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Granulocyte elastase activation and degradation of factor XIII
THROMBOSIS RESEARCH (c)
Pergamon
Press
GWWJLOCYTE
18; Ltd.
343-351 1980.
ELASTASE OF
Printed in the U.S.A. oo49-3848/80/100~4?-0Q$o2.o0/0
ACTIVATIOP! FACTOR
ATTD DEGRADATIO::
XIII
Per Henriksson, Inga Marie Nilsson, Kjell Ohlsson and PBl Stenberg Department of Paediatrics, Coagulation Laboratory, Departments of Clinical Chemistry and Surgery, and Hospital Pharmacy, General Hospital, Malmo, Sweden (Received Received
18.12.1979; Accepted by by Executive
in revised form 25.1 . 1980. Editor H.C. Godal.. Editorial Office 3.3. 1980)
ABSTRACT
The effect of a pure human granulocyte elastase preparation on purified human plasma and placenta factor XIII was studied with agarose gel electrophoresis combined with activity staining, based on the transamidase catalysed incorporation of monodansylthiacadaverine into casein. The factor XIII zymogens were'degraded by elastase as judged from the time dependent decrease of the potential transamidase activities during incubation. No other enzymatically active fragments with electrophoretic migration rates different from that of the original protein were formed during the incubation. However, elastase activated the plasma zymogen in the absence of thrombin. The mechanism of activation and subsequent degradation by granulocytic elastase might help to explain why the plasma factor XIII is occasionally low in patients with abnormal proteolysis. Studies on a commercially available placenta factor XIII preparation revealed that it contained at least three proteins with transamidase activity, two of which were thrombin independent. Quantification by the transamidase catalysed incorporation of 14C-putrescine into casein showed that the thrombin independent fraction constituted approximately 40 % of the total potential transamidase activity observed in the presence of thrombin. Key words: g:-nnu: IY:Y~~ elastase, plasma and placenta factor XIII, activity staining 343
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F XIII
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INTRODUCTION
Factor XIII is the zymoqen of a transamidatinq enzyme operating in the final step of blood coagulation. It catalyses the covalent cross-linking of fibrin and thereby stabilises the fibrin clot and makes it more resistant to mechanical and proteolytic forces. Any defect in this process may lead to dissolution of the clot and bleeding (1). A variety of proteases, including trypsin and pancreatic elastase, are known to degrade factor XIII (2, 3). Similarly, purified elastase-like and chymotrypsin-like proteases from human qranulocytes (4), leukocyte extracts and plasmin (5) degrade factor XIII in vitro. Low plasma factor XIII activity has recently been reported in clinical conditions with abnormal proteolysis in blood due probably to release of lysosomal enzymes (6-9). The present investigation with a new activity staining technique (10) further elucidates the effect of qranulocyte elastase on factor XIII.
MATERIALS
AND
METHODS
Human plasma factor XIII was prepared according to the method of Lorand and Gotoh (11) as modified by Curtis et al (12 . The (A 1 % at zymogen was stored at -200C as a 5.5 mq/ml solution 280 nM = 13.8) in 50 mM Tris chloride/l mM EDTA, pH 7.5.1 cm Placenta factor XIII was purchased from Behringwerke, GFR (Faktor-XIII-Konzentrat OBKH 10 lot No 434023). The lyophilised powder was stored at 4OC and used before the recommended expiration date. Just before use a stock solution of the placenta extract . was made up in 25 mM Tris acetate (pH 7.5) containing EDTA (2 mMJ and sodium chloride (130 mM). Human qranulocyte elastase was isolated from extracts of the lysosome-like granules according to the method of Ohlsson and Olsson (13). It was stored at -2OOC as a 2 mq/ml solution in 10 mM sodium acetate buffer (pH 4.0). Before use, it was diluted with Tris acetate (25 mM; pH 7.5) containing EDTA (2.0 mM) and saline (130 mM). Human alphal-antitrypsin, kindly supplied by Professor C.-B. Laurell, was dissolved in 50 mM Tris chloride (pH 7.4).
Procedures Incubation. The factor XIII zymoqens were incubated with ---------elastase at 37OC in a total volume of 2.0 ml containing the placenta factor XIII extract (2.5 mq/ml) or human plasma factor XIII (67 gq/ml), elastase (2.5 pq/ml), EDTA (2 mM), Tris acetate (25 mM, pH 7.5) and sodium chloride (130 mM). Appropriate controls were run with saline instead of elastase. At different intervals (5, 25, 50, 90, 150, 210, 270 min) aliguots (100 pl) of the reaction mixtures were added to alpha,-
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antitrypsin (100 pl of a solution of 200 pg/ml in saline). The samples were carefully mixed and stored at -2OOC until subjected to electrophoresis (about 24-48 h). Aqarose gel electroEhoresis was performed as described by Johangg,,-~laj-,~-i~8~ ana'Tb'V/cm in 1 mm thick agarose (LitexHSA, Denmark) gels supported by a polyester film (Cronar). The buffer used was 75 mM sodium-barbital (pH 8.6) containing 2.0 mM EDTA. The samples (20 pl) treated with elastase and the corresponding control samples were applied to the same gel in two parallel slit lines 5.4 cm apart. The activity ----~-----------_ was visualised by -------staining the electrophoreto----------grams at 370C for about 2 hours, as described in detail by Stenberg and Stenflo (10). The staining solution (0.1 ml/cm2 gel) consisted of 0.7 mM monodansylthiacadaverine (N-(5-amino-3thiapentyl)-5-dimethylamino-l-naphthalenesulfon~ide l/2 fumarate) (15), 7 g/l of casein (Merck 2244), 9 NIH U/ml of bovine thrombin (Topostasin Roche) or saline, 9 mM of dithiothreitol (Sigma), and 7 mM calcium chloride in 50 mM Tris chloride (pH 7.5). Excess fluorescent monodansylthiacadaverine was removed by repeated washing of the gels with aqueous trichloroacetic acid, acetic acid and Tris buffer, as described by Stenberg and Stenflo (10). Fluorescent bands on the washed and neutralised agarose gels were illuminated at 254 nm and photographed with Polaroid type 107 film and a Polaroid MP-3 camera equiped with a Wratten No 15 filter. The thrombin independent transamidase activity in the pla----------------____________________------centa factor XIII extract was measured with a modification of the filter paper assay described by Lorand et al (16). The ,reaction mixture (90 pl) consisted of placenta factor XIII extract (0.57 mg/ml), N,N-dimethylcasein 0.5 mg/ml (17), dithiothreitol (6.5 mM), 1.4-14C-putrescine (The Radiochemical Centre, 0.33 mM, specific activity 2J!_f3 Ci/mole), calcium chloride (5.6 mM) and Tris chloride (50 mM, pH 7.5). In the control experiments thrombin (2.5 NIH U/ml) was included and the mixture was incubated at 370C for 40 min before the addition of substrates and calcium chloride. In another control EDTA (2 mM) was used instead of calcium. Ten and 20 min after the calcium addition, aliquots (5 pl) of the reaction mixtures were spotted onto pieces of filter paper and washed and counted as described by Lorand et al (16).
RESULTS At 37OC, a pH of 7.5 and in the absence of calcium, the zymogen of the placenta factor XIII preparation was rapidly degraded in the presence of elastase (Fig. la,c). No other enzymatically active fragments with electrophoretic migration rates different from that of the original protein were formed during the incubation. Potential transamidase activity in the control series did not change during the incubation period (Fig. lb,d).
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F XIII
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FIG. 1 a-d Activity stained agarose gel electrophoretograms with placenta-factor XIII. Incubation times, minutes (gels l-7): 5, 25, 50, 90, 150, 210, 270. Temperature: 370C. Thrombin dependent transamidase activity after incubation with (a) and without (b) elastase. Thrombin independent transamidase activity after incubation with (c) and without (d) elastase. Note: For practical reasons, only parts of the gels carrying transamidase activity, have been included in these figures. During photography, different exposure conditions have been used for the various series. Consequently the relative levels of transamidase activity should only be estimated with the same series.
FIG. 2 a-c Activity stained agarose gels electrophoretograms with plasma factor XIII. Incubation times, minutes (gels l-7): 5, 25, 50, 90, 150, 210, 270. Temperature: 37oc. Thrombin dependent transamidase activity after incubation with (a) and without (b) elastase. Transamidase activity after incubation with elastase without (c) thrombin in the activity staining solution. The arrows denote the sample application sites and + the position of the anode. See note in the legend of Figure 1.
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When thrombin was omitted from the activity staining solution, both elastase treated placenta factor XIII (Fig. lc) and the control (Fig. ld) displayed transamidase activity although the activity of the elastase treated samples was significantly reduced and appeared in a single band. In contrast with the elastase treated zymogen, thrombin-independent transamidase activity of the control appeared in two separate bands unaffected by the incubation (Fig. ld). The same pattern with two thrombin-independent bands was observed in separate experiments with freshly prepared solutions of the placenta factor XIII preparation and where no alphal-antitrypsin was added to the sample. Quantification, based on the enzyme catalysed incorporation of 14C-putrescine into casein, revealed that about 40 % of the apparent total potential transamidase activity in a freshly prepared solution of Behringwerke placenta factor XIII extract was independent of thrombin. However, practically all the transamidase activity in the solution was calcium dependent (Table I). The potential transamidase activity in human plasma factor XIII was reduced by treatment with elastase (Fig. 2a), while the control (Fig. 2b) was unaffected. After incubation of the plasma zymogen with elastase, the thrombin-independent transamidase activity increased progressively and reached its highest level after about 50 min incubation (Fig. 2~). In contrast, plasma factor XIII in the control series did not display any activity in the absence of thrombin (results not shown).
TABLE I Incorporation of 14C-putrescine into casein catalysed by a placenta factor XIII preparation in the presence and absence of thrombin and calcium. For details, see Materials and Methods
Activating agents
Relative transamidase activity (cpm/lO min incubation/5 ~1 sample)
Thrombin and calcium
1068
Calcium; no thrombin
435
No thrombin; no calcium (EDTA)
19
Thrombin; no calcium (EDTA)
17
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GRANULOCYTE
ELASTASE
AND
F XIII
Vol.ld,.\o.~/S
DISCUSSION
Granulocyte elastase is ,a calcium-independent serine protease with a neutral pH optimum (13). It degrades not only elastin, but also a variety of protein substrates, including some specific factors of the complement (18), coagulation (4) and kinin systems (19). The introduction of the specific fluorescent activity staining procedure (lo), which is very sensitive (201, has made it possible to investigate in greater detail the effect of isolated elastase on plasma and placenta factor XIII. Our experiments in vitro demonstrate the ability of elastase to degrade the factor XIII zymogens of human plasma and placenta. No fragments with electrophoretic mobilities differing from that of the native zymogens and carrying transamidase activity were formed during the incubation. A different situation was recently reported by Stenberg and Stenflo (lo), who found that, after storage and thereby probably due to proteolysis, guinea pig liver transglutaminase can be electrophoretically separated into two protein bands with transamidase activity. In the absence of thrombin, the placenta factor XIII preparation displayed a significant transamidase activity (Fig. Id and Table II. The elastase treated placenta factor XIII displayed one thrombin independent transamidating band (Fig. lc) , while the control samples (with no elastase) exhibited two (Fig. Id). Since this pattern was found also in the samples taken at the beginning of the incubation it is obvious that this batch of placenta factor XIII concentrate consists of at least three proteins with transamidating activities, two of which are thrombin-independent. The reason is not obvious for the difference in electrophoretic pattern between the two placenta factor XIII control series (Fig. lb and ld) with and without thrombin in the activity staining solutions. Schwartz et al (3) observed that during incubation of the platelet zymogen with thrombin, some of the a'- chains initially formed are cleaved. According to Schrode et al (21) this proteolysis results in a concomitant loss of transamidase activity which offers a possible explanation for our finding. Based on the 14C-putrescine incorporation into casein, thrombin-independent transamidase activity amounted to approximately 40 8 of the activity observed with thrombin (2.5 NIH U/ml) present during the preincubation. Since this thrombin concentration mightalso degrade the enzymatically active fraction of the placenta factor XIII preparation the amount of thrombin-independent activity might be less than 40 % of the total potential transamidating activity in the preparation. Due to the existing thrombin-independent activity in the placenta factor XIII preparation no unambiguous conclusions can be drawn from this study concerning an elastase-catalysed activation of this zymogen. The activated forms of factor XIII are rapidly cleared from the circulation as shown in animal studies (22). This should be borne in mind when prescribing substitution therapy with this type of factor XIII preparation. This was illustrated by Zimmerman et al (231, who injected 4 ml of a preparation containing 250 units of placenta factor XIII (corresponding to the factor
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XIII activity in 250 ml of normal plasma) into a factor XIII deficient child with a plasma volume of approximately 250 ml. Within two hours the factor XIII plasma level was found to be only about 23 % of normal i.e. a loss of 77 %. The activity recovered had a plasma half life of 4.7 days. The transamidase activity of the elastase-treated plasma fattor XIII preparation in the absence of thrombin (Fig. 2~) may be interpreted as an elastase catalysed activation of the zymogen. Also other proteolytic enzymes, such as trypsin and reptilase, can activate factor XIII (3). The activation by these two enzymes involves cleavage of lysyl or arginyl peptide bonds. Granulocyte elastase, however, is known to split preferentially valyl peptide bonds and secondly alanyl bonds (24). The peptide released from human plasma factor XIII on activation by thrombin contains valyl as well as alanyl peptide bonds (25). But it is not known whether any of these bonds is involved in the elastase catalysed activation. Elastase and other granulocytic proteases are released from dying or phagocytising leukocytes (26). Our incubation mixtures contained 2.5 pg/ml of elastase, a concentration which might well be reached within a local inflammatory process when considering that one million.leukocytes contain 3-4 pg elastase (26). The activity of elastase released into the circulation is effectively inhibited by alpha1 -antitrypsin and alpha2-macroglobulin (27, 28). However, in extravascular inflammatory processes or disorders with tissue injuries a local infiltration of granulocytes might be so massive as to result in the proteolytic activity exceeding the neutralising power of the inhibitors thus making the action of free proteases possible (29, 30). The reduction of plasma factor XIII activity observed in various clinical conditions with abnormal proteolysis might well be explained by the mechanism of activation and subsequent degradation shown in our experiments.
ACKNOWLEDGEMENT This investigation was supported by grants from the Swedish Medical Research Council (B79-19X-00087-15B, B79-13X-05194-02, B79-17X-03910-07A), the Swedish Association against Cancer (1300-B78-01X) and the Medical Faculty, University of Lund.
REFERENCES 1. DUCKERT,F. Documentation of the plasma factor XIII deficiency 190-199, 1972. in man. Ann. N.Y. Acad. Sci., 202, 2. KOPEC,M., LATALLO,Z.S., STAHL,M. and WEGRZYNOWICZ,S. The effect of proteolytic enzymes on fibrin stabilizing factor. Biochim. Biophys. Acta, l&, 437-445, 1969.
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3. SCHWARTZ,M.L., PIZZO, S.V., HILL,R.L. and MCKEE,P.A. Human factor XIII from plasma and platelets. J. Biol, Chem., && 1395-1407, 1973. 4. SCHMIDT,W., EGBRING,R. and HAVEMANN,K. Effect of elastaselike and chymotrypsin-like neutral proteases from human granulocytes on isolated clotting factors. Thromb. Res., 5, 315-326, 1975. 5. HENRIKSSON,P. and NILSSON,I.M. Effects of leukocytes, plasmin and thrombin on clotting factors. A comparative in vitro 16, 301-312, 1979. study. Thromb. Res.. 6. EGBRING,R., SCHMIDT,W., FUCHS,G. and HAVEMANN,K. Demonstration of granulocytic proteases in plasma of patients with acute leukemia and septicemia with coagulation defects. Blood, 49, 219-231, 1977. 7. HENRIKSSON,P., HEDNER,U., NILSSON,I.M. and NILSSON,P.-G. Generalized proteolysis in a young woman with WeberChristian disease (nodular nonsuppurative panniculitis). Stand. J. Haematol., 14, 355-360, 1975. 8. HENRIKSSON,P., HEDNER,U. and NILSSON,I.M. Factor (fibrin stabilising factor) in Henoch-mnlein's Acta Paediat. Stand., 66,273-277, 1977.
XIII purpura.
9. HENRIKSSON,P., STENBERG,P., NILSSON,I.M., RASOVIC,N., KEZIC,J. and STENBJERG,S. A specific, fluorescent activity staining procedure applied to plasma and red blood cells in congenital factor XIII deficiency. _-_Br. J. Haematol. 1979 (In press). 10. STENBERG,P. and STENFLO,J. A rapid and specific fluorescent activity staining procedure for transamidating enzymes. Analyt. Biochem. -93, 445-452, 1979. 11. LORAND,L. and GOTOH,T. Fibrinoligase. The fibrin stabilising factor system. In: .---. Methods in enzymology Perlman,G.E. and Lorand,L. eds , -19, 770-782 (Academic Press, New York). 12.
CURTIS,C.G., BROWN,K.L., CREDO,R.B., DOMANIK,R.A., GRAY,A., STENBERG,P. and LORAND,L. Calcium-dependent unmasking of active center cysteine during activation of fibrin stabilizing factor; Biochemistry, 2, 3774-3780, 1974.
13. OHLSSON,K. and OLSSON,I. The neutral proteases of human granulocytes. Isolation and partial characterization of granulocyte elastase. Europ. J. Biochem., 42, 519-527, 1974. 14. JOHANSSON,B.G. Agarose gel electrophoresis. Lab. Invest., 29, Suppl. 124, 7-19, 1972.
Stand. J. Chin.
15. LJUNGGREN,C., HOFFMAN,K.-J., STENBERG,P., SVENSSON,U., NILSSON,L., HAKCKOPN,A. and LUNDEN,R. Fibrin-stabilizing factor inhibitors. Monodansylated weak aliphatic diamines. J. Med. Chem .,L7, 649-65., 1974. 16. LORAND,L., CAMPBELL-WILKES,L.K. and COOPERSTEIN,L. A filter paper assay for transamidating enzymes using radioactive amine substrates. Analyt. Biochem., 50, 623-631, 1972.
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17. LIN,Y., MEANS,G.E. and FEENEY,R.E. The action of proteolytic enzymes on N,N-dimethyl proteins. J. Biol. Chem., 244, 789-793, 1969. 18. JOHNSON,U., OHLSSON,K. and OLSSON,I. Effects of granulocyte neutral proteases on complement components. Stand. J. Immunol., _5, 421-426, 1976. 19. DITTMAN,B., WIMMER,R., LINDERMANN,R. and OHLSSON,K. The effect of human granulocyte proteinases on kininogens.In: Internat. Symp. Kinins, Tokyo, Japan 1978. 20. HENRIKSSON,P., NILSSON,L., NILSSON,I.M. and STENBERG,P. tal iron intoxication with multiple coagulation defects degradation of factor VIII and factor XIII. Stand. J. Haemat. -22, 235-240, 1979. 21. SCHRODE,J., CHUNG,S.I. and FOLK,J.E. Thrombin cleavage ducts of human placental transglutaminase. Fed. Proc., 1487, 1976 (Abstr 659).
Faand
pro35,
22. LEE,S.Y. and CHUNG,S.I. Biosynthesis and degradation of plasma protransglutaminase (factor XIII). Fed. Proc., -35, 1486, 1976 (Abstr 658). MEINRENKENIW., EGBRING,R. '23. ZIMMERMANN,R., EHLERS,W.;MANZ,F., and GEMMEKE,H. Ein neuer Fall von schwerem Faktor XIIIMangel. Untersuchungen zum Verhalten der Untereinheiten und des Turnover des Fibrinstabilisierenden Faktors. -Blut, 35, 457-464, 1977. 24. BLOW,A.M.J. Action of human lysosomal oxidized B chain of insulin. Biochem.
elastase on the J., 161, 13-16, 1977.
25. TAGAKI,T. and DOOLITTLE,R.F. Amino acid sequence studies on factor XIII and the peptide released during its activation by thrombin. Biochemistry, 13, 750-756, 1974. 26. OHLSSON,K. and OLSSON,I. The extracellular release of granulocyte collagenase and elastase during phagocytosis inflammatory processes. Stand. J. Haematol.,g, 145-152, 1977.
and
27. OHLSSON,K. and OLSSON,I. Neutral proteases of human granulocytes. III. Interaction between human granulocyte elastase and plasma protease inhibitors. Stand. J. Clin. Lab. Invest. 34, 349-355, 1974. 28. OHLSSON,K. and OLSSON,A.-S. Immunoreactive granulocyte elastase in human serum. Hoppe-Seyler's Zschr. Phys. Chem. ,359, 1531-1539, 1978. 29. OHLSSON,K. Collagenase and elastase released during peritonitis are complexed by plasma protease inhibitors. Surgery, 79, 652-657, 1976. 30. DELSHAMMAR,M. and OHLSSON,K. Granulocyte collagenase and elastase and the plasma protease inhibitors in human pus. Surgery., 83, 323-327, 1978.
Pergamon
Press
GWWJLOCYTE
18; Ltd.
343-351 1980.
ELASTASE OF
Printed in the U.S.A. oo49-3848/80/100~4?-0Q$o2.o0/0
ACTIVATIOP! FACTOR
ATTD DEGRADATIO::
XIII
Per Henriksson, Inga Marie Nilsson, Kjell Ohlsson and PBl Stenberg Department of Paediatrics, Coagulation Laboratory, Departments of Clinical Chemistry and Surgery, and Hospital Pharmacy, General Hospital, Malmo, Sweden (Received Received
18.12.1979; Accepted by by Executive
in revised form 25.1 . 1980. Editor H.C. Godal.. Editorial Office 3.3. 1980)
ABSTRACT
The effect of a pure human granulocyte elastase preparation on purified human plasma and placenta factor XIII was studied with agarose gel electrophoresis combined with activity staining, based on the transamidase catalysed incorporation of monodansylthiacadaverine into casein. The factor XIII zymogens were'degraded by elastase as judged from the time dependent decrease of the potential transamidase activities during incubation. No other enzymatically active fragments with electrophoretic migration rates different from that of the original protein were formed during the incubation. However, elastase activated the plasma zymogen in the absence of thrombin. The mechanism of activation and subsequent degradation by granulocytic elastase might help to explain why the plasma factor XIII is occasionally low in patients with abnormal proteolysis. Studies on a commercially available placenta factor XIII preparation revealed that it contained at least three proteins with transamidase activity, two of which were thrombin independent. Quantification by the transamidase catalysed incorporation of 14C-putrescine into casein showed that the thrombin independent fraction constituted approximately 40 % of the total potential transamidase activity observed in the presence of thrombin. Key words: g:-nnu: IY:Y~~ elastase, plasma and placenta factor XIII, activity staining 343
344
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ELASTASE
AND
F XIII
voua,xo.3/4
INTRODUCTION
Factor XIII is the zymoqen of a transamidatinq enzyme operating in the final step of blood coagulation. It catalyses the covalent cross-linking of fibrin and thereby stabilises the fibrin clot and makes it more resistant to mechanical and proteolytic forces. Any defect in this process may lead to dissolution of the clot and bleeding (1). A variety of proteases, including trypsin and pancreatic elastase, are known to degrade factor XIII (2, 3). Similarly, purified elastase-like and chymotrypsin-like proteases from human qranulocytes (4), leukocyte extracts and plasmin (5) degrade factor XIII in vitro. Low plasma factor XIII activity has recently been reported in clinical conditions with abnormal proteolysis in blood due probably to release of lysosomal enzymes (6-9). The present investigation with a new activity staining technique (10) further elucidates the effect of qranulocyte elastase on factor XIII.
MATERIALS
AND
METHODS
Human plasma factor XIII was prepared according to the method of Lorand and Gotoh (11) as modified by Curtis et al (12 . The (A 1 % at zymogen was stored at -200C as a 5.5 mq/ml solution 280 nM = 13.8) in 50 mM Tris chloride/l mM EDTA, pH 7.5.1 cm Placenta factor XIII was purchased from Behringwerke, GFR (Faktor-XIII-Konzentrat OBKH 10 lot No 434023). The lyophilised powder was stored at 4OC and used before the recommended expiration date. Just before use a stock solution of the placenta extract . was made up in 25 mM Tris acetate (pH 7.5) containing EDTA (2 mMJ and sodium chloride (130 mM). Human qranulocyte elastase was isolated from extracts of the lysosome-like granules according to the method of Ohlsson and Olsson (13). It was stored at -2OOC as a 2 mq/ml solution in 10 mM sodium acetate buffer (pH 4.0). Before use, it was diluted with Tris acetate (25 mM; pH 7.5) containing EDTA (2.0 mM) and saline (130 mM). Human alphal-antitrypsin, kindly supplied by Professor C.-B. Laurell, was dissolved in 50 mM Tris chloride (pH 7.4).
Procedures Incubation. The factor XIII zymoqens were incubated with ---------elastase at 37OC in a total volume of 2.0 ml containing the placenta factor XIII extract (2.5 mq/ml) or human plasma factor XIII (67 gq/ml), elastase (2.5 pq/ml), EDTA (2 mM), Tris acetate (25 mM, pH 7.5) and sodium chloride (130 mM). Appropriate controls were run with saline instead of elastase. At different intervals (5, 25, 50, 90, 150, 210, 270 min) aliguots (100 pl) of the reaction mixtures were added to alpha,-
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antitrypsin (100 pl of a solution of 200 pg/ml in saline). The samples were carefully mixed and stored at -2OOC until subjected to electrophoresis (about 24-48 h). Aqarose gel electroEhoresis was performed as described by Johangg,,-~laj-,~-i~8~ ana'Tb'V/cm in 1 mm thick agarose (LitexHSA, Denmark) gels supported by a polyester film (Cronar). The buffer used was 75 mM sodium-barbital (pH 8.6) containing 2.0 mM EDTA. The samples (20 pl) treated with elastase and the corresponding control samples were applied to the same gel in two parallel slit lines 5.4 cm apart. The activity ----~-----------_ was visualised by -------staining the electrophoreto----------grams at 370C for about 2 hours, as described in detail by Stenberg and Stenflo (10). The staining solution (0.1 ml/cm2 gel) consisted of 0.7 mM monodansylthiacadaverine (N-(5-amino-3thiapentyl)-5-dimethylamino-l-naphthalenesulfon~ide l/2 fumarate) (15), 7 g/l of casein (Merck 2244), 9 NIH U/ml of bovine thrombin (Topostasin Roche) or saline, 9 mM of dithiothreitol (Sigma), and 7 mM calcium chloride in 50 mM Tris chloride (pH 7.5). Excess fluorescent monodansylthiacadaverine was removed by repeated washing of the gels with aqueous trichloroacetic acid, acetic acid and Tris buffer, as described by Stenberg and Stenflo (10). Fluorescent bands on the washed and neutralised agarose gels were illuminated at 254 nm and photographed with Polaroid type 107 film and a Polaroid MP-3 camera equiped with a Wratten No 15 filter. The thrombin independent transamidase activity in the pla----------------____________________------centa factor XIII extract was measured with a modification of the filter paper assay described by Lorand et al (16). The ,reaction mixture (90 pl) consisted of placenta factor XIII extract (0.57 mg/ml), N,N-dimethylcasein 0.5 mg/ml (17), dithiothreitol (6.5 mM), 1.4-14C-putrescine (The Radiochemical Centre, 0.33 mM, specific activity 2J!_f3 Ci/mole), calcium chloride (5.6 mM) and Tris chloride (50 mM, pH 7.5). In the control experiments thrombin (2.5 NIH U/ml) was included and the mixture was incubated at 370C for 40 min before the addition of substrates and calcium chloride. In another control EDTA (2 mM) was used instead of calcium. Ten and 20 min after the calcium addition, aliquots (5 pl) of the reaction mixtures were spotted onto pieces of filter paper and washed and counted as described by Lorand et al (16).
RESULTS At 37OC, a pH of 7.5 and in the absence of calcium, the zymogen of the placenta factor XIII preparation was rapidly degraded in the presence of elastase (Fig. la,c). No other enzymatically active fragments with electrophoretic migration rates different from that of the original protein were formed during the incubation. Potential transamidase activity in the control series did not change during the incubation period (Fig. lb,d).
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FIG. 1 a-d Activity stained agarose gel electrophoretograms with placenta-factor XIII. Incubation times, minutes (gels l-7): 5, 25, 50, 90, 150, 210, 270. Temperature: 370C. Thrombin dependent transamidase activity after incubation with (a) and without (b) elastase. Thrombin independent transamidase activity after incubation with (c) and without (d) elastase. Note: For practical reasons, only parts of the gels carrying transamidase activity, have been included in these figures. During photography, different exposure conditions have been used for the various series. Consequently the relative levels of transamidase activity should only be estimated with the same series.
FIG. 2 a-c Activity stained agarose gels electrophoretograms with plasma factor XIII. Incubation times, minutes (gels l-7): 5, 25, 50, 90, 150, 210, 270. Temperature: 37oc. Thrombin dependent transamidase activity after incubation with (a) and without (b) elastase. Transamidase activity after incubation with elastase without (c) thrombin in the activity staining solution. The arrows denote the sample application sites and + the position of the anode. See note in the legend of Figure 1.
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AND F XIII
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When thrombin was omitted from the activity staining solution, both elastase treated placenta factor XIII (Fig. lc) and the control (Fig. ld) displayed transamidase activity although the activity of the elastase treated samples was significantly reduced and appeared in a single band. In contrast with the elastase treated zymogen, thrombin-independent transamidase activity of the control appeared in two separate bands unaffected by the incubation (Fig. ld). The same pattern with two thrombin-independent bands was observed in separate experiments with freshly prepared solutions of the placenta factor XIII preparation and where no alphal-antitrypsin was added to the sample. Quantification, based on the enzyme catalysed incorporation of 14C-putrescine into casein, revealed that about 40 % of the apparent total potential transamidase activity in a freshly prepared solution of Behringwerke placenta factor XIII extract was independent of thrombin. However, practically all the transamidase activity in the solution was calcium dependent (Table I). The potential transamidase activity in human plasma factor XIII was reduced by treatment with elastase (Fig. 2a), while the control (Fig. 2b) was unaffected. After incubation of the plasma zymogen with elastase, the thrombin-independent transamidase activity increased progressively and reached its highest level after about 50 min incubation (Fig. 2~). In contrast, plasma factor XIII in the control series did not display any activity in the absence of thrombin (results not shown).
TABLE I Incorporation of 14C-putrescine into casein catalysed by a placenta factor XIII preparation in the presence and absence of thrombin and calcium. For details, see Materials and Methods
Activating agents
Relative transamidase activity (cpm/lO min incubation/5 ~1 sample)
Thrombin and calcium
1068
Calcium; no thrombin
435
No thrombin; no calcium (EDTA)
19
Thrombin; no calcium (EDTA)
17
j48
GRANULOCYTE
ELASTASE
AND
F XIII
Vol.ld,.\o.~/S
DISCUSSION
Granulocyte elastase is ,a calcium-independent serine protease with a neutral pH optimum (13). It degrades not only elastin, but also a variety of protein substrates, including some specific factors of the complement (18), coagulation (4) and kinin systems (19). The introduction of the specific fluorescent activity staining procedure (lo), which is very sensitive (201, has made it possible to investigate in greater detail the effect of isolated elastase on plasma and placenta factor XIII. Our experiments in vitro demonstrate the ability of elastase to degrade the factor XIII zymogens of human plasma and placenta. No fragments with electrophoretic mobilities differing from that of the native zymogens and carrying transamidase activity were formed during the incubation. A different situation was recently reported by Stenberg and Stenflo (lo), who found that, after storage and thereby probably due to proteolysis, guinea pig liver transglutaminase can be electrophoretically separated into two protein bands with transamidase activity. In the absence of thrombin, the placenta factor XIII preparation displayed a significant transamidase activity (Fig. Id and Table II. The elastase treated placenta factor XIII displayed one thrombin independent transamidating band (Fig. lc) , while the control samples (with no elastase) exhibited two (Fig. Id). Since this pattern was found also in the samples taken at the beginning of the incubation it is obvious that this batch of placenta factor XIII concentrate consists of at least three proteins with transamidating activities, two of which are thrombin-independent. The reason is not obvious for the difference in electrophoretic pattern between the two placenta factor XIII control series (Fig. lb and ld) with and without thrombin in the activity staining solutions. Schwartz et al (3) observed that during incubation of the platelet zymogen with thrombin, some of the a'- chains initially formed are cleaved. According to Schrode et al (21) this proteolysis results in a concomitant loss of transamidase activity which offers a possible explanation for our finding. Based on the 14C-putrescine incorporation into casein, thrombin-independent transamidase activity amounted to approximately 40 8 of the activity observed with thrombin (2.5 NIH U/ml) present during the preincubation. Since this thrombin concentration mightalso degrade the enzymatically active fraction of the placenta factor XIII preparation the amount of thrombin-independent activity might be less than 40 % of the total potential transamidating activity in the preparation. Due to the existing thrombin-independent activity in the placenta factor XIII preparation no unambiguous conclusions can be drawn from this study concerning an elastase-catalysed activation of this zymogen. The activated forms of factor XIII are rapidly cleared from the circulation as shown in animal studies (22). This should be borne in mind when prescribing substitution therapy with this type of factor XIII preparation. This was illustrated by Zimmerman et al (231, who injected 4 ml of a preparation containing 250 units of placenta factor XIII (corresponding to the factor
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XIII activity in 250 ml of normal plasma) into a factor XIII deficient child with a plasma volume of approximately 250 ml. Within two hours the factor XIII plasma level was found to be only about 23 % of normal i.e. a loss of 77 %. The activity recovered had a plasma half life of 4.7 days. The transamidase activity of the elastase-treated plasma fattor XIII preparation in the absence of thrombin (Fig. 2~) may be interpreted as an elastase catalysed activation of the zymogen. Also other proteolytic enzymes, such as trypsin and reptilase, can activate factor XIII (3). The activation by these two enzymes involves cleavage of lysyl or arginyl peptide bonds. Granulocyte elastase, however, is known to split preferentially valyl peptide bonds and secondly alanyl bonds (24). The peptide released from human plasma factor XIII on activation by thrombin contains valyl as well as alanyl peptide bonds (25). But it is not known whether any of these bonds is involved in the elastase catalysed activation. Elastase and other granulocytic proteases are released from dying or phagocytising leukocytes (26). Our incubation mixtures contained 2.5 pg/ml of elastase, a concentration which might well be reached within a local inflammatory process when considering that one million.leukocytes contain 3-4 pg elastase (26). The activity of elastase released into the circulation is effectively inhibited by alpha1 -antitrypsin and alpha2-macroglobulin (27, 28). However, in extravascular inflammatory processes or disorders with tissue injuries a local infiltration of granulocytes might be so massive as to result in the proteolytic activity exceeding the neutralising power of the inhibitors thus making the action of free proteases possible (29, 30). The reduction of plasma factor XIII activity observed in various clinical conditions with abnormal proteolysis might well be explained by the mechanism of activation and subsequent degradation shown in our experiments.
ACKNOWLEDGEMENT This investigation was supported by grants from the Swedish Medical Research Council (B79-19X-00087-15B, B79-13X-05194-02, B79-17X-03910-07A), the Swedish Association against Cancer (1300-B78-01X) and the Medical Faculty, University of Lund.
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