THE CONTRIBUTIONS OF CA-2+, PHOSPHOLIPIDS AND TISSUE-FACTOR APOPROTEIN TO THE ACTIVATION OF HUMAN BLOOD-COAGULATION FACTOR-X BY ACTIVATED FACTOR-VII

In the extrinsic pathway of blood coagulation, Factor X is activated by a complex of tissue factor, factor VII(a) and Ca2+ ions. Using purified human coagulation factors and a sensitive spectrophotometric assay for Factor Xa, we could demonstrate activation of Factor X by Factor VIIa in the absence of tissue-factor apoprotein, phospholipids and Ca2+. This finding allowed a kinetic analysis of the contribution of each of the cofactors. Ca2+ stimulated the reaction rate 10-fold at an optimum of 6 mM (V(max) of 1.1 x 10-3 min-1) mainly by decreasing the K(m) of Factor X (to 11.4 μM). In the presence of Ca2+, 25 μM-phospholipid caused a 150-fold decrease of the apparent K(m) and a 2-fold increase of the apparent V(max) of the reaction; however, both kinetic parameters increased with increasing phospholipid concentration. Tissue-factor apoprotein contributed to the reaction rate mainly by an increase of the V(max), in both the presence (40 500-fold) and absence (4900-fold) of phospholipid. The formation of a ternary complex of Factor VIIa with tissue-factor apoprotein and phospholipid was responsible for a 15 million-fold increase in the catalytic efficiency of Factor X activation. The presence of Ca2+ was absolutely required for the stimulatory effects of phospholipid and apoprotein. The data fit a general model in which the Ca2+-dependent conformation allows Factor VIIa to bind tissue-factor apoprotein and/or a negatively charged phospholipid surface resulting into a decreased intrinsic K(m) and an increased V(max) for the activation of fluid-phase Factor X.