THE THROMBOPLASTIC PROTEIN: STRUCTURE, PROPERTIES, DISINTEGR,4TION*

BY ERWIN CHARGAFF, AARON BEKDICH, AND SEYMOUR S. COHEN

(From the Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York)

(Received for publication, July 29, 1944)

The thromboplastic protein from beef lungs has formed the subject of a number of communications from this laboratory. These investigations included studies of the isolation of this agent by fractional salt precipita- tion (1) and by high speed sedimentation (Z), of the composition of the lipids attached to the protein (3), of its electrophoretic behavior (2, 4), its particle weight (2), its immunological properties (I), and its reaction with heparin (5). A preliminary report on experiments aiming at the disintegration of this material by mild means was published recently (6).

The present article will contribute informat.ion on the following topics: the isolation of the thromboplastic protein from beef lung extracts by various centrifugal methods; the characterization of the nucleic acid and of the lipids contained in the complex; its disintegration by the action of ether, alcohol, and proteolytic enzymes; its act,ivity as a clotting factor; and some of its enzymatic properties.

EXPERIMENT;1L

Isolation

The isolation of the thromboplastic protein by the fractional ultracen- trifugation of beef lung extracts has been described previously (2). This procedure leads, under proper conditions, t,o very active and homogeneous preparations (with respect to electrophoretic mobility and sedimentation velocity), but the isolation of monodisperse preparations is time consuming and suitable for the processing of only small lots. The sedimentation of lung extracts in a refrigerated International centrifuge equipped with a multispeed attachment or, if larger volumes are to be worked up, the com- bination of this procedure with a preliminary sedimentation in a Sharples supercentrifuge yields products that, although heterogeneous with respect to particle size, are equal in activity and electrophoretic homogeneity to the fractions obtained by the more laborious method.

The description of one experiment will be sufficient. Beef lungs, freshly

* This work has been supported by a grant from the John and Mary R. Markle Foundation. This is Paper XVIII of a series of studies on the chemistry of blood coagulation.

161

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162 STRUCTURE OF THROMBOPLSSTIC PROTEIN

obtained from the slaughterhouse, were ground and the material, which weighed 3870 gm., was extracted with 3900 cc. of physiologica saline for 1 hour at 4”. The mixture was pressed out through several layers of cotton gauze and the filtrate (3320 cc.) passed rapidly through a turbine-driven Sharples laboratory supercentrifuge rotating at 50,000 R.P.M. The centri- fuge was equipped with cooling coils (7) through which ice mater circulated, whereby the contents of the clarifier bowl mere maintained at a low tem- perature. The inside of the bowl was lined with a celluloid sheet. This operation, in which the widest delivery jet was employed, required about 8 minutes. The fluid remaining in the bowl was displaced by 200 cc. of saline. The combined effluents lvere adjusted to a concentration of l/15 M phosphate buffer by the addition of M buffer of pH 7. The sediment was suspended in the liquid remaining in the bowl and the mixture, similarly adjusted to pH 7, centrifuged in a refrigerated angle centrifuge at 4800 R.P.M. (2700 g) for 30 minutes. The supernatant was united with the main solution which then was passed through the Sharples centrifuge, a fine delivery jet being used, at a very slow rate (16 cc. per minute). 500 cc. of the saline-phosphate buffer mixture were introduced for washing while the centrifuge was running. The copious pink sediment was re- moved from the celluloid liner and suspended in 200 cc. of ice-cold 0.1 M

borate buffer of pH 8.5. The suspension was centrifuged for 90 minut,es at 20,000 R.P.M. (31,000 g) in a refrigerated International centrifuge equipped with a multispeed att’achment. The suspension of the sediments in 100 cc. of borate buffer was again subject,ed to centrifugation at the same speed. The pel1et.s were suspended in 500 cc. of borate buffer and the suspension was spun for 30 minut,es at 8000 R.P.M. (5000 g), in order to remove the more easily sedimentable material. This fraction was twice washed with borate buffer in the centrifuge at 5000 g, resuspended in borate buffer, and freed from a small amount of coarse material by cen- trifugation at 4000 R.P.M. (1900 g) for 30 minutes. The supernatant was dialyzed for 24 hours against running tap water and for 96 hours against ice-cold distilled water. The evaporation of the water from the frozen suspension in a vacuum yielded the material sedimentable at 8000 R.P.M.

(5000 g) as 7.02 gm. of a slightly yellowish voluminous felt. This fraction contained N 5.8, P 2.0 per cent; N:P ratio 6.4. The reaction for acetal phosphatides (8) was positive.

The supernatant from this sedimentation which contained the thrombo- plastic protein was subjected to three more centrifugation cycles at 31,000 g and 5000 g alternately. The final solution, adjusted to an exact volume of 100 cc. with borate buffer of pH 8.5, contained 1.75 mg. of N and 0.38 mg. of P per cc. (N: P ratio 10.2). The total yield of the thromboplastic protein approximated 2.2 gm. Portions of this solution were used in ex- periments which will be described later; t,he remaining measured aliquot

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E. CHARGAFF, A. BENDICH, AND S. S. COHE?; 163

~-as dialyzed for 48 hours each against running and ice-cold distilled water and was dried from the frozen state in DQCUO, when Preparation 3 (Table I) was obtained as a white felt. This material was homogeneous in the electrophoresis cell, but, polydisperse when examined in the analytical ultracentrifuge. Its electrophoretic mobility in borate buffer of pH 8.5 was -7.6 X low5 sq. cm. per volt per second (descending and ascending boundaries).

Analytical data on other preparations of the thromboplastic protein, isolated by various centrifugal methods, will also be found in Table I. All substances gave a strongly positive reaction for acetal phosphatides (8).

TABLE I Composition and Properties of Thromboplastic Protein Preparations

PreparLl- tion So.

1 U. 2 M., U. 3 s., M. 4 M.

Yield per kilo

tissue

WC.

470 440 535 480

-

N

per cent

7.4 I 7.8 I

2 /

P

per cm1

1.6 1.5 1.6 1.6

S:P

10.2 11.5 10.5 10.8

Phosphatase activity

Phospha- tase umts

I perm3.

Y

0.003 1.6 0.008 1.6 0.003 2.2

Initial activity

Al00

2.8 4.5 3.8

* U. = air-driven vacuum ultracentrifuge; M. = International multispeed centri- fuge; S. = Sharples laboratory supercentrifuge.

t Expressed as the smallest amount clotting 0.1 cc. of rooster plasma (normal clotting time above 90 minutes) within 30 minutes.

Composition

The thromboplastic protein preparations discussed here formed white or almost white voluminous fabrics which dispersed readily in slightly alkaline buffers l;o give markedly opalescent solutions, similar to the preparations described previously (2). All fractions gave a positive Molisch reaction. The fuchsin test for acetal phosphatides (8) \vas invariably positive, the diphenylamine reaction for desoxyribose nucleic acid (9) negative. A few, but not all, preparations exhibited a reddish brown color on the addition of iodine, resembling that given by glycogen.

Lipids*-For the extraction of the lipids, 398.0 mg. of Preparation 3 (Table I) were suspended in 90 cc. of a mixture of equal parts of absolute alcohol and ether, and the mixture was refluxed for 24 hours. The extrac- tion residue, Fraction 3-Pr, weighed 188.9 mg. (47.5 per cent of the throm-

1 The solvents used were purified and rectified by distillation. Ether was freed of peroxides. Whenever possible, the operations were carried out in a nitrogen at- mosphere.

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164 STRPCTURE OF THROMBOPLASTIC PROTEIN

boplastic protein) and formed an almost white powder. It w-ill be dis- cussed in the next section.

The alcohol-ether extract was concentrated t,o dryness in vacua and the solution of the residue in ether extracted twice with 10 per cent aqueous sodium chloride. The ethereal solution was dried with anhydrous sodium sulfate and the evaporation residue of t.he filtrate taken up in chloroform. The solution was filtered, evaporated to dryness in vac?Lo, and the residue dried to constant weight over P,Os in vacua. The total lipids, Fraction 3-L, weighed 154.2 mg. (38.8 per cent of the thromboplastic protein) and formed a light brown soft paste. The analytical composition of this frac- tion (in per cent) was found as follows: X 1.35, P 2.52, atomic N:P ratio 1.19, amino nitrogen (10) 0.17, amino nitrogen following hydrolysis w&h 5 N HCI for 18 hours at 100” 0.54, iodine value (11) 50.6, total cholesterol (12) 19.1 (there mere practically no cholesterol esters), acetal phosphatides (13) 0.8 (calculated as palmitaldehyde).2

In another experiment, 214.0 mg. of Preparation 1 (Table I) were ex- tracted with 30 cc. of absolute alcohol-ether (1: 1) for 23 hours. The extraction residue, Fraction l-Pr, weighed 106.5 mg. (49.8 per cent of the thromboplastic protein). The evaporation residue of the extract, was taken up in 2 cc. of chloroform. The addit’ion of 4 volumes of acetone to the solution, following centrifugation and concentration, precipitated the acetone-insoluble lipid fraction weighing 64.9 mg. (30.3 per cent of the thromboplastic protein) and containing 1; 2.1, P 3.6 per cent. The ace- tone-soluble fraction weighed 35.0 mg. (16.3 per cent of the thromboplastic protein). The separation had not been complete, a,s the acetone-soluble lipids contained an appreciable amount of P, oiz. 0.84 per cent.

Protein Moiety-The residues from the alcohol-ether extraction of the thromboplastic protein gave the following analytical figures : Fraction I-Pr, N 12.3, P 0.44; Fraction 3-Pr, N 13.4, P 0.38, amino sugar (14) 0.99 (calculated as glucosamine). Studies of the amino acid composition of these substances will be presented in a later report. Following hydrolysis, the presence of reducing substances in these preparations could be demon- strated. When, for instance, Fraction 3-Pr was subjected to hydrolysis at 100” with 1 N HCI for 1 hour, 13.3 per cent of reducing sugars (calculated as glucose) was found by the Hagedorn-Jensen method. This would cor- respond to a sugar content of 6.3 per cent in the thromboplastic protein Preparation 3.

Nucleic Acid--=i suspension of 53 mg. of Preparation 1 (Table I) in 5 cc. of 0.1 M acetate buffer of pH 4.9 was heated to boiling for 5 minutes

2 We are greatly indebted to Dr. W. $1. Sperry for the cholesterol determinations, to Dr. H. Waelsch for the estimation of the acetal phosphatides, and to Mr. D. B. Sprinson for the determinations of amino nitrogen.

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E. CHARGAFF, A. BEh-DICH, AND S. S. COHERT 165

(compare (15)). Th e examination of the supernatant after centrifugation showed that 3.7 per cent of the total P of the thromboplastic protein had been liberated by this treatment. When examined in a Hilger spectro- graph (at a concentration of 6.5 y of P per cc.), the solution exhibited the ultraviolet absorption spectrum shown in Fig. 1, with a maximum at 2610 A.

Disintegration

Freezing in Presence of Ether-A preliminary account of some of these experiments was published recently (6). More complete data are as- sembled in Table II. All experiments were carried out in borate buffer of pH 7.7 (Experiments 1 and 5) or pH 8.5 (Experiments 2 to 4). Fresh

DENSITY

LO-

.9

2400 25 26 27 28 29 30 31 3200

FIG. 1. Ultraviolet absorption spectrum of nucleic acid isolated from the thrombo- plastic protein (Preparation 1) by heat denaturation.

solutions of the thromboplastic protein preparations, as obtained following the final centrifugal purification, were employed in these operations. These samples had not undergone dialysis and drying.

In a typical experiment (Experiments 2, 3, Table II), the solution of 330 mg. of Preparation 3 in 15 cc. of borate buffer of pH 8.5 was mixed with 10 cc. of ether, cooled for 4 minutes to -3O”, and permitted to thaw. The clear colorless ether layer was removed, 5 cc. of fresh ether were added, and the freezing treatment was repeated six to eight times. The ether layer was replaced by fresh solvent each time. The mixture then was centri- fuged, to effect a more complete separation of the layers, and the aqueous phase washed twice with ether. At this stage, most of the protein had collected in the interface, but after the removal of the remaining ether by careful evacuation, a homogeneous aqueous suspension resulted. It was

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166 STRCCTURE OF THROMBOPLASTIC PROTEIN

subjected to cent,rifugation at 4000 R.P.M. (1900 g) for 30 minutes, when Fraction A (Table II) precipitated. Subsequent centrifugation at 20,000 R.P.M. (31,000 g) for 90 to 120 minutes effected the sedimentation of small amounts of Fraction R, which appeared to be practically unaltered throm- boplastic protein. The supernatant contained Fraction C, which was

TABLE II Disintegration of Thromboplastic Protein by Free& ng 1 in Presence of Ether

T

--

- - / -

Exper~nen t

1

2

3

4 (Control experiment

5 )

-

rhrombo- plastic protein

prepara- t,on ?;a.’

Fraction K0.i

Propor- tion of

starting material

2-A 2-c 3-A 3-B 3-c

per cent per cent I; per cent

50.0 8.1 1.3 14.0 11.6 0.86 50.4 8.3 1.4

7.5 8.1 1.5 15.8 12.1 0.70

3-AA 3-BB 3-cc

48.3 8.0 1.2 11.8 8.4 1.2 15.9 12.3 0.49

C-3-B 98.1 c-3-c 1.7 4-A 50.8 4-c 16.7

-

I\‘

7.2

8.1

-

P

1.6

1.4

rhromboplastic activlty$

Y

0.001 2 0.003 0.008

Inactive up to 6

Y 0.003 0.008

Inactive up to 6 Y 0.003 0.02 0.008 2

-

Phosphatase activity

Phos- phatac

unit: Per ml

/ ie I i a :.

1.6 4.1 2.7 2.9 2.0

1.5 4.8 2.5

0.9 0.7 1.2 1.7

I- :nitial ctivity

AIM

3.0 5.3 5.6 4.4 4.5

3.4 8.0 3.7

1.8 1.2 1.8 1.7

* The numbers of the preparations refer to Table I. t The disintegration products are designated by the numeral corresponding to

the preparation used, followed by letters defining the centrifugal characteristics of the fraction: A, almost complete sedimentation at 1900 g; B, no sedimentation at 1900 g, complete sedimentation at 31,000 g; C, no sedimentation at 31,000 g. Duplication of the letters denotes the repetition of an experiment. The control experiment in which ether was omitted is designated by C preceding the number.

$ Expressed as the smallest amount clotting 0.1 cc. of rooster plasma (normal clotting time above 90 minutes) within 30 minutes.

found to consist of a mixture of non-sedimentable proteins. In one case, viz. Fraction 3-C (Table II), an electrophoretic study was carried out which revealed the presence of three components with the following mobil- ities and relative proportions (borate buffer of pH 8.5, descending bound- aries) : I, -3.3 (25 per cent) ; II, -6.6 (46 per cent); III, -8.1 (29 per cent) X 10-j sq. cm. per volt per second. It mill be remembered that the

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E. CHARGAFF, A. BENDICH, AND S. S. COHEN 167

thromboplastic protein Preparation 3, which formed the starting material, was homogeneous electrophoretically and had a mobility of -7.6. The A fractions gave a positive, the C fractions a negative Feulgen test for acetal phosphatides (8).

Control experiments, carried out simultaneously, in which the ether was omitted (e.g. Experiment 4, Table II), failed to show any gross changes in the protein due t,o the freezing and thawing; no appreciable disruption or aggregation was observed. The sedimentation of the protein (Fraction C-3-B), negligible at 1900 g, became almost complete at 31,000 g. The supernatant contained only traces of non-sedimentable protein (Fraction C-3-C).

All fractions were finally suspended in buffer, dialyzed against running and ice-cold distilled water, and recovered by the evaporation of the water in the frozen state in a vacuum.

For the recovery of the liberated lipids, the ethereal extracts from Ex- periments 2 and 3 (Table 11) were combined, extracted several times with 10 per cent aqueous sodium chloride, and dried with anhydrous sodium sulfate. The lipid mixture was taken up in chloroform and then again in ether and the solutions lvere clarified each time. The lipid preparation weighed 120.8 mg. (18.3 per cent of the thromboplastic protein) and con- tained N 0.85, P 1.5. It gave a strong reaction for acetal phosphatides (8). The comparison of yield and composition of this fraction with those of the total lipid preparation obtained by extraction with hot alcohol- ether, discussed in a preceding section, shows that t,he lipids removed by freezing in the presence of ether amounted to about one-third of the total lipids present. Their composition was significantly different, inasmuch as a relatively larger proportion of non-phospholipid material appeared to be removed by the freezing process.

The treatment of the disintegration products wit’h hot alcohol and ether removed additional lipid material, as exemplified by the following analyt- ical figures: Fraction 3-A:1 (extracted), N 12.8, P 0.46; Fraction 3-CC (estracted), N 14.3, P 0.22.

Action of Alcohol-A solution of 110 mg. of thromboplastic protein (Preparation 3) in 5 cc. of borate buffer of pH 8.5 was shaken with 15 cc. of absolute alcohol-ether (1:9) for 1 minute in the cold. Centrifugation of the mixture at 4000 R.P.M. (1900 g) for 30 minutes effected the separa- tion of an only slightly turbid aqueous phase, a thick jelly which collected at the interface, and a yellow ether layer. The ether layer was removed and the aqueous layer, together with the jelly, was repeatedly washed with ether in the centrifuge. The removal of the remaining ether by careful evacuation was followed by centrifugation at 20,000 R.P.M. (31,000 g) for 90 minutes, dialysis of the suspended sediment and of the supernatant, and vacuum concentration of the frozen solutions.

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168 STRUCTURE OF THROMBOPLASTIC PROTEIN

The sedimentable material, Fraction 3-D, formed 76.4 mg. (69.5 per cent of the thromboplastic protein) of a slightly yellowish felt; the non- sedimentable portion from the supernatsnt, Fraction 3-E, consisted of a voluminous fluff, weighing 20.0 mg. (18.2 per cent). The lipid fraction, isolated from the ether extract in the usual manner, weighed 15.7 mg. (14.3 per cent) and formed a brown paste which gave a positive reaction for acetal phosphatides. FracCon 3-D likewise gave a positive Feulgen reaction, whereas Fraction 3-E gave none. AAdditional evidence of the presence of lipids in Fraction 3-D was afforded by the effect of hot alcohol- ether on this material. The resulting Fraction 3-D (extracted) had a sig- nificantly changed composition. Analytical data on these fractions are compared in Table III.

Action of Proteolytic Enzymes-In one set of experiments, the action of crystalline trypsin and chymotrypsin3 on the thromboplastic protein

TABLE III Disintegration of Thromboplastic Protein (Preparation 3) by Means of Alcohol

Phosphatase activity Proportion Thrombo-

Fraction Ko. of starting N P material

plastic activity* Phospha-

tase units Initial per mg. activity

_______ per cent per cent per cent 7 Al00

3-D. . . 69.5 8.0 1.4 0.2 1.8 2.5 3-D (Extracted). . 12.8 ’ 0.41 3-E 18.2 10.4 0.63 6 0 0 Lipids.................. 14.3 0.73

* Expressed as the smallest amount clotting 0.1 cc. of rooster plasma (normal clot- ting time above 90 minutes) within 30 minutes.

Preparation 1 (Table I) and on the corresponding extraction residue (Frac- tion 1-Pr), resulting from the removal of lipids by means of hot alcohol- ether, was examined. The suspensions of the dried substances in 0.1 M

borate buffer of pH 7.8 contained per cc. 0.73 mg. of Preparation 1 or 1.3 mg. of Fraction I-Pr and 20 y of trypsin or chymotrypsin respectively. The mixtures were incubated at 37” and the disaggregation of the sus- pended particles was followed turbidimet,rically in a Klett-Summerson photoelectric calorimeter. The turbidity curves obtained with trypsin* are reproduced in Fig. 2.5 An experiment with a freshly prepared throm- boplastic protein preparation (similar to Preparation 3, Table I), which had

3 These enzyme preparations were kindly placed at our disposal by Dr. M. Kunits of the Rockefeller Institute, Princeton.

4 The curves observed with chymotrypsin were almost completely identical. 6 The instrument used gave a reading of 118 for No. 2 and of 234 for No. 4 of the

nephelometric barium sulfate scale of McFarland (16).

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E. CHARGAFF, h. BENDICH, AND S. S. COHEN 169

not undergone drying, is likewise included as Curves I, a, b (3.2 mg. of protein, 20 y of trypsin per cc. of borate buffer of pH 7.8). It will be seen that the decrease in turbidity was much greater with the lipid-free Frac-

Turbidity

:,': I-. . . . . Jl . . _ ,. * . . . .x . . . . . . . . . . . . . . . . j#.. . . . . . . . . . . . . . . . . . . . * . . . . . . . . . . . . . . . . . . . . . * 1,

I b

FIG. 2. Influence of crystalline trypsin on turbidity of thromboplastic protein preparations. Curve I, 0.3 per cent solution of thromboplastic protein; Curve II, 0.07 per cent suspension of Preparation 1; Curve III, 0.13 per cent suspension of lipid-free Fraction I-Pr. Control experiments are shown as a, tryptic digestion ex- periments as b. Borate buffer, pH 7.8,37”.

TABLE IV Action of Crystalline Trypsin and Chymotrypsin on Lipid-Free Thromboplastic Protein

(Fraction I-Pr)

Trypsin Chymotrypsin s”bbtr;te

digestion Before After Dialyz- Before After dialysis dialysis po”Ikn dialysis dialysis “k,:‘-

portion ___----

y per cc. y per cc. y per CG. per ce?d y per cc. y per cc. per Gent

N.. . . . . . . . . 161 133 84 37 137 95 31 P ._._.__.................. 5.7 4.5 3.6 Xl 4.5 4.1 9 Carbohydrates (as glucose) 85 76 11 85 81 5

tion 1-Pr (Curves III, a, b) than with the intact thromboplastic protein fractions (Curves I, a, b and II, a, b).

The mixtures containing the lipid-free Fraction 1-Pr and trypsin or chymotrypsin were, after 4 hours at 37”, centrifuged at 4000 R.P.M. and the supernatants, following the removal of aliquots for analysis, dialyzed overnight. The results are summarized in Table IV. The carbohydrate

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170 STRUCTURE OF THROMBOPLASTIC PROTEIN

determinations mere carried out by the orcinol method (17, 18). The values in Table IV, corrected for changes in concentration during dialysis, show that a large portion of the suspended substrate was brought into solution by either enzyme. It is worthy of note that a considerable pro- portion of the nitrogen contained in the prot.ein was converted into a dialyz- able form, whereas most of the phosphorus and carbohydrates remained undialyzable.

Thromboplastic Activity

Clotting of Plasma-The assays were carried out with rooster plasma in the usual arrangement (2, 19). The experiments were performed at 30.6” by mixing 0.1 cc. of fresh plasma with 0.03 cc. of the solution of the protein in borate buffer of pH 8.5. For purposes of comparison, the activities are expressed as the smallest amount clotting 0.1 cc. of plasma within 30 minutes. The results obtained with various preparations of the thrombo- plastic protein will be found in Table I. The act,ivities of the disintegra- tion products obtained by freezing in the presence of ether and by the ac- tion of alcohol are summarized in Tables II and III respectively. It may be of interest to present here the assay protocol for one highly active frac- tion, viz. Fraction 3-A (Table II, Experiment 2), in order to illustrate the truly remarkable potency of these preparations which in this case permitted the demonstration of 3 X lo-lo gm.

Thromboplastic protein in experiment

Clotting time, min.. .

The extremely heavy material which, as described in the first section of the experimental part, is removed from the solution of the thromboplastic protein by sedimentat.ion at 8000 R.P.M. (5000 g) possesses little thrombo- plastic activity. The smallest amount of this fraction that clotted 0.1~~. of plasma within 30 minutes was found to be 2 y.

Action on Prothrombin-The preparations of the thromboplastic protein were free of thrombin. The conversion of prothrombin to thrombin under the influence of the purified thromboplastic protein was followed in a number of instances. The purified prothrombin used, for which we are highly indebted t.o Dr. Mr. H. Seegers, Parke, Davis and Company, De- troit, had an activity of about 2000 units per mg. of nitrogen (compare (20, 21)). Human fibrinogen was employed in a technique essentially similar to that of previous experiments (22). To 0.1 cc. of a 0.1 per cent solution of prothrombin in physiological saline, 0.1 cc. of the thromboplas-

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E. CHARGAFF, A. BENDICH, ilND S. S. COHEN 171

tic protein (Table I, Preparation 3) dilution in saline containing 0.15 per cent calcium nitrate were added. After 20 minutes at room temperature (30”), 0.03 cc. of the mixture was placed in a small test-tube and 0.2 cc. of a 1.4 per cent fibrinogen solution in phosphate buffer of pH 7 was added. In the following clotting time determinations, which represent the average of a number of tests carried out at 30”, each tube contained the equivalent of 15 y of prothrombin, 2.8 mg. of fibrinogen, and the indicated amounts of the thromboplastic protein.

Thromboplastic protein in experiment

Clotting time, sec.. . . .

In other experiments, the heat stability of the thromboplastic protein was studied. The experimental arrangement was similar to the one men-

TABLE V

Stability of Thromboplastic Protein

Thromboplastic protein in experiment

3-Y 1-Y 0.33 y 0.11 y 0.037 y 0-Y

Se‘. *cc. sec. SeG. sec. sec.

Unheated. . 9 11 13 18 27 >72QO Heated.. 10 10 10 15 30 >7200

tioned above, with the exception of the temperature (25”) and the fibrino- gen concentration (0.7 per cent). A portion of the 0.02 per cent solution of the thromboplastic protein (Preparation 3) was heated to 80” for 20 minutes. Dilutions of both the unheated and the heated solutions were then incubated with prothrombin and tested as described before. (See Table V.)

The stability of the thromboplastic protein was much less marked when more dilute solutions of the substance (20 y per cc. and less) were exposed to heat. In this case, complete or partial inactivation appeared to take place.

Phosphatase Activity

The phosphatase activity of the thromboplastic. protein preparations and their disintegration products is summarized in Tables I to III. The determinations were carried out in the presence of magnesium ions with sodium @-glycerophosphate as the substrate. For the experimental ar- rangement and the definition of the units employed, reference should be

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172 STRUCTURE OF THROMBOPLASTIC PROTEIN

made to a previous publication (2). The determination of the phosphatase units is based on the work of Albers (23), the computation of the initial activity on studies of Bodansky (24).

The fraction sedimentable at 8000 R.P.M. (5000 g) contained 1.4 phos- phatase units per mg. and had an initial activity Aloo = 3.0.

The incubation of thromboplastic protein preparations with crystalline trypsin or chymotrypsin in the experimental arrangement described before was found to be without influence on the phosphatase activity.

Outflow Time, Seconds

250 _ a

110 -

100 - 90. 1 , ! 1 I I / I I I I

5 10 I5 20 25 30 35 40 45 50 55 60

Minutes

FIG. 3. Effect on viscosity of gelatin. Curve I, thromboplastic protein Prepara- tion 1,5.0 mg. in 5 cc. of gelatin; Curve II, crystalline trypsin, 55 yin 5 cc. of gelatin; Curve III, thromboplastic protein Preparation 1 (1.7 mg.) and crystalline trypsin (55 y) in 5 cc. of gelatin. Phosphate buffer, pH 7.4,34”.

Examination for Proteolytic Action

A preceding article (2) included a report, on attempts to test for the pres- ence of a trypsin-like activity in the thromboplastic protein by means of a titrimetric procedure wi-ith benzoylargininamide as substrate. The results were, however, inconclusive, since the slow decomposition of the protein itself prevented accurate determinations. For this reason, the experi- ments were repeated with a method that did not rely on changes in acidity. The procedure adopted finally was that of Northrop and Hussey (25) which employs the decrease in viscosity of a gelatin solution as the criterion of proteolytic action. A number of preparations (Preparations 1 and 3 in Table I, Fractions 3-AA and 3-CC in Table III) was tested in concentra- tions from 1 to 5 mg. per 5 cc. of gelatin solution. The arrangement was

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E. CHARGAFF, A. BENDICH, AND S. S. COHEN 173

similar to the one described by Northrop and Hussey (25). The experi- ments were carried out in phosphate buffer of pH 7.4 at 34” by means of an Ostwald viscosimeter (outflow time 68.7 seconds for water at 34”).

None of the substances tested had even the slightest influence on the viscosit,y of gelatin. An example will be found as Curve I in Fig. 3. The adequacy of the experimental procedure was proved by the viscosity de- crease produced by crysta.lline trypsin, chymokypsin, and commercial trypsin (Fairchild). The effect of cryst,alline trypsin is illustrated by Curve II in Fig. 3. Peculiar results were obtained when mixtures of thromboplast.ic protein and trypsin were examined: the tryptic action was invariably found to be inhibited to a certain extent. An example is shown as Curve III in Fig. 3. The thromboplastic protein was, however, without effect on the action of chymotrypsin.

DISCUSSION

The experiments here presented demonstrate the possibility of isolating large quantities of the thromboplastic protein from beef lung extracts by means of a cooled Sharples supercentrifuge. This was to be expected, in view of the very high particle weight of the substance (Z), since a similar instrument has been used for the isolation of the tobacco mosaic virus (7). The further purification of the thromboplastic protein preparations thus obtained requires the employment of a refrigerated high speed centrifuge.

The protein preparations which, it should be remembered, are isolated by very mild methods and are not in contact. with organic solvents in the course of their preparation lose about one-half of their weight by exhaustive extraction with hot alcohol-ether. Between 40 and 45 per cent of the material may be recovered as purified lipids. The composition of the total lipid fraction could, on the basis of the analytical figures, be tentatively expressed as follows (in per cent of tota’ lipids): cholesterol 19 (almost ex- clusively in the free state), fat 18, phospholipids 63 (“lecithin” 26, “ceph- alin” 25, “sphingomyelin” 12). The acetal phosphatide content of the lipid fraction may be estimated as about 1.5 per cent. The lipids attached to the thromboplastic protein preparation obtained by fractional salt pre- cipitation (l), which perhaps was contaminated with the coarsely par- ticulate fract,ion sedimentable at 8000 R.P.M., formed the subject of a pre- vious more detailed study (3).

The residue remaining from the estraction of the lipid portion consists in the main of proteins, some carbohydrates, and a nucleic acid of the ribose nucleic acid type. If the phosphorus content of this material (0.38 per cent) is assumed to be entirely due to nucleic acid, the presence of about 1.8 per cent of ribose nucleic acid in the intact thromboplastic protein would be indicated. The electrophoretic homogeneity of the complex

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174 STRUCTURE OF THROMBOPLSSTIC PROTEIN

speaks against the assumption that nucleic acid is present as an impurity. It appears to be atta-ched to the protein by fairly labile bonds. In prep- arations that had undergone more extensive chemical manipulation than the ones discussed here, the electrophoretic examination did reveal the existence of a small amount of a separate faster moving compound which carried most of the non-lipid phosphorus and probably consisted of nu- cleic acid liberated in the course of the isolation of the complex (4). The lability to heat of the ribose nucleic acid-protein bond was demonstrated in recent work on the tobacco mosaic virus (15) and has also been observed in the present studies of the thromboplastic protein.

The question may be raised, whether one is entitled to regard the throm- boplastic protein and similar lipoproteins as compounds between proteins and lipids or whether the latter occur in purely physical association. The appearance of complexity in structure of the macromolecular t,hrombo- plastic protein, which this paper serves to emphasize, is mainly a reflec- tion of our lack of understanding of the modes of linkage prevailing be- tween its various constituents. To give even a partial catalogue of our ignorance on this subject would require more space than can be afforded here, and reference should be made to a recent review article on lipopro- teins (26). But there are certain features that serve to distinguish genuine lipid-protein complexes from simple mixtures or loose adsorpt.ion systems. In general, the term lipoprotein may be said to designate a group of com- pounds whose properties, e.g. biological reactivity, solubility, physical characteristics, etc., differ from those of the sum of their components. In the case of the thromboplastic protein, for instance, it would be hard to understand how the large amounts of water-insoluble lipids and steroids present could escape separation from the protein portion, in the course of the elaborate centrifugal fractionation procedure, unless they are in chem- ical combination with the protein. The behavior of these complexes towards organic solvents (26) may be mentioned as another remarkable characteristic.

The thromboplastic protein of beef lungs resembles in certain respects, e.g. the presence of ribose nucleic acid and of acetal phosphatides, the submicroscopic particles isolated from a number of tissues (compare (27, 28)). Its nitrogen content appears to be considerably lower than that of the liver particles for which a figure of 9.1 per cent has been re- ported (27). Whether the similarity between these substances, which are probably of cytoplasmic origin, is more than superficial will have to be decided by detailed chemical and immunological studies. Data on some immunological properties of particulate tissue proteins, including lung particles, have been reported (29, 30).

The assays of the thromboplastic potency of the preparations reported

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E. CHARGAFF, A. BENDICH, AND S. 53. COHEN 175

in Tables I to III and in the section on the activation of purified prothrom- bin revealed a very high degree of activity. In some cases, amounts as small as 0.0003 y of the thromboplastic protein still exhibited a noticeable potency. This agent appears to be comparat#ively stable to heat. It is possible that the high lipid content has a protective function in this regard. The heat stability of thromboplastically active crude tissue extracts was remarked upon more than 30 years ago ((31) p. 529). The old distinction bet,ween thermostable thromboplastic agents (lipid fact.or) and thermo- labile ones (protein factor) would, therefore, seem to lose some of its sharpness.

The experiments on the disintegration of the thromboplastic protein by freezing in the presence of ether are based on an observation of McFarlane (32). This author found that a large proportion of the serum lipids, or- dinarily not extractable with ether, was transferred into the ether phase when ether-containing serum was frozen to a temperature below-25” and then allowed to thaw. By a similar treatment, the thromboplastic protein has been found to break into four principal fractions: (1) About one-half of the material is aggregated to form coarse particles sedimentable at. 4000 R.P.M. (Fractions A, Table II). The N : P ratio and the phospha- tase activity of this fraction are changed only slightly; the thromboplastic potency is somewhat higher t,han that of the starting material. (2) A small fraction (Fractions B, Table II) shows the centrifugal characteristics and the thromboplastic activity of the unaltered sta.rting material, but has, in general, a higher phosphatase potency. (3) The supernatant from these two fractions contains a considerable proportion (14 to 17 per cent) of a mixture of non-sedimentable proteins (Fractions C, Table II). This fraction, which in one experiment could be shown to consist of three dis- tinct electrophoretic components, is devoid of thromboplastic activity, but, is quite active as phosphatase. (4) h lipid fraction amounting to about 18 per cent of the starting material, i.e. roughly one-third of the total lipids of the thromboplastic prot,ein, is recovered from the ether phase. Control experiments, in which the freezing was carried out in the absence of ether, failed to reveal any evidence of a similar disintegration (compare Experiment 4, Table II).

The disintegration experiments with ether containing 10 per cent of al- cohol (Table III) do not require any ext,ended comment. They showed that even brief contact wiith alcohol sufficed to bring a,bout an almost com- plete destruction of the thromboplastic potency and a far reaching inter- ference with the phosphatase activity. In this case, too, the disruption of the thromboplastic protein was accompanied by the detachment of a non-sedimentable protein fraction and of lipids.

The experiments on the effect of trypsin and chymotrypsin demonstrate

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176 STRUCTURE OF THROMBOPLASTIC PROTEIN

that a portion of the thromboplastic protein can be digested by proteolytic enzymes. It should be of interest, as part of a general study of the physio- logical function of lipoprot,eins, to investigate whether the lipids contained in the intact complex are able to protect the protein moiety from the en- zymatic attack.

The phosphatase activity of the thromboplastic protein (2) and of its disintegration products was followed, not because of any possible con- nection between phosphat,ase action and the activation of prothrombin (there probably is none) but as an indicator of the adequacy of the methods used for the splitting of the complex. It is known that the phosphatase present in kidney particles may be released in a non-sedimentable form by autolysis (33). Similarly, tryptic digestion has been used to effect the liberation of intestinal phosphatase from tissue particles (34). The ex- periments, mentioned in the present article, on the digestion of the throm- boplastic protein by proteolytic enzymes in the course of which the phos- phatase activity remained unchanged, are reminiscent of these findings.

The examination of the thromboplastic protein for a trypsin-like action, which had previously led to inconclusive results (2), was resumed in the present studies by a different technique. No indication of any effect on gelatin, which served as the substrate, could be found. The thromboplas- tic protein had, in fact, an inhibiting action on crystalline trypsin, perhaps because of the competition between two substrates (gelatin and thrombo- plastic protein) for the enzyme.

The findings regarding the disruption of the thromboplastic protein by freezing in the presence of ether may perhaps throw some light on the structure of this lipoprotein. X-ray studies of similar substances have shown that these complexes occur as thin protein layers inserted between bimolecular lipid leaflets (35). This view permits the assumption that these units could arrange in a regular manner to form large complexes whose size would perhaps be limited by the intracellular spaces in which their formation takes place. The importance of the lipids in maintaining an automat.ic uniformity of particle size and electrophoretic mobility could thus be understood. Once the prot,ective water barrier is frozen away, the uniformity of the ostensibly homogeneous complex disappears owing to the removal of lipids by the ether, and separation into discrete components takes place.

The authors are highly indebted to Dr. D. H. Moore for the electro- phoresis experiments. They are very grateful to Miss Helen Fabricant for technical assistance and for help with some of the analytical determina- tions.

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E. CHARG.4FF, A. BENDICH, AND S. S. COHEN 177

SUMMARY

This paper describes continued work on the thromboplastic protein of beef lungs, an agent representative of the tissue factors which initiate the blood-clot.ting process by their action on prothrombin. The isolation of this high molecular lipoprotein by various centrifugal means is discussed. Studies of the composition of the lipid fraction and indications of the presence of ribose nucleic acid, both in combination with a protein com- ponent, are presented.

This substance is shown to be disintegrated into a number of fractions by freezing in the presence of ether. The disruptive effect of alcohol and of prot,eolytic enzymes has likewise been studied. The activities of the products are discussed with respect to thromboplastic and phosphatase potencies. A remarkably high thromboplastic activity (tested with both plasma and prothrombin) has been found in some of the fractions.

No indication of a trypsin-like activity of the thromboplastic protein could be found when the decrease in viscosity of a gelatin solution was used as the criterion of enzyme action.

The article concludes with a discussion of the possible function of the lipids in maintaining the architecture of the thromboplastic protein.

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S. CohenErwin Chargaff, Aaron Bendich and Seymour

DISINTEGRATIONSTRUCTURE, PROPERTIES,

THE THROMBOPLASTIC PROTEIN:

1944, 156:161-178.J. Biol. Chem. 

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