STUDIES ON THE CHEMISTRY OF BLOOD COAGULATION

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1 STUDIES ON THE CHEMISTRY OF BLOOD COAGULATION IV. LIPID INHIBITORS OF BLOOD CLOTTING OCCURRING IN MAMMALIAN TISSUE* BY ERWIN CHARGAFF (From the Departmenls of Biological Chemistry and Surgery, College oj Physicians and Surgeons, Columbia University, New York) (Received for publication, July 31, 1937) Recently it has been found and reported in a brief note (1) that cerebroside fractions obtained from brain of sheep and pigs and from spinal cord of cattle contain a substance of apparently lipid nature which a&s as inhibitor of the clotting of blood and plasma. Since that time a fraction of similar properties has also been isolated from blood cells. This apparently new substance or group of substances is of interest, since any finding relative to the controlling factors of blood clotting, which function in thr body, will contribute to a final elucidation of the extremely complicated problem of blood coagulation. It is proposed here to describe as concisely as possible the isolat,ion of the lipid inhibit,ors from various sources and their properties. EXPERIMENTAL In the course of this work a very large number of fratitions were collected and assayed as to their action in blood clotting. It obviously will be possible to give only a few instances. Preparation of Lipid Inhibitor from Sheep Brain Fractionation of Brain Lipids-The first organ to be examined, sheep brain, proved one of the best sources of the inhibiting lipid fraction. 50 brains, weighing about 4.5 kilos, were three times passed through the meat grinder. The material then was ex- * Study of the mechanism of thrombosis and embolism supported by the Carnegie Corporation of New York. 175

2 176 Chemistry of Blood Coagulation. IV tracted with 6 lit,ers of acetone at room temperature for 24 hours. The acetone treatment was repeated twice with fresh solvent and the organ powder was subsequently treated four times for 24 hours with freshly distilled ether, 3 liters of the solvent being used each time. In the last extraction only a small amount of material was removed. The air-dried powder was suspended in 3 liters of absolute alcohol and kept at boiling temperature for 5 minutes. The hot solution was filtered. The filtrate on being chilled deposited a large amount, of cerebrosides which were removed by filtration. The tissue powder was reextracted with the alcoholic mother liquor of the cerebroside precipitates. Five extractions sufficed for the almost complete removal of alcohol-soluble cerebrosides. To the crude material thus extracted a second cerebroside crop was added which had precipitated from the ethereal brain extracts on extended cooling. The cerebrosides, the total weight of which was gm., were dissolved in a warm mixture of 1700 cc. of chloroform and absolute alcohol (2: l), and filtered. On cooling Fraction A-l crystallized as gm. of snow-white protagon crystals. Analysis, found, N 2.2, P 0.5, S 0.8. The precipitate obtained on concentrat,ion of the mother liquor of Fraction A-l was recrystallized from chloroform-alcohol (2:1), yielding Fraction A-2, gm. of a white, not definitely crystalline powder. Analysis, found, N 2.0, P 1.3, S Determination of Clotting Time-The anticoagulant activity of the various fractions was assayed according to the technique recently developed in this laboratory (2). All the determinations were carried out in a water thermost.at at 30. The usual substrate was chicken plasma, either unactivated or activated by the addition of muscle extract (2,3). In a few cases human blood and recalcified oxalated human plasma were also used. It was quite easy to obtain relatively stable emulsions of the lipids examined by converting them into a homogeneous paste with a small amount of physiological saline, followed by dilut,ion to the desired concentration. The volumes used throughout were 0.1 cc. of plasma, 0.03 cc. of the lipid emulsions, and 0.02 cc. of an approximately 0.35 per cent solution of the muscle extract preparation in physiological saline. The readings were carried out at intervals of bct,ween 6 and 10 minutes with activated plasma

3 E. Ghargaff 177 and somewhat less frequently when unactivated plasma was used. All measurements were made in duplicate. Action of Cerebrosides in Blood Clotting-Fraction A-l which, as it would appear from the analytical results, consisted mainly of cerebron, kerasin, and the sulfur-containing lipids, was found completely inactive in blood clotting. Subfractions obtained by recrystallization from various solvents also proved inactive. On the other hand, Fraction A-2, which was considerably richer in phosphatides, exerted a very marked anticoagulant act,ivity, as shown in Table I. Isolation of Lipid khibitor--a solution of 7.43 gm. of Fraction -4-2 in 35 cc. of warm dry pyridine was cooled to - 12, and the TABLE Fraction A-d from Sheep Brain Amount in 0.1 cc. activated chicken plasma Clotting time ml. I precipitate filtered off and washed with cold acetone. Fraction A-3 consisted of 3.64 gm. of a white powder. This substance showed still more marked anticoagulant activity than Fraction A-2. Analysis, found, N 2.1, P 2.1, S From the pyridine filtrate of Fraction A-3 Fraction A-4 was obtained by precipitation with acetone as 3.16 gm. of a slightly yellow powder, which showed some anticoagulant properties. Analysis, found, N 1.7, P 0.5, S Fraction A-3 had about twice the anticoagulant potency of A-2, whereas Fraction A-4 was only half as potent as A-2. This experiment shows that the inhibitor is but sparingly soluble in pyridine. Fraction A-3 was twice recrystallized from 20 cc. and 10 cc.

4 178 Chemistry of Blood Coagulation. IV portions of glacial acetic acid. The resulting fraction, A-5, weighed 1.99 gm. and was devoid of any inhibiting properties. It consisted mainly of cerebron and kerasin. In the same manner, a similar, inactive fraction, A-6, weighing 2.60 gm. was obtained from Fraction A-4. The combined acebic acid mother liquors from Fraction A-5 on addition of acetone yielded Fraction A-7, 0.55 gm. of a yellowish powder which retained most of the anticoagulant activity of Fraction A-3. Analysis, found, N 2.3, P 3.0, S From this experiment the inhibitor appears t.o be easily soluble in glacial acetic acid. Fraction A-7 (480 mg.) was recrystallized from 8 cc. of methyl Amount in 0.1 cc. plasma or blood - w > >250.- TABLE Fraction A-8 jrom Sheep Brain i II Clotting Recalcified time oxalsted human plasma 2 s ,- - Human blood alcohol in the presence of norit. The resulting fraction, A-8, consisted of 230 mg. of a faintly yellow powder. The inhibiting effect as exerted by this fraction on the clotting of chicken plasma, human plasma, and human blood is shown in Table II. Analysis, found, N 2.5, P 2.7. From the methyl alcohol mother liquor Fraction A-9 was recovered in the usual manner, 160 mg. of a white powder, which was only slightly less active as inhibitor than was Fraction A-8. Analysis, found, N 2.8, P 3.5. The properties of the lipid anticoagulant (Fractions A-8 and A-9) may be summarized as follows: It is insoluble in acetone, slightly soluble in cold pyridine and ether, easily soluble in cold glacial acetic acid, and can be recrystallized from methyl alcohol or

5 E. Chargaff 179 ethyl acetate. With water the typical myelin forms are observed. All fractions examined contained nitrogen and phosphorus, and small amounts of sulfur. The compounds gave no Molisch reaction and failed to reduce Fehling s solution even after acid hydrolysis. From the data given above it will be clear that the inhibitor will be found in the crude sphingomyelin fraction of brain and possibly other organs. In order to estimate the amounts of sphingomyelin in our fractions and to ascertain whether purified sphingomyelin has any action on blood clotting, use was made of the crystalline compound between sphingomyelin and Reinecke acid, which recently has been described by Thannhauser and Setz (4). From mg. of Fraction A mg. of a pink crystalline reineckate were obtained; from 78.0 mg. of Fraction A mg. of a similar compound. These salts were slightly soluble in cold methyl alcohol, insoluble in cold acetone. Assuming that the conversion factor of 0.877, as given by Thannhauser and Setz (5), can be applied in this case, Fraction A-8 would have contained 71.3 per cent and Fraction A per cent of sphingomyelin. The purified sphingomyelin preparations which were recovered from their reineckates by decomposition with silver nitrate (4) were found to be entirely inactive in blood clotting. Because of the very small amounts available, it could not yet be determined whether that portion which was not precipitated by Reinecke acid had retained the anticoagulant activity. Levene (6) has shown that an impurity which tenaciously clings to the sphingomyelin can be removed by precipitation with ethyl alcohol from the solution of the phosphatide in ligroin. Accordingly, a further purification of the inhibitor was attempted. The combined remainder of Fractions A-8 and A-9 (150 mg.) was twice recrystallized from ethyl acetate. The resulting fraction, A-10, 138 mg. of a white powder, was slightly more active than Fraction A-8. To a solution of mg. of Fraction A-10 in 2 cc. of a mixture of ligroin (b.p ) and absolute alcohol (5:l) were added 6 cc. of absolute alcohol; the mixture was cooled and the precipitate which had formed was removed by centrifugation, washed with alcohol, and dried. It weighed 12.7 mg. From the ligroin-alcohol solution the dissolved material was recovered (117.6 mg.) and subjected to a second precipitation with alcohol

6 180 Chemistry of Blood Coagulation. IV from ligroin. The second precipimtc, which weighed 13.7 mg., was combined with the first and designated Fraction A-11. It formed a fine slight,ly yellow powder which on contact with water readily formed an opalescent, solution. Analysis, found, N 1.9, P 2.5, S 1.1. The soluble portion was rccovcred by concentration of the solution in vacua and precipit,at,ion with acetone. Fraction A-12 after crystallization from ethyl acetate weighed 98.9 mg. and formed a white powder. Its solubility properties were similar to those of Fraction A-8. Analysis, found, C 62.4, H 10.8, N 2.6, P 3.0, s 0.7. Both Fractions A-11 and A-12 had anticoagulant properties, TABLE Fractions A-11 and A-12 jrom Sheep Brain III Amount in 0.1 cc. activated chicken plasma nq I- i - Clotting time Fraction A-11 Fraction A Fraction A-12 being considerably more active, as shown in Table III. It should, however, be noted that in comparison with Fraction A-8 the purification process applied did not result in any marked increase in activity. This fact will be discussed later in this paper. Experiments on Lipid Inhibitors jrom Other Sources Pig Brain-With one batch of this organ results essentially similar to those reported for sheep brain were observed, although the various fractions showed a considerably lower activity. Some of the cruder fractions had to be assayed with unactivated chicken plasma, in order to demonstrate the anticoagulant action. With another portion of pig brains it was observed that the clot-

7 E. Chargaff 181 ting activator, which is known to be associated with the cephalin group (cj. (7)) and ordinarily can be extracted by exhaustive treatment of the tissue with ether, was removed with the utmost difficulty. Most of the cerebrosidc fractions collected still showed some thrombokinase activity. In some fractions the simultaneous presence of an inhibiting substance could be demonstrated. This is due to the fact that the activator and the inhibitor do not seem to neutralize each other. This question will be taken up later in this paper. It was attempted to bring about the removal of cephalin by treating the cadmium chloride complexes of the various fractions with benzene, which dissolves the cadmium chloride complex of cephalin. These experiments were discontinued when it was found that the cerebrosides purified TABLE IV Fraction E-6 from Beef Spinal Cord Amount in 0.1 cc. plssma w Clotting time Chicken plasma Activated chicken plasma over t heir cadmium double salts still contained traces of cadmium, even after repeated treatment with 20 per cent ammonia in methyl alcohol. The presence of small amounts of cadmium could be detected by means of the spot test with diphenylcarbazide (8). Cadmium salts are known to exert anticoagulant act,ivity (9), and we have found that 1 mg. of CdClz.2H& corresponded to 90 inhibit or units. Beef Spinal Cord-We are indebted to Dr. D. Klein, of The Wilson Laboratories, Chicago, for 450 gm. of material representing the total benzene extract of spinal cord of cattle. After extraction with ether, recrystallization from chloroform-alcohol (2: l), and treatment with glacial acetic acid, as described above, Fraction E-6 was obtained in a yield of 0.18 per cent of the starting

8 182 Chemistry of Blood Coagulation. IV material, which had the anticoagulant properties shown in Table IV. Sheep Blood-A mixture of 3.5 liters of blood and a solution of 9.4 gm. of sodium oxalate in 1.2 liters of isotonic saline was centrifuged for 30 minutes. The supernatant plasma was drawn off and worked up separately. The sedimented blood cells were several times washed with saline containing 0.1 per cent of sodium oxalate and with pure saline in the cold, and dehydrated with acetone. They then were extracted in a continuous all-glass extractor (fitted with a porous glass filt,er plate) with acetone, ether, and a mixture of 3 parts of methyl alcohol and 1 part of chloroform. The defatted cells weighed gm. From the methyl alcohol-chloroform extract Fraction F-l was obtained, TABLE V Fraction P-6 from Blood Cells of Sheep Amount in 0.1 cc. activated chicken plamm Clotting time LT mg. of a brown powder. This preparation was found to contain both activator and inhibitor. The blood cells were then twice extracted with a total volume of 1000 cc. of boiling absolute alcohol. In this way Fraction F-6 was obtained as 460 mg. of a brown powder. This substance was a pot,ent anticoagulant, as shown in Table T. Treatment with warm glacial acetic acid resulted in the removal of some insoluble inert material. From the acetic acid solution an active substance was obtained, Fraction F-7, weighing mg. Preliminary experiments indicated that the active substance was soluble in chloroform-ethyl acetate (1:3). The coloring impurities can be removed by slow filtration of a dilute solution of the inhibitor in methyl alcohol-ligroin (9: 1) through an adsorption column con-

9 E. Chargaff 183 taining aluminum oxide (activated according to Brockmann). The inhibitor itself is not adsorbed. The oxalated plasma which had been freed of blood cells was worked up separately. All the lipid fractions obtained showed activating effects only. Influence of Cephalin on Anticoagulant Action of Lipid Inhibitor-It has been mentioned above that fractions were ob- TABLE Lipid Fractions Containing Activator and Inhibitor. Tested on Unactivated Chicken Plasma Fmotion D-3 (pig brain) D-15 (pig brain) E-4 (beef spinal cord) F-l (sheep blood cells) VI homt in 0.1 cc. plasma Clotting time w. Control Control Control Control tained quite frequently which seemed to contain both activators and inhibitors of blood clotting. This inference is based on a number of tests in which characteristic results were obtained, a selection of which is arranged in Table VI. It will be seen that, while the fractions tested had a certain activating effect inasmuch as the controls invariably clotted last, the clotting times observed were directly proportional to the amounts used, instead of inversely proportional, as would be the case for an ordinary acti-

10 184 Chemistry of Blood Coagulation. IV vator (cf. (7) Table II). In the case of inhibitors the clotting times evidently are directly proportional to the amount of substance applied. For the time being, the only explanation which can be advanced is that these fractions consist of a mixture of activator and inhibitor having different reaction mechanisms or reaction speeds of a different order of magnitude, so that by the superimposition of two antagonistic effects the peculiar picture here discussed is produced. It. will be the subject of fut,ure work to develop a method for the dissociation of such mixt,urcs. So far, experiments along this line have been unsuccessful. DISCIJSSION It is not yet possible to decide whether the lipid inhibitor described in the present article has any important physiological function among the fact,ors which may be assumed to control the normal behavior of blood in the body. The fact that it has been found in various organs and even in blood, although interesting, does not necessarily imply that it plays an essential role. The same reservation, of course, applies equally to heparin, as has been recently pointed out (1). Purified heparin, when tested in V&O, shows a higher anticoagulant effect than our lipid fractions. On the other hand, the fact that the activator of blood coagulation associated with the cephalin group and the inhibitor here described, which is associated with the sphingomyelin group, both have the general characteristics and properties of a lipid subst,ance may appear of significance. It is highly unlikely that the activity of our preparations is due to a contamination with heparin. The solubility properties of the latter compound are entirely different; for instance, glacial acetic acid is a very good solvent for the lipid inhibitor, while it is extensively used in the purification of hcparin as a precipitating agent (10). It will he possible to gain an insight into the chemical nature of the lipid inhibitor only after a larger amount of purified material has been accumulated. For the present, t.wo points should be mentioned. One concerns the fact, which has often been observed, that a drop in act ivity occurs when the solutjion of the inhibitor in ligroin is treated with alcohol. This decrease in anticoagulant, potency appears to be irreversible, since even a mixt,urc of the two fractions obtained is less active t,han the ori-

11 E. Chargaff 185 ginal material. -1 plausible explanation may bc found in the application of an idra familiar in enzyme rhemist,ry : that of a specific carrier substance which is essential for the activity of a pa,rticular enzyme. Although in genrral the carriers arc considered to be proteins, thcrc is no rcason to exclude the possibility of the cxistcwcc of lipid carriers. Sphingomyclin, for instanw, Iwcauw of its physical propertics and its ampliotcric character could \wy ~11 act as a carrier substancr. The swond question which should br discussed is n-hethcr hhc lipid inhibitor cont,ains sulfur. The occurrence of sulfuric acid cxstclrs of wrtbrosides in brain has often been demonstratrd (cj. (11)). That sulfuric acid esters of polysaccharidcs and other substanws of high molecular weight art as extremely potent anticoagulants is a ~11 established fact (12-14). The arguments which speak aga,inst the assumption that the lipid inhibitor is a sulfuric acid derivative are: (1) Those brain lipid fractions which caontain most of the sulfur, e.g. Fra,ction ;Z-1 from sheep brain, arc devoid of antiwagulant activity. On the basis of its sulfur content Fraction &1-l contains about 23 per cent of ccrebrosidc monosulfuric acids. (2) Tl 1c solubility properties of the lipid inhibitors dcscribcd in this paper are different from those of cerebron sulfuric acid isolatc>d by Blix (11). In answer to these objections it could bc argued that bherc may occur in nature complex sulfurcontaining acids of different types; e.g., sulfuric acid esters and sulfonic* acids. It is by no means established that the anticoagulant activity of hrparin, as compared with the inactivity of chondroit,inw~lfuric ac*id, is due merely to a higher percentage of combined sulfuric acid. The arguments in favor of the lipid inhibitor being a derivative of sldfuric acid arc: (1) In the course of t,hr purification of the lipid inhibitor thrrc is a marked incwasr in the sulfur contents of t,hc wwcutirc fractions. \Vhwcas thr starting material, Frartion.\-2, wntaincd 0.13 per writ sulfur, Fra,ction A-3 contained 0.32 per cent, Fraction A\-T 0.68 per cent, Fraction h par cent, and Fraction A per rent. (2) Sphingomyelin sulfuric acid, which has not yet been isolated, is likely to have the solubility properties of sphingomyelin and would not be found in the original fractions richest in sulfur, the bulk of which consists of ccrebron, kerasin, and their derivatives. (3) In Paper T7 (15), following, it is shown that. crrehroside sulfuric acids can be

12 186 Chemistry of Blood Coagulation. IV synthesized which possess a high degree of anticoagulant potency. (4) It has recently been found (16) that the anticoagulant effect exert,cd by heparin and certain other sulfuric acid derivatives is almost completely stopped by the addition of a protamine, e.g. salmirw. This is probably due to the formation of insoluble salts with the sulfuric acid derivatives. It has been observed that both the lipid inhibitors and the synthetic wrcbroside sulfuric acids arc equally affected by the addition of salmine, whereas anticoagulants which do not belong to this group (sodium citrate, sodium oxalate) arc not. Thanks arc due to Dr. 31. Stanley-Brown and Dr. K. B. Olson for help in the preparation of the chicken plasma used in the tests, to Mr. W. Saschek for a large number of microanalyses, and to Mr. L. Hammer for general assistance in the course of these experiments. SUMMAHT A liljid fraction inhibiting the coagulation of blood and plasma has been found in the brain of sheep and pigs, in the spinal cord of cattle, and in the blood cells of sheep. The isolation and purification of the mat.erial are described, and the significance of the findings is discussed. 1. Chargaff, 15., Scicncc, 86, 548 (1937). 2. Chargaff, J5., J3ancroft, J?. R., and Stanley-Brown, M., J. Biol. Chem., 116, 149 (1936). 3. Fischer, A., Biochctu. Z., 240, 357 (1931). 4. Thannhauser, 8..J., and S&z, P., J. Biol. Chena., 116,527 (1936). 5. Thannhauser, S. J.. and Setz, P., J. Biol. Chem., 116, 533 (1936). 6. I,evenc, P. A., J. Bid. Chwn., 24, 69 (1916). 7. ( hargaff, 15., Bancroft, 1. I\., and Stanley-Brown, &I., J. Bid. Chcm., 116, 237 (1936). 8. Feigl, F., Qualitative Antlysc mit Hilfc von T~lpfelretllitionen, J,cipsic, 2nd edition, 176 (1935). 9. Hiiusler, H., and Schnetz, H., Biochen~. Z., 276, 187 (1935). 10. Charles, A. F., and Scott, D. A., Biochem. J., 30,1927 (1936). 11. Blix, G., Z. physiol. Chem., 219, 82 (1933). 12. Jorpes, E., Biochem. J., 29, 1817 (1935). 13. Bergstriim, S., Z. physiol. Chem., 238, 163 (1936). 14. Chargaff, E., Bancroft, F. W., and Stanley-Brown, M., J. Biol. Chcna. 116, 155 (1936). 15. Chargaff, E., J. B,iol. Cheva., 121, 187 (1937). 16. Chargaff, E., and Olson, K. B., unpuhlished data.