Effects of a Synthetic Factor Xa Inhibitor (JTV-803) on Various Laboratory Tests

Abstract

Synthetic direct inhibitors of factor Xa are capable of prolonging the global anticoagulant assay times in a concentration-dependent fashion. The relative degree of thrombin generation inhibition at an equivalent prolongation is not similar to the results observed with heparins and oral anticoagulant drugs. In addition, the direct factor Xa inhibitors prolong the Russell' viper venom test (RVVT) and Heptest clotting times. Ecarin clotting time (ECT) and thrombin time (TT) remain unaffected. The kinetics of factor Xa inhibition are markedly different than those observed with pentasac-charide and heparins. Therefore, the methods developed for heparins and pentasaccharides may not be applicable for the monitoring of factor Xa inhibitors. To test the feasibility of using the prothrombin time (PT), International Normalized Ratio (INR), activated partial thromboplastin time (aPTT), Heptest, thrombin time, RVVT, ECT, and a modified anti-Xa amidolytic assay, to monitor a synthetic factor Xa inhibitor, normal human pool plasma samples were spiked with a synthetic factor Xa inhibitor in the concentration range of 0 to 1 μg/mL and 0 to 25 μg/mL. Different laboratory tests were performed and INR and other ratios were calculated. The anticoagulant effects on whole blood were measured using the activated clotting time (ACT). Further studies on the effect of factor Xa inhibitor on platelet aggregation; factor II, VII, and X functional levels; and fibrinopeptide A (FPA) generation were carried out at equivalent INR levels in comparison to oral anticoagulant and antithrombin agents. FPA generation at equivalent anticoagulant level in comparison to heparin (twice the baseline) was also carried out. Factor Xa inhibitor produced a concentration-dependent prolongation of the ACT. ACT was doubled at a concentration of 4 to 5 μg/mL. There was a marked difference in the prolongation of the PT by a synthetic factor Xa inhibitor dependent on the ISI of the PT reagent used. When the results were calculated to determine INR, marked variations were noted between the recombinant thromboplastin and rabbit brain thromboplastin. The rabbit brain thromboplastin reagent gave markedly high INR values. Similar results were observed when different aPTT reagents were studied. In the anti-Xa assay, modification of the incubation time was employed to extend the proper sensitivity range. These studies warrant further investigation to understand the mechanism of action of factor Xa inhibitors.

Keywords

Factor Xa inhibitor Amidolytic anti-Xa assay Fibrinopeptide A Tissue factor pathway inhibitor International Normalized Ratio

References

1. Friberger P , Erberg N , Holmer E , et al. Antithrombin assay-the use of human or bovine thrombin and the observation of a second heparin cofactor. Thromb Res 1982;25:433-433.

2. Griffith MJ . Measurements of the heparin enhanced antithrombin III/thrombin reaction rate in the presence of synthetic substrate. Thromb Res 1982;25:245-245.

3. Hoppensteadt DA , Fareed J , Walenga JM , et al. Validity of serine protease inhibition tests in the in vitro and in vivo assessment of the action of heparin and its fractions. Fed Proc 1984;43:604-604.

4. Van Wijk EM , Kahle IH , Wiebosch M , et al. Optimization of a mechanized amidolytic factor Xa assay; influence of reaction conditions and reagents. Clin Chem 1981;27:918-918.

5. Walenga JM , Fareed J , Hoppensteadt DA . In vitro coagulation and amidolytic methods for evaluating the activity of heparin and a low molecular weight derivative (PK 10169). Semin Thromb Hemost 1985;11:17-17.

6. Kaiser B , Paintz M , Scholz O , et al. A synthetic inhibitor of factor Xa DX-9065a, reduced proliferation of vascular smooth muscle cells in vivo in rats. Thromb Res 2000;98:175-175.

7. Yamazaki M , Asakura H , Aoshima K , et al. Effect of DX-9065a, an orally active, newly synthesized and specific inhibitor of factor Xa, against experimental disseminated intravascular coagulation in rats. Thromb Haemost 1994;72:393-393.

8. Kitchen S , Lampierto R , Woolley AM , et al. Anti-Xa monitoring during treatment with low molcular weight or danaparoid: Inter-assay variability. Thromb Res 1999;82:1289-1289.

9. Rai R , Sprengeler PA , Elrod KC , et al. Perspectives on factor Xa inhibition. Curr Med Chem 2001;8:101-101.

10.

10. Smythe M . The heparin Anti-Xa therapeutic range. Are we there yet? Chest 2002;121:303-303.

11.

11. Fareed J , Walenga JM , Hoppensteadt DA , et al. Factor Xa inhibitors in the control of thrombogenesis (review article). Hamostaseologie 1999;19:55-55.