Abstract

Abstract 4170

Introduction

Rivaroxaban and apixaban are selective, reversible but structurally different direct Factor Xa inhibitors that are in late-stage clinical development for the prevention and treatment of venous and arterial thrombosis. Studies in animal models demonstrated antithrombotic efficacy with these new agents (Perzborn E et al. J Thromb Haemost 2005;3:514–521; Wong PC et al. J Thromb Haemost 2008;6:820–829). The objective of this study was to characterize and compare in vitro rivaroxaban and apixaban in functional assays.

Methods

Factor Xa activity, rate constants (kon/koff), and prothrombinase activity were measured using purified Factor Xa and measuring the cleavage of chromogenic (Factor Xa), fluorogenic substrates (kon/koff), or using prothrombin as a substrate in a reconstituted prothrombinase complex and measuring the activity of the generated thrombin in the presence of a thrombin-specific chromogenic substrate. Clotting times and thrombin generation (TG) were measured using commercially available kits. Tissue factor (TF)-mediated platelet aggregation was measured in defibrinated plasma.

Results

Rivaroxaban and apixaban showed similar affinity for free Factor Xa (Ki 0.4 and 0.6 nM, respectively), comparable association (kon 1.7 × 107 M–1 s–1 and 0.88 × 107 M–1 s–1, respectively) and dissociation (koff 5 × 10–3 s–1 and 2.4 × 10–3 s–1, respectively) rates, and inhibition of prothrombinase-bound Factor Xa (2.1 nM and 2.7 nM, respectively; Table). However, in human plasma-based systems, the 2 agents showed different potency. Despite the reported comparable plasma protein binding for apixaban and rivaroxaban (87% and 92–95%, respectively), higher concentrations of apixaban were needed to inhibit TG and TF-mediated platelet aggregation, and to prolong clotting time, compared with rivaroxaban. The concentrations needed to double clotting times, such as prothrombin time, activated partial thromboplastin time, clotting times in thromboelastometric measurements triggered either by the extrinsic (ex-TEM®) or intrinsic (in-TEM®) coagulation pathway, and prothrombinase-induced clotting time, were 3- to 8-fold higher for apixaban than for rivaroxaban (Table). The IC50 values for reducing the peak TG and endogenous thrombin potential in the TG assay were 3-fold higher for apixaban (0.20 and 4.96 μM, respectively) compared with rivaroxaban (0.06 and 1.48 μM, respectively; Table). The IC50 values for inhibiting TF-mediated platelet aggregation were 8-fold higher for apixaban (0.51 μM) versus rivaroxaban (0.06 μM; Table).

Conclusions

These results demonstrate that structurally different Factor Xa inhibitors may differ in their antihemostatic potency in spite of comparable affinity to Factor Xa, and suggest that rivaroxaban may be a more potent anticoagulant and indirect inhibitor of platelet aggregation than apixaban.

Table

Characteristics of rivaroxaban and apixaban in in vitro studies

 Rivaroxaban Apixaban Difference in potency 
Affinity constant (Ki0.4 nM 0.6 nM  
Rate constants (kon, koff1.7 × 107 M–1 s–1, 5 × 10–3 s–1 0.88 × 107 M–1 s–1, 2.4 × 10–3 s–1  
Prothrombinase (IC502.1 nM 2.7 nM  
Thrombin generation (5 pM tissue factor)    
Peak thrombin generation (IC500.06 μM 0.20 μM 3-fold 
Endogenous thrombin potential (IC501.48 μM 4.96 μM 3-fold 
Coagulation assays (concentrations needed to double clotting times)    
Prothrombin time 0.69 μM 5.43 μM 8-fold 
Activated partial thromboplastin time 2.07 μM 7.14 μM 3-fold 
Prothrombinase-induced clotting time 0.17 μM 0.68 μM 4-fold 
Thrombelastography (ex-TEM®) 0.34 μM 1.46 μM 4-fold 
Thrombelastography (in-TEM®) 0.87 μM 6.22 μM 7-fold 
Tissue factor-induced platelet aggregation (IC500.06 μM 0.51 μM 8-fold 
 Rivaroxaban Apixaban Difference in potency 
Affinity constant (Ki0.4 nM 0.6 nM  
Rate constants (kon, koff1.7 × 107 M–1 s–1, 5 × 10–3 s–1 0.88 × 107 M–1 s–1, 2.4 × 10–3 s–1  
Prothrombinase (IC502.1 nM 2.7 nM  
Thrombin generation (5 pM tissue factor)    
Peak thrombin generation (IC500.06 μM 0.20 μM 3-fold 
Endogenous thrombin potential (IC501.48 μM 4.96 μM 3-fold 
Coagulation assays (concentrations needed to double clotting times)    
Prothrombin time 0.69 μM 5.43 μM 8-fold 
Activated partial thromboplastin time 2.07 μM 7.14 μM 3-fold 
Prothrombinase-induced clotting time 0.17 μM 0.68 μM 4-fold 
Thrombelastography (ex-TEM®) 0.34 μM 1.46 μM 4-fold 
Thrombelastography (in-TEM®) 0.87 μM 6.22 μM 7-fold 
Tissue factor-induced platelet aggregation (IC500.06 μM 0.51 μM 8-fold 
Disclosures:

Perzborn:Bayer Schering Pharma AG: Employment. Tersteegen:Bayer Schering Pharma AG: Employment. Harwardt:Bayer Schering Pharma AG: Employment. Lange:Bayer Schering Pharma AG: Employment.

Author notes

*

Asterisk with author names denotes non-ASH members.