Haemostasis 1991;21:111-116
© 1991 S. Karger AG, Basel 0301-0147/91/0212-0111S2.75/0
Degradation of Human Factor X by Human Polymorphonuclear Leucocyte Cathepsin G and Elastase1 P. T. Turkington Kuwait University, Health Sciences Centre, Department of Medical Laboratory Technology, Sulaibikhat, Kuwait
Key Words. Calcium • Cathepsin G • Factor X • Pancreatopeptidase • Proteinase inhibitor
Introduction Human polymorphonuclear leucocytes are known to contain large amounts of ca thepsin G (EC 3.4.21.20) and elastase (EC 3.4.21.11) in their azurophilic granules [1]. Although both proteinases have been shown to destroy the coagulant activity of several 1 Supported by grants MDH188 and MFH001, Kuwait University.
blood clotting factors in vitro [2, 3], the pro teolytic action of cathepsin G on some clot ting factors is different from that of elastase. Cathepsin G produces distinct fragments by attacking different cleavage sites and is par tially inhibited by calcium ions [4], Although plasma has naturally occurring inhibitors to protect the body from the action of elastase and cathepsin G in vitro [5], it has been reported that coagulant destruction can oc cur even in the presence of excess inhibitor
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Abstract. Cathepsin G and elastase from human polymorphonuclear leucocytes were used in vitro to digest human factor X. Clotting assays showed that both proteinases affected a rapid loss in the coagulant activity of factor X. Calcium ions almost totally protected the coagulant activity of factor X against the action of cathepsin G but not elastase. Polyacryl amide gel electrophoresis (in nonreducing conditions and in the presence of SDS) indicated that the proteolytic action of cathepsin G led to the removal of a peptide of low molecular mass (pX) with the consequent formation of a single stable high molecular mass product (PX). SDS electrophoresis (under reducing conditions and in the presence of SDS) indicated that a pX was derived from the light chain of factor X. The proteolytic action of elastase led to the formation of numerous degradation products. Analysis of the products generated by the action of cathepsin G indicated that cathepsin G cleaved position Phe40:Trp41 in the light chain of factor X. In the presence of citrated plasma, cathepsin G but not elastase, was responsible for a loss in coagulant activity.
potential in vitro [6]. These in vitro observa tions do not agree with clinical reports which show that an increased release of elastase is not always associated with clotting factor changes [7]. In this report we examine the effect of human cathepsin G and elastase on factor X, a simpler structural model, and describe the effect of calcium ions and human citrated plasma on this proteolysis. Materials and Methods Cathepsin G and elastase were prepared from hu man peripheral blood polymorphonuclear leucocytes as previously described [8]. The specific activity of elastase against Boc-Ala-ONp was 136 nkat/mg and the specific activity of cathepsin G against Boc-TyrONp was 410 nkat/mg [9, 10]. Human factor X was prepared as previously described and had a specific coagulant activity of 108 units/mg [11, 12]. The mo lecular mass (Mr) estimation [13] of 29 kD for elas tase [8], 24 kD for cathepsin G [14] and 58 kD for factor X [11] were in agreement with published re sults. The concentration of elastase di-proteinase in hibitor complex (E-di-PI) was estimated as previously described [ 15]. The biological activities of antithrom bin III and di-antiplasmin were estimated using kits supplied by Kabi Diagnostics (UK). The activation of factor X and PX by Russell’s viper venom was mea sured against the substrate H-D-Ile-Glu-Gly-Arg pnitroanilide (S2222). The hydrolysis of the substrate (200 mmol/1 final concentration) was performed at 25 °C in 150 mmol/1 Tris-HCl, pH 7.4, containing 1 mol/1 NaCl and 1 mg/ml bovine serum albumin. The hydrolysis was measured at 405 nm. A standard line relating the rate of hydrolysis of S2222 against the concentration of factor X or PX showed that the amidolytic activity of factor X and PX were indistin guishable. The ester activity of each proteinase was measured against the substrates Boc-Tyr-ONp and Boc-Ala-ONp, respectively, in the presence and ab sence of calcium ions. Calcium at a concentration of up to 40 mmol/1 had no inhibitory or potentiating effect on the ester activity. Blood (9 ml) was collected from healthy donors (n = 30) into plastic tubes con taining 0.15 M trisodium citrate (1 ml). Each sample
Turkington
was centrifuged at 2,500# for 10 min at room temper ature and the plasma pooled. The plasma was stored in plastic containers at -20 °C and used within 1 week. Degradation o f Factor X Factor X (2.3 mg) was incubated with elastase (2.4 pg) or cathepsin G (2.4 pg) and made up to a total volume of 4 ml in 50 mmol/1 Tris-HCl, pH 7.4, con taining 500 mmol/1 NaCl. Serial volumes were with drawn at intervals and assayed for changes in coagu lant activity [16] and Mr [13]. The changes in clotting activity brought about by the proteases were ex pressed as a percentage of a control solution that lacked the proteinase. Factor X was preincubated with calcium ions at final concentrations of 1, 2.5, 5, 10, 20 and 40 mmol/1 for 5 min prior to the addition of cathepsin G or elastase. Serial volumes were with drawn at intervals and assayed for changes in clotting activity and Mr. The results shown here are the aver age of nine experiments at an enzymexlotting factor molar ratio of 1:500. Separation and Characterization o f the Degradation Products When preparing the degradation products gener ated by the action of cathepsin G on factor X, the amount of factor X was increased to a concentration of 20 mg/4 ml. After incubation, cathepsin G was inactivated by the addition of phenyl-methyl-sulphonyl-fluoride (PMSF) at a final concentration of 50 pmol/1. The high molecular mass product (PX) was separated from the low molecular mass degradation product (pX) by an established procedure [17]. N-terminal amino acids on factor X and PX were identi fied as their dansylated derivatives obtained from acid hydrolysates of dansylated proteins on thinlayer chromatography [18-20]. The COOH-terminal amino acids of the pX fragment were determined using agarose carboxypeptidase A and B [17]. Effect o f Cathepsin G and Elastase on the Coagulant Activity o f Plasma Fresh plasma (1 ml) was incubated with cathepsin G or elastase (50 pi) at 37 °C to give final concentra tions in the range 0.1-20.0 pmol/1 (3-600 mg/1) of plasma. At serial intervals, samples were withdrawn and assayed for factor X, VIII, VII and II coagulant activity, antithrombin III activity, cti-antiplasmin ac tivity and E-oti-PI concentration.
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Results Cathepsin G and elastase rapidly de stroyed the coagulant activity of human fac tor X (fig. 1). When the degradation of factor X by elastase was followed on polyacryl amide electrophoresis (in the presence of SDS and under nonreducing conditions) nu merous degradation products were observed (fig. 2a). In contrast to the action of elastase, the degradation of factor X by cathepsin G produced a single major product (PX) with an Mr of approximately 52 kD (fig. 2b). While calcium failed to protect the coagulant activity of factor X from the action of elas tase, it almost totally protected the coagulant activity of factor X from the action of ca thepsin G (fig. 1). This was confirmed by the presence of a clotting factor that was resis tant to the action of cathepsin G (fig. 2c). Polyacrylamide electrophoresis (in the pres ence of SDS and under reducing conditions) indicated that the pX was derived from the light chain of factor X (fig. 3). While hydrolysis of dansylated factor X by HC1 revealed alanine and serine as the N-terminal amino acids, hydrolysis of the PX product revealed serine only. Hydrolysis
of PX by p-toluene sulphonic acid revealed tryptophan and indicated that the action of cathepsin G had exposed tryptophan as a new N-terminal amino acid. Treating the pX product with agarose carboxypeptidase A led to the release of Phe, Glu and Asn in in creasing concentrations and in this order. These results indicated that cathepsin G had cleaved the light chain at position Phe40:Trp41 (fig. 4). The slope relating the hydrolysis of increasing concentrations PX and increasing concentrations of factor X was indistinguishable. Elastase at concentra tions up to 15 pmol/1 plasma (450 mg/1) pro duced no change in the clotting activity of factor X in the presence of citrated plasma. In contrast, the addition of cathepsin G at a concentration of 0.1 pmol/1 plasma (3 mg/1) resulted in a rapid decrease in the coagulant activity of factor X (fig. 5). The addition of elastase was accompanied by dose-depen dent rise in the concentration of E-a,-PI while the antithrombin III level and ot2-antiplasmin level did not change throughout the incubation period. At an elastase concentra tion of 20 pmol/1 plasma (600 mg/1) a 10% decrease in the clotting activity of factor VIII was observed.
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Fig. 1. Effect of cathepsin G and elastase on the coagulant activity of human factor X. • = Cathepsin G on factor X; ▼= cathepsin G on factor X + 2.5 mmol/1 cal cium; ■ = elastase on factor X; ▲= elastase on factor X + 2.5 mmol/1 calcium.
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5
W
-----— 25
O
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T im e,m in
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t i me , mi n
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Fig. 2. Effect of cathepsin G and elastase on factor X when followed on SDS electrophoresis (nonreduc ing conditions): (a) elastase on factor X; (b) cathepsin G on factor X; (c) cathepsin G on factor X + 2.5 mmol/l calcium. Fig. 3. Effect of cathepsin G on factor X when fol lowed on SDS electrophoresis in reducing conditions. H = Heavy chain of factor X; L = light chain of factor X; pX' = the portion of the light chain that remained after cleavage by cathepsin G.
Discussion Although cathepsin G and elastase were both capable of the rapid destruction of the coagulant activity of factor X, their mecha nisms were different. The action of elastase was responsible for a generalized cleavage of the molecule. This was in contrast to the pro
teolytic action of cathepsin G which inacti vated factor X by the singular removal of the gla domain present in the light chain by cleaving position Phe40:Trp41 (fig. 4). The gla-containing domain which is necessary for coagulant activity is not required for amide or ester activity [22], Although calcium ions almost totally protected factor X from ca-
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Fig. 4. Diagram illustrating the structure of human factor X [21] and where it is cleaved by cathepsin G. Y = y-Carboxyglutamic acid res idue region.
Degradation of Factor X
thepsin G it did not protect the zymogen from the action of elastase. It is highly likely that the binding of calcium ions to the ycarboxyglutamic acid region of factor X in duced conformational changes that rendered the zymogen almost totally resistant to the action of cathepsin G [23]. The failure of cal cium to inhibit the action of elastase sug gested that the y-carboxyglutamic acids were destroyed at an early stage of the proteolysis which was in keeping with the electropho retic profile (fig. 2a). Whereas a small amount of cathepsin G induced a rapid destruction of several blood clotting factors, it took 200 times as much elastase to reduce the clotting activity of fac tor VIII by 10% (fig. 5). Although other workers have reported that low concentra tions of elastase (10 mg/1 plasma) induced coagulation changes, even in the presence of excess inhibitor potential [6], in clinical situ ations high concentrations of elastase can occur with corresponding changes in clotting factors [5], The failure of elastase, even at
high concentrations, to induce coagulation changes suggested that plasma inhibitors were capable of a rapid and irreversible inac tivation of elastase without a concomitant loss in clotting activity. This observation may help to explain the lack of correlation between coagulation changes and the in creased elastase levels that are observed in septicaemia and sepsis. The data presented here showed that ca thepsin G and elastase were capable of the rapid destruction of factor X although by different methods. Whereas calcium almost totally protected factor X from the action of cathepsin G, it did not protect the zymogen from elastase. It appears that plasma inhibi tors are capable of the rapid inactivation of elastase without concomitant loss in coagu lant activity.
References 1 Plow E: The major fibrinolytic proteases of hu man leucocytes. Biochim Biophys Acta 1980;630: 47-56. 2 Schmidt W, Egbring R, Havemann K: Effect of elastase-like and chymotrypsin-like neutral pro teases from human granulocytes on isolated clot ting factors. Thromb Res 1975;6:315-326. 3 Karpati J, Varadi K, Elodi S: Effect of granulocyte proteases on human coagulation factors IX and X. The protective effect of calcium. Hoppe Seylers Z Physiol Chem 1982;363:521-525. 4 Bingenheimer C, Gramse M, Egbring R, et al: Influence of calcium on the fibrinogen degrada tion products with anticlotting properties. HoppeSeylers Z Physiol Chem 1981;362:853-863. 5 Travis J, Salvesen GS: Human plasma proteinase inhibitors. Annu Rev Biochem 1983;52:655— 709. 6 Egbring R, Schmidt W, Fuchs G, et al: Demon stration of granulocytic proteases in plasma of patients with acute leukaemia and septicaemia with coagulation defects. Blood 1977;49:219— 231.
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Fig. 5. Changes in clotting activity that occur in factor II (•), VII (▲), IX (■) and X (▼) following the addition of cathepsin G (0.1 nmol or 3 pg) per milli litre of plasma.
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17 Wylie ARG, Lonsdale-Eccles JD, Blumsom NL, et al: Proteolysis of bovine and human prothrombin and of bovine factor X by rat mast cell protein ases. Thromb Res 1986;44:327-337. 18 Benson BJ, Hanahan DJ: Structural studies on bovine prothrombin. Isolation and partial charac terization of the Ca2+ binding and carbohydratecontaining peptides of the N-terminal region. Bio chemistry 1975;14:3265-3277. 19 Weiner AM, Platt T, Weber K: Amino-terminal sequence analysis of proteins purified on a nano mole scale by gel electrophoresis. J Biol Chem 1972;247:3242-3251. 20 Liu T-Y, Chang YH: Hydrolysis of proteins with p-toluene sulphonic acid. J Biol Chem 1971 ;246: 2842-2848. 21 Kaul RK, Hildebrand B, Roberts S, et al: Isolation and characterization of human blood-coagulation factor X cDNA. Gene 1986;41:311-314. 22 Esmon NL, DeBault LE, Esmon CT: Proteolytic formation and properties of y-carboxyglutamic acid domainless protein C. J Biol Chem 1983,258: 5548-5553. 23 Furie B, Furie BC: Spectral changes in bovine fac tor X associated with activation by the venom coagulant protein of Vipera russelli. J Biol Chem 1976;251:6807-6814.
Received: December 22, 1990 Accepted by K. Lechner: January 29, 1991 P.T. Turkington, PhD University of Malta Pharmacy Department Msida (Malta)
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7 Jochum M, Fritz H, Duswald K, et al: Plasma lev els of human granulocytic elastase-alpha-1-pro teinase inhibitor complex in patients with septi cemia and acute leukemia; in Selected Topics in Clinical Enzymology. Berlin, de Gruyter, 1983, pp 85-99. 8 Baugh RJ, Travis J: Human leucocyte granule elastase: Rapid isolation and characterization. Biochemistry 1976;15:836-841. 9 Gupton BF, Carroll DL, Tuhy PM, et al: Reaction of azapeptides with chymotrypsin-like enzymes. J Biol Chem 1984;259:4279-4287. 10 Powers JC, Boone R, Carroll DL, et al: Reaction of azapeptides with human leucocyte elastase and porcine pancreatic elastase. J Biol Chem 1984; 259:4288-4294. 11 DiScipio RG, Hermodson MA, Yates SG, et al: A comparison of human prothrombin, factor IX (Christmas Factor), factor X (Stuart factor) and protein S. Biochemistry 1977;16:698-706. 12 DiScipio RG, Hermodson MA, Davie EW: Acti vation of human factor X (Stuart factor) by a pro tease from Russell’s viper venom. Biochemistry 1977;16:5254-5260. 13 Weber K, Osborn M: The reliability of molecular weight determinations by dodecyl sulphate-polyacrylamide gel electrophoresis. J Biochem 1969; 244:4406-4412. 14 Travis J, Bowen J, Baugh R: Human ai-antichymotrypsin: Interaction with chymotrypsin-like proteinases. Biochemistry 1978;17:5651-5656. 15 Neumann S, Gunzer G, Hennrich N: PMN-elastase assay: Enzyme immunoassay for human poly morphonuclear elastase complexed with alpha-1proteinase inhibitor. J Clin Chem Clin Biochem 1984;22:693-697. 16 Austin DEG, Rhymes IL: A Laboratory Manual of Blood Coagulation. Oxford, Blackwell Scientific, 1975.
© 1991 S. Karger AG, Basel 0301-0147/91/0212-0111S2.75/0
Degradation of Human Factor X by Human Polymorphonuclear Leucocyte Cathepsin G and Elastase1 P. T. Turkington Kuwait University, Health Sciences Centre, Department of Medical Laboratory Technology, Sulaibikhat, Kuwait
Key Words. Calcium • Cathepsin G • Factor X • Pancreatopeptidase • Proteinase inhibitor
Introduction Human polymorphonuclear leucocytes are known to contain large amounts of ca thepsin G (EC 3.4.21.20) and elastase (EC 3.4.21.11) in their azurophilic granules [1]. Although both proteinases have been shown to destroy the coagulant activity of several 1 Supported by grants MDH188 and MFH001, Kuwait University.
blood clotting factors in vitro [2, 3], the pro teolytic action of cathepsin G on some clot ting factors is different from that of elastase. Cathepsin G produces distinct fragments by attacking different cleavage sites and is par tially inhibited by calcium ions [4], Although plasma has naturally occurring inhibitors to protect the body from the action of elastase and cathepsin G in vitro [5], it has been reported that coagulant destruction can oc cur even in the presence of excess inhibitor
Downloaded by: Nagoya University 133.6.82.173 - 1/15/2019 1:51:55 AM
Abstract. Cathepsin G and elastase from human polymorphonuclear leucocytes were used in vitro to digest human factor X. Clotting assays showed that both proteinases affected a rapid loss in the coagulant activity of factor X. Calcium ions almost totally protected the coagulant activity of factor X against the action of cathepsin G but not elastase. Polyacryl amide gel electrophoresis (in nonreducing conditions and in the presence of SDS) indicated that the proteolytic action of cathepsin G led to the removal of a peptide of low molecular mass (pX) with the consequent formation of a single stable high molecular mass product (PX). SDS electrophoresis (under reducing conditions and in the presence of SDS) indicated that a pX was derived from the light chain of factor X. The proteolytic action of elastase led to the formation of numerous degradation products. Analysis of the products generated by the action of cathepsin G indicated that cathepsin G cleaved position Phe40:Trp41 in the light chain of factor X. In the presence of citrated plasma, cathepsin G but not elastase, was responsible for a loss in coagulant activity.
potential in vitro [6]. These in vitro observa tions do not agree with clinical reports which show that an increased release of elastase is not always associated with clotting factor changes [7]. In this report we examine the effect of human cathepsin G and elastase on factor X, a simpler structural model, and describe the effect of calcium ions and human citrated plasma on this proteolysis. Materials and Methods Cathepsin G and elastase were prepared from hu man peripheral blood polymorphonuclear leucocytes as previously described [8]. The specific activity of elastase against Boc-Ala-ONp was 136 nkat/mg and the specific activity of cathepsin G against Boc-TyrONp was 410 nkat/mg [9, 10]. Human factor X was prepared as previously described and had a specific coagulant activity of 108 units/mg [11, 12]. The mo lecular mass (Mr) estimation [13] of 29 kD for elas tase [8], 24 kD for cathepsin G [14] and 58 kD for factor X [11] were in agreement with published re sults. The concentration of elastase di-proteinase in hibitor complex (E-di-PI) was estimated as previously described [ 15]. The biological activities of antithrom bin III and di-antiplasmin were estimated using kits supplied by Kabi Diagnostics (UK). The activation of factor X and PX by Russell’s viper venom was mea sured against the substrate H-D-Ile-Glu-Gly-Arg pnitroanilide (S2222). The hydrolysis of the substrate (200 mmol/1 final concentration) was performed at 25 °C in 150 mmol/1 Tris-HCl, pH 7.4, containing 1 mol/1 NaCl and 1 mg/ml bovine serum albumin. The hydrolysis was measured at 405 nm. A standard line relating the rate of hydrolysis of S2222 against the concentration of factor X or PX showed that the amidolytic activity of factor X and PX were indistin guishable. The ester activity of each proteinase was measured against the substrates Boc-Tyr-ONp and Boc-Ala-ONp, respectively, in the presence and ab sence of calcium ions. Calcium at a concentration of up to 40 mmol/1 had no inhibitory or potentiating effect on the ester activity. Blood (9 ml) was collected from healthy donors (n = 30) into plastic tubes con taining 0.15 M trisodium citrate (1 ml). Each sample
Turkington
was centrifuged at 2,500# for 10 min at room temper ature and the plasma pooled. The plasma was stored in plastic containers at -20 °C and used within 1 week. Degradation o f Factor X Factor X (2.3 mg) was incubated with elastase (2.4 pg) or cathepsin G (2.4 pg) and made up to a total volume of 4 ml in 50 mmol/1 Tris-HCl, pH 7.4, con taining 500 mmol/1 NaCl. Serial volumes were with drawn at intervals and assayed for changes in coagu lant activity [16] and Mr [13]. The changes in clotting activity brought about by the proteases were ex pressed as a percentage of a control solution that lacked the proteinase. Factor X was preincubated with calcium ions at final concentrations of 1, 2.5, 5, 10, 20 and 40 mmol/1 for 5 min prior to the addition of cathepsin G or elastase. Serial volumes were with drawn at intervals and assayed for changes in clotting activity and Mr. The results shown here are the aver age of nine experiments at an enzymexlotting factor molar ratio of 1:500. Separation and Characterization o f the Degradation Products When preparing the degradation products gener ated by the action of cathepsin G on factor X, the amount of factor X was increased to a concentration of 20 mg/4 ml. After incubation, cathepsin G was inactivated by the addition of phenyl-methyl-sulphonyl-fluoride (PMSF) at a final concentration of 50 pmol/1. The high molecular mass product (PX) was separated from the low molecular mass degradation product (pX) by an established procedure [17]. N-terminal amino acids on factor X and PX were identi fied as their dansylated derivatives obtained from acid hydrolysates of dansylated proteins on thinlayer chromatography [18-20]. The COOH-terminal amino acids of the pX fragment were determined using agarose carboxypeptidase A and B [17]. Effect o f Cathepsin G and Elastase on the Coagulant Activity o f Plasma Fresh plasma (1 ml) was incubated with cathepsin G or elastase (50 pi) at 37 °C to give final concentra tions in the range 0.1-20.0 pmol/1 (3-600 mg/1) of plasma. At serial intervals, samples were withdrawn and assayed for factor X, VIII, VII and II coagulant activity, antithrombin III activity, cti-antiplasmin ac tivity and E-oti-PI concentration.
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Results Cathepsin G and elastase rapidly de stroyed the coagulant activity of human fac tor X (fig. 1). When the degradation of factor X by elastase was followed on polyacryl amide electrophoresis (in the presence of SDS and under nonreducing conditions) nu merous degradation products were observed (fig. 2a). In contrast to the action of elastase, the degradation of factor X by cathepsin G produced a single major product (PX) with an Mr of approximately 52 kD (fig. 2b). While calcium failed to protect the coagulant activity of factor X from the action of elas tase, it almost totally protected the coagulant activity of factor X from the action of ca thepsin G (fig. 1). This was confirmed by the presence of a clotting factor that was resis tant to the action of cathepsin G (fig. 2c). Polyacrylamide electrophoresis (in the pres ence of SDS and under reducing conditions) indicated that the pX was derived from the light chain of factor X (fig. 3). While hydrolysis of dansylated factor X by HC1 revealed alanine and serine as the N-terminal amino acids, hydrolysis of the PX product revealed serine only. Hydrolysis
of PX by p-toluene sulphonic acid revealed tryptophan and indicated that the action of cathepsin G had exposed tryptophan as a new N-terminal amino acid. Treating the pX product with agarose carboxypeptidase A led to the release of Phe, Glu and Asn in in creasing concentrations and in this order. These results indicated that cathepsin G had cleaved the light chain at position Phe40:Trp41 (fig. 4). The slope relating the hydrolysis of increasing concentrations PX and increasing concentrations of factor X was indistinguishable. Elastase at concentra tions up to 15 pmol/1 plasma (450 mg/1) pro duced no change in the clotting activity of factor X in the presence of citrated plasma. In contrast, the addition of cathepsin G at a concentration of 0.1 pmol/1 plasma (3 mg/1) resulted in a rapid decrease in the coagulant activity of factor X (fig. 5). The addition of elastase was accompanied by dose-depen dent rise in the concentration of E-a,-PI while the antithrombin III level and ot2-antiplasmin level did not change throughout the incubation period. At an elastase concentra tion of 20 pmol/1 plasma (600 mg/1) a 10% decrease in the clotting activity of factor VIII was observed.
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Fig. 1. Effect of cathepsin G and elastase on the coagulant activity of human factor X. • = Cathepsin G on factor X; ▼= cathepsin G on factor X + 2.5 mmol/1 cal cium; ■ = elastase on factor X; ▲= elastase on factor X + 2.5 mmol/1 calcium.
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Fig. 2. Effect of cathepsin G and elastase on factor X when followed on SDS electrophoresis (nonreduc ing conditions): (a) elastase on factor X; (b) cathepsin G on factor X; (c) cathepsin G on factor X + 2.5 mmol/l calcium. Fig. 3. Effect of cathepsin G on factor X when fol lowed on SDS electrophoresis in reducing conditions. H = Heavy chain of factor X; L = light chain of factor X; pX' = the portion of the light chain that remained after cleavage by cathepsin G.
Discussion Although cathepsin G and elastase were both capable of the rapid destruction of the coagulant activity of factor X, their mecha nisms were different. The action of elastase was responsible for a generalized cleavage of the molecule. This was in contrast to the pro
teolytic action of cathepsin G which inacti vated factor X by the singular removal of the gla domain present in the light chain by cleaving position Phe40:Trp41 (fig. 4). The gla-containing domain which is necessary for coagulant activity is not required for amide or ester activity [22], Although calcium ions almost totally protected factor X from ca-
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Fig. 4. Diagram illustrating the structure of human factor X [21] and where it is cleaved by cathepsin G. Y = y-Carboxyglutamic acid res idue region.
Degradation of Factor X
thepsin G it did not protect the zymogen from the action of elastase. It is highly likely that the binding of calcium ions to the ycarboxyglutamic acid region of factor X in duced conformational changes that rendered the zymogen almost totally resistant to the action of cathepsin G [23]. The failure of cal cium to inhibit the action of elastase sug gested that the y-carboxyglutamic acids were destroyed at an early stage of the proteolysis which was in keeping with the electropho retic profile (fig. 2a). Whereas a small amount of cathepsin G induced a rapid destruction of several blood clotting factors, it took 200 times as much elastase to reduce the clotting activity of fac tor VIII by 10% (fig. 5). Although other workers have reported that low concentra tions of elastase (10 mg/1 plasma) induced coagulation changes, even in the presence of excess inhibitor potential [6], in clinical situ ations high concentrations of elastase can occur with corresponding changes in clotting factors [5], The failure of elastase, even at
high concentrations, to induce coagulation changes suggested that plasma inhibitors were capable of a rapid and irreversible inac tivation of elastase without a concomitant loss in clotting activity. This observation may help to explain the lack of correlation between coagulation changes and the in creased elastase levels that are observed in septicaemia and sepsis. The data presented here showed that ca thepsin G and elastase were capable of the rapid destruction of factor X although by different methods. Whereas calcium almost totally protected factor X from the action of cathepsin G, it did not protect the zymogen from elastase. It appears that plasma inhibi tors are capable of the rapid inactivation of elastase without concomitant loss in coagu lant activity.
References 1 Plow E: The major fibrinolytic proteases of hu man leucocytes. Biochim Biophys Acta 1980;630: 47-56. 2 Schmidt W, Egbring R, Havemann K: Effect of elastase-like and chymotrypsin-like neutral pro teases from human granulocytes on isolated clot ting factors. Thromb Res 1975;6:315-326. 3 Karpati J, Varadi K, Elodi S: Effect of granulocyte proteases on human coagulation factors IX and X. The protective effect of calcium. Hoppe Seylers Z Physiol Chem 1982;363:521-525. 4 Bingenheimer C, Gramse M, Egbring R, et al: Influence of calcium on the fibrinogen degrada tion products with anticlotting properties. HoppeSeylers Z Physiol Chem 1981;362:853-863. 5 Travis J, Salvesen GS: Human plasma proteinase inhibitors. Annu Rev Biochem 1983;52:655— 709. 6 Egbring R, Schmidt W, Fuchs G, et al: Demon stration of granulocytic proteases in plasma of patients with acute leukaemia and septicaemia with coagulation defects. Blood 1977;49:219— 231.
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Fig. 5. Changes in clotting activity that occur in factor II (•), VII (▲), IX (■) and X (▼) following the addition of cathepsin G (0.1 nmol or 3 pg) per milli litre of plasma.
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Received: December 22, 1990 Accepted by K. Lechner: January 29, 1991 P.T. Turkington, PhD University of Malta Pharmacy Department Msida (Malta)
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