Proteolysis of bovine and human prothrombin and of bovine factor x by rat mast cell proteinase

THROMBOSIS RESEARCH 44; 327-337, 1986 0049-3848/86 $3.00 t .OO Printed in the USA. Copyright (c) 1986 Pergamon Journals Ltd. All rights reserved.

PROTEOLYSIS

OF BOVINE AND HUMAN X BY RAT MAST Alistair R.G. Wylie, Nigel L. Blumsom

PROTHROMBIN AND CELL PROTEINASE

OF BOVINE

FACTOR

John D. Lonsdale-Eccles, and Donald T. Elmore

Medical Biology Centre, Queen's Department of Biochemistry, University of Belfast, Belfast BT9 7BL, N. Iireland, UK (Submitted to Editor S. Magnusson April 1985; Accepted by Editors-in-Chief B. BlombBck and A.L. Copley 25.7.1986)X ABSTRACT The Cr-chymotrypsin-likeproteinase from rat peritoneal mast cells (RMCP I) rapidly destroyed the normal clotting activity of purified, calcium-free, bovine prothrombin, human prothrombin and bovine factor X and simultaneously removed similar N-terminal peptides (M approximately 5000) from both prothrombin and the 'light' chain ofrfactor X. The amino acid composition of the peptides agreed with the known composition of the regions of the respective parent molecules where y-carboxyglutamic acid residues are situated. Ca2+ ions protected each of the proteins from proteolysis and loss of procoagulant activity. Prolonged incubation in the standard physiological assay medium used for prothrombin or treatment with Echis carinatus venom indicated that the thrombogenic portion of prothrombin survived proteolysis by RMCP I. This restricted proteolysis was confirmed by electrophoretic analysis.

INTRODUCTION Chymotrypsin-like proteinases occur in peritoneal mast cells (l), tissue mast cells (2), neutrophil granulocytes (3) and basophils (4) are assumed to act upon accessible extracellular substrates. Thus, the behaviour of human neutrophil granulocyte proteinase towards certain coagulation factors (5-7) and of rat skin tissue mast cell proteinase towards fibrin and elastin (8) have been investigated. Tissue mast cells are distributed perivascularly and, like peritoneal mast cells, they contain an ionic complex of heparin, histamine and at least g?y

words:

Prothrombin,

factor

X, mast

z- The acceptance

cell

proteinase,

proteolysis

of this communication by the Editors-in-Chief is exceptional and caused by the utter negligence of the Editor, to whom it was originally submitted. 327

328

PROTHROMBIN, FX AND RMCP-I

Vol. 44, No. 3

one proteinase within their granules. The increase in vascular permeability following mast cell degranulation must facilitate the access of plasma proteins to both heparin and proteinase. We have briefly reported (9) that a rapid and extensive loss of procoagulant activity occurs when prothrombin and factor X are exposed to RMCP I; thrombin and factor Xa are much more stable to proteolysis. It is possible that mast cell proteinases may have a role in the regulation of localised extravascular blood coagulation following events such as tissue injury and inflammation.

MATERIALS

AND METHODS

Reagents were purchased as follows: rabbit brain cephalin, plasma deficient in factors VII and X and Echis carinatus venom (Sigma London Chemical Co, Ltd, Poole, U.K.), Russell's viper venom (Wellcome Research Laboratories, Beckenham, U.K.), bovine pancreatic carboxypeptidase A immobilised on agarose (Miles Laboratories, Ltd., Slough, U.K.) and Ficoll 400 (Pharmacia (Great Britain), Ltd., Hounslow, U.K.). Isolation

and purification

of clotting factors

Prothrombin and factor X were obtained from oxalated bovine blood according to Esnouf et al. (10) and from titrated bovine blood according to Bajaj and Mann (11). The latter method was adapted for the isolation of human prothrombin from a small volume of human acid-citrate-dextrose plasma. Benzamidine hydrochloride was added to both anticoagulants and to the chromatographic buffers at a final concentration of 1mM for the method of Esnouf et al (11). The purity of prothrombin and factor X was assessed by polyacrlamide gel electrophoresis (PAGE). Molecular weights were determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE). Prothrombin, freed from buffer salts by exhaustive dialysis against distilled water, was stored at -2OOC in 5m Factor X was stored at -2OOC in O.OZM-phosphate or tris aqueous glycerol. chloride buffer, pH 7.2, containing 0.1 M NaCl. Isolation

and purification

of RMCP I

RMCP I was isolated from the combined peritoneal washings of 60 freshly killed rats using 0.34 M sucrose containing 8 units of heparin/ml (lOml/rat). Mixed peritoneal cells were collected, washed free of sucrose and separated by centrifugation for 20 min at 330 x q through layers of 30, 40 and 50% Ficoll 400 in balanced salt solution (0.03M Na HP0 /KH2P04 buffer containing 0.137M NaCl, 4.7 mM KCl, 2.5 mM CaC12 and 1.2 4 blgi04 adjusted to pH 7.2). Mast cells were recovered at the 30/4Oqb Ficoll interface. Washed mast cells were ultrasonicated on ice in 0.34 M sucrose at 8 p peak-to-peak amplitude for five periods of 1 min at 1 min intervals to release granules. RMCP I was extracted from centrifuged granules between 0.5 M and 1.2 M NaCl with the aid of mild sonication (8~ peak-to-peak amplitude for 30s) and purified by gel filtration on a column (43 x 2.5 cm) of Sephadex G-100 with 0.02 M-phosphate a-N-Methoxycarbonyl-Lbuffer, pH 7.0, containing 1.2 M-NaCl as eluant. phenylalanine p-nitrophenyl ester was used as a substrate to detect active The proteinase solution was concentrated by ultrafiltration into enzyme. 0.02 M phosphate buffer, pH 7.0, containing 0.1 M NaCl and stored at -2O'C. The operational molarity of RMCP I was determined using 4-methylumbelliferyl-p-N,N,N-trimethylammonium cinnamate (12).

Vol. 44, No. 3

Proteolysis -

of clotting

PROTHROMBIN, FX AND RMCP-I

329

factors

Prothrombin and factor X were digested with RMCP I at 28'C and 25OC respectively in 0.02 M tris chloride buffer, pH 7.2, containing 0.1 M NaCl Digests for electrophoretic when samples were assayed for clotting activity. analysis were performed in 0.02 M phosphate buffer, pH 7.2, (for SDS-PAGE) or in 0.02 M tris chloride buffer, pH 7.2, (for PAGE) both containing 0.1 M NaCl. An enzyme: substrate molar ratio of 1:500 was found by experiment to provide a suitable rate of reaction for investigations over a 30 min period and the total volume of digest was adjusted $0 allow withdrawal of aliquots at intervals. Prothrombin was assayed at 28 C by a modification of the twostage procedure of Ware and Seegers (13) and also with the purified procoagulant of Echis carinatus venom (14). In both methods, prothrombin was first converted into thrombin which was then assayed by its ability to clot fibrinogen. Factor X was assayed at 25OC by the method of Bachmann et al (15). No effort was made to inactivate RMCP I before assaying for activity of prothrombin or factor X. Cylindrical gels (10 x 0.6 cm) of 7.5% polyacrylamide (for PAGE) (16) or 10% polyacrylamide containing 0.1% sodium dodecyl sulphate for (SDS-PAGE) (17) were used to examine charge and molecular weight alterations to prothrombin and factor X following digestion with RMCP I. Serial aliquots were drawn at intervals for electrophoretic analysis; they were stored on ice and in the presence of SDS and/or mercaptoethanol where appropriate before loading on to gels. All gels were stained with Coomassie Brilliant Blue R-250. Amino acid analyses of peptides and proteins were performed on an LKB 4102 analyser; samples were prepared by hydrolysis with 6M HCl at 105OC. Y-Carboxyglutamic acid was not specifically determined. Amino acids liberated by agarose-carboxypeptidase A were similarly assayed. N-Terminal amino acids were identified as their dansyl derivatives obtained from acid hydrolysates of dansylated peptides and proteins (18) by thin layer chromatography on polyamide sheets (19). N-Terminal dansyl-Trp was identified after hydrolysis with p-toluenesulphonic acid (20).

RESULTS The disappearance of potential clotting activity as a result of the action of RMCP I on bovine and human prothrombin followed approximately first order kinetics. Clotting assays indicated, however, that prothrombin was never completely exhausted but fell to 5-10x of the original level (Fig.1). This observation apparently disagreed with the results of SDS-PAGE analysis of prothrombin digests which showed that bovine prothrombin (Mr. 69,500) had been completely converted into a single product (Mr 63,800) (Fig.la). Human prothrombin (Mr. 74,000) was similarly converted into a single product (Mr.68,800) (Apparent molecular weights are mean values determined from 32 and 20 gel electropherograms of bovine and human prothrombin digests respectively). The small product (Mr approximately 5,OOO)was not observed during SDS-PAGE. In the absence of SDS, however, PAGE revealed both proteolytic products (Fig. lb) the larger of which was more cationic and the smaller more anionic than bovine prothrombin. SDS-PAGE in the presence of 2-mercapto-ethanol of digests of bovine thrombin revealed only one band (M, 64,000), indicating that proteolysis was restricted to one or other end of the prothrombin molecule.

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PROTHROMBIN, FX AND RMCP-I

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la1

Time Iminl

Time lminl

0

Fig. 1

5

10

15 Time lminl

20

25

30

The effect of RMCP I on the clotting activity of bovine prothrombin determined after 6 min (H) and after 30 min (H> activation in a two stage assay, and (u) after generation of thrombin by Echis carinatus venom CRMCP ~I:Cprothrombinl= 1:500; T = ZS'C. Also shown diagramatically is the separation of digests ofprothrombin by electrophoresis on polyacrylamide gels in the presence (a) and absence (b) of SDS.

In view of the structural characteristics of the bovine prothrombin molecule (21), the combined electrophoretic analyses suggested that RMCP I had removed an N-terminal anionic peptide (Mr approximately 5,000) from This hypothesis accounts for the slow and incomplete generation prothrombin. of thrombin from the cleaved prothrombin in the two-stage assay, since the smaller fragment contains all of the residues of Y-carboxyglutamic acid. The larger fragment was shown to possess the full thrombogenic activity of prothrombin in two ways. Prolongation of the time allowed for activation of prothrombin to thrombin in the standard two-stage assay from 6 min to 30 min increased the yield of thrombin (Fig. 1). In addition, full thrombinactivity was generated when the RMCP I digest of prothrombin was activated with the procoagulant of Echis carinatus venom (Fig. 1). These results support the view that only the acceleratory and not the thrombogenic function of prothrombin had been cleaved by RMCP I.

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PROTHROMBIN, FX AND RMCP-I

331

Prcteolysis of bovine factor X by RMCPI also followed first order kinetics but resulted in almost total loss of clotting activity (Fig. 2). SDS-PAGE revealed that inactivation proceeded concurrently with the conversion Jf a molecule M, 55,500) into a produce (Mr 50,200 indicating that With reduced samples of factor X a fragment (Mr 5,300) had been cleaved. digests. SDS-PAGE demonstrated that proteolysis was limited and involved only the light chain (Fig. 2).

- Ibl

la1

:II H

75

L L’

+

35x

)1235lC

Time IminI

Time Iminl

0

0

0

5

10

15

20

25

30

Tim0 lminl

Fig. 2

The effect of rat mast cell proteinase on the clotting activity of seen in relation to the diagrammatic bovine factor X behaviour of digests of factor X on electrophoresis on SDS-polyacrylamide gels (a) before and (b) after reduction with dithiothreitol.

Prcthrombin and its degradation products were separated by chromatography on DEAE-cellulose (Fig. 3). The smaller fragment exhibited normal light absorption at 217 nm but an unusually low absorption at 280 nm (Fig. 3), indicating a low content of aromatic amino acids. This is consistent with the view that the smaller fragment is derived from the N-terminal region of prothrombin. The products of factor X proteolysis were also separated on DEAE-cellulose (Fig. 3). The factor X used as substrate was a mixture of Although factor Xl and factor X2 which co-chromatographed on DEAE-cellulose. the large and small products of proteolysis by RMCP I were each partially resolved on this column (Fig. 3), the partially resolved peaks were pooled for subsequent analysis.

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PROTHROMBIN, FX AND RMCP-I

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Lo.0

Fig. 3

The separation on a column (2.2 x 20 cm) of DEAE-cellulose, equilibrated in 0.1 M-HCl/Tris buffer pH 7.5, of 3MCP I digests of (A) bovine prothrombin ([gl:[Sl= 1:2400; T ; 37OC; in 0.1 M-HCl/Tris, pH 7.5 containing 0.02 M.NaCl), eluted by an 800 nl linear gradient (------) of 0.0-1.0 M-NaCl in 0.1 M-HCl/Tris buffer pH 7.5 and collecting 5 ml fractions and (b) bovine Factor X ([EI:cSl = 1:1600; T=25OC; in 0.1 M-HCl/Tris pH 7.5, containing 0.05 M-SaCl), eluted by a 1000 ml linear gradient (-----) of 0.0-0.5 M-NaCl in 0.1 M-HCl/Tris buffer pH 7.5, and collecting 8 ml fractions. Absorption (E) at 280 nm (-)

and 217 nm (-)

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PROTHROMBIN, FX AND RMCP-I

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TABLE 1 The Amino-acid

Amino acid

Composition

of Fragments P" and FX"

Mols/mol of peptide P"

FX"

Ala

5

4

Arg

3

2

Asp

3

4

* cys

2

2

Glu

10

14

Gly

2

1

His

0

0

Ile

0

0

Leu

5

4

LYS

2

2

Met

0

0

Phe

3

3

Pro

1

0

Ser

3

3

Thr

1

1

Trp

0

+

Tyr

0

1

1

2

Val

The above values represent rounded means of values obtained for 24, 48 and 72 h hydrolysates, except for Ser and Thr which are values extrapolated to zero time. Trp was identified qualitatively in FX".

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PROTHROMBIN, FX AND RMCP-I

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Hydrolysis of the dansylated derivatives of bovine prothrombin, factor X the large fragments P' and FX' and the small fragments P" and FX" by hydrochloric acid identified Ala, Ala, none, Lys, Ala, and Ala respectively as N-terminal residues. Hydrolysis of dansylated Pr in p-toluenesulphonic acid (20) revealed the presence of Trp as the N-terminal residue. Agarose-carboxypeptidase A progressively released Phe, Ala, Asp and traces of Thr from purified P" establishing the C-terminal sequence: -Asp-Ala-Phe-OH. Similar studies on purified FX" established the C-terminal sequence: -Ser-Lys-Tyr-OH. The release of Lys was attributed to the presence of carboxypeptidase B as impurity in the immobilised carboxypeptidase A. The above evidence indicates that P" comprises the sequence Ala1._...Phe41 of bovine prothrombin and that FX" consists of the sequence Ala1.....Tyr44 from the light chain of bovine factor X. Confirmation was achieved by amino acid analysis of Prr and FX" (Table 1). Trp was qualitatively demonstrated in FX" but not in PI'. Met, Ile, His and Tyr were completely absent from P" whereas Met, Ile, His and Pro were detectable at trace levels in FX" indicating the presence of a minor contaminant. All of the preceding digestions of prothrombin and factor 5 by RMCP I were performed in the absence of added calcium ions. A comparison of the ratio of prothrombin to larger fragment (P:P') determined densitometrically from SDS gels of 45 min digests (pH 7.2, 28OC, I = 0.12) of bovine prothrombin by RMCP I in the presence of Ca2+ ions showed the latter to be inhibitory Inhibition was complete in the presence in a concentration-dependent manner. of 3-10 mM CaC12.

DISCUSSION

The procoagulant activity of prothrombin and factor X was destroyed following the cleavage by RMCP I of an N-terminal polypeptide of about The fragment from factor X originated from the light chain. 5000 daltons. Amino-acid analysis No further cleavage was observed on longer digestion. and identification of N- and C-terminal residues have established that the smaller fragments comprise residues 1-41 from bovine prothrombin and l-44 from bovine factor X. We reported in our preliminary communication (9) that a second cleavage occurred in factor X at the Trp41 - Ser42 bond, but we were unable to isolate the fragment comprising residues 141 in the present study. 2+ a-Chymotrypsin in the absence of Ca , ions has been found to cleave the Tyr44 - Thr45 bond of human prothrombin and the Trp41 - Ser42 and Tyr44 LYS~~ bonds of bovine factor X and Xa (22-27). The proteolysis of prothrombin and factor X by RMCP I is similarly inhibited by Ca2+ ions. It is significant that Ca2+'induces aconformational change in prothrombin and factor X (28-33). The protection afforded by Ca2' ions in a concentration-dependent mode against proteolysis by RMCP I may be relevant to the situation within the microenvironment of degranulated mast cells. Heparin, which binds Ca2+ It ions co-operatively (34), is bound to the proteinase within the granule. is possible that prothrombin-bound Ca2+ ions could be shared by heparin, enforcing a localisation of prothrombin and perhaps exerting a degree of conformational change on the prothrombin molecule and leaving it susceptible If this mechanism is correct, it to cleavage by the mast-cell proteinase. would represent an economic control of excessive extravascular coagulation

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PROTHROMBIN, FX AND RMCP-I

A somewhat similar intravascular negative feedback within damaged tissues. control mediated by the action of thrombin on prothrombin has been proposed (35). The foregoing hypothesis is not only relevant to events within the inflammatory response, but also in those conditions characterised by basophil chronic basophilic accumulation (eg. the Arthus and Schwartzmann reactions, leukaemia) as a result of the presence of a chymotrypsin-like enzyme in basophils.

ACKNOWLEDGEMENT ARGW was the holder of a Medical Research Council (U.K.) postgraduate studentship; JDL-E was supported by a grant from the Wellcome Trust.

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