ABSTRACT
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.
Subject(s)
Chromatography, Affinity/methods , Thromboplastin/isolation & purification , Brain , Calcium/pharmacology , Electrophoresis, Polyacrylamide Gel , Factor VII/pharmacology , Humans , Molecular Weight , Sepharose/pharmacology , Thromboplastin/physiology , Tissue ExtractsABSTRACT
Tissue thromboplastin apoprotein was partially purified from human brain. The apoprotein was recombined with mixed phospholipids to yield active thromboplastin. The recombined thromboplastin induced proteolytic activation of isolated human factor IX in the presence of factor VII and Ca2+. The clotting times of various deficient plasmas were determined as a function of apoprotein concentration, keeping the phospholipid concentration constant. The clotting times of a factor XII-deficient plasma were the same as those of a factor XII/factor IX-deficient plasma, except at very low apoprotein concentrations. However, under those conditions the difference in clotting times was independent of the presence of anti-factor VII serum. Similar observations were made for factor XI-deficient plasma in comparison with factor XI/factor IX-deficient plasma. These results indicate that activation of factor IX by factor VII/tissue thromboplastin does not significantly contribute to plasma coagulation.
Subject(s)
Apoproteins/pharmacology , Blood Coagulation , Factor IX/analysis , Thromboplastin/pharmacology , Apoproteins/isolation & purification , Brain Chemistry , Calcium/pharmacology , Factor IXa , Factor VII , Humans , Thromboplastin/isolation & purificationABSTRACT
Pooled plasma of patients under stable oral anticoagulation has been analysed with respect to the presence of the vitamin-K dependent factors (factors II, VII, IX and X). Of all factors 1.5-2 times more antigen than procoagulant activity was present. The concentration of factors II, X (measured spectrophotometrically) and VI is about 0.25 U/ml while factor IX is slightly higher. Coagulation assays of factor X always gave lower values than the spectrophotometric assay. This discrepancy was not influenced by the removal of either factor II-, factor VII- or factor IX antigen. However, when the factor X antigen was replaced by normal factor X, all factor X assay gave identical results, indicating that PIVKA X is responsible for these discrepancies. Using the technic of the Thrombotest-dilution curve it was shown that PIVKA X is the factor that causes the abnormal prolongation of ox-brain prothrombin time in these plasmas.
