Prothrombin Salakta : An abnormal prothrombin characterized by a defect in the active site of thrombin

THROMBOSIS RESEARCH 34; 507-518, 1984 0049-3848184 $3.00 + .OO Printed in the USA. Copyright (c) 1984 Pergamon Press Ltd. All rights reserved.

PROTHROMBIN SALAKTA : AN ABNORMAL PROTHROMBIN CHARACTERIZED BY A DEFECT IN THE ACTIVE SITE OF THROMBIN

A. BEZEAUD*

, L. DROUET**

, C. SORIA**, M-C. GUILLIN*

*Departement d'Hematologie, Faculte Xavier Bichat, 16 rue Henri Huchard. 75018. Paris. ** Laboratoire d'H&natolooie. Haoital Lariboisiere, 2 rue Ambroise Pare, 75010, Paris. - . ’

(Received 8.3.1984; Accepted in original form 26.3.1984 by Editor J. Stenflo) ABSTRACT An abnormal prothrombin has been detected in a 17 yr-old female originating from Tunisia. There was no history of excessive bleeding. Prothrombin time and activated partial thromboplastin time were moderately prolonged. Prothrombin activity was 15-18 % when measured using either the classical one-stage and two-stage assays, or assays with Echis carinatus venom or staphylocoagulase, whereas prothrombin antigen was 100 %. In keeping with current nomenclature practices, the abnormal molecule has been designated prothrombin Salakta. The electrophoretic behaviour and calcium binding properties of the abnormal prothrombin did not differ significantly from normal, as assessed by crossed immunoelectrophoresis. Prothrombin Salakta was isolated by chromatography on DEAE-Sephadex and Dextran sulphate sepharose. Electrophoretic migration of purified prothrombin Salakta on SDS polyacrylamide gels or alkaline disc gels was normal. Upon activation by either bovine factor Xa or Echis carinatus venom, thrombin activity produced by prothrombin Salakta was only 15 % of normal, even when the incubation period was prolonged for 24 hours. The pattern of factor Xa-catalyzed proteolysis of prothrombin Salakta, investigated by SDS polyacrylamide gel electrophoresis, was found to be normal. These results indicated that prothrombin Salakta was characterized by a defective thrombin enzymatic activity. Thrombin Salakta was therefore isolated by heparin-sepharose chromatography. Affinity for heparin and molecular weight of thrombin Salakta were found to be normal. Biological activity of thrombin Salakta, determined by clotting assay, was 535 u/mg versus 3 200 u/mg for normal thrombin. Amidolytic activity of thrombin Salakta parallelled its clotting activity, suggesting that the defect resides either in the catalytic site or in the residues adjacent to the catalytic site and implicated as contact residues, rather than in the fibrinogen recognition site.

KEY

WORDS:

Prothrombin-salakta,

abnormal 507

prothrombin,

thrombin.

508

ABNORMAL PROTHROMBIN

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INTRODUCTION

Inherited qualitative disorders of prothrombin are very rare coagulation disorders. Only 12 variants have been reported (l-12), all of them being characterized by a clear-cut discrepancy between the prothrombin level as determined by coagulation methods and immunological methods. Although in none of the cases the structural defect has been yet identified, the characterization of an abnormal prothrombin provides a unique opportunity for investigation of prothrombin and thrombin functions, and may lead to the identification of critical structural features.

Under physiological conditions, formation of thrombin is obtained by limited proteolysis of prothrombin by the serine protease factor Xa. The sites of peptide-bond splitting and order of bonds cleavages during prothrombin activation have been established (13 and references herein). The functional defect of two variants, prothrombin Barcelone (14) and Madrid (15) has been demonstrated to be related to an imoairement of proteolysis at Arg 273-Thr whereas prothrombin Quick (16') and Metz (17)s are characterized by a defect in the active site of thrombin. The present report concerns a new dysprothrombinemia, characterized by an abnormality in the active site of thrombin.

CASE

REPORT

The patient is a 22 yr-old female, originating from Tunisia, who was first referred to us at age 17 because of an isolated prothrombin deficiency, discovered before a caesaraen. Physical examination of the patient was normal. There was no clinical or laboratory evidence of hepatic disease, and no history of bleeding. The caesaraen was performed under substitutive therapy (PPSB) without bleeding manifestations. The family was not available for study.

MATERIALS

AND METHODS

Materials c Echzs carinatus venom was purchased from Sigma and staphylocoagulase from Stago laboratories. DEAE-Sephadex A 50, Dextran sulphate, Cyanogen bromide activated-Sepharose 4B and Heparin-sepharose were obtained from Pharmacia. S 2238 was from AB Kabi Diagnostica. Anti human prothrombin anti-serum.was purchased from Atlantic antibodies. Human brain cephalin was prepared according to Bell and Alton (18). All reagents used were of the highest grade commercially available.

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Coagulation studies Coagulation studies were performed by standard procedures (19), and included : platelet count, bleeding time, prothrombin time, activated partial thromboplastin time (APTT), screening for the presence of an inhibitor in plasma, and specific assays for factors II, V, VII, X, VIII, IX, XI, fibrinogen.

Plasma prothrombin activity was measured using the classical onestage assay (20), the two-stage assay described by Ware and Seegers (21) slightly modified (22) and one-stage assays using staphylocoagulase (23) or Echis carinatus venom (1) as activators. Prothrombin antigen was measured by electroimmunoassay(24). Two dimensional crossed immunoelectrophoresis was carried out according to Laurel1 (25).

Proteins purification Prothrombin was isolated from acid-citrate-dextrose plasma by barium citrate adsorption and elution, DEAE-Sephadex chromatography and dextran sulphate-sepharosechromatography, as previously described (1).

The prothrombin activating principle present in E&is was purified according to Franza et al (26).

carinatus

venom

d-thrombin was obtained by activating purified prothrombin with purified Ecbis carinatus venom. Prothrombin (5 mg, in 0.02 M Tris-HCl, pH 7.5, 0.05 M NaCl) was incubated with the venom (50 pg) for 60 minutes, at 20" C, and chromatographed on a heparin-sepharose column (1.5 x 8 cm). Thrombin was eluted with a linear gradient of NaCl (0.05 M to 1 M) in 0.02 M Tris-HCl, pH 7.5 (2 x 50 ml). Trace amounts of heparin, which contaminated the preparations, were removed by running &-thrombin through a DEAE-Sephadex column equilibrated in 0.02 M Tris-HCl, pH 7.5,0.15 M NaCl.

Studies on purified prothrombin activation Activity of purified prothrombin was determined by two-stage assays using either factor Xa or Echis carinatus venom as activators. Purified prothrombin (ID Pg in 1 ml 0.02 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.01 M CaCl containing 50 pg/ml human brain cephalin) was incubated at 37' 8 with 1 pg of purified bovine factor Xa (kindly provided by C.M. Jackson, Washington University, St Louis, MO, U.S.A.). Prothrombin activation by Echis carinatus venom was studied in the same manner, by incubating 10 pg prothrombin in 0.02 M Tris-HCl, pH 7.5, 0.15 M NaCl with 0.1 pg of purified Echis carinatus venom. Production of thrombin was measured by both clotting and chromogenic assay, at timed intervals until a plateau was reached. Prothrombin activation products were identified by SDS gel electrophoresis.

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Clotting activity of thrombin was determined using purified human fibrinogen (27) that was greater than 95 % clottable. Dilutions were made in 0.15 M NaCl, 0.01 M CaC12, 0.01 M Imidazole, pH 7.4, 0,l % PEG 6 000. A reference curve was prepared using human thrombin, lot 83, kindly supplied by Dr D.L. Aronson, Bureau of Biologics, Food and Drug Administration, Bethesda, Md, U.S.A..

Amidolytic activity of thrombin was determined using H-D-Phe-PipArg-pNA (S 2238). Thrombin was diluted in 0.01 M Hepes, 0.01 M Tris-HCl, pH 7.8, 0.5 M NaCl, 0,l % PEG 6 000. S 2238 was used at a final concentration of 0.1 mM in 0.01 M Hepes, 0.01 M Tris-HCl, pH 7.8, 0.1 M NaCl, 0,l % PEG 6 000. Assays were performed at 37" C in a final volume of followed at 405 nm. Purified 0.5 ml and the changes in absorbance humand-thrombin of known clotting activity was used to prepare the reference curve.

Polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) was performed according either to Weber and Osborn (28) or to Laemmli (29). Polyacrylamide disc gel electrophoresis at alkaline pH was performed according to Davis (30).

Measurement of protein concentration Protein concentrations were determined by absorbance reading at 280 nm. The molar extinction coefficients for human prothrombin (Br : 72 000) and thrombin (plr : 36 500) were assumed to be respectively 13.2 (31) and 18.3 (32).

RESULTS

The bleeding time, platelet counts and levels of factors V, VII, VIII, IX, X, fibrinogen were normal. The prothrombin time was 25 % of normal and APTT time 69 sec. (control, 45 sec.). Tests for the presence of a circulating anticoagulant were negative. Prothrombin activity was measured using different techniques (Tab. I). Whatever the activator used (thromboplastin, &his carinatus venom or sts;;;locoagulase), the apparent amount of prothrombin was 15-18 % of . In contrast, prothrombin antigen was 100 % of normal.

ABNOML

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PROTHR~BIN

TABLE I

Prothrombin

F. II (%) /

level in the propositus

One-stage assay

Two-stage assay

Echis carinatus

15

If

17

plasma

Staphylocoagulase 18

Immunoassay 100

The electrophoretic migration of the abnormal prothrombin, studied by crossed immunoelectrophoresis, was normal, either in the presence or absence of calcium ions during the first electrophoresis run (not shown).

Prothrombin Salakta was shown to adsorb normally onto barium citrate, and therefore a standard procedure (1) was used for its isolation. The elution profile on DEAE-Sephadex chrcmatography was normal. When compared by polyacrylamide gel electrophoresis, either at alkaline pH (fig. lA), or in the presence of SDS (fig. lB), mobilities of normal prothrombin and prothrombin Salakta were identical,

FIG.

1

Polyacrylamide gel electrophoresis of purified prothrombin, A. Disc gel electrophoresis in 7 % acrylamide, at pH 8.9 B. SDS gel electrophoresis in 10 % acrylamide, 0.1 4;SD.5.a:normal prothrombin b : prothrombin Salakta - c : mixture w/w normal prothrombin and prothrombin Salakta.

Kinetics of prothrombin activation by factor Xa, in the presence of calcium and phosphdlipids, are shown on Fig. 2. Thrombin activity production by prothrombin Salakta was impaired. When the plateau was reached, apparent specific activity of prothrombin Salakta was 177 and 160 thrombin units/mg, when measured respectively as clotting or amidolytic activi ty, versus 1 200 and 1 150 units/mg for normal prothrombin. Prolongation of the incubation period for 24 hours did not result in an increase in the enzymatic activity produced.

ABNOR~L

512

PROTHROFl6IN

A *_*-t--+-.--.

3

,‘to?

,r ,d

n Z

,’

:

?’

1:

E

*--

,’

4’ &-

FIG.

2

Production of thrombin activity upon the action of factor Xa. 10 pg prothrombin were incubated at 37" C in 1 ml 0.02 M TrisHCl, pH 7.5, 0.15 M NaCl, 0.01 M CaC12 containing 1 pg factor .%'a and 50 pg human brain cephalin. Clotting (A) and amidolytic (3) activities were measured at timed intervals. normal prothrombin. prothrombin Salakta c--+

Purified prothrombin was activated by the enzyme isolated from E&is carinatus venom (Fig. 3). Amidolytic activity was produced faster than clotting activity, as a consequence of meizothrombin and meizothrombin des Fl formation (26,33), both when normal prothrombin and prothrombin Salakta were incubated with the venom. When the plateau was reached, apparent specific activity of prothrombin Salakta was 165 thrombin units/mg (clotting and amidolytic activities) versus 1 150 and 1 060 thrombin units/mg for normal prothrombin.

FIG. 3

Production of thrombin activity upon the action of Echis carinatus venom. 30 pq prothrombin were incubated at 37' C in 1 ml 0-02 ~'4 Tris-HCl, pH 7.5, 0.15 M NaCl with 0.1 pq Echis carinatus venom, Clotting (A) and amidolytic (B) activities were measured at timed intervals. e--3 prothrombin Salakta +--+ normal prothrombin,

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PROT~ROM~~N

one reslults obtained suggested that the functional defect of prothrombin Salakta was due either to an icpairement of prothrombin proteolysis at Arg 322-Ile, or to a defec t in the active site of thromoin. The activation products obtained upon Xa action were therefore analyzed by SDS gel electrophoresis. Proteolysis of prothronbin Salakta .qas she+;? to oroceed at a normal rate (Fig. 4), yielding the transitory product, prethrombin 1 and final products, thrombin, fragment 1, fragment 2, w i t b, T

molecular wei.ghts identical to those of t5eir normal counterparts.

-II ?Pre

Tt

-T

-Fl cF2

-11 ?PreTt TB -Fl -f2

0

30

40

120

;2*a

:

FIG. 4

SDS gel electrophoresis analysis of prothrombin activation by facteur Xa. 0.5 my normal prothrombin (A and C) or prothrombin Salakta (3 and D) were incubated in 0.5 ml 0.02 IV Tris-HCl, pH 7.5, 0.15 M NaCl, 0.01 M CaCIZ containing 50 ,uq factor Xa and 50 ,uq human brain cephalin. Incubation times are indicated in minutes at the bottom of the gels. A and B : unreduced samples - C and D : reduced samples.

Thrombin Salakta was isolated by heparin-sepharose chromatography, after prothrombin activation by purified EchiT carinatus venom, as described in "methods". Thrombin Salakta was eluted at the same ionic strength as normal thrombin. Mobility of thrombin Salakta, as determined

by polyacrylarii:? gel electrophoresis in the presence of SZ5 ;Y3j ri;r-72: 5) both in non reducing and rsLd;cing conditions. Less ;har 7 % ,of (Fiq. degraded forms (/ or 8 thrombinf were deteciable on the ;?ls. Llsftirg activity of normal thrombin and thronbin Salakta were resoectively 3 200 and 535 u/zig. Same results were obtained tihen assayin? thrcz-bin activity using S 2238.

FIG.

5

Electr~o~,,-horetic mobility of purified thrombin Salak~a. Analysis by slab gel efectrophoreais on 7-20 % polyacrylamide gradient gels containing 0.1 % SDS. A: unreduced samples - B : reduced samples. aand c : normal thrombin. b and d : thrombin Salakta.

DISCUSSION

Under physiological conditions, limited proteolysis of prothrombin, catalyzed by facteur Xa, yields thrombin, which is a critical enzyme in the coagulation system, interacting with platelets, endothelial cells, fibrinogen and other plasma proteins to effect the hemostatic plug formation. The maximum rate of conversion of prothrombin to thrombin by factor Xa requires the additional presence of calcium ions, phospholipids, and factor Va. Functional defects of prothrombin may therefore be related either to an impairement of prothrombin proteolysis by factor Xa (14, IS), to a defective prothrombin binding to phospholipids (34) or factor Va, or to an abnormality in the thrombin active site (16, 17).

Abnormal prothrombins 12) or acquired conditions,

have been described as congenital disorders (loccuring in vitamin K deficiencies or during

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vitamin K antagonists administration. Although the family was not available for study, there is a high probability that the dysprothrombinemia described here is congenital. That is, it was discovered at age 17, there was no medical history and no drug ingestion, and the laboratory findings remained unchanged over a 5 years period. The abnormal prothrombin was therefore designated prothrombin Salakta.

Prothrombin activity in the patient's plasma was found to be abnormal whatever the activator used for the assay, namely factor Xa, &his carinatus venom or staphylocaogulase. The mode of action of these enzymes in unmasking the thrombin active site is quite different : factor Xa cleaves prothrombin at Arg 273-Thr and Arg 322-Ile (13 and references herein), Echis carinatus venom splits the peptide bond Arg 322-Ile (26, 33), whereas staphylocoagulase forms a stoichiometric complex with prothrombin, inducing thrombin activity without proteolysis taking place (35, 36). The results obtained here suggested therefore that the functional defect of prothrombin Salakta was most probably due to an abnormality in the thrombin active site, rather than to an impairement of the proteolytic process unmasking the active site. Accordingly, the rate and extent of prothrombin Salakta proteolysis by factor Xa were found to be normal, as assessed by SDS gel electrophoresis.

The abnormality of thrombin Salakta was further demonstrated, using purified material. Clotting activity of thrombin Salakta was decreased (17%) as compared to normal thrombin. This was not due to a contamination of the preparation by prethrombin 2 or degraded forms of thrombin (P orb'), as assessed by polyacrylamide slab gel electrophoresis. Enzymatic activity of thrombin Salakta towards the tripeptide S 2238 was also 17 % of normal, indicating that the functional defect of thrombin Salakta resides most probably in the catalytic site or its vicinity, i.e. in the arginine side-chain pocket or apolar binding sites located immediately to the left of the pocket (37), rather than in the fibrinogen recognition site.

ACKNOWLEDGEMENTS

This study was supported by grants from Institut National de la Sante et de la Recherche Medicale (82 50 11) and from Faculte de Medecine Xavier Bichat, Universite Paris VII,

The authors are indebted to MS Laurence Bennegent for expert technical assistance and to MS Martine Billaut for typing the manuscript.

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