Journal of Thrombosis and Thrombolysis

, Volume 24, Issue 1, pp 43–51

Razaxaban, a direct factor Xa inhibitor, in combination with aspirin and/or clopidogrel improves low-dose antithrombotic activity without enhancing bleeding liability in rabbits

Authors

    • Department of Thrombosis ResearchBristol-Myers Squibb Company
  • Earl J. Crain
    • Department of Thrombosis ResearchBristol-Myers Squibb Company
  • Carol A. Watson
    • Department of Thrombosis ResearchBristol-Myers Squibb Company
  • Ruth R. Wexler
    • Department of Thrombosis ResearchBristol-Myers Squibb Company
  • Patrick Y. S. Lam
    • Department of Thrombosis ResearchBristol-Myers Squibb Company
  • Mimi L. Quan
    • Department of Thrombosis ResearchBristol-Myers Squibb Company
  • Robert M. Knabb
    • Department of Thrombosis ResearchBristol-Myers Squibb Company
Article

DOI: 10.1007/s11239-007-0017-9

Cite this article as:
Wong, P.C., Crain, E.J., Watson, C.A. et al. J Thromb Thrombolysis (2007) 24: 43. doi:10.1007/s11239-007-0017-9

Abstract

Coactivation of platelets and the blood coagulation cascade contributes to the pathophysiology of arterial thrombosis. Combination therapy with antiplatelet and anticoagulant drugs may be needed for maximizing the prevention and treatment of arterial thrombosis. Few studies have thoroughly investigated the combined antithrombotic and bleeding effects of these antithrombotic agents. We, therefore, evaluated the antithrombotic and bleeding profiles of dual and triple therapy with razaxaban, a direct factor Xa inhibitor, plus aspirin and/or clopidogrel in rabbit models of electrolytic injury-induced carotid artery thrombosis and cuticle bleeding time, respectively. Compounds were infused either IV or into the portal vein from 1 h before arterial injury or cuticle transection to the end of experiment. Carotid blood flow was used as a marker of antithrombotic effect. We first evaluated the antithrombotic potency of razaxaban, and examined its ex vivo effects on coagulation parameters to confirm its selectivity. Antithrombotic ED50 of razaxaban averaged 0.22 ± 0.05 mg/kg/h (n = 6). Razaxaban at 3 mg/kg/h IV produced full antithrombotic efficacy, increased significantly ex vivo activated partial thromboplastin time and prothrombin time by 2.2 ± 0.1- and 2.3 ± 0.1-fold, respectively, and inhibited ex vivo factor Xa activity significantly by 91 ± 5% (n = 6, P < 0.05) without affecting ex vivo thrombin activity. Razaxaban at concentrations up to 10 μM did not alter in vitro platelet aggregation responses to ADP, γ-thrombin or collagen. To identify additive or synergistic antithrombotic effects of the various combination therapies, we purposefully used marginally effective doses of razaxaban at 0.1 mg/kg/h, aspirin at 0.3 mg/kg/h and clopidogrel at 1 mg/kg/h. Dual combination of threshold doses of razaxaban and aspirin or clopidogrel produced an enhanced antithrombotic effect without further increases in bleeding time. When compared with dual therapy with aspirin and clopidogrel (38 ± 5% increase in blood flow), addition of razaxaban increased blood flow to 75 ± 5% without additional bleeding time effects (n = 6/group, P < 0.05). In summary, razaxaban was an effective antithrombotic agent in a rabbit model of arterial thrombosis. Low-dose razaxaban was useful in combination with sub-optimal doses of aspirin and/or clopidogrel for the prevention of occlusive arterial thrombosis without excessive bleeding.

Keywords

Antithrombotic agentsAspirinClopidogrelFactor Xa inhibitors

Introduction

Previously we reported a novel series of potent and orally-active small-molecule factor Xa (FXa) inhibitors [1], exemplified by DPC423 [2, 3]. Modification of the DPC423 series has led to additional orally bioavailable FXa inhibitors, including razaxaban (DPC906, BMS-561389, Fig. 1) [4]. In this study we evaluated the efficacy of this potent and selective FXa inhibitor in prevention of arterial thrombosis in a rabbit model of the electrolytic injury-induced carotid artery thrombosis (ECAT) [5] and examined its ex vivo effects on coagulation parameters to confirm its selectivity. We selected rabbits as the animal model because razaxaban has similar potency in inhibiting human and rabbit FXa (Ki = 0.19 nM in human and 0.16 nM in rabbit) [4].
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Fig. 1

Top panel: Structural formula for razaxaban (1-(3′-aminobenzisoxazol-5′-yl)-3-trifluoromethyl-N-[2-fluoro-4- [(2′-dimethylaminomethyl)imidazol-1-yl]phenyl]-1H-pyrazole-5-carboxyamide hydrochloride). Bottom panel: Effects of vehicle and razaxaban on carotid blood flow (expressed as % control carotid blood flow) after thrombus induction in ECAT rabbits. Means ± SE and n = 6 per group. *P < 0.5, compared to vehicle

Recognizing that coactivation of platelets and the blood coagulation cascade contributes to the pathophysiology of arterial thrombosis, monotherapy with either an antiplatelet agent or an anticoagulant may be insufficient for maximizing the prevention and treatment of platelet-rich arterial thrombosis [6, 7]. In fact, the combination of warfarin and aspirin has been shown to be more effective than aspirin alone in improving the clinical outcomes in patients with acute coronary syndrome but at an increased risk of major bleeds [8]. Despite its proven efficacy, long-term warfarin therapy remains underutilized in the clinic due to a narrow therapeutic index leading to an increased risk of bleeding [7]. Oral FXa inhibitors may be an alternative to warfarin in the long term treatment of thrombosis if they can offer a wider therapeutic index. Thus, the second objective of this study was to evaluate to what extent addition of a selective FXa inhibitor, such as razaxaban, to aspirin would provide additional antithrombotic efficacy and increase bleeding time in rabbits. Clopidogrel is another widely used antiplatelet agent in the clinic, and in combination with aspirin is the standard of care for the secondary prevention of cardiovascular events in patients with arterial thrombosis [6, 7, 9]. Therefore, we also evaluated effects of the dual therapy with razaxaban and clopidogrel, and the triple therapy with the FXa inhibitor, aspirin and clopidogrel on arterial thrombosis and hemostasis in rabbits.

Materials and methods

All experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and the regulations of the Animal Care and Use Committee of the Bristol-Myers Squibb Company.

Reagents

The following drugs and chemicals were used in this study: chromogenic substrates, S-2222 and S-2238 (Chromogenix AB products distributed by DiaPharma Group, Inc., West Chester, OH); human α-thrombin and FXa (Enzyme Research Laboratories, Inc., South Bend, IN); human γ-thrombin (ICN Biomedicals, Inc., Costa Mesa, CA); and activated partial thromboplastin time (aPTT) reagent, ADP, collagen and thromboplastin with calcium (Sigma Chemical Co., St. Louis, MO), and clopidogrel (Plavix®, Bristol-Myers Squibb/Sanofi Pharmaceuticals, New York, NY). Razaxaban was synthesized at Bristol-Myers Squibb Company.

Antithrombotic studies

The rabbit ECAT model, described by Wong et al. [5], was used in this study. Briefly, male New Zealand White rabbits (2.5–3.5 kg) (Covance, Denver, PA) were anesthetized with ketamine (50 mg/kg + 50 mg/kg/h IM) and xylazine (10 mg/kg + 10 mg/kg/h IM). An electromagnetic flow probe was placed on a segment of an isolated carotid artery to monitor blood flow. Thrombus formation was induced by electrical stimulation of the carotid artery for 3 min at 4 mA using an external stainless-steel bipolar electrode. Carotid blood flow was measured continuously over a 45-min period to monitor thrombus-induced occlusion. Razaxaban or vehicle was infused IV 1 h before the electrical stimulation of the carotid artery and continuously during the 45 min period.

The ED50 (effective dose) of razaxaban that increased blood flow level at 45 min to 50% were determined using the 4-parameter logistic equation, y = A + ((BA)/(1 + ((C/x)D))) where A = minimum y value, B = maximum, C = Log ED50 or Log EC50 and D = slope factor, and the logistic fit was analyzed by XLfit© (Microsoft, Redmond, WA).

Combination studies on arterial thrombosis and hemostasis

Since clopidogrel is a prodrug and requires hepatic activation in vivo [9], it was given directly into the liver via portal vein infusion for maximal activation. For this procedure, we surgically implanted a portal vein cannula by exposing and cannulating a side-branch of the main mesenteric vein via an abdominal incision. Care was taken not to advance the tip of portal vein cannula into the main mesenteric vein.

Compounds and vehicle were infused IV (razaxaban, aspirin or vehicle) or into the portal vein (IPV) (clopidogrel or vehicle) 1 h prior to the electrical stimulation of the carotid artery and continuously to the end of the study. Carotid blood flow was measured as described above. In addition, at the end of the study, the injured segment of the carotid artery was isolated, excised, and rinsed in saline. Using a pair of forceps, the artery was gently squeezed to remove the thrombus onto a piece of standard weigh paper. Then the thrombus was blotted to remove excess fluid and weighed. The vehicle used for razaxaban and aspirin was 5% dextrose in water. The vehicle used for clopidogrel was acidified 5% dextrose in water (pH 3.5).

The cuticle bleeding time model has been commonly used in rabbits to monitor the bleeding potential of inhibitors of FXa, FIXa, tissue factor and thrombin [1013], and was used for bleeding evaluation in these combination studies. Briefly, the hind paws of a separate group of anesthetized rabbits were shaved. A standard cut was made at the apex of the cuticle with a razor blade. Blood was allowed to flow freely by keeping the bleeding site in contact with 37°C Lactated Ringer’s solution. Bleeding time was defined as the time after transection when bleeding was ceased. It was measured by averaging the bleeding time of three nail cuticles both in the control and treatment periods. Compound or vehicle was given, as described above, i.e., 1 h prior to the cuticle bleeding and continuously during the bleeding time measurement period.

Coagulation assays

Ex vivo clotting times, including aPTT and prothrombin time (PT), anti-FXa and antithrombin activities were measured as described previously by Wong et al. [5]. Clotting times were measured with a fibrometer (BBL Fibrosystem, Becton Dickinson, Cockeysville, MD). Ex vivo FXa and thrombin activities were determined using the chromogenic substrates S-2222 and S-2238, respectively. Assays were performed in a Spectramax Plus spectrophotometer (Molecular Devices, Sunnyvale, CA).

In vitro platelet aggregation

Platelet rich plasma was prepared with the centrifugation of fresh citrated blood collected from rabbits. Platelet aggregation was measured with a platelet aggregometer (Model PAP-4D, BioData, Horsham, PA). Two hundred microliter of platelet rich plasma was mixed with 20 μl of saline, DMP802 at 3 μM in saline or razaxaban at 1–10 μM in saline and incubated for 3 min at 37°C. DMP802 is a GPIIb/IIIa receptor antagonist (IC50 = 29 nM against human platelet aggregation response to 10 μM ADP) [14] and was included as a positive control. Percentages of platelet aggregation were determined 4 min after the addition of 20 μl of the agonist (ADP at 10 μM, γ-thrombin at 35 nM and collagen at 10 μg/ml, final concentration).

Statistical analysis

Statistical analyses used were analysis of variance and the Student-Newman–Keuls Test using the SAS system (SAS for Windows release 8.02A, Cary, NC). A value of P < 0.05 was considered statistically significant. All data are means ± SE.

Results

Antithrombotic effect of razaxaban in rabbits

Figure 1 shows effects of vehicle and razaxaban on carotid blood flow after electrical stimulation. Control carotid blood flow in these animals averaged 26 ± 2 ml/min. After the initiation of thrombosis, blood flow was gradually decreased, and the artery was totally occluded in about 30 min in vehicle-treated animals. Razaxaban at 0.1–3 mg/kg/h IV produced a dose-dependent increase in duration of the patency of the injured artery with ED50 of 0.22 ± 0.05 mg/kg/h.

Effects of razaxaban on coagulation markers in rabbits

Figure 2 (top panel) shows ex vivo effects of raxazaban on aPTT and PT. Control aPTT and PT in vehicle-treated rabbits averaged 21 ± 1 and 8 ± 0.2 sec, respectively. Razaxaban increased aPTT and PT in a dose-dependent manner. We observed a good correlation between the antithrombotic effects of razaxaban and its ex vivo aPTT (r = 0.80, P < 0.0001) and PT (r = 0.85, P < 0.0001). Figure 2 (bottom panel) shows the ex vivo effect of razaxaban on FXa and thrombin activity. Razaxaban produced a dose-dependent inhibition of ex vivo FXa activity and did not change ex vivo thrombin activity. We also observed a good correlation between the antithrombotic effect of razaxaban and its ex vivo anti-FXa activity (r = 0.77, P < 0.0001).
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Fig. 2

Top panel: ex vivo aPTT and PT effects of razaxaban. Bottom panel: ex vivo anti-FXa and anti-thrombin effects of razaxaban. Means with the different letter are significantly different at P < 0.05 when compared with each other. Means ± SE and n = 6 per group

In vitro effects of razaxaban on platelet aggregation

Razaxaban at 1, 3 and 10 μM did not change the in vitro platelet aggregation responses to ADP, γ-thrombin or collagen (Fig. 3). The platelet aggregation responses to ADP, γ-thrombin and collagen in the presence of the GPIIb/IIIa antagonist DMP802 at 3 μM were significantly reduced (Fig. 3).
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Fig. 3

Effects of vehicle, razaxaban and DMP 802 on the in vitro platelet aggregation responses to ADP (10 μM), γ-thrombin (35 nM) and collagen (10 μg/ml). Means ± SE and n = 5 per group. *P < 0.5, compared to vehicle

Antithrombotic dose-dependent effects of clopidogrel and aspirin

Clopidogrel and aspirin produced dose-dependent increases in antithrombotic effects in rabbits (Fig. 4). Compared with the vehicle, clopidogrel at 3 and 10 mg/kg/h IPV and aspirin at 1 and 3 mg/kg/h IV significantly increased carotid blood flow at 45 min after electrical stimulation (P < 0.05).
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Fig. 4

Effects of vehicle (n = 4), clopidogrel (top panel, n = 5–6 per dose) and aspirin (bottom, n = 6 per dose) on carotid blood flow (expressed as % control carotid blood flow) after thrombus induction in ECAT rabbits. Means ± SE *P < 0.5, compared to vehicle

Antithrombotic effects of razaxaban, clopidogrel, aspirin and combination

In order to detect additive or synergistic antithrombotic effects of the various combination therapies, we deliberately used marginally effective doses of razaxaban, aspirin and clopidogrel in this combination study (i.e., razaxaban at 0.1 mg/kg/h IV, clopidogrel at 1 mg/kg/h IPV and aspirin at 0.3 mg/kg/h IV). These doses were picked based on pilot studies, which showed that at these low doses, razaxaban inhibited ex vivo FXa activity by 29 ± 7% (n = 6), aspirin inhibited the ex vivo 250 μM arachidonic acid-induced platelet aggregation by 37 ± 17% (n = 6), and clopidogrel inhibited the 10 μM ADP-induced platelet aggregation by 39 ± 13% (n = 6).

As shown in Fig. 5, razaxaban prevented the occlusion of the thrombotic carotid artery in animals treated with aspirin, clopidogrel or the combination of aspirin and clopidogrel. Triple combination of razaxaban, aspirin and clopidogrel maintained carotid blood flow at about 80% level for at least 45 min. Figure 6 shows effects of razaxaban, clopidogrel, aspirin or their combination on carotid blood flow at 45 min and thrombus weight. Control thrombus weight averaged 10.4 ± 0.97 mg in vehicle-treated rabbits (n = 6), The addition of razaxaban in animals treated with the vehicle, aspirin, clopidogrel or the combination with aspirin and clopidogrel produced significantly greater antithrombotic effects, as shown by higher levels of carotid blood flow or greater reduction of thrombus weight, than their corresponding treatment alone. Dual combinations of razaxaban plus aspirin, razaxaban plus clopiodgrel and clopidogrel plus aspirin produced similar antithrombotic effects as shown in Fig. 6. Triple combination of razaxaban, aspirin and clopidogrel produced close to 80% carotid blood flow and 60% thrombus reduction.
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Fig. 5

Effects of vehicle, razaxaban, aspirin, aspirin + razaxaban, clopidogrel, clopidogrel + razaxaban, aspirin + clopidogrel and aspirin + clopidogrel + razaxaban on carotid blood flow (expressed as % control carotid blood flow) after thrombus induction in ECAT rabbits. Means ± SE and n = 6 per group. *P < 0.5, compared to the corresponding control group. Doses (mg/kg/h): razaxaban 0.1, aspirin 0.3, clopidogrel 1. All injected IV except IPV for clopidogrel. ASA, aspirin; Clopi, Clopidogrel; Raza, razaxaban

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Fig. 6

Effects on carotid blood flow and thrombus weight in each treatment group. Means with the different letter are significantly different at P < 0.05 when compared with each other. Means ± SE and n = 6 per group. Doses (mg/kg/h): razaxaban 0.1, aspirin 0.3, clopidogrel 1. All injected IV except IPV for clopidogrel. ASA, aspirin; Clopi, Clopidogrel; Raza, razaxaban

Bleeding time effects of razaxaban, clopidogrel, aspirin and combination

In the following bleeding time studies, we evaluated razaxaban at 0.1 mg/kg/h IV, clopidogrel at 1 mg/kg/h IPV and aspirin at 0.3 mg/kg/h IV.

Control bleeding time averaged 154 ± 4 sec in vehicle-treated rabbits. As shown in Fig. 7, razaxaban alone did not increase cuticle bleeding time significantly. Aspirin slightly increased cuticle bleeding time, but the change was not statistically significant. Razaxaban plus aspirin increased bleeding time (23 ± 5%; n = 6) significantly when compared to vehicle or razaxaban alone, but not to aspirin alone. Clopidogrel alone increased bleeding time significantly by 41 ± 11% (n = 6) when compared to vehicle. The combination of razaxaban and clopidogrel did not cause a greater increase in bleeding time than clopidogrel alone (40 ± 7%for razaxaban plus clopidogrel vs. 41 ± 11% for clopidogrel). The combination of aspirin plus clopidogrel did not increase bleeding time significantly than clopidogrel alone. We also observed that the bleeding time effect of razaxaban plus aspirin (23 ± 5%) was significantly less than that of clopidogrel plus aspirin (51 ± 4%) (P < 0.05). The addition of razaxaban to the combination of clopidogrel and aspirin did not cause any significant additional increase in bleeding time when compared to clopidogrel plus aspirin.
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Fig. 7

Effects on bleeding times in each treatment group. Means with the different letter are significantly different at P < 0.05 when compared with each other. Means ± SE and n = 6 per group. Doses (mg/kg/h): razaxaban 0.1, aspirin 0.3, clopidogrel 1. All injected IV except IPV for clopidogrel. ASA, aspirin; Clopi, Clopidogrel; Raza, razaxaban

Discussion

Razaxaban is a small-molecule, reversible, active-site, direct inhibitor of FXa with potency similar to that of our previously reported FXa inhibitor DPC423 [1, 2]. Unlike DPC423, razaxaban does not inhibit trypsin and plasma kallikrein [2, 4]. As a more selective inhibitor of FXa, razaxaban is a good representative of the direct FXa inhibitor class. This study demonstrated that this selective FXa inhibitor was effective in preventing arterial thrombosis in the rabbit ECAT model. Although razaxaban and other FXa inhibitors were shown to be efficacious in the prevention of human venous thrombosis [1519], their efficacy in arterial thrombosis has not been reported. Our study, therefore, suggests the potential opportunities for a direct FXa inhibitor in arterial thrombosis.

Since FXa is at the convergent point of the intrinsic and extrinsic pathways of coagulation, it was expected that inhibition of FXa by razaxaban would prolong aPTT, the intrinsic pathway-dependent clotting time, and PT, the extrinsic pathway-dependent clotting time. Our study showed that both aPTT and PT tracked well with the antithrombotic efficacy of razaxaban in rabbits. Similar to descriptions of other small-molecule direct FXa inhibitors, we observed significant antithrombotic efficacy of razaxaban at less than 2-fold increases in aPTT and PT [13, 20], suggesting that the antithrombotic efficacy of direct FXa inhibitors could be achieved at doses that produce moderate increases in systemic anticoagulation. Similar to aPTT and PT, anti-FXa activity tracked well with the antithrombotic activity of razaxaban in rabbits.

Both in vitro and ex vivo studies support that razaxaban is a selective FXa inhibitor. Razaxaban inhibited ex vivo FXa activity but not ex vivo thrombin activity. It also did not alter in vitro platelet aggregation response to thrombin, supporting that the antithrombotic effect of razaxaban is consistent with FXa inhibition but not related to thrombin inhibition. Razaxaban did not alter in vitro platelet aggregation responses to ADP and collagen, further supporting that razaxaban lacks direct antiplatelet activities. However, by inhibiting FXa, razaxaban is expected to inhibit thrombin generation in vivo, thus reducing thrombin-induced platelet activation and producing an indirect antiplatelet effect.

To date, few studies have thoroughly investigated the antithrombotic and bleeding profiles of different combinations of antiplatelet and anticoagulant drugs in humans or in animals. Our study is the first evaluation of the antithrombotic and bleeding profiles of dual and triple therapy with a direct FXa inhibitor plus aspirin and/or clopidogrel in rabbit models of thrombosis and bleeding. To identify additive or synergistic antithrombotic effects of the various combination therapies, we purposefully used marginally effective doses of razaxaban at 0.1 mg/kg/h, aspirin at 0.3 mg/kg/h and clopidogrel at 1 mg/kg/h.

We observed that aspirin at 0.3 mg/kg/h IV did not prevent arterial thrombosis and increased bleeding time slightly in rabbits. However, the combination of this ineffective antithrombotic dose of aspirin and a low dose of razaxaban produced a significant greater antithrombotic effect than razaxaban alone, and this combination has a similar increase in bleeding time compared to aspirin alone. Furthermore, this low-dose combination of aspirin plus razaxaban matched the antithrombotic effect of the combination of low-dose aspirin plus low-dose clopidogrel, but did so with less increase in bleeding time. This result is consistent with our previous study, which demonstrated that co-administration of low doses of aspirin and the direct FXa inhibitor DPC423 enhanced antithrombotic efficacy but did not prolong bleeding time [13]. Other investigators have also reported that aspirin enhances the antithrombotic effect of anticoagulants such as heparin, low molecular weight heparin, FIX antibody and a direct thrombin inhibitor in animals [2123].

Clopidogrel at 1 mg/kg/h IPV was marginally effective in preventing arterial thrombosis in the rabbit ECAT model. In the rabbit cuticle bleeding model, this low dose of clopidogrel significantly increased bleeding time. Interestingly, the addition of this low-dose clopidogrel to the low-dose razaxaban enhanced their antithrombotic effects but did not elicit additional increases in bleeding time over that of clopidogrel. Similarly, greater antithrombotic effects were also noted when clopidogrel was added to melagatran, a direct thrombin inhibitor, and SanOrg123781A, an antithrombin-dependent inhibitor of FXa and thrombin, in animals [23, 24]. Unlike razaxaban plus clopidogrel, a greater increase in bleeding times was observed with melagatran plus clopidogrel [23], whereas the bleeding time effect of SanOrg123781A plus clopidogrel was not reported [24].

The current study showed that the addition of a low-dose of razaxaban to the combination of low doses of aspirin and clopidogrel produced a very significant antithrombotic effect without an additional increase in the bleeding time in rabbits. Likewise, to our knowledge, this study may be the first report of the effects of the triple combination of a direct FXa inhibitor, aspirin and clopidogrel on arterial thrombosis and hemostasis. Unlike our findings, Hong et al. [23] reported that the addition of melagatran to the combination of aspirin and clopidogrel did not result in additional antithrombotic efficacy and bleeding. Factors such as different mechanisms of drug action, experimental condition, species, and dose may account for some of the differences between these findings. It should be noted that the persistence of thrombin generation was observed in patients despite long term therapy with clopidogrel plus aspirin, which suggests the need of a more intensive antithrombotic therapy [25]. Thus, it seems to be reasonable that an anticoagulant should be added to the dual therapy with aspirin plus clopidogrel to suppress this persistence of thrombin production. Our finding with the triple combination, therefore, suggests that a FXa inhibitor may be a suitable anticoagulant to be added to the dual antiplatelet therapy with aspirin plus clopidogrel to provide additional efficacy without additional bleeding.

The mechanism for the enhanced antithrombotic effect of the direct FXa inhibitor in combination with aspirin, clopidogrel or the combination of aspirin plus clopidogrel is not known. Aspirin inhibits platelets by inhibiting cyclooxygenase and was shown to reduce platelet-dependent thrombin generation [26], whereas clopidogrel inhibits platelet activity by blocking the platelet ADP P2Y12 receptors [9]. Thus, it is not unexpected to observe additive efficacy when razaxaban is added to aspirin, clopidogrel or the combination of aspirin and clopidogrel since these agents all have different mechanisms of antithrombotic action. However, it is not clear why the low dose of razaxaban did not increase the bleeding time effects of low doses of aspirin, clopidogrel, or aspirin plus clopidogrel. A possible explanation is that the low-dose razaxaban does not totally suppress the production of thrombin, and the remaining thrombin may be at sufficient levels to activate the high affinity thrombin receptors on platelets to induce hemostasis. The clinical implication of this finding remains to be investigated.

In summary, our study demonstrates that razaxaban was an effective antithrombotic agent in a rabbit model of arterial thrombosis. Ex vivo aPTT, PT and anti-FXa activity were good markers to monitor the antithrombotic efficacy of razaxaban. In addition the low-dose razaxaban combined with low doses of aspirin and/or clopidogrel prevented arterial thrombosis without excessively increasing bleeding time. If these findings extend to other FXa inhibitors and translate into the clinic, oral FXa inhibitors may be an alternative to warfarin to be used alone or in combination with aspirin and/or clopidogrel for the long-term prevention and treatment of human arterial thrombosis.

Acknowledgments

We thank Dr. Lucius T. Rossano for a sample of razaxaban, Dr. Baomin Xin for plasma level determinations, Alverna Staus and Ming Y. He for technical assistance, and Drs. William A. Schumacher and Martin L. Ogletree for review of this manuscript.

Copyright information

© Springer Science+Business Media, LLC 2007