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Diagnostic and Genetic Studies on Fibrin-Stabilizing Factor with a New Assay Based on Amine Incorporation

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Diagnostic and Genetic Studies on Fibrin-Stabilizing Factor with a New Assay Based on Amine Incorporation Diagnostic and Genetic Studies on Fibrin-Stabilizing Factor with a New Assay Based on Amine Incorporation L. Lom~r., T. URAYAMA, J. W. C. DE KIEwIwT, and H. L. NossEL From the Biochemistry Division, Department of Chemistry, Northwestern University, Evanston, Illinois 60201 and the Department of Medicine, College of Physicians and Surgeons, Columbia University, New York 10032 A B S T R A C T Fibrinoligase, the fibrin cross-linking en- The amine incorporation test was applied to a clinical zyme, transiently appearing during the course of coagu- case of hereditary total fibrin-stabilizing factor defi- lation in normal blood, was shown to catalyze the incor- ciency. The effect of transfusion therapy was studied, poration of a fluorescent amine, monodansylcadaverine and some of the patient's relatives were examined. [or N- ( 5-aminopentyl) -5-dimethylamino-1-naphthalene- Whereas a paternal aunt and uncle gave values well sulfonamide] into casein. The reaction provided the within the normal range, a brother and the mother basis of a sensitive fluorimetric method for measuring proved to be partially deficient and could be considered as the activity of the enzyme (and also of similar other heterozygous carriers. The father appeared to have a transpeptidases, such as transglutaminase). reduced level of fibrin-stabilizing factor, though not In tests involving plasma, certain difficulties had to be quite as low as the other two relatives. overcome which were mainly due to the fact that the Two infusions (1 liter each) of fresh normal plasma, enzyme itself does not occur in citrated plasma. Only its administered about 26 hr apart, brought levels in the precursor (fibrin-stabilizing factor or factor XIII) is patient's plasma close to those found in the mother present, still requiring limited proteolytic activation by and brother. The corrective power of the transfusions, thrombin. Thus, in order to measure amine incorpora- however, rapidly declined within 5-6 days. Futility of tion with plasma as a source of the factor, thrombin the last transfusion could be ascribed to the appearance must be added. This necessitated a differential desensi- of a neutralizing antibody directed against the precursor tization of the intrinsic fibrinogen so that the latter stabilizing factor, a serious complication. could not clot and could not thereby interfere with amine General diagnostic versatility and potential of the incorporation. Also, the thrombin-inactivating capacity quantitative amine incorporation assay with plasma is of plasma had to be saturated to enable full conversion discussed. of the factor to the transpeptidase. Concentrations of casein, monodansylcadaverine, calcium, and hydrogen ions were chosen to permit almost maximal velocity of INTRODUCTION amine incorporation. A linear relationship with regard Covalent cross-linking of fibrin by transpeptidation con- to plasma concentration could be obtained only under stitutes the last step in normal blood coagulation (1). such conditions. No similar assay is presently available It involves the selective formation of y-glutamyl-E-lysine for quantitatively evaluating fibrin-stabilizing factor bridges (approximately 1 mole/100,000 g of protein) levels in plasma. between fibrin units (2-4) and results in the production of more stable structures which can no longer be dis- An account of this work was given as a lecture at the persed in 1 % monochloroacetic acid (5) or in 5 M urea Federation Meetings, Atlantic City, 1968. (Urayama, T., and L. Lorand. 1968. Fed. Proc. 27: 693; Abstract No. 2682.) (6). Some important aspects of this fibrin-stabilizing Received for publication 22 November 1968 and in revised system have already been uncovered. Normal plasma form 7 February 1969. contains a precursor protein (7) (called fibrin-stabilizing 1054 The Journal of Clinical Investigation Volume 48 1969 factor or factor XIII [8]) which is converted to the mate the corrective power (16) of a given plasma active transpeptidase (fibrinoligase) by the action of (normal or partially deficient in the factor) when mixed thrombin. In this limited proteolytic process, the electro- with one which is totally deficient in fibrin-stabilizing phoretic mobility of the factor (bovine) is changed, but factor. sedimentation characteristics (3) are not altered appre- Meaningful quantitative tests for fibrin-stabilizing fac- ciably. Fibrinoligase functions as a transpeptidating (i.e. tor in plasma would, of course, be of great clinical value. transamidating) enzyme catalyzing a reaction between Using amine incorporation as a tool, we have now (donor) E-amino-lysine (9) and (acceptor) .y-glutamyl developed a suitable assay and applied it for studying (10) carbonyl groups of adjacent fibrin molecules. the inheritance pattern of the fibrin-stabilizing factor Cross-linking of fibrin was shown to be selectively deficiency disease (17) and for evaluating transfusion inhibited by certain amines (2, 9, 10-12) which are therapy in a totally deficient individual with a severe themselves substrates for the enzyme. As such, these bleeding disorder. Though studies at Northwestern Uni- compounds become incorporated into fibrin and block versity now encompass six totally deficient unrelated its acceptor groups for normal cross-linking. The amine- individuals and their families, the present paper will incorporating activity of the enzyme forms the basis of deal only with the first such case which was initially a quantitative analysis. In specific terms, the incorpora- investigated at Columbia University. The other studies tions of hydroxylamine, hydrazine, glycine ethyl ester, will be reported within the framework of a more exten- monodansylcadaverine,' and histamine have been studied sive genetic survey in due course. (Table I). The first two served as chemical labels, forming fibrin hydroxamate and hydrazide, respectively. METHODS Glycine ethyl ester and histamine were used in the form The sulfate salt of monodansylcadaverine or N-(5-amino- of radioisotopes. As a fluorescent compound, mono- pentyl)-5-dimethylamino-1-naphthalenesulfonamide was pre- dansylcadaverine proved to be a very sensitive tracer pared by Dr. N. G. Rule as described previously (2). and was chosen as a donor substrate for the investiga- Approximately 30 mg of this compound was dissolved in tions in the 3 ml of 0.05 M tris (hydroxymethyl) amino methane (Trizma reported present paper. base, Sigma Chemical Co., St. Louis, Mo.) adjusted to pH Hitherto, assay methods (13) for the stabilizing sys- 7.5 with hydrochloric acid. Concentration of the stock solu- tem depended on the ability of the activated factor to tion is determined by measuring the absorbancy (at 326 mIu) convert simple fibrin gels into the acid- or urea-insoluble of a suitable (10 .1) aliquot in (2.0 ml) 20% (v/v) dioxane- form. The extent of "insoluble" clot formation was taken water. We assume that the molar extinction is identical (4.64 X 106 cm' mole-1) with that of dansylamide (see ref- as an index of cross-linking. Dilution of the factor to a erence 18). threshold level of activity provided the basis for empiri- Bovine thrombin was purified from Parke Davis & Co. cal quantitation. In its most recently developed version Thrombin Tropical by a modification (Lorand and Urayama, (14), a multistage assay consisted of (a) the activation unpublished data) of the Rasmussen procedure (19). Activity of the enzyme was matched against a National Institutes of of fibrin-stabilizing factor by thrombin, followed (b) by Health (NIH) standard (lot No. B-3). the quenching of thrombin (unless water-insoluble en- Acid citrate dextrose (ACD) human plasma for the zyme was used (3), which could be removed by filtra- isolation of fibrin-stabilizing factor (3, 20) was obtained tion); (c) the activated factor was then mixed with through the courtesy of Dr. J. N. Shanberge of Evanston isolated fibrin and (d) cross-linking was allowed to Hospital. Factor activity was expressed in terms of fibrin cross-linking units, based on the visual disappearance of proceed. Finally, (e) 1% monochloroacetic acid was clots in monochloroacetic acid as previously described (13). added to test for the extent of cross-linking either by Solutions of the factor were kept at 0°C in 0.05 M Tris- visual inspection or by quantitatively analyzing the acid- 0.1 M NaCl, 1 mm ethylenediaminetetraacetate at pH 7.5. insoluble residue. Though this and related methods Casein (Hammarsten), lot no. T-1371, was purchased from Mann Research Laboratories, Inc., New York, re- could be applied usefully for studying purified factor duced glutathione from Cyclo Chem. Corp., Los Angeles, preparations, assays based on clot solubility are particu- Calif., and sodium dodecylsulfate from K. and K. Labora- larly difficult to interpret quantitatively when direct tories, Jamaica, N. Y. Other compounds were reagent grade. testing of the fibrin-stabilizing factor content of plasma All solutions must be clear and, if necessary, filtered. Fluorescence intensities were obtained with an Aminco- is required. Since the thrombin-activated factor is sensi- Bowman spectrophotofluorimeter (excitation at approxi- tive to sulfhydryl-blocking reagents, efforts have been mately 355 m/u; emission at approximately 525 m/u). In directed towards a method based on "iodoacetate toler- order to provide a suitable solvent system for redissolving the trichloroacetic acid-precipitated proteins, all fluorescence ance" in relation to the formation of urea- or acid- measurements were carried out in a mixture
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