Inhibition Of Human Factor Xa By Various Plasma Protease Inhibitors

The second order rate constants for the interaction of human factor Xa with antithrombin III (in the absence of heparin) α₁ antitrypsin and α₂ macroglobulin have been determined at pH 7.4 (50mM Tris HCl, 0.1M NaCl) in the presence of 3mM CaCl₂. Factor Xa (free of activation peptide) and α₁ antitrypsin were purified from outdated human plasma. Pure human preparations of antithrombin III and α₂ macroglobulin were obtained elsewhere. The rate of inhibition was measured by determining the residual factor Xa activity at time intervals using S2337. In the case of the inhibition by α₂ macroglobulin, measurement was made of bound factor Xa by carrying out the assay in the presence of soybean trypsin inhibitor, The rates were determined at molar concentrations of inhibitor to factor Xa of 1:1, 2:1, 4:1, and 8:1 and were concentration dependent indicating second order kinetcis. Suitable plots according to the full second order rate equation yielded straight lines from which the rates constants were calculated.

Calculated values for the inhibition of factor Xa by antithrombin III, α₁ antitrypsin and α₂ macroglobulin were 5.80×10⁴ M^-1 min^-1, 1.36×10⁴ M^-1 min^-1 and 4.00×10⁴ M^-1 min^-1 respectively. The potential effectiveness of the inhibition for the inactivation of factor Xa in plasma can be calculated from these rate constants and the plasma levels of the inhibitors. Assuming plasma concentrations of the inhibitors to be 4.46μM, 42.4μM and 3.11μM respectively, the relative order of effectiveness under plasma conditions is α₁ antitrypsin > antithrombin III > α₂ macroglobulin in the proportions 4.64 : 2.08 : 1.00. These results are in contrast to the calculated effectiveness in plasma of these inhibitors against thrombin (of which antithrombin III is the most effective inhibitor). Cephalin was found to inhibit the interaction between factor Xa (2.8×10⁴ M^-1 min^-1 at 100 μg/ml) and antithrombin III but not between factor Xa and α₁ antitrypsin.