute myocardial infarction (AMI), but also in patients with massive pulmonary embolism, ischemic stroke, deep vein thrombosis and peripheral arterial occlusion. Genetically modified variants of t-PA have been developed for bolus administration in patients with AMI.

Synthetic selective inhibitors of coagulation factor Xa strongly inhibit thrombin generation without affecting initial thrombin forming time necessary for platelet activation in hemostasis

DX-9065a and JTV-803, synthetic selective inhibitors of activated factor X (FXa), have recently been demonstrated as strongly effective antithrombotic agents in animal thrombosis models, yet with a low risk of bleeding. The aim of the present study was to elucidate these characteristics. Using a chromogenic assay with purified coagulation factors, 73.9% of thrombin generation was suppressed by the addition of DX-9065a (0.20 microm) and 75.7% by JTV-803 (0.18 microm). Inhibition by argatroban (0.19 microm) was less (36.0%) and initial thrombin forming time (T50), the time required to generate 50% thrombin activity in vitro, which is considered important for platelet aggregation in hemostasis, was significantly prolonged by argatroban. In contrast, DX-9065a and JTV-803 had no apparent influence on T50, suggesting that initial thrombin was formed immediately, as in the control. We also investigated platelet aggregation in defibrinated plasma induced by tissue factor, to clarify whether initial thrombin contributes to hemostasis. Aggregation was not affected by the addition of either FXa inhibitor, whereas it was significantly reduced by argatroban. Our results suggest that initial thrombin, which is formed despite the presence of a FXa inhibitor, can activate platelets. We concluded that DX-9065a and JTV-803 are able to inhibit thrombin generation significantly without affecting the formation of initial thrombin for platelet activation, which may contribute to hemostasis through the preservation of normal bleeding time.

The role of sulfatide in thrombogenesis and haemostasis

In 1961, Wago et al. reported a potential anticoagulant role for sulfatide using animal experiments. Since then there have been many studies of sulfatide in the field of thrombogenesis/haemostasis, yielding contradictory conclusions. Some report that sulfatide has anti-thrombotic activity because it prolongs clotting time, inhibits platelet adhesion, and prolongs bleeding. Others report that sulfatide induces thrombosis in animal models. This mini-review is a chronologic review of reports examining the role of sulfatide in thrombogenesis/haemostasis together with the introduction of data from our laboratory and a discussion of the possible mechanisms underlying these curious phenomena.

Development and applications of animal models of thrombosis.

Development and applications of animal models of thrombosis.

A prospective, nonrandomized, open-labeled pilot study investigating the use of magnesium in patients undergoing nonacute percutaneous coronary intervention with stent implantation.

Magnesium has recently been shown to inhibit acute stent thrombosis in animal models. This study tested the feasibility of magnesium administration in patients undergoing nonacute percutaneous coronary intervention with stent implantation. Twenty-one patients undergoing nonemergent percutaneous coronary intervention were enrolled and received intravenous magnesium sulfate (2-g bolus over 20 minutes prepercutaneous coronary intervention, followed by 14 g over 12 hours infusion). safety outcomes--hypotension, bradycardia, bleeding complications and heart block within first 24 hours; angiographic outcomes--acute thrombotic occlusion and need for platelet glycoprotein IIb/IIIa inhibitor bailout; and clinical outcomes--death, myocardial infarction, recurrent ischemia, and need for urgent revascularization at 48 hours and 30 days. No significant effects on heart rate or blood pressure, major bleeding complication, or new electrocardiographic changes were observed. Angiographic thrombus was visualized in two cases, and coronary artery dissection in one case poststent deployment. None of these cases required the use of glycoprotein inhibitors for bailout. Death, myocardial infarction, recurrent ischemia, and need for urgent revascularization were not observed. The serum magnesium level increased from 2.1 +/- 0.3 mg/dL at baseline to 3.5 +/- 0.8 mg/dL at the end of the infusion (P <.0001). Platelet activation was significantly inhibited at the end of the magnesium sulfate infusion. Intravenous magnesium sulfate has been demonstrated as a feasible and safe agent in patients undergoing nonacute percutaneous coronary intervention with stent implantation. A randomized clinical trial comparing magnesium with glycoprotein inhibitors during percutaneous coronary intervention is warranted.

Activated thrombin-activatable fibrinolysis inhibitor attenuates spontaneous fibrinolysis of batroxobin-induced fibrin deposition in rat lungs.

Studies have shown that inhibition of TAFI by small peptides enhances pharmacological effects of tPA in animal models of thrombosis, suggesting that TAFI modulates the fibrinolytic system. In this study, we investigated the effect of activated human TAFI (TAFIa) on endogenous fibrinolysis in a rat model of intravascular fibrin deposition. (125)I-labeled fibrinogen was injected intravenously followed by a bolus injection of batroxobin, a thrombin-like enzyme. Batroxobin cleaved fibrinogen to form insoluble fibrin that was deposited in tissues, including the lungs. This was shown by a decrease of radioactivity in the blood as a result of consumption of (125)I-labeled fibrinogen and an elevation of radioactivity in the lungs 5 min following batrox-obin administration. Endogenous fibrinolysis was detected by a gradual increase in radioactivity in the blood and a decrease in radioactivity in the lungs at 30 min, an indication of radiolabeled fibrin degradation products (FDPs) being released into the circulation from the tissues. Intravenous administration of human TAFIa dose-dependently attenuated the later phase reduction of radioactivity in the lungs. When the dose of TAFIa was 218 micro g/kg, giving a peak plasma level of TAFIa 0.9 +/- 0.05 micro g/ml, the spontaneous fibrinolysis was completely prevented. These results provide direct evidence that an increase in circulating TAFIa impairs endogenous clot lysis in a rat model of fibrin deposition.

Overview of anticoagulant drugs for the future

More efficacious, safer, and easier to use anticoagulants are under development. Multiple agents have been shown to be effective in ex vivo or animal thrombosis models and several have progressed to clinical studies. Investigators have not yet determined if pharmaceuticals that inhibit coagulation factor activity earlier in the cascade (for example, inhibitors of tissue factor/factor VIIa, factor IXa, or Xa) are superior to those that block the cascade at a later point. Orally bioavailable drugs for the long-term treatment of thrombotic disorders, particularly those that do not require monitoring, are needed and are under development. Local delivery of anticoagulants or genes modulating anticoagulant control at sites of increased thrombogenicity, such as in diseased arteries, is a promising treatment modality that may decrease systemic bleeding problems. Much about the initiating pathophysiologic events leading to venous thrombotic disease needs to be elucidated before such local therapy can be tested in the venous vasculature. While awaiting better anticoagulants to become routinely available, we need to improve patient management with existing drugs by instituting anticoagulation clinics, promoting patient self-monitoring, and improving efforts to educate patients and health care providers about the use of anticoagulant drugs. Semin Hematol 39:145-157. Copyright 2002, Elsevier Science (USA). All rights reserved.

Function of Tissue-type Plasminogen Activator Releaser on Vascular Endothelial Cells and Thrombolysis In Vivo

The effect of monosodium[2-(6-hydroxynaphthalen-2-yl)-6-methylpyrimidin-4-yloxy]acetate dihydrate (JTV-926) on fibrinolysis was investigated in vitro and in vivo. JTV-926 released tissue-type plasminogen activator (t-PA) from human vascular endothelial cells in a dose-dependent manner. The thrombolytic effect of JTV-926 was studied using three animal thrombosis models; a photo-irradiation-induced mouse carotid artery thrombosis model, a photo-irradiation-induced rat femoral artery thrombosis model and a thrombin-induced rat venous thrombosis model. In the mouse thrombosis model, t-PA deficient mice (t-PA-/- mice) and their wild-type (t-PA+/+) were used. JTV-926 was injected as a bolus 30 min after the interruption of blood flow by an occlusion thrombi. Blood flow was continuously monitored for 180 min after intravenous administration of JTV-926 (1 mg/kg). Although the recanalization rate of the occluded artery was 37.5% in t-PA +/+ mice with the vehicle control, it increased to 75% in t-PA+/+ mice after JTV-926 administration. However, when JTV-926 was administrated in t-PA-/- mice, vascular recanalization was not observed in any arteries. In the photo-irradiation-induced rat femoral artery thrombosis model, intra-duodenal administration of JTV-926 induced thrombolysis. Moreover, in the thrombin-induced rat venous thrombosis model, the dose-dependent thrombolysis was also observed by oral administration of JTV-926. It was suggested that JTV-926 revealed a sufficient thrombolytic effect through the absorption from the intestine. Thus, a newly synthesized compound, JTV-926 induced t-PA release from vascular endothelial cells and effective thrombolysis in vivo.

Orally active heparin and low-molecular-weight heparin.

The heparins, (unfractionated heparin (UFH) and low-molecular-weight heparins (LMWH)) given by subcutaneous or intravenous injection have been used extensively in the prevention and treatment of both venous and arterial thromboembolic disorders. The increasing use of the heparins, LMWHs in particular, in the out of hospital setting has stimulated interest in the development of orally absorbable antithrombotic agents that require little or no monitoring, and this includes the heparins. UFH or LMWH delivered orally has been shown to have an antithrombotic effect in animal thrombosis models although there is little change in plasma coagulation tests. The addition of a simple organic chemical N -(8-(2-hydroxybenzoyl)amino)caprylate (SNAC) to UFH markedly enhances its absorption. A phase II study in patients undergoing total hip replacement indicated that SNAC heparin in two different doses was as effective and safe as UFH given subcutaneously. A phase III clinical trial comparing two doses of SNAC heparin given orally with LMWH by subcutaneous injection for the prevention of venous thromboembolism in patients undergoing total hip replacement is currently underway.

A Novel Platelet-Rich Arterial Thrombosis Model in Rabbits: Simple, Reproducible, and Dynamic Real-Time Measurement by Using Double-Opposing Inverted-Sutures Model

Though numerous animal thrombosis models have been introduced, an easy, reliable, and reproducible arterial thrombosis model remains a continuing challenge prior to a thrombolytic study. In an effort to evaluate the efficiency of various recombinant thrombolytic agents with specific affinity to activated platelets in vivo, we developed a novel double-opposing inverted-sutures model to create a platelet-rich thrombus in the femoral artery of rabbits. The arteriotomy was done semicircumferentially, and variously sized microsurgical sutures were introduced intraluminally in a double-opposing inverted manner. The animals were divided into three groups according to the double-opposing inverted-sutures used: Group 1 with 10-0 nylon ( n=6), Group 2 with 9-0 nylon ( n=6), and Group 3 with 8-0 nylon ( n=22). The superficial epigastric branch was cannulated with a thin polyethylene (PE) tube for intraarterial administration of the studied thrombolytic agent. The blood flow was continuously measured with a real-time ultrasonic flow meter. Within 2 h of installation of the sutures, there was no thrombus formation in either Group 1 or 2. In Group 3, the thrombosis rate was 91% (20 of 22) under a steady baseline flow (with an average of 12.23±2.40 ml/min). It was highly statistically significant with a P-value of .0000743 using Fisher's Exact Test. The averaged time to thrombosis was 21.8±9.8 min. The ultrasonic flow meter to record the dynamic real-time measurement of blood flow was a guideline for thrombus formation or dissolution, which was correlated with the morphological findings of stenotic status of the vessel detected by the Doppler sonography. The components of the thrombus were proven to be platelet-rich predominant by histological examination via hematoxylin and eosin (H&E) stain and transmission electron microscopy (TEM). To confirm that the double-opposing inverted-sutures model would be useful for a study of thrombolytic agents, we evaluated the effects of recombinant tissue-type plasminogen activator (rt-PA) and streptokinase-human plasminogen (SK-HPlg). The average time to thrombolysis post rt-PA infusion was 16.0±8.2 min and that of SK-HPlg was 79.6±23.1, which were similar to the previous reports. In conclusion, the novel double-opposing inverted-sutures (8-0 nylon) model provides a simple, reliable, and reproducible platelet-rich arterial thrombosis model with noninvasive and dynamic real-time measurement. It may be applied in assessing the efficiency of the recombinant thrombolytic agents and offers many advantages of an arterial platelet-rich in vivo thrombosis model.

Discovery of transition state factor Xa inhibitors as potential anticoagulant agents.

Factor Xa is an attractive biological target in the discovery and development of either parenteral or orally active anticoagulant agents. Several strategies have been utilized at COR Therapeutics in the pursuit of tri-peptide based transition state mimetic factor Xa inhibitors with high aqueous solubility. Some of these inhibitors have displayed excellent in vitro potency in inhibiting factor Xa in the prothrombinase complex. More importantly, these compounds showed strong in vivo antithrombotic efficacy without significant bleeding complications in several animal thrombosis models. These results demonstrated that small molecule factor Xa inhibitors could be advantageous over Warfarin and LMWH. For the discovery and development of orally active anticoagulant agents, small organic molecules as reversible factor Xa inhibitors were explored. From a medicinal chemistry perspective, significant insight has been gained regarding the in vivo antithrombotic efficacy and pharmacokinetic behaviors of each class of factor Xa inhibitors. This review will focus on the design and discovery of transition state factor Xa inhibitors as potential parenteral anticoagulant agents. Several excellent comprehensive review articles on factor Xa inhibitors have appeared recently [1-4].

Nitric oxide insufficiency, platelet activation, and arterial thrombosis.

Nitric oxide (NO) was originally discovered as a vasodilator product of the endothelium. Over the last 15 years, this vascular mediator has been shown to have important antiplatelet actions as well. By activating guanylyl cyclase, inhibiting phosphoinositide 3-kinase, impairing capacitative calcium influx, and inhibiting cyclooxygenase-1, endothelial NO limits platelet activation, adhesion, and aggregation. Platelets are also an important source of NO, and this platelet-derived NO pool limits recruitment of platelets to the platelet-rich thrombus. A deficiency of bioactive NO is associated with arterial thrombosis in animal models, individuals with endothelial dysfunction, and patients with a deficiency of the extracellular antioxidant enzyme glutathione peroxidase-3. This enzyme catalyzes the reduction of hydrogen and lipid peroxides, which limits the availability of these reactive oxygen species to react with and inactivate NO. The complex biochemical reactions that underlie the function and inactivation of NO in the vasculature represent an important set of targets for therapeutic intervention for the prevention and treatment of arterial thrombotic disorders.

Effect of Activated Prothrombin Complex Concentrate or Recombinant Factor VIIa on the Bleeding Time and Thrombus Formation During Anticoagulation with a Direct Thrombin Inhibitor

Melagatran is the active form of the oral, direct thrombin inhibitor H 376/95. In several animal models of thrombosis, the antithrombotic properties of melagatran have been demonstrated, without any increase in experimental bleeding. However, as with all anticoagulants, in emergency situations, reversal of the anticoagulation may be necessary. In this study, increasing doses of activated prothrombin complex concentrate (APCC, Feiba) or recombinant factor VIIa (r-F VIIa, NovoSeven) were superimposed on high doses of melagatran, or saline, in anaesthetised rats. The haemostatic effect was evaluated in two bleeding time models and a potential prothrombotic effect was evaluated in an arterial thrombosis model. Compared with melagatran alone (0.5 μmol/kg/h), Feiba in doses of ≥25 U/kg significantly shortened the prolonged bleeding time and reduced blood loss. In addition, Feiba ≥50 U/kg when added to melagatran (2 μmol/kg/h), significantly reduced bleeding time. No potentiation of thrombus formation was observed when Feiba was added to melagatran, compared with controls. NovoSeven at high doses (2–10 mg/kg) produced a nonsignificant trend in reduction of blood loss and with the highest dose (10 mg/kg) producing only a mild nonsignificant reduction in bleeding time. The prolonged prothrombin time (PT) and the ecarin clotting time (ECT) were more effectively shortened by Feiba than by NovoSeven. In contrast, whole blood clotting time (WBCT) was more effectively shortened by NovoSeven than by Feiba. Activated partial thromboplastin time (APTT) was shortened by NovoSeven but was prolonged by Feiba. Thrombin–antithrombin (TAT) complex formation was increased in a dose-dependent fashion more effectively by Feiba than by NovoSeven. Conclusion: Feiba (APCC) reversed prolonged bleeding time and blood loss in rats treated with high doses of melagatran and compared with the control group thrombus formation was not potentiated. NovoSeven (r-F VIIa) at high doses had less pronounced effects on blood loss and bleeding times compared with Feiba.

Perspectives on factor Xa inhibition.

Blood coagulation involves a complex cascade of enzymatic reactions, ultimately generating fibrin, the basis of all blood clots. This cascade is comprised of two arms, the intrinsic and extrinsic pathways which converge at factor Xa to form the common pathway. Factor Xa activates prothrombin to thrombin, which in turn catalyzes the conversion of fibrinogen to fibrin. Recently, both natural and synthetic factor Xa inhibitors have shown promising pharmacological effects in animal models of thrombosis. Accordingly, factor Xa has emerged as a compelling target for pharmacological intervention and much recent effort has focused on selective and potent inhibition of this key enzyme. Factor Xa and other enzymes in the coagulation cascade belong to the trypsin-like serine protease family, the various members of which are involved in numerous physiological functions in the body. Hence, to avoid toxicity and adverse side effects, it is important to selectively inhibit the target enzyme. Achieving the needed selectivity has proved challenging due to the high degree of structural homology around the active site of this class of enzymes. This article provides a brief review of the strategies currently being employed to develop oral anticoagulants and, more specifically, the structural features of protein-ligand binding that have been utilized to achieve potency and selectivity toward factor Xa. Additionally, selected lead molecules will be discussed to highlight binding motifs used to attain both potency and selectivity in drug candidates.

Discovery of 1-[3-(aminomethyl)phenyl]-N-3-fluoro-2'-(methylsulfonyl)-[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC423), a highly potent, selective, and orally bioavailable inhibitor of blood coagulation factor Xa.

Factor Xa (fXa) plays a critical role in the coagulation cascade, serving as the point of convergence of the intrinsic and extrinsic pathways. Together with nonenzymatic cofactor Va and Ca2+ on the phospholipid surface of platelets or endothelial cells, factor Xa forms the prothrombinase complex, which is responsible for the proteolysis of prothrombin to catalytically active thrombin. Thrombin, in turn, catalyzes the cleavage of fibrinogen to fibrin, thus initiating a process that ultimately leads to clot formation. Recently, we reported on a series of isoxazoline and isoxazole monobasic noncovalent inhibitors of factor Xa which show good potency in animal models of thrombosis. In this paper, we wish to report on the optimization of the heterocyclic core, which ultimately led to the discovery of a novel pyrazole SN429 (2b; fXa K(i) = 13 pM). We also report on our efforts to improve the oral bioavailability and pharmacokinetic profile of this series while maintaining subnanomolar potency and in vitro selectivity. This was achieved by replacing the highly basic benzamidine P1 with a less basic benzylamine moiety. Further optimization of the pyrazole core substitution and the biphenyl P4 culminated in the discovery of DPC423 (17h), a highly potent, selective, and orally active factor Xa inhibitor which was chosen for clinical development.

Thromboxane-B2levels in serum of rabbits receiving a single intravenous dose of aqueous extract of garlic and onion

We have shown previously that fresh garlic extract is effective in reducing thromboxane formation by platelets both in vivo and in vitro animal models of thrombosis. In the present study, the effect of different concentrations of a single dose of aqueous extracts of garlic and onion were evaluated on serum thromboxane-B2synthesis in rabbits. Different concentrations of garlic and onion were administered as single doses in the ear vein of rabbits. Rabbits were bled before and at different intervals after the infusion of garlic or onion extracts. Venous blood was collected and allowed to clot at 37°C for 1 h. Thromboxane-B2level was measured in the serum by radioimmunoassay. It was observed that garlic inhibits the thrombin-induced platelet synthesis of TXB2in a dose-and time-dependent manner. Maximum inhibition of TXB2occurred between 0.5 h and 6 h at 25 and 100 mg kg−1garlic. At 24 h post-garlic infusion TXB2inhibition was reduced to 15% of the control and TXB2levels were comparable to that of the control values at 72 h pots-garlic infusion. Infusion of 100 mg kg−1onion extract did not elicit any inhibitory effect on TXB2synthesis in the serum of rabbit during the treatment period. The rapid recovery of platelet cyclooxygenase activity after infusion of a single dose of garlic suggests that garlic should be taken more frequently in order to achieve beneficial effects in the prevention of thrombosis.

Novel inhibitors of factor X for use in cardiovascular diseases.

The complementary roles of platelets and thrombin in the pathophysiology of acute coronary syndromes suggests that for treatment to be effective, both mediators must be targeted. Although great strides have been made in the development of antiplatelet therapies, attempts to inhibit thrombin have been less successful. Unfractionated heparin is limited by a number of pharmacologic shortcomings as well as an inability to meaningfully suppress thrombin generation. The low molecular weight heparins have yielded encouraging results in large-scale clinical trials, but it remains unclear whether their benefit stems from a superior pharmacologic profile to unfractionated heparin or is determined by an enhanced ability to suppress thrombin generation (by virtue of a direct anti-Xa effect). Regardless, investigators have become increasingly interested in factor Xa as a potential target for antithrombotic therapy. A number of naturally occurring Xa antagonists have been identified. Work with recombinant forms of these proteins confirms that factor Xa inhibition can suppress thrombin generation in a variety of animal thrombosis models. Accordingly, a number of synthetic direct and indirect Xa antagonists are under development for the prevention and treatment of thrombotic disorders. The following review summarizes the evolution of factor Xa antagonists.

The design and synthesis of thrombin inhibitors: the introduction of in vivo efficacy and oral bioavailability into benzthiazolylalanine inhibitors

The further optimisation of the novel lead compound CGH752 (Fig. 1) is described. By introducing various substituents into the 6-position of the 3,3-dimethyltetrahydroquinoline (DMTHQS) ring we have been able to favourably affect the in vitro and in vivo activity, and the pharmacokinetics of such compounds. One of the inhibitors synthesised (CGH1484) is bioavailable and shows efficacy in animal models of thrombosis.

Effect of allicin from garlic powder on serum lipids and blood pressure in rats fed with a high cholesterol diet

The use of fresh aqueous garlic extract is known to be effective in reducing thromboxane formation by platelets in both in vivo and in vitro animal models of thrombosis. In the present study, we studied the effect of Lichtwer garlic powder (containing 1.3% alliin equivalent to 0.6% allicin) on the serum cholesterol, triglyceride, glucose, protein, and systolic blood pressure in rats fed with a high cholesterol diet. Experimental rats were fed a 2% high cholesterol diet with and without garlic powder for 6 weeks. Control rats were fed a normal diet. The aqueous garlic powder extract was given orally to rats on a daily basis. It was observed that cholesterol-fed animals had a significant increase in serum cholesterol compared to the control group of rats fed on a normal diet. However, when the rats were fed with a high cholesterol diet mixed with garlic powder, there was a significant reduction in their serum cholesterol levels compared with the group which were on a diet containing high cholesterol without garlic powder. Serum triglyceride levels were also significantly lowered by garlic powder when compared to control and high cholesterol diet group rats. The blood pressure of the high cholesterol diet animals was significantly higher compared to the animals receiving the control diet. The blood pressure of the animals receiving garlic powder and high cholesterol diet was significantly lower as compared to the high cholesterol and control diet group. No significant changes were observed in the serum glucose and protein in all of the rats. These results show that garlic is beneficial in reducing blood cholesterol, triglycerides levels and systolic blood pressure in hypercholesterolemic rats. Our experimental results show that garlic may beneficially affect two risk factors for atherosclerosis – hyperlipidemia and hypertension.

Contribution of in vivo models of thrombosis to the discovery and development of novel antithrombotic agents

Cardiovascular and cerebrovascular diseases continue to be the leading cause of death throughout the world. Over the past two decades, great advances have been made in the pharmacological treatment and prevention of thrombotic disorders (e.g., tissue plasminogen activators, platelet GPIIb/IIIa antagonists, ADP receptor antagonists such as clopidogrel, low-molecular weight heparins, and direct thrombin inhibitors). New research is leading to the next generation of antithrombotic compounds such as direct coagulation FVIIa inhibitors, tissue factor pathway inhibitors, gene therapy, and orally active direct thrombin inhibitors and coagulation Factor Xa (FXa) inhibitors. Animal models of thrombosis have played a crucial role in discovering and validiting novel drug targets, selecting new agents for clinical evaluation, and providing dosing and safety information for clinical trials. In addition, these models have provided valuable information regarding the mechanisms of these new agents and the interactions between antithrombotic agents that work by different mechanisms. This review briefly presents the pivitol preclinical studies that led to the development of drugs that have proven to be effective clinicallly. The role that animal models of thrombosis are playing in the discovery and development of novel antithrombotic agents is also described, with specific emphasis on FXa inhibitors. The major issues regarding the use of animal models of thrombosis, such as the use of positive controls, appropriate pharmacodynamic markers of activity, safety evaluation, species-specificity, and pharmacokinetics, are highlighted. Finally, the use of genetic models in thrombosis/hemostasis research and pharmacology is presented using gene-therapy for hemophilia as an example of how animal models have aided in the development of these therapies that are now being evaluated clinically. In summary, animal models have contributed greatly to the discovery of currently available antithrombotic agents and will play a primary role in the discovery and characterization of the novel antithrombotic agents that will provide safe and effective pharmacological treatment for life-threatening thrombotic diseases.