Activation of Bovine Factor IX By Bovine Factor XIa . Active Site Titration Of Factor IXa and Development of A spectro-Photometric assay for Factor IX

Activation of factor IX by factor XIa occurs via an intermediate which has no esterase activity towards synthetic arginine esters or coagulant activity as determined with a clotting assay. Factor IXa can be active site titrated using p-nitrophenyl-p1-guanidinobenzoate (p-NPGB) as a titrant. The rate and equilibrium constants describing the active site titration will be presented. To determine whether the intermediate occurring during factor IX activation by factor XIa has its active site exposed for p-NPGB the time course of activation of factor IX by factor XIa was followed l) by active site titration of the active sites generated, 2) by gel- electrophoretic analysis in the presence of sodium dodecyl sulfate, 3) by a clotting assay for factor IXa and 4) by measurement of factor IXa using a spectrophotomefric assay. It will be shown that the intermediate occurring during activation of factor IX by factor XIa does not interact with p-NPGB indicating that the active site is not available in the intermediate. Factor IXa can be determined spectrophotometrically by measurement of the rate of factor X activation by factor IXa in the presence of phospholipid and CaCl2. The factor Xa generated is measured using the chromogenic substrate S222. Since human factor IX can be activated with bovine factor XIa and since human factor IXa can activate bovine factorX the bovine clotting factors factor XIa and factor X can be used to construct an assay for factor fx in plasma samples. Experiments will be presented in which it is shown that the spec- trophotometric assay for human factor IXa can be used to determine levels of factor IX in plasma samples of healthy individuals, in plasma samples deficient in various clotting factors and in plasma samples from patients under anti-coagulant therapy. The results are in agreement with a factor IX clotting test.