Extrinsic activation of human coagulation factors IX and X on the endothelial surface.

In previous kinetic studies, the catalytic efficiency of the activation of human coagulation factors IX and X by factor VIIa in the presence of purified tissue factor apoprotein was found to be essentially equal. These activation reactions were now studied on the surface of human umbilical vein endothelial cells. The cells were stimulated with endotoxin to express tissue factor. This tissue factor activity was saturable with factor VIIa and could be inhibited by rabbit antibodies against human tissue factor apoprotein. Only stimulated cells supported factor VIIa activity. No difference in the reactivity of factor VII and VIIa was observed in the presence of factor X, due to rapid feedback activation of factor VII by factor Xa. However, the activation of factor IX by factor VII shows a 10 min lag-phase, which reflects that the activation of factor VII by factor IXa is a less efficient process. The kinetic parameters for the factor VIIa dependent activation of factor IX and factor X on the endothelial surface were: Km 0.09 microM, Vmax 0.13 pmol/min, and Km 0.071 microM, Vmax 0.41 pmol/min, respectively. The same ratio between the Vmax for factor X and factor IX activation was observed as in a cell free system. However, the Km of factor IX was 4-fold higher on the endothelial surface than in the cell free system. Together, these kinetic parameters will favour factor X activation 5-fold over factor IX activation at physiological concentrations of these proteins. The activation of factor X by factor VIIa on the endothelial surface was characterized by a short lag-phase, which was absent in factor IX activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutations in hemophilia Bm occur at the Arg180-Val activation site or in the catalytic domain of factor IX.

Hemophilia Bm is characterized by a strikingly prolonged plasma ox brain prothrombin time. In an attempt to find an explanation for this phenomenon we have analyzed various aspects of the Bm variants factor IX Deventer, factor IX Milano, factor IX Novara, and factor IX Bergamo. Proteolytic cleavage by factor XIa was normal in two Bm variants, but absent at the Arg180-Val bond in the other two. In the latter variants Arg180 was replaced by either Trp or Gln, whereas Val181----Phe and Pro368----Thr replacements have occurred in the variants that were normally cleaved by factor XIa. In all four variants the Bm effect could be neutralized with a single monoclonal antibody against factor IX. Also, after treatment with factor XIa, none of the Bm variants reacted with antithrombin III (in contrast to normal factor IXa). Purified factor IX Deventer (one of the variants with a replacement of Arg181), either with or without pretreatment with factor XIa, was found to be a more effective competitive inhibitor of the factor VIIa-tissue factor-induced factor X activation than similarly treated normal factor IX. In addition, this inhibitory effect was much more pronounced when bovine tissue factor was used instead of human tissue factor. We propose that the normal activation of factor IX not only produces a conformational change around the active site serine that allows efficient substrate binding and catalysis, but that the same conformational change is instrumental in effectively dissociating factor IXa from the activating factor VIIa-tissue factor complex. Amino acid replacements that disrupt this conformational transition directly (e.g. Pro368----Thr near the catalytic center) or indirectly (mutations at the Arg180-Val activation site) therefore lead to a combination of 1) the loss of coagulant activity and 2) an inhibitory effect in the ox brain prothrombin time assay.

Application of factor VII-Sepharose affinity chromatography in the purification of human tissue factor apoprotein.

Coagulation factor VII covalently coupled to Sepharose proved to be an effective binding ligand for human tissue factor apoprotein, the specific cofactor of factor VII for the activation of factor X and IX. This interaction is completely calcium-dependent and the calcium ions cannot be replaced by magnesium or barium ions. The binding of the apoprotein to immobilized factor VII seems to be independent of the presence of phospholipid. When factor VII-Sepharose column chromatography is combined with a mild extraction procedure, tissue factor apoprotein could be purified approximately 40,000-fold from an acetone powder of human brain. SDS-PAA gel electrophoresis revealed that with this simple purification scheme human tissue factor apoprotein can be purified to apparent homogeneity and that the apoprotein migrates at a molecular weight of 47,000. The isolated human protein is heterogeneously glycosylated; the two different forms of the apoprotein function as cofactor of factor VII in the activation of both factor X and factor IX.