Regulation of factor IX(a) in vitro in human and mouse plasma and in vivo in the mouse. Role of the endothelium and the plasma proteinase inhibitors

Abstract

The regulation of human factor IX(a) was studied in vitro in human and mouse plasma and in vivo in the mouse. In human plasma, ~60% of the 125I-Factor IX(a) was bound to antithrombin III (ATIII) by 2 h, with no binding to α2-macroglobulin or α1-proteinase inhibitor, as assessed by gel electrophoresis and IgG-antiproteinase inhibitor-Sepharose beads. In the presence of heparin, virtually 100% of the 125I-Factor IX(a) was bound to ATIII by 1 min. The distribution of 125I-Factor IX(a) in mouse plasma was similar. The clearance of 125I-Factor IX(a) was rapid (50% clearance in 2 min) and biphasic and was inhibited by large molar excesses of ATIII-thrombin and α1-proteinase inhibitor-trypsin, but not α2-macroglobulin-trypsin; it was also inhibited by large molar excesses of diisopropylphosphoryl- (DIP-) Factor X(a), DIP-thrombin, and Factor IX, but not by prothrombin or Factor X. The clearance of Factor IX was also rapid (50% clearance in 2.5 min) and was inhibited by a large molar excess of Factor IX, but not by large molar excesses of Factor X, prothrombin, DIP-Factor X(a), or DIP-thrombin. Electrophoresis and IgG-antiproteinase inhibitor-Sepharose bead studies confirmed that by 2 min after injection into the murine circulation, 60% of the 125I-Factor IX(a) was bound to ATIII. Organ distribution studies with 125I-Factor IX(a) demonstrated that most of the radioactivity was in the liver. These studies suggest that Factor IX(a) binds to at least two classes of binding sites on endothelial cells. One site apparently recognizes both Factors IX and IX(a), but not Factor X, Factor X(a), prothrombin, or thrombin. The other site recognizes thrombin, Factor X(a), and Factor IX(a), but not the zymogen forms of these clotting factors. After this binding, Factor IX(a) is bound to ATIII and the complex is cleared from the circulation by hepatocytes.