title: Diagnostic and genetic studies on fibrin-stabilizing factor with a new assay based on amine incorporation.
abstract: Fibrinoligase, the fibrin cross-linking enzyme, transiently appearing during the course of coagulation in normal blood, was shown to catalyze the incorporation of a fluorescent amine, monodansylcadaverine [or N-(5-aminopentyl)-5-dimethylamino-1-naphthalene-sulfonamide] into casein. The reaction provided the basis of a sensitive fluorimetric method for measuring the activity of the enzyme (and also of similar other transpeptidases, such as transglutaminase). In tests involving plasma, certain difficulties had to be overcome which were mainly due to the fact that the enzyme itself does not occur in citrated plasma. Only its precursor (fibrin-stabilizing factor or factor XIII) is present, still requiring limited proteolytic activation by thrombin. Thus, in order to measure amine incorporation with plasma as a source of the factor, thrombin must be added. This necessitated a differential desensitization of the intrinsic fibrinogen so that the latter could not clot and could not thereby interfere with amine incorporation. Also, the thrombin-inactivating capacity of plasma had to be saturated to enable full conversion of the factor to the transpeptidase. Concentrations of casein, monodansylcadaverine, calcium, and hydrogen ions were chosen to permit almost maximal velocity of amine incorporation. A linear relationship with regard to plasma concentration could be obtained only under such conditions. No similar assay is presently available for quantitatively evaluating fibrin-stabilizing factor levels in plasma.The amine incorporation test was applied to a clinical case of hereditary total fibrin-stabilizing factor deficiency. The effect of transfusion therapy was studied, and some of the patient's relatives were examined. Whereas a paternal aunt and uncle gave values well within the normal range, a brother and the mother proved to be partially deficient and could be considered as heterozygous carriers. The father appeared to have a reduced level of fibrin-stabilizing factor, though not quite as low as the other two relatives. Two infusions (1 liter each) of fresh normal plasma, administered about 26 hr apart, brought levels in the patient's plasma close to those found in the mother and brother. The corrective power of the transfusions, however, rapidly declined within 5-6 days. Futility of the last transfusion could be ascribed to the appearance of a neutralizing antibody directed against the precursor stabilizing factor, a serious complication. General diagnostic versatility and potential of the quantitative amine incorporation assay with plasma is discussed.
mesh_terms: D000328:Adult; D000588:Amines / Q000378:metabolism*; D000906:Antibodies / Q000032:analysis; D001778:Blood Coagulation Disorders / Q000175:diagnosis* / Q000235:genetics* / Q000378:metabolism / Q000628:therapy; D001803:Blood Transfusion; D002118:Calcium / Q000494:pharmacology; D002364:Caseins / Q000096:biosynthesis; D005169:Factor VIII / Q000032:analysis; D005260:Female; D005337:Fibrin / Q000096:biosynthesis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D008297:Male; D008722:Methods; D013917:Thrombin / Q000032:analysis
chemical_list: D000588:Amines; D000906:Antibodies; D002364:Caseins; D005169:Factor VIII; D005337:Fibrin; D013917:Thrombin; D002118:Calcium