Factor VII activity, as measured in a one-stage clotting assay, increases when whole blood is clotted in glass. Prior studies in this laboratory using factor-deficient plasmas suggested that this factor VII activation was due to activated factor IX (IXa). We therefore studied activation of VII by IXa and by activated factor X (Xa) in purified systems. Human factors II, VII, IX, and X were each purified to homogeneity as judged by SDS-polyacrylamide gel electrophoresis. Reaction mixtures of VII, IXa or Xa, and other cofactors and enzymes were made, and subsampled for VII activity. The activation state of VII was judged by comparison of one-stage clotting assay to a coupled amidolytic assay using a synthetic substrate. In the presence of phospholipid (PL) and calcium (Ca), both IXa and Xa activated VII 25 fold; however, Xa was roughly 800 times more efficient than IXa. In the absence of PL, Xa was roughly 20 times more efficient than IXa, in Ca-containing solutions. Only slight activation of VII by either enzyme occurred in the absence of Ca. The addition of prothrombin (II) markedly slowed activation of VII by both Xa and IXa; however, this effect did not occur if fully-decarboxylated II was used. The addition of anti thrombin III and thrombin-modified factor VIII at physiologic concentrations did not change rates of VII activation by IXa or Xa.These results confirm the ability of IXa and Xa to activate factor VII at physiologic concentrations in purified systems. The higher relative efficiency of Xa over IXa under all conditions studied contrasts strikingly with observations in whole plasma systems where the VII activation measurable after clotting is greater in X-deficient than in IX-deficient plasma. The activation of VII by Xa and IXa may serve as an amplification loop in the generation of clotting by either “intrinsic” or “extrinsic” cascades.
Activation of human factor VII by activated factors IX and X
