ON THE COAGULATION OF FIBRINOGEK’ BY ERWIN CHARGAFF AND AARON BENDICH (Prom the Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York) (Received for publication, April 26, 1943) The nature of the chemical reactions that, in the presence of thrombin, lead to the formation of fibrin, the insoluble coagulation product of fibrino- gen, is largely unknown. Because of the lack of reliable comparative analyses of the composition of pure fibrinogen and fibrin and of the amino acid distribution in these proteins, it is not even possible to decide whether Downloaded from the products obtained from fibrinogen by heat coagulation and by the action of thrombin are different. To speak, as is sometimes done, of throm- bin as a “denaturase” (1, 2) is, at the present state of our knowledge, an addition to our terminology rather than to the understanding of the reac- tions involved. www.jbc.org Papain has been shown to coagulate fibrinogen directly (3), but this in itself is not sufficient indication of the proteolytic nature of the physiolog- ical coagulation phenomenon. It is, however, conceivable that with cer- tain enzymes the formation of fibrin represents the first step in the pro- by guest, on September 5, 2010 teolytic degradation of fibrinogen and that this initial phase is particularly protracted with thrombin as clotting agent. The literature contains re- ports on the fibrinolytic action of thrombin (4, 5), and experiments on the formation and lysis of fibrin by staphylococci may also be cited (6). For the time being, it would appear advisable to limit the definition of the action of thrombin to the statement that it brings about changes in the structure of fibrinogen that lead to the formation of the insoluble protein fibrin1 and to defer speculation on the nature of these changes to a time when more experimental data are available. It has recently been found and briefly reported from this laboratory (7) that ninhydrin (1,2,3-indantrione hydrate) (I) coagulated fibrinogen and formed clots which in some respects closely resembled fibrin. Alloxan (II) and salicylaldehyde were likewise, although much less markedly, ac- tive. This observation led to a search for other synthetic agents having a * This work has been supported by a grant from the John and Mary R. Markle Foundation. This is Paper XVI of a series of studies on the chemistry of blood coagulation. r It will avoid misunderstandings if the term fibrin is reserved for the coagulation product obtained with thrombin. Insoluble products formed from fibrinogen with other clotting agents will be referred to as coagulated fibrinogen. 93 94 COAGULATION OF FIBRINOGEN NH-CO I I co co I I NH-CO (0 (II) similar action. The sodium salt of 1,2-naphtizoquinone-4-suljonic acid (III) and the potassium salt of 1,4-naphthoquinone-W-suZjonic acid (IV) have been found to produce firm clots from solutions of purified fibrinogen at 30” and pH 7. Ch!loramine-T (sodium p-toluenesulfonchloroamide) Downloaded from (V), while acting even more rapidly under similar conditions, forms a loose coagulum. 0 0 CH, www.jbc.org by guest, on September 5, 2010 Neither the thromboplastic factor nor calcium was required in this reac- tion which proceeded with fibrinogen preparations free of prothrombin. The clotting times observed depended on the concentrations of both the fibrinogen and the clotting agent. In fibrinogen solutions of the strength used in the present investigation, the clotting agents, with the exception of chloramine-T, produced firm coherent clots. The retraction of the clots appeared to proceed at a faster rate than with fibrin produced by thrombin: a solid opaque core of protein began to form underneath the transparent gel a short time after the coagulation by the clotting agent in high con- centration. In a number of experiments, the recovery of nitrogen in the coagulation products was studied (Tables III and IV). With ninhydrin and the LY-and p-naphthoquinone sulfonates, the amounts of fibrinogen N recovered in the clots were of the same order of magnitude as found in the correspond- ing fibrin samples. Alloxan, and perhaps also chloramine-T, appeared to attack the protein more energetically. The low sulfur content of the co- agulation product obtained with alloxan (Table III, Experiment 4) may be significant. Compound formation between ninhydrin and fibrinogen E. CHARGAFF APiD A. BENDICH 95 seemed to have occurred to only a slight extent, if at all. The somewhat higher sulfur contents of the fibrinogen samples clotted by the napht,ho- quinone sulfonates may indicate the presence of these agents in the clots. It is, however, so difficult to wash these coagulation products satisfactorily that mechanical adsorption cannot be excluded. It would undoubtedly be of interest to study the x-ray diffraction patterns of the coagulation prod- ucts of fibrinogen. The clotting activity of the ketones and quinones mentioned in the pre- ceding paragraphs made it appear of interest to st,udy a number of biologi- cally occurring compounds that could, under physiological conditions, con- ceivably give rise to active substances. One of the systems studied was Downloaded from mesoinositol and the series of oxidation products derived from it; viz., tetrahydroxyquinone (VI), rhodizonic acid (VII), croconic acid (VIII),z leuconic acid (IX). None of these compounds, however, exhibited any ii o=c- C-OH o=c- c=o www.jbc.org OH O- , -OH I II I I o=c C-OH O=C c=o ..+ OH 0 Q OH ‘C’ ‘C/ I Ii- II II by guest, on September 5, 2010 0 0 0 0 (VI) (VII) (VIII) (IX) clotting activity. Solutions containing l-ascorbic acid oxidized with iodine (S-10) or by the catalytic action of the ascorbic oxidase from squash (11) likewise had no effect on fibrinogen. Another biological syst,em studied, which could be considered most immediately related to the subject of this communication, was that of com- pounds belonging to the vitamin K group, of which Z-methyl-l ,4-naphtho- quinone is the most active representative (compare (12-14)). The ex- amination of the possible direct role in clotting of compounds displaying vitamin K activity is, unfortuna,tely, difficult. The quinones having vita- min K activity are not sufficiently soluble in water for clotting tests and all known water-soluble vitamin K analogues are derivatives of either the related hydroquinone or of naphthalene. Two water-soluble derivatives of 2-methyl-1,4-naphthoquinone were examined; viz., the potassium salts of 2-methyl-1,4-naphthoquinon&-sulfonic acid (X) (15) and the new com- pound3 2-methyl-i ,&naphthoquinone-8-sulfonic acid (XI). The anti- hemorrhagic activity of compound (X) is only slight ((15), compare also (16)) and that of compound (XI) has not yet been established, but it is 2 The position of the hydroxyls in compounds (VII) and (VIII) is not quite certain. 3 Chargaff, E., and Bendich, A., unpublished results. 96 COAGULATION OF FIBRINOGEN 0 SOaK 0 A -CIIz / \’ -CH, IA- I if) \ ( SosK \n A II 0 0 co (XI) known that the introduction of a methyl group into position 8, as in 2,8- dimethyl-1,4-naphthoquinone (17), destroys the antihemorrhagic activity almost completely (13). Compound (X) proved inactive as a clotting Downloaded from agent. As this was possibly due to the absence of a free 3 position, com- pound (XI) was prepared, but it, too, failed to coagulate fibrinogen. It thus cannot be proposed at present that vitamin K, or a compound to which it gives rise in the body, is directly involved in the coagulation of fibrinogen; e.g., as a prosthetic group in the prothrombin or thrombin www.jbc.org molecule. The contrary may, in fact, be true; viz., that quinones display- ing vitamin K activity are structurally protected from entering into reac- tions with the body proteins (of which the clotting of fibrinogen may be only one instance) that would prevent them from reaching the liver. A by guest, on September 5, 2010 similar suggestion was first put forward by Fieser and collaborators ((13) p. 668). A list of other carbonyl compounds which were tested for fibrinogen clotting and found inactive will be given in the experimental part. It is, perhaps, noteworthy that the triketone d,S, 4-triketopentane, which may be considered the simplest nliphatic analogue of ninhydrin, failed to coagu- late fibrinogen. The discussion of the possible mechanism of action of the synthetic clotting agents on fibrinogen will best start from the fact that most of the compounds found to be active are able to oxidize amino acids and peptides containing free amino groups. This effect has been shown for chlor- amine-T (18), 1,2-naphthoquinone-4-sulfonic acid (19)) ninhydrin (20, 21), and alloxan (22). For purposes of comparison the action on alanine of some of the clotting agents and also of several carbonyl compounds devoid of coagulant activity was studied. It will be seen from the data presented in this paper that, with one significant exception, the ability of the compounds to decarboxylate the amino acid paralleled their activity in the coagulation of fibrinogen. Generally, substances that failed to liberate COz from alanine were inactive in clotting, with the exception of 1,4-naphthoquinone-2-sulfonic acid which, although an active clotting agent, did not decarboxylate alanine. The very pronounced reactivity of 1,2-naphthoquinone and its 4-sulfonic 31. CHARQAFF AND A. BDNDICH 97 acid is well known and has been particularly stressed in the procedures developed by Ehrlich and Herter (23) and by Folin (24). Rmines are added in position 4 (25, 26), in the case of the 4-sulfonate with the simul- taneous liberation of bisulfite (27).