Characterization of Rat Factors X and Xa: Demonstration of Factor Xa in Rat Plasma

Abstract

We have found that rat plasma corrected the non-activated PT of human normal or factor-X deficient plasma, and the factor Xa-like activity being constantly detected in every 1 ml of blood collected via the cannulated carotid artery of rats. The present study was undertaken to characterize the factor Xa-like activity in rat plasma by preparing rat factor X and a monoclonal antibody against it. Factor X was purified from a BaCl₂ eluate of rat plasma by chromatographies on columns of DEAE-Sepharose CL-6B and Sulfate Cellulofine or on a column of Affi-Gel 10 conjugated with a monoclonal antibody against rat factor X. Factor Xa-like activity in rat plasma was eliminated by the treatment of rat plasma with a monoclonal antibody which recognized the heavy chain portions of rat factors X and Xa. A kinetical study demonstrated that rat factor Xa was strongly inhibited by rat antithrombin III, with a K₁ of 2.2×10^-11M, in the presence of heparin. However, in the absence of heparin, the second order rate constant for the inhibition of rat factor Xa by rat antithrombin III was 2.6×104M^-1•min^-1, which was one forty-third that for the inhibition of human factor Xa by human antithrombin III. Furthermore, rat factor Xa was resistant to the inhibition by rat α-1-antitrypsin and α-2-macroglobulin. These results indicate that the small amount of factor Xa (1.7% of total factor X) in rat plasma was derived through the continuous activation of factor X in the circulation or through the instantaneous activation of factor X through the interaction of blood with the vascular wall during the collection, and remained stable in the plasma after the collection.