THROMBOSIS RESEARCH 67; 147-155,1992 0049-3848/92 $5.00 + .OOPrinted in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
CATHEPSIN G, A REGULATOR OF HUMAN VITAMIN K, DEPENDENT CLOTTING FACTORS AND INHIBITORS
P.T. Turkington Kuwait University, Health Sciences Centre Department of Medical Technology, PO Box 31470,
Kuwait
(Received 11.1.1992; accepted in revised form 145.1992 by Editor O.N. Ulutin)
ABSTRACT Cathepsin G was used in vitro to digest human factor VII and Clotting assays indicated that the proteinase factor IX. affected a rapid loss in coagulant activity while in the presence calcium ions the activity was almost totally protected. of SDS-polyacrylamide gel electrophoresis indicated the removal of a peptide from each zymogen, VII-L from factor VII and IX-L from factor IX. This lead to the formation of VII-H and IX-H N-terminal analysis of the VII-H and IX-H respectively. products and COOH-terminal analysis of the VII-L and IX-L products confirmed that cathepsin G had cleaved position Phe4g:Trp41 in factor VII and factor IX. The cleavage site is the same as that when cathepsin G is reacted with factor II, factor X and protein C. The unique action of cathepsin G may be part of a regulatory system for controlling the coagulant activity of vitamin K dependent clotting in vivo.
INTRODUCTION Cathepsin azurophilic shown, in Key words
G (EC.3.4.21.20) is a serine proteinase that is found in the granules of polymorphonuclear leucocytes [l]. It has been vitro, to decrease the coagulant and anticoagulant activity of : Cathepsin G, Factor VII, Factor IX
147
148
II, VII, IX, X, PC REGULATION
Vol. 67, No. 2
three vitamin K dependent clotting zymogens by removing the calcium binding region (gla-domain). Cathepsin G removes the calcium binding region of prothrombin [2], factor X [3] and protein C [4] by cleaving a specific phenylalanyl-tryptophan residue in the aromatic amino acid stack region of the non catalytic portion of the zymogen [5]. The removal of the gla-domain may help to explain the haemorrhagic manifestations that are associated with qualitative and quantitative defects of In this report we examine the effect of cathepsin G on leucocytes [6]. factor VII and factor IX to see if it removed the gla-domain by cleaving the same specific phenylalanyl-tryptophan bond.
Cathepsin G was prepared from human polymorphonuclear leucocytes [7]. The specific activity of cathepsin G was 410 nkat/mg against the substrate Boc-Tyr-ONp [8]. Human factor VII and factor IX were prepared as previously described and had a specific coagulant activity of 108 units/mg and 111 units/mg respectively [9,10]. The molecular mass (Mr) [l l] of 24 kD for cathepsin G [12], 55 kD for factor VII [9] and 56 kD for factor IX [lo] were in agreement with published results. Ion of factor VW factor IX Each clotting factor (2.5 mg) was incubated with cathepsin G (3.0 ug) and made up to a total volume of 4 ml in 50 mmol/l Tris-HCI, pH 7.4 containing 500 mmol/l NaCI. Serial volumes were withdrawn at intervals The change in and assayed for coagulant activity [9,10] and Mr [ll]. clotting activity brought about by the proteinase was expressed as a percentage of a control solution that lacked the proteinase. Factor VII or factor IX were each preincubated in 50 mmol/l Tris-HCI, pH 7.4 containing calcium ions at final concentrations of 1, 2, 5, 10, 20 and 40 mmol/l for addition of cathepsin G. 5 min prior to the Serial volumes were assayed for clotting activity and Mr. The withdrawn at intervals and results shown here are the average of nine experiments at an enzyme: clotting factor molar ratio of approximately 1:500. .
.
on and characten7etron of the deor&&on prow When preparing the degradation products for N-terminal and COOHterminal analysis the amount of zymogen was increased to a concentration of 20 mg/4 ml. At this concentration the incubation time was increased After incubation, cathepsin G was to allow for complete degradation. inactivated by the addition of phenyl-methyl-sulphonyl fluoride (PMSF) at a final concentration of 50 umol/L. The degradation products were separated on salt gradient ranging from 0.2 M NaCI-1 .O M NaCl [2]. N-
Vol. 67, No. 2
149
II, VII, IX, X, PC REGULATION
terminal amino acids on the zymogens and the VII-H and IX-H products were identified on thin layer chromatography as dansylated derivatives obtained from acid hydrolysates of dansylated proteins 113, 141. The COOH-terminal amino acids of VII-L and IX-L were determined using agarose carboxypeptidase A and B [15]. Measu eme t of ester activitv of thL7vmooens and ala-domainless The ratio of the amidolytic activity of each zymogen against oroducls.n The estimation was its gla-domainless product was calculated. performed by the method of Esmon [16] except that the substrate and the activator varied according to the zymogen. S2222 for factor VII, carbobenzyl-trp-arg-isobutylthioester for factor IX, S2238 for factor II, S2222 for factor X and S2238 for protein C. The zymogens and their gladomainless products were prepared as previously described [2,3,4].
Although human polymorphonuclear leucocyte cathepsin G affected a rapid loss in coagulant activity (Fig.1) it did not indicate whether the loss was due to generalized proteolysis or through limited cleavage.
L 0
10
20
JO
40
50
60
70
time, min
Fig. 1
Effect of cathepsin G on the coagulant activity of human factor VII and factor IX. The activity was measured against a control that lacked cathepsin G. Factor VII (no calcium) [ 0 1, factor VII (2.5 mmol/l calcium) [ m 1, factor IX (no calcium) [ A 1, factor IX (2.5 mmol/l calcium) [ + 1.
150
Vol. 67, No. 2
II, VII, IX, X, PC REGULATION
To determine the extent of proteolysis we followed the reaction on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The degradation lead to the formation of VII-H from factor VII (Fig. 2a) and IXH from factor IX (Fig. 2c) with an Mr of 49 kD and 50 kD respectively. The electrophoretic profile indicated the removal of the peptides VII-L from factor VII, IX-L from factor IX, with an Mr of approximately 6-8 kD.
-VII ,.
-VIM
__“Li.-*
sokD-45
kD_
0
Fig. 2
--,
time,min
60
Effect of cathepsin G on human factor VII (2a) and factor IX when followed on SDS-polyacrylamide electrophoresis (2b) under reducing conditions. Fig 2c and 2d shows the electrophoretic profile when cathepsin G is added to factor VII and factor IX in the presence of 2.5 mmol/l calcium.
To establish the position of the cleavage site(s) we first separated the products on ion exchange chromatography. N-terminal amino acids of the high molecular mass products were identified on thin layer chromatography by their dansylated derivatives obtained from acid Hydrolysis with p-toluenesulphonic hydrolysates of dansylated proteins
Vol. 67, No. 2
II, VII, IX, X, PC REGULATION
151
revealed tryptophan as the new N-terminal amino acid on VII-H and IX-H. The COOH-terminal amino acids of VII-L and IX L were determined following their release by solid phase carboxypeptidase. Treatment of each peptide with solid phase carboxypeptidase lead to the release of Phe, Leu and Lys from VII-L and Phe, Glu and Thr from IX-L. The amino acids were released in increasing concentration and in this order. From examining the known sequence of each clotting factor we concluded that cathepsin G had cleaved position Phe4o:Trp41 in factor VII and factor IX and thereby had removed the calcium binding region (gla-domain). The cleavage site is the same as that observed when cathepsin G is incubated with protein C, factor X and factor II (Fig. 3). acid
CathepsinG
Factor II Factor VII Factor IX Factor X Protein C
+
A-N-T-F-L-E-E-V-R-K-G-N-L-E-R-E-C-V-E-E-T-C-S-Y-E-E-A-F-E-A-L-E-S-S-T-A-T-D-V-F-W-A A-N-A-F-L-E-E-L-R-P-G-S-L-E-R-E-C-K-E-E-Q-C-S-F-E-E-A-R-E-I-F-K-D-A-E-R-T-K-L-F-W-I Y-N-S-G-K-L-E-E-F-V-Q-G-N-L-E-R-E-C-M-E-E-K-C-S-F-E-E-A-R-E-V-F-E-N-T-E-K-T-T-E-F-W-L A-N-S-F-L-E-E-M-K-K-G-H-L-E-R-E-C-M-E-E-T-C-S-Y-E-E-A-R-E-V-F-E-D-S-D-K-T-N-E-F-W-N A-N-S-F-L-E-E-L-R-H-S-S-L-E-R-E-C-I-E-E-I-C-D-F-E-E-A-K-E-I-F-Q-N-V-D-D-T-L-A-F-W-S
A=Ala.E=Glu,K=Lys,W=Trp,R=Arg,G=Gly,M=Met,Y=Tyr,N=Asn,H=His,F=Phe, V=Val,D=Asp.I =Ile, P=Pro,Q=Gln,C=Cys,T=Thr, S=Ser, L=Leu,E=Glu
Fig. 3
Shows the sites cleaved in human factor VII (present work), factor IX (present work) factor X [3], factor II [2] and protein C [4] by cathepsin G. Cathepsin G cleaves the light chain of factor X and protein C. The amino acid sequence is taken from the previous reports [17,18,19,20,21].
Although the loss of the gla-domain by a single cleavage explained the decrease in coagulant activity the loss could also have occurred from cleavages in the catalytic domain of each zymogen. To see if the catalytic products remained intact we measured the domain of the degradation ester or amidolytic activity of each product and compared it to that of its parent zymogen. We observed that the rate of hydrolysis of the gladomainless product was no different from that of the parent zymogen (Table 1). The retention of ester or amidolytic activity was the same as that observed when cathepsin G was incubated with protein C, factor X and factor II (Table 1). From these results we concluded that cathepsin G had domain of any of the degradation products not destroyed the catalytic activity or anticoagulant activity in each and that the loss in coagulant
152
II, VII, IX, X, PC REGULATION
zymogen was due to the removal of the calcium binding domain) by cleaving Phe40:Trp41 in all five zymogens.
Vol. 67, No. 2
region
(gla-
TABLE The Ratio of the Ester or Amidolytic Activity of each Zymogen against its Gla-domainless Product. The Assay was Performed essentially by the Method of Esmon [16] except that the Buffer, the Substrate and the Activator varied according to the Zymogen. Factor II, Factor X and Protein C and their Gla-domainless Products were prepared as previously described [2,3,4]. Activator
Echis carinatus Thrombin Vipera russelli Vipera russelli Vipera russelli
Substrate
Zymogen
S2236 S2236 s2222 s2222 CBZ-T-A-BTE’
Factor II Protein C Factor X Factor VII Factor IX
Nomenclature of of gla-domainless Product Pl’ and P2’ PC-P PX VII-H IX-H
Activity Ratio 0.92 0.86 1.06 0.94 0.90
* carbobenzyl-trptophan-arginine-isobutylthioester
Dl=JssloN Our results show that the removal of the gla-domain by cathepsin G decreases the activity of five vitamin K dependent zymogens. In as much as the coagulant activity of factors II, VII, IX, X and protein C is a function of the gla residues, the proteolytic action of cathepsin G in removing the gla-domain may represent a balancing regulatory system. This hypothesis is supported firstly by the selective action of cathepsin G which does not attack the non-catalytic portion of any of the zymogens, secondly the unique specificity of cathepsin G in contrast to the action of polymorphonuclear leucocyte elastase which destroys the coagulant multiple sites in the zymogen and thirdly by the way activity by attacking nature has conserved the aromatic stack region [5]. Whether this control occurs in the fluid phase of blood is not clear since naturally occurring inhibitors such as alpha-l -antichymotrypsin can inactivate the proteinase [22] and calcium has an inhibitory effect (Fig.1, Fig 2b, Fig 2d), [2,3,4]. It is not known how the calcium inhibitory mechanism works in vivo but it is known that calcium can induce conformational changes in the zymogens in vitro which renders them almost resistant to the action of cathepsin G 1231.
Vol. 67, No. 2
II, VII, IX, X, PC REGULATION
P-
153
miEM~m
This work was supported by Kuwait University grant MH014.
1.
The major fibrinolytic proteases Plow E. Biochim Biophys Acta 1980; 630:47-56.
2.
Turkington PT, Blumsom NL, Elmore DTE. The degradation of bovine by human polymorphonuclear leucocyte and human prothrombin cathepsin G. Thromb Res 1986;44:339-345.
3.
Turkington PT. The degradation of human factor X by polymorphonuclear leucocyte cathepsin G and elastase. Haemostasis 1991;21 :l 1 l-l 16.
4.
Turkington PT. Cathepsin G can produce a gla-domainless protein in vitro. Thromb Res 1991;63:399-406.
5
Furie B, Furie BC. The molecular basis of blood coagulation. Cell 1988;53:505-518.
6.
Jochum M, Fritz H, Duswald K, Hiller E. Plasma levels of human granulocytic elastase- o( -1 -proteinase inhibitor complex (E-d -1 -PI) in patients with septicemia and acute leukemia: in Selected Topics in
Clinical
Enzymology.
Berlin,
of human
Walter de Gruyter,
leucocytes.
human
C
1983, pp 85-99.
7.
Baugh RJ, Travis J. Human leukocyte granule elastase; and characterization. Biochemistry 1976;15:836-841.
8.
Gupton BF, Carroll DL, Tuhy PM, Kam CM, Powers azapeptides with chymotrypsin-like enzymes. J Biol Chem 1984;259:4279-4287.
9.
Broze GJ, Majerus PW. (Human factor VII). Methods Enzymol 1976;80:228-236.
Rapid
JC.
isolation
Reaction
of
154
II, VII, IX, X, PC REGULATION
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10.
Miletich JP, Braze GJ, Majerus PW. Purification of human factors II, IX and X using sulphated dextran beads. Methods Enzymol 1976;80:221-228.
11.
of molecular Weber K, Osborn M. The reliability determinations by dodecyl sulphate-polyacrylamide electrophoresis. J Biochem 1969;244:4406-4412.
12
Travis J, Bowen J, Baugh R. Human d -1 -antichymotrypsin:lnteraction with chymotrypsin-like proteinases. Biochemistry1 978;17:5851-5856.
13.
Weiner AM, Platt T, Weber K. Amino-terminal sequence proteins purified on a nanoscale by gel electrophoresis. J Biol Chem 1972;247:3242-3251.
14
Liu T-Y, Chang YH. Hydrolysis of proteins with p-toluenesulphonic acid. J Biol Chem 1971;246:2842-2848.
15
Ambler RP, Carboxypeptidases A and B. In. Methods in Enzymol. Vol Xl. Edited by CHW Hirs 1967. Academic Press, New York pp 436-445.
16
Esmon NL, DeBault LE, Esmon CT. Proteolytic formation and properties of 70carboxyglutamic acid domainless protein C. J Biol Chem 1983;258:5548-5553.
17
Hagen FS, Gray CL, 0,Hara P, Grant FJ, Saari GC, Woodbury RG, Hart CE, lnsley M, Kisiel W, Kurachi K, Davie EW. Characterization of a cDNA coding for human factor VII. Proc Natl Acad Sci USA 1986;83:2412-2416.
18
Kurachi K, Davie EW. Isolation and characterization of a cDNA coding for human factor IX. Proc Natl Acad Sci USA 1982;79:6461-6464.
weight gel
analysis
of
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155
19
Friezner-Degan SJ, MacGillivray RTA, Davie EW. Characterization of the complementary deoxyribonucleic acid and gene coding for human Biochemistry 1983;22:2087-2097. prothrombin.
20
McMullen BA, Fujikawa K, Kisiel W, Sasagawa T, Howald WN, Kwa EY, Weinstein B. Complete amino acid sequence of the light chain of human blood coagulation factor X: Evidence for the identification of the residue 63 as 6 - hydroxyaspartic acid. Biochemistry 1983;22:2875-2884.
21
Foster DC, Yoshitake S, Davie EW. The nucleotide sequence of the gene for human protein C. Proc Natl Acad Sci USA1985;82:4673-4677.
22
Travis J, Salvesen GS. Human plasma proteinase inhibitors. Ann Rev Biochem 1983;52:655-709.
23
Furie B, Furie BC. Spectral changes in bovine factor X associated with activation by the venom coagulant protein of Vipera russelli. J Biol Chem 1976:251:6807-6814.
CATHEPSIN G, A REGULATOR OF HUMAN VITAMIN K, DEPENDENT CLOTTING FACTORS AND INHIBITORS
P.T. Turkington Kuwait University, Health Sciences Centre Department of Medical Technology, PO Box 31470,
Kuwait
(Received 11.1.1992; accepted in revised form 145.1992 by Editor O.N. Ulutin)
ABSTRACT Cathepsin G was used in vitro to digest human factor VII and Clotting assays indicated that the proteinase factor IX. affected a rapid loss in coagulant activity while in the presence calcium ions the activity was almost totally protected. of SDS-polyacrylamide gel electrophoresis indicated the removal of a peptide from each zymogen, VII-L from factor VII and IX-L from factor IX. This lead to the formation of VII-H and IX-H N-terminal analysis of the VII-H and IX-H respectively. products and COOH-terminal analysis of the VII-L and IX-L products confirmed that cathepsin G had cleaved position Phe4g:Trp41 in factor VII and factor IX. The cleavage site is the same as that when cathepsin G is reacted with factor II, factor X and protein C. The unique action of cathepsin G may be part of a regulatory system for controlling the coagulant activity of vitamin K dependent clotting in vivo.
INTRODUCTION Cathepsin azurophilic shown, in Key words
G (EC.3.4.21.20) is a serine proteinase that is found in the granules of polymorphonuclear leucocytes [l]. It has been vitro, to decrease the coagulant and anticoagulant activity of : Cathepsin G, Factor VII, Factor IX
147
148
II, VII, IX, X, PC REGULATION
Vol. 67, No. 2
three vitamin K dependent clotting zymogens by removing the calcium binding region (gla-domain). Cathepsin G removes the calcium binding region of prothrombin [2], factor X [3] and protein C [4] by cleaving a specific phenylalanyl-tryptophan residue in the aromatic amino acid stack region of the non catalytic portion of the zymogen [5]. The removal of the gla-domain may help to explain the haemorrhagic manifestations that are associated with qualitative and quantitative defects of In this report we examine the effect of cathepsin G on leucocytes [6]. factor VII and factor IX to see if it removed the gla-domain by cleaving the same specific phenylalanyl-tryptophan bond.
Cathepsin G was prepared from human polymorphonuclear leucocytes [7]. The specific activity of cathepsin G was 410 nkat/mg against the substrate Boc-Tyr-ONp [8]. Human factor VII and factor IX were prepared as previously described and had a specific coagulant activity of 108 units/mg and 111 units/mg respectively [9,10]. The molecular mass (Mr) [l l] of 24 kD for cathepsin G [12], 55 kD for factor VII [9] and 56 kD for factor IX [lo] were in agreement with published results. Ion of factor VW factor IX Each clotting factor (2.5 mg) was incubated with cathepsin G (3.0 ug) and made up to a total volume of 4 ml in 50 mmol/l Tris-HCI, pH 7.4 containing 500 mmol/l NaCI. Serial volumes were withdrawn at intervals The change in and assayed for coagulant activity [9,10] and Mr [ll]. clotting activity brought about by the proteinase was expressed as a percentage of a control solution that lacked the proteinase. Factor VII or factor IX were each preincubated in 50 mmol/l Tris-HCI, pH 7.4 containing calcium ions at final concentrations of 1, 2, 5, 10, 20 and 40 mmol/l for addition of cathepsin G. 5 min prior to the Serial volumes were assayed for clotting activity and Mr. The withdrawn at intervals and results shown here are the average of nine experiments at an enzyme: clotting factor molar ratio of approximately 1:500. .
.
on and characten7etron of the deor&&on prow When preparing the degradation products for N-terminal and COOHterminal analysis the amount of zymogen was increased to a concentration of 20 mg/4 ml. At this concentration the incubation time was increased After incubation, cathepsin G was to allow for complete degradation. inactivated by the addition of phenyl-methyl-sulphonyl fluoride (PMSF) at a final concentration of 50 umol/L. The degradation products were separated on salt gradient ranging from 0.2 M NaCI-1 .O M NaCl [2]. N-
Vol. 67, No. 2
149
II, VII, IX, X, PC REGULATION
terminal amino acids on the zymogens and the VII-H and IX-H products were identified on thin layer chromatography as dansylated derivatives obtained from acid hydrolysates of dansylated proteins 113, 141. The COOH-terminal amino acids of VII-L and IX-L were determined using agarose carboxypeptidase A and B [15]. Measu eme t of ester activitv of thL7vmooens and ala-domainless The ratio of the amidolytic activity of each zymogen against oroducls.n The estimation was its gla-domainless product was calculated. performed by the method of Esmon [16] except that the substrate and the activator varied according to the zymogen. S2222 for factor VII, carbobenzyl-trp-arg-isobutylthioester for factor IX, S2238 for factor II, S2222 for factor X and S2238 for protein C. The zymogens and their gladomainless products were prepared as previously described [2,3,4].
Although human polymorphonuclear leucocyte cathepsin G affected a rapid loss in coagulant activity (Fig.1) it did not indicate whether the loss was due to generalized proteolysis or through limited cleavage.
L 0
10
20
JO
40
50
60
70
time, min
Fig. 1
Effect of cathepsin G on the coagulant activity of human factor VII and factor IX. The activity was measured against a control that lacked cathepsin G. Factor VII (no calcium) [ 0 1, factor VII (2.5 mmol/l calcium) [ m 1, factor IX (no calcium) [ A 1, factor IX (2.5 mmol/l calcium) [ + 1.
150
Vol. 67, No. 2
II, VII, IX, X, PC REGULATION
To determine the extent of proteolysis we followed the reaction on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The degradation lead to the formation of VII-H from factor VII (Fig. 2a) and IXH from factor IX (Fig. 2c) with an Mr of 49 kD and 50 kD respectively. The electrophoretic profile indicated the removal of the peptides VII-L from factor VII, IX-L from factor IX, with an Mr of approximately 6-8 kD.
-VII ,.
-VIM
__“Li.-*
sokD-45
kD_
0
Fig. 2
--,
time,min
60
Effect of cathepsin G on human factor VII (2a) and factor IX when followed on SDS-polyacrylamide electrophoresis (2b) under reducing conditions. Fig 2c and 2d shows the electrophoretic profile when cathepsin G is added to factor VII and factor IX in the presence of 2.5 mmol/l calcium.
To establish the position of the cleavage site(s) we first separated the products on ion exchange chromatography. N-terminal amino acids of the high molecular mass products were identified on thin layer chromatography by their dansylated derivatives obtained from acid Hydrolysis with p-toluenesulphonic hydrolysates of dansylated proteins
Vol. 67, No. 2
II, VII, IX, X, PC REGULATION
151
revealed tryptophan as the new N-terminal amino acid on VII-H and IX-H. The COOH-terminal amino acids of VII-L and IX L were determined following their release by solid phase carboxypeptidase. Treatment of each peptide with solid phase carboxypeptidase lead to the release of Phe, Leu and Lys from VII-L and Phe, Glu and Thr from IX-L. The amino acids were released in increasing concentration and in this order. From examining the known sequence of each clotting factor we concluded that cathepsin G had cleaved position Phe4o:Trp41 in factor VII and factor IX and thereby had removed the calcium binding region (gla-domain). The cleavage site is the same as that observed when cathepsin G is incubated with protein C, factor X and factor II (Fig. 3). acid
CathepsinG
Factor II Factor VII Factor IX Factor X Protein C
+
A-N-T-F-L-E-E-V-R-K-G-N-L-E-R-E-C-V-E-E-T-C-S-Y-E-E-A-F-E-A-L-E-S-S-T-A-T-D-V-F-W-A A-N-A-F-L-E-E-L-R-P-G-S-L-E-R-E-C-K-E-E-Q-C-S-F-E-E-A-R-E-I-F-K-D-A-E-R-T-K-L-F-W-I Y-N-S-G-K-L-E-E-F-V-Q-G-N-L-E-R-E-C-M-E-E-K-C-S-F-E-E-A-R-E-V-F-E-N-T-E-K-T-T-E-F-W-L A-N-S-F-L-E-E-M-K-K-G-H-L-E-R-E-C-M-E-E-T-C-S-Y-E-E-A-R-E-V-F-E-D-S-D-K-T-N-E-F-W-N A-N-S-F-L-E-E-L-R-H-S-S-L-E-R-E-C-I-E-E-I-C-D-F-E-E-A-K-E-I-F-Q-N-V-D-D-T-L-A-F-W-S
A=Ala.E=Glu,K=Lys,W=Trp,R=Arg,G=Gly,M=Met,Y=Tyr,N=Asn,H=His,F=Phe, V=Val,D=Asp.I =Ile, P=Pro,Q=Gln,C=Cys,T=Thr, S=Ser, L=Leu,E=Glu
Fig. 3
Shows the sites cleaved in human factor VII (present work), factor IX (present work) factor X [3], factor II [2] and protein C [4] by cathepsin G. Cathepsin G cleaves the light chain of factor X and protein C. The amino acid sequence is taken from the previous reports [17,18,19,20,21].
Although the loss of the gla-domain by a single cleavage explained the decrease in coagulant activity the loss could also have occurred from cleavages in the catalytic domain of each zymogen. To see if the catalytic products remained intact we measured the domain of the degradation ester or amidolytic activity of each product and compared it to that of its parent zymogen. We observed that the rate of hydrolysis of the gladomainless product was no different from that of the parent zymogen (Table 1). The retention of ester or amidolytic activity was the same as that observed when cathepsin G was incubated with protein C, factor X and factor II (Table 1). From these results we concluded that cathepsin G had domain of any of the degradation products not destroyed the catalytic activity or anticoagulant activity in each and that the loss in coagulant
152
II, VII, IX, X, PC REGULATION
zymogen was due to the removal of the calcium binding domain) by cleaving Phe40:Trp41 in all five zymogens.
Vol. 67, No. 2
region
(gla-
TABLE The Ratio of the Ester or Amidolytic Activity of each Zymogen against its Gla-domainless Product. The Assay was Performed essentially by the Method of Esmon [16] except that the Buffer, the Substrate and the Activator varied according to the Zymogen. Factor II, Factor X and Protein C and their Gla-domainless Products were prepared as previously described [2,3,4]. Activator
Echis carinatus Thrombin Vipera russelli Vipera russelli Vipera russelli
Substrate
Zymogen
S2236 S2236 s2222 s2222 CBZ-T-A-BTE’
Factor II Protein C Factor X Factor VII Factor IX
Nomenclature of of gla-domainless Product Pl’ and P2’ PC-P PX VII-H IX-H
Activity Ratio 0.92 0.86 1.06 0.94 0.90
* carbobenzyl-trptophan-arginine-isobutylthioester
Dl=JssloN Our results show that the removal of the gla-domain by cathepsin G decreases the activity of five vitamin K dependent zymogens. In as much as the coagulant activity of factors II, VII, IX, X and protein C is a function of the gla residues, the proteolytic action of cathepsin G in removing the gla-domain may represent a balancing regulatory system. This hypothesis is supported firstly by the selective action of cathepsin G which does not attack the non-catalytic portion of any of the zymogens, secondly the unique specificity of cathepsin G in contrast to the action of polymorphonuclear leucocyte elastase which destroys the coagulant multiple sites in the zymogen and thirdly by the way activity by attacking nature has conserved the aromatic stack region [5]. Whether this control occurs in the fluid phase of blood is not clear since naturally occurring inhibitors such as alpha-l -antichymotrypsin can inactivate the proteinase [22] and calcium has an inhibitory effect (Fig.1, Fig 2b, Fig 2d), [2,3,4]. It is not known how the calcium inhibitory mechanism works in vivo but it is known that calcium can induce conformational changes in the zymogens in vitro which renders them almost resistant to the action of cathepsin G 1231.
Vol. 67, No. 2
II, VII, IX, X, PC REGULATION
P-
153
miEM~m
This work was supported by Kuwait University grant MH014.
1.
The major fibrinolytic proteases Plow E. Biochim Biophys Acta 1980; 630:47-56.
2.
Turkington PT, Blumsom NL, Elmore DTE. The degradation of bovine by human polymorphonuclear leucocyte and human prothrombin cathepsin G. Thromb Res 1986;44:339-345.
3.
Turkington PT. The degradation of human factor X by polymorphonuclear leucocyte cathepsin G and elastase. Haemostasis 1991;21 :l 1 l-l 16.
4.
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