THROMBOSIS RESEARCH 20; 281-283, 1980 0049-3848/80/200281-03$02.00/O Printed in the USA. All rights reserved. Copyright (c) 1980 Pergamon Press Ltd

NOTE ON THE PREPARATION OF BOVINE THROMBIN

Abha Ghosh and Walter H. Seegers

Department of Phvsioloav, Wayne State University School of Medicjne, D&oit-, Michigan, U.S.A. -

(Received 27.6.1980. Accepted by Editor N. Alkjaersig. Received in final form by Executive Editorial Office 6.10.1980)

INTRODUCTION

Pure bovine a-thrombin was first obtained in 1958 (1,2). In that work, purified prothrombin complex was converted to thrcmbin and the latter was iso- lated by using an amberlite IRC-50 ion exchange resin column. Since that time other methods for purification have been employed in different laboratories. One of the simplest approaches is to buy commercial thrombin and pass it through an amberlite IRC-50 column, This presents the difficulty of not know- ing to what extent unspecified enzymes in comnercial preparations have modi- fied the thrombin molecular structure,

Currently the main problem in the purification of thrombin resides with finding an efficient procedure for the conversion of prothrombin to thrombin. Our procedure consists of preparing bovine prothrombin complex as described (3). The prothrombin yield is excellent, It is activated with crude Echis carinatus venom (Sigma Chemical Co.), and the activation mixture is pax through an amberlite IRC-50 column as described (1). Some Factor Xa‘can be obtained as a by-product,

Thrombin generation in

PROCEDURE

the prothrombin complex at 37'C, is complete in 30 to 60 minutes with a yield near 100% (Fig, 1). The activation mixture is arranged as follows:

Prothrombin complex (90,000 U/ml) , , , , , 5.5 ml Echis carinatus venom (1.0 mg/ml) , , . , . 6.3 ml Calcium chloride (l.OM) , , . , . , . , . . 0.7 ml Imidazole buffer (O.O5M, pH 7.2). , . , , . 50.0 ml

Key Words: Thrombin purification ?? Prothrombin complex . Amberlite IRC-50 ?? Echis carinatus venom

281

282 PREPARATION OF THROMBIN Vo1.20, No.2

lpi,, , . 0 IO to 30 TIME IN MINUT’ES

FIG. 1

Activation of prothrombin (8,000 U/ml) in bovine prothrombin com- plex with Echis carinatus venom (0.1 mg/ml) at pH 7.2 and 37'C.

The activation mixture is then mixed with 200 ml of settled amberlite IRC-50 resin previously equilibrated with 0.05M imidazole buffer (pH 7.2). This is stirred at 4°C for 30 minutes, and filtered by using a sintered glass Buchner funnel. The filtrate is saved for Factor Xa isolation. The resin is then washed thoroughly with 0.05M potassium phosphate buffer pH 7.0, and the resin slurry is then poured into a 5 x 12 cm column. The thrombin is eluted with 0.3M potassium phosphate buffer pH 8.0. The second elution fraction ob- tained corresponds to a-thrombin. The thrombin fractions are pooled, pre- cipitated with ammonium sulfate (4Og/lOO ml), stirred for 30 minutes at 4'C, and centrifuged at 8,000 rpm for 30 minutes. The supernatant fluid is dis- carded. The thrombin is dissolved in 30 ml of 0.05M sodium acetate buffer pH 5.6 and dialyzed efficiently for an hour at 4°C against 4 liters of 0.05M sodium acetate buffer pH 5.6. Excessive dialysis produces a precipitate with associated loss of activity.

DISCUSSION

The procedure outlined uses reagents, methods, and assays previously described (1,3,4). Large or small scale production can be equally efficient. The specific activity of the thrombin is near 6,000 Iowa U/mg, and a single component is indicated by SOS-polyacrylamide gel electrophoresis. The yield from the original prothrombin complex is near 45 to 50%. The same method can be used successfully with human prothrombin complex. The product is stable for many weeks at 4"C, even though thrombin is more stable near pH 5.2 (5).

ACKNOWLEDGEMENTS

This work was supported by grants HL-03424-22 National Heart, Lung and Blood Institute, National Public Health Service.

and HL-18435-03 from the Institutes of Health, U.S.

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REFERENCES

1. SEEGERS, W.H., LEVINE, W.G. and SHEPARD, R.S. Further studies on the purification of thrombin. Can. J. Biochem. Physiol. 36, 603-611, 1958.

2. SEEGERS, W.H., CASILLAS, G.C., SHEPARD, R.S., THOMAS, W.R. and HALICK, P. Some properties of thrombin preparations. Can, J. Biochem. Physiol. 37, 775-785, 1959.

3. MCCOY, L.E. and SEEGERS, W.H. Preparation of purified prothrombin complex and purified prothrombin. Thromb. Res. 1, 461-472, 1972.

4. NOVOA, E., SEEGERS, W.H. and HASSOUNA, H.I. Improved procedures for the purification of selected vitamin K-dependent proteins. Prep. Biochem. 6, 307-338, 1976.

5. SEEGERS, W.H. and MCCOY, L.E. Stability conditions related to a pre- viously unrecognized form of thrombin, Thromb. Diath. Haemorrh. 27, 361- 362, 1972,