Production of prothrombin fragment 1–44 with acutin and some effects on thrombin generation

0049-3848/80/200271-09$02.00/O THROMBOSIS RESEARCH 20; 271-279, 1980 Printed in the USA. AI1 rights reserved. Copyright (c) 1980 Pergamon Press Ltd

PRODUCTION OF PROTHROMBIN FRAGMENT l-44 WITH ACUTIN AND SOME EFFECTS ON THROMBIN GENERATION Che-Ming Teng and Walter H. Seegers Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan, U.S.A.

(Received 18.7.1980; in revised form 20.10.1980. Accepted by Editor N. Alkjaersig)

ABSTRACT The thrombin-like enzyme from Agkistrodon acutus snake venom is called acutin. It cleaves three peptide bonds in bovine prothrombin; namely, Lys,,-Tyr 5; Arg,, -Ser digesting prothrombin 4ragment B wit~5~~u~?~,A~~~~td~~e~~~~a$ 45 156 were obtained and purified. Peptide 1-44 contains all ten y-carboxyglutamic acid residues of prothrombin. Working with the prothrombinase system, thrombin generation was inhibited by peptide l-44 when the substrate was prothrombin, accelerated when the substrate was prethrombin 1, and there was no effect with prethrombin 2 even if peptide 45-156 and prothrombin fragment 2 were also present. When peptide l-44 was covalently bound as in prothrombin fragment 1 it did function with fragment 2 in prethrombin 2 activation. Peptide 45-156 thus serves a function in thrombin generation. INTRODUCTION The amino terminal end of the prothrombin molecule is of special interest because it functions in calcium ion binding and the formation of complexes with phospholipids, platelet and red cell membranes. The first 44 amino acid residues of prothrombin are removed by acutin (1) which is a thrombin-like enzyme isolated (2) from Agkistrodon acutus snake venom. In the work described in this paper we used this enzyme to break prothrombin fragment 1 into two portions thus making it possible to isolate the two fragments. The one containing the ten y-carboxyglutamic acid residues functions as an accelerator or inhibitor of thrombin generation depending upon the nature of the thrombin zymogen.

Key Words:

Prothrombin - Acutin - Thrombin formation * Prothrombin Fragment l-44 271

272

PROTHROMBIN FRAGMENT l-44

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Some clarification of word meanings seems essential. The 44 amino terminal residue polypeptide of prothrombin has been named P fragment (3). In this paper we call it prothrombin fragment 1-44 or simply peptide l-44. It contains the ten y-carboxyglutamic acid (Gla) residues of the prothrombin mole. cule (Fig. 1). The polypeptide stretch in prothrombin comprising residues 45 through 156 was isolated and called R fragment (3). We refer to it as prothrombin fragment 45-156. The latter, when covalently bound to peptide l-44, is generally called prothrombin fragment 1 and is readily cleaved from prothrombin by thrombin. In 1939 this digestion was refered as the inactivation of prothrombin by thrombin (4).

FIG.

1

Amino acid sequence of bovine prothrombin fragment 1.2 arranged from several publications (5-10). Large arrow indicates three bonds broken by acutin at LYSL,~,Arglss and Arg2T4. Thrombin cleaves at Argls and Factor Xa at Arg274. Prothrombin fragment 1 represents the sequence Prom residues 1-156 and fragment 2 from 157-274.

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273

PROTHROMBIN FRAGMENT 1-44

Prothrombin fragment 1 and prothrombin fragment 2 are covalently bound to each other as prothrombin fragment 1.2 (Fig, 1). The latter is the nonthrombin portion of prothrombin and is the fragment-in serum comprising the . _ main fragment produced during blood coagulation (11). Acutin breaks three bonds in prothrombin (Fig. 1); namely, Lys44-Tyrbs; Arg1s6-Serls ; and Argz74-Thr275 (1). By usin acutin to di est prothrombin it is possib7e to obtain the thrombin portion 9prethrombin 23 of prothrombin. The same enzyme cleaves only Lys44-Tyr45in purified prothrombin fragment 1. Consequently one can perform the digestion and isolate two fragment corresponding to prothrombin fragments l-44 and 45-156. The latter has been isolated (3). To our knowledge no one has purified peptide l-44. Part of it, however, has been obtained as fragment 12-44 and contains eight Gla residues (12) as compared with ten in prothrombin or peptide l-44. Most of the procedure used for the isolation of fragment 12-44 can be applied for the purification of peptide l-44. Carlisle, Morita and Jackson (13) have isolated peptides l-42 and l-40 and presented studies of circular dichroism spectra as well as ultraviolet. EXPERIMENTAL RESULTS Materials and methods. These were the same as those outlined in a recent publication in this journal (1). Digestion of prothrombin fragment 1. In preliminary experiments the proportion of enz.vmeto substrate was studied. The buffer used was 0.05M tris (hydroxymethyl)aminomethane hydrochloride in O.lM sodium chloride at pH 7.4. About 45 mg purified prothrombin fragment 1 plus 1.3,mg acutin were dissolved in 10 ml tris-HCl buffer. The digestion proceeded at 37PC for 24 hours (Fio. 2). At that time a small amount of orothrombin fraament 1 remained-and was separated from peptide l-44 with h Sephadex G-50-column.

FIG,

2

SDS-polyacrylamide gel (7.5%) electrophoresis of prothrombin fragment 1 digestion with acutin. 5Opg protein applied. Gel 1 is zero time, gels 2, 3, 4 and 5 correspond to 1, 3, 5 and 24 hr digestion. Peptide l-44 passed ..:. through the gel. Molecular weight of fragment 45-156 was found to be 18,000.

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PROTHROMBIN FRAGMENT l-44

274

Isolation of peptides l-44 and 45-156. In principle we followed the procedure used for isolating fragment 12-44 (11). This involves adsorption on barium citrate, elution with sodium sulfate, desalting with a Sephadex G-15 column, and drying from the frozen state. To separate residual prothrombin fragment 1 we used a Sephadex G-50 column (Fig. 3). With the third passage through that column a single component elution pattern was obtained. The yield of the peptide from prothrombin fragment 1 was 65% of the theoretical. An amino acid analysis also corresDonded to the theoretical composition. By SDS polyacrylal mjde gel (10%) electrophoresis the peptide was unquestionably clean. Fragment 45-156 remained n the supernatant solution when peptide l-44 was adsorbed on barium citrate In this solution fragment 45-156 was practically a single component For final purification it was first desalted with a Sephadex G-15 co11 umn. The protein was dried from the frozen state, applied to a Sephadex G 50 column (1.5 x 80 cm) and eluted with 0.1 M ammonium bicarbonate. After two further gel filtrations a single symmetical peak was obtained. This purified fragment 45-156 was homogeneous as judged by SDS-polyacrylamide gel (10%) electrophoresis analysis.

FIG.

0

0

10 20 3Q 40

TUBE

NUMBER

SO 60

3

Separation of peptide l-44 from residual prothrombin fragment 1 on Sephadex G-50 column (1.5 x 80 cm) conditioned with O.lM ammonium bicarbonate. Flow rate 25 ml/hr. Three ml/tube. .-e---e--280 nm -o-o206 nm. Peptide l-44 contains no Tyr and only one Trp. Tubes 25-40 pooled for refractionation.

THROMBIN GENERATION EXPERIMENTS General arrangement. Purified thrombin zymogen was placed in a standard activation mixture at 3ToC. Periodically samples were taken from thrombin analysis. The reaction mixture was as follows: Thrombin zymogen (12,000 U/ml). . . Crude "cephalin" (0.3% suspension). Factor Xa (200 U/ml). . . . . , . . AC-Globulin (Factor V, 500 U/ml). . Calcium chloride (O.lM) . . . . , . Imidazole buffer (pH 7.2)*. , . . .

. , . . . .

. . . . . .

. . , . . .

. . . . . .

. . . . , .

0.1 0.1 0.1 0.1 0.1 0.5

ml ml ml ml ml ml

*Test materials incorporated Prothrombin as zymogen. At a molar ration of 1 to 1.5 (prothrombin to peptide l-44), a significant increase in rate and yield of thranbin was observed (Fig. 4). In higher concentrations of peptide l-44 the reaction was inhibited and at a 1-15 molar ratio practically no thrombin formed in 30 minutes.

vol. 20,

PROTHROMBIN FWiMENT

No. 2

275

1-44

Prethrombin 1 as zvmoqen. As recorded frequently in the literature, prethrombin 1 was only partially converted to thrombin (Fig. 4). Addition of peptide l-44 supplied the deficiency in the activation mixture. A molar ratio of 1:l was sufficient. Considering the numerous variables involved, our result is approximately the same as when prothrombin fragment 1 was used (14). With further experimentation it might be found that peptide l-44 is less active than profragment 1. In fact we were able to reduce the concentration ofpeptide l-44, and then demonstrate a strong synergistic effect by the addition of purified fragment 45-156. FVO~HROBIN ml

‘PEPTIOE

PRETMOh46IN

44

1

+ PEPTIDE 44

4 /:/ 1

;4w s sit90 8 E

TIME

k

Oo

IO

TIME

IN MINUTES FIG.

CONTROL

f

al

30

IN MINUTES

4

Generation of thrombin from prothrombin and prethrombin 1 in standard activation mixture. LEFT, Small amounts of peptide had a procoagulant effect. Large quantities inhibited. One to four molar ratio of prothrombin to peptide l-44 designated as 1:4 etc. Zymogen concentration was constant. RIGHT. Prethrombin 1 concentration was kept constant. At least one mol of peptide l-44 was required for each mol of prethrombin 1 as shown by the top curve marked 1:l. Lesser amounts (1:0.5 and 1:0.25 molar ratio) were associated with reduced yields of thrombin as indicated by the two middle curves. Prethrombin 2 as zymogen. In previous work (1,15), prothrombin fragment 1.2 easily corrected the deficiency of the reaction mixture when prethrombin 2 was the zymogen. Likewise a combination of prothrombin fragment 1 plus fragment 2 functioned equally well (1). Peptide l-44 proved to be inactive. It did not function by itself nor in combination with purified prothrombin fragment 2. The following combination was also inactive: peptide l-44 plus pe tide 45-156 plus prothrombin fragment 2 (157-274). Previously it was found Cl! that a combination of prothrombin fraoment 1 (l-156) and orothrombin fragment 2 (157-274) is active. There is-thus a requirement for peptide 45156 to be covalently linked. The prothrombin fragments contribute procoagulant effects as follows: l-44 plus 45-156 plus 157-274 = Inactive l-156 . . . . , .plus 157-274 = Active l-274 . , . , , . , . . , . . = Active

PROTHROMBIN FRAGMENT l-44

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DISCUSSION The thrombin-like enzyme from Agkistrodon acutus snake venom is called acutin and is uniquely useful for the study of prothrombin because it fulfills requirements not known to be met by other enzymes. It degrades prothrombin down to the prethrombin 2 portion of the molecule. After the degradation, prethrombin 2 can be isolated without very much contamination with thrombin (1). In this oaoer we describe its usefulness for isolating peptide bond. This l-44. To'produce peptihe'l-44 a&tin digests the Lys,,-Tyr bond is nearly refractory to thrombin, but each enzyme break: the Arg,s6Ser,,, bond. With prethrombin 2 as substrate we found the following in previous work (1):

Prethrombin 2

Ca2+ AC-globulin (Factor V) Phospholipid Prothrombin fragment 1 Prothrombin fragment 2 Factor Xa

> a- thrombin

On the basis of the experiments continued in this report we found that peptide l-44 cannot be substituted for prothrombin fragment 1 even if peptide 45-156 is also present. The two must be bound covalently. Thus, it follows that the 45-156 sequence of prothrombin fragment 1 has a function (Fig. 5). It is interesting, from the erspective of evolution, that the 45-156 sequence stretch (s ecifically 66-144P is more conserved than the vitamin K-dependent region (16P for which there is a function; namely, calcium binding. With prethrombin 1 as the substrate. Prothrombin fragment 1 has a procoagulant effect (14) and peptide l-44 functioned as a substitute for prothrombin fragment 1 piovided there was a sufficient uantity. When the concentration was reduced a synergistic effect of peptiae 45-156 could be demonstrated. We can then summarize (Fig. 5): Ca*+ AC-globulin (Factor V) Phospholipid Peptide l-44* Factor Xa Prethrombin 1 *Or prothrombin fragment 1 or peptide l-44 + peptide 45-156

z a-

thrombin prothrombin fragment 2

With prothrombin as substrate. Peptide l-44 is already a part of the molecule and additional quantities in the form of peptide 1-44 inhibited thrombin formation. The inhibition was complete with sufficient amounts.

PROTHROMBIN FRAGMENT l-44

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277

By contrast small amounts had a procoagulant effect. In previous reports prothrombin fragment 1 was used and only inhibition was observed (1,15). Our work is surrmarizedas follows (Fig. 5).

Prothrombin

Ca2+ AC-globulin (Factor V) Phospholipid Peptide l-44* Factor Xa

> a-thrombin + prothrombin fragment 1.2 or no products *Low concentration accelerates, High inhibits.

PROTHROMBIN Substrate

??

Anticoagulant PRETHROMBIN

1 I Substrate

? ?(I-44) ??

Procoagulant

(Strong)

Procoagulant

(Weak)

Procoagulant

(Medium)

PRETHROMBIN W

2

‘.:

Substrate

Procoagulant

FIG. 5 Thrombin generation in the standard activation mixture consisting of thrombin zymogen (either prothrombin, prethrombin 1, or prethrombin 2) in the presence of optimum amounts of AC-globulin (Factor V), phospholipid, Factor Xa, and calcium ions. This activation mixture was supplemented with prothrombin fragments; namely, peptide l-44, peptide 45-156, prothrombin fragment 1, and prothrombin fragment 2. Prothrombin itself served as a supplement to prethrombin 2 as indicated in the diagram.

278

PROTHROMBIN FRAGMENT 1-44

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When studying a thrombin zymogen it is usually essential to have data on whether the maximum possible yield of thrombin is known. One of us has paid attention to this problem over forty years. The original well known two-stage method for the study of this question underwent successive modifications in association with the discovery and availability of AC-globulin, Factor Xa, prothrombin fragments, Protein M, the procoagulant effect of trypsin, snake venoms etc. In work from this laboratory we used qualitative and quantitative variations of such materials to be certain of "complete conversion of prothrombin to thrombin," or "complete conversion of prothrombin to prethrombin 1," or "complete conversion of prothrombin to prethrombin 2," or "complete conversion of prethrombin 1 to meizothrombin 1" etc. There are innumerable possibilities for verification, including SDS polyacrylamide gel electrohoresis, immunological procedures, chromatography, specific activity, and other ways. Standardized conditions have been very helpful. It is also important to remember that esterolytic and proteolytic activity of the enzyme are not always the same (17). All of these considerations were taken into account in the work reported in this paper. ACKNOWLEDGMENTS This work was supported by grants HLB-03424-22 and HLB-18435-03 from National Heart, Lung and Blood Institute, HIH, U.S. Public Health Service. Che-Ming Ten was recipient of a Fogarty International Research Fellowship (TW-02743-013 . Amino acid analyses were performed by June Snow. REFERENCES 1.

SEEGERS, W.H., TENG, C.M. and NOVOA, E. Preparation of bovine prethrombin 2: Use of acutin and activation with prothrombinase or ecarin. Thromb. Res. 2; 11-20, 1980.

2.

OUYANG, C., HONG, J.S. and TENG, C.M. Purification and properties of the thrombin-like principle of Agkistrodon acutus venom and its comparison with bovine thrombin. _(ThromDiath. Haemorrh.)s, 224-234, 1971.

3.

SEEGERS, W.H., WALZ, D.A., REUTERBY, J. and MCCOY, L.E. Isolation and some properties of thrombin-E and other prothrombin derivatives. Thromb. Res. 4, 829-859, 1974.

4.

MERTZ, E.T., SEEGERS, W.H. and SMITH, H.P. Inactivation of prothrombin by purified thrombin solutions, Proc. Sot. Exp. Biol. Med. 4l_, 657-661, 1939.

5.

REUTERBY, J., WALZ, D.A., MCCOY, L.E. and SEEGERS, W.H. Amino acid sequence of 0 fragment of bovine prothrombin. Thromb. Res. 4, 885-890, 1974.

6.

MAGNUSSON, S., SOTTRUP-JENSON, L., PETERSEN, T.E., MORRIS, H.R. and DELL, A. Primary structure of the vitamin K-dependent part of prothrombin. FEBS Letters 44, 189-193, 1974.

7.

HEWETT-EMMETT, D., MCCOY, L.E., HASSOUNA, H.I., REUTERBY, J., WALZ, D.A. and SEEGERS, W.H. A partial gene duplication in the evolution of prothrombin? Thromb. Res. 5_, 421-430, 1974.

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279

8.

HEWETT-EMMETT, D., WALZ, D,A,, REUTERBY, J,, MCCOY, L.E. and SEEGERS, W.H. The amino acid sequence of PR fragment (NHz-terminal fragment) of bovine prothrombin. Thromb. Res. _7, 227-234, 1975.

9.

MAGNUSSON, S., PETERSEN, T.E., SOTTRUP-JENSEN, L. and CLAEYS, H. Complete primary structure of prothrombin: Isolation, structure and reactivity of ten carboxylated glutamic acid residues and regulation of prothrombin activation by thrombin. In: Proteases and Biological Control, Cold Spring Harbor, 1975, pp. 123-149.

10.

WALZ, D.A., HEWETT-EMMETT, D, and SEEGERS, W.H. Amino acid sequence of human prothrombin fragments 1 and 2. Proc. Natl. Acad. Sci. U.S.A. 74, 1969-1972, 1977.

11.

ARONSON, D.L., STEVAN, L., BALL, A.P., FRANZA, JR., B.R. and FINLAYSON, J.S. Generation of the combined prothrombin activation peptide (F 1.2) during the clotting of blood and plasma. J, Clin. Invest, 6B, 1410-1418, 1977.

12.

FURIE, B.C., BLUMENSTEIN, M. and FURIE, B. Metal binding sites of a Ycarboxyglutamic acid-rich fragment of bovine prothrombin. J. Biol. Chem. 254, 12521-12530, 1979,

13.

CARLISLE, T.L., MORITA, T. and JACKSON, C,M, Gla-region secondary structure in prothrombin and Factor X. In: Vitamin K Metabolism and Vitamin K-Dependent Proteins. Suttie, J.W, (Ed.) University Park Press, 1979, pp. 58-61.

14.

SEEGERS, W.H,,NOVOA, E., WALZ, D.A., ANDARY, T.J. and HASSOUNA, H.I. Effects of prothrombin fra ments on thrombin formation, and separation from Ac-globulin (Factor V3 . Thromb. Res. 8, 83-97, 1976.

15.

ESMON, C.T. and JACKSON, C.M. The conversion of prothrombin to thrombin. IV. The function of the fragment 2 region during activation in the presence of factor V. J. Biol. Chem. 249, 7791-7797, 1974.

16.

HEWETT-EMMETT, D., WALZ, D.A. and SEEGERS, W.H. Evolutionary and functional observations on the primary structure of the non-thrombin region (residues l-273) of human prothrombin. Biochem. Sot. Trans. 6, 1452-1455, 1977.

17.

NOVOA, E. and SEEGERS, W.H. Mechanism of a-thrombin and B-thrombin-E formation: Use of ecarin for isolation of meizothrombin 1. Thromb. Res. 18, 657-668, 1980.