A B S T R A C T 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-naphthalenesulfonamide] 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.

Summary. The biochemical defect in a patient suffering from very severe bleeding episodes was shown to be related to the presence of an inhibitor which specifically interfered only with the thrombin‐catalysed conversion of the fibrin stabilizing factor zymogen (factor XIII) into fibrinoligase, but had no effect on the transamidase activity of the latter enzyme. As such, it differed from other known inhibitors of the fibrin stabilizing factor system. Inhibition could be demonstrated against both human and bovine factor XIII. In terms of neutralizing equivalents, the patient's plasma contained about a tenfold excess of the inhibitor over the factor‐XIII content found in average normal plasma. This high potency was thought to be the reason for the patient's unresponsiveness to transfusions. Precipitation with ammonium sulphate, followed by chromatographic purification on DEAE‐cellulose, allowed recovery of the inhibitor in good yield in the ‘IgG immunoglobulin’ fraction. Since, prior to the onset of bleeding episodes at about 50 yr of age the patient received no transfusions, it could be assumed that the inhibitor was of autoimmune origin. Though the cause of the disease remains unknown, the question is raised whether previous treatment with isoniazid could be involved. Accidental modification of fibrin stabilizing factor by isoniazid could have provoked a breakdown of immunological tolerance against this component in the patient's plasma. Lorand et al (1972) have recently demonstrated the modification of proteins with isoniazid in enzymatic reactions.

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