THROMBOSIS

RESEARCH

61;433-440,1991

0049-3848/91 $3.00 + .OOPrinted in the USA. Copyright (c) 1991 Pergamon Press plc. All rights reserved.

FIBRINOGEN DOES NOT PROTECT VON WILLEBRANO FACTOR AGAINST PROTEOLYSIS BY HUMAN CATHEPSIN G *** , I. Scharrer I. sopata*+, Zs.Vigh M. Kopet*, M. Jelerlska*, +*+ . and K. Breddin and

** Institute

Medicine,

University

*Institute of Hematology Warsaw, Poland ***

Center Germany

of

Internal

(Received 18.10.1990;accepted

of

***

Rheumatology,

Hospital,

Frankfurt/M.

in revisedform 10.12.1990 by EditorH. Vinazzer)

ABSTRACT The susceptibility of von Willebrand factor /vWF/ to digestion by human neutrophil cathepsin G in highly /BHS/ and in cryoprecipurified FVIII/vWF concentrate In contrast to human neutropitate has been studied. cathepsin G inactivates and degrades phi1 elastase, vWF not only in BHS but also in cryoprecipitate. Fibrinogen added in excess to purified FVIII/vWF concentrate does not protect vWF against cathepsin G proteolysis. Biological significance of this phenomena are discussed.

INTRODUCTION It is well documented human polymorphonuclears trophil elastase /HNE/

like

proteinase,

degrade

that two main proteolytic /HPN/ active at neutral

and cathepsin and inactivate

enzymes

of

pH: human neuG called also chymotrypsin the purified factor VIII

/FVIII/ and von Willebrand factor /vWF/ preparations (1,2,3). Activity of factor VIII was found much more sensitive to those granulocytic enzymes than vWF activity (1,2). Correlation of proteolysis of vWF highest molecular weight multimers with vWF inactivation was documented for HNE (3) but was not tested for cathepsin G. Two main HPN proteinases appeared to act cooperatively and to be many times more potent in inactivating FVIII and vWF thananapancreatic enzymes (1). aogous The role of HPN derived proteinases in the degradation of vWF multimers has been implicated in pathogenesis of the acquired

von Willebrand me (4,5_,6), in Key

words:

disease associated the generation of

cathepsin

G,

with myeloproliferative the altered form of

FVIII/vWF 433

concentrate,

syndrovWF

in

cryoprecipitate

the

434

majority of as in stored

FIBRINOGEN DOES NOT PROTECT vWF...

commercial concentrates cryoprecipitates (9).

of

FVIII/vWF

Vol. 61, No. 4

(7,8)

as

well

Recently we have reported that FVIII and vWF were much more susceptible to proteolysis by HNE in a highly purified concentrate than in the cryoprecipitate (10). The evidence has been provided that a high content of fibrinogen in a cryoprecipitate may protect FVIII and vWF against HNE mediated alterations. In the present study we have compared the effects of a human neutrophil cathepsin G preparation, free of HNE, on vWF and on factor VIII coagulant activity /VIII:C/ in a highly purified FVIII/vWF concentrate and in the cryoprecipitate. We have also examined the effects of fibrinogen addition to the purified FVIII/vWF concentrate on the proteolysis by cathepsin G.

MATERIALS AND METHODS

Concentrate Cryoprecipitate,

of

FVIII/vWF, Ristofact,

Haemate-HS Behring,

1000 (‘BHS),Behring,Germany. Germany.

Cathepsin G was isolated from human neutrophils according to KopeC et al(Z). The activity of cathepsin G determined with the chromogenic substrate BTEE (N-benzoyl-L-tyrosine ethyl ester, Calbiochem) was 160 U/mg protein. The cathepsin G preparation was checked using the chromogenic substrate S-2484 to be free of HNE activity. Cathepsin G wa8 incubated with FVIII/vWF concentrate or cryoprewith 0.5 M NaCl, pH 7.5. cipitate at 37 C in a 50 mM Tris buffer Proteolysis was terminated by addition of SST1 (Soybean Trypsin Inhibitor, Sigma) to a final concentration 0.1 mg/ml. Multimeric composition factor VIII coagulant bed earlier (10).

of vWF, ristocetin activity /VIII:C/

cofactor activity/RCof/, were measured as descri-

RESULTS During incubation of FVIII/vWF concentrate with cathepsin G the progressive disappearance of large size vWF multimers occurThis effect was dependent on digestion time /Fig. l/, and ed. 2/. Also in cryoprecipitate on cathepsin G concentration /Fig. vWF multimers appeared vulnerable to cathepsin G degradation /Fig. 3/.

Vol. 61, No. 4

FIBRINOGEN DOES NOT PROTECT vWF...

1

Fig.

1.

2.

3

4

5

6

Time course of cathepsin G induced changes in vWF multimeric composition of BHS. BHS was incubated with cathepsin G added at enzyme: vWF protein ratio (w/w> 1:250 for various time periods. BHS - 1; BHS with cathepsin G incubated for: 15 min -2; 30 min -3; 60 min -4; 120 min -5; 180 min -6.

1

Fig.

2

435

2

3

4

5

6

7

Changes in vWF multimeric composition of BHS induced by decreasing cathepsin G concentrations. BHS -1; BHS for 2 hrs with incubated for 2 hrs -2; BHS incubated vWF protein ratio (w/w> of:1 to cathepsin G at enzyme: to 5000 100 -3; 1 to 250 -4; 1 to 500 -5; 1 to 2500 -6;l -7.

436

FIBRINOGEN DOES NOT PROTECT vWF...

1 Fig.3.

3

Effect of cathepsin G on vWF multimeric pattern of cryoprecipitate. Control plasma -1; cryoprecipitate -2; cryoprecipitate after incubation with cathepsin G at the enzyme to vWF protein ratio (w/w> 1:lOO -3. In

activation

2

Vol. 61, No. 4

both BHS and of RCof and

cryoprecipitate VIII:C /Table

cathepsin 1 and 21.

G provoked

in-

TABLE 1 Effects of concentrate

cathepsin (BHS)

G on RCof

and

VIII:C

in

Time (min) BHS BHS BHS BHS

+ + + +

buffer buffer cathepsin cathepsin

Cathepsin

G to

0 60 15 60

G G

vWF protein

ratio

the

FVIII/vWF

RCof (%>

VIII:C (%>

100 98 10 4

100 73 3 3

(w/w)

was

1:lOO

TABLE 2 Effects

of

cathepsin

G on RCof

cryoprecipiatet cryoprecipitate cryporecipitate Cathepsin

and

+ buffer + buffer + cathepsin G to

vWF protein

VIII:C

G ratio

in

cryoprecipitate

Time (min)

RCof (%I

VIII:C (%I

0 60 60

100 93 2

100 74 1

(w/w>

was

1:lOO

Vol.61,

No. 4

FIBRINOGEN DOES NOT PROTECT vWF...

437

In

further experiment we have studied the influence of fibrinogen addition to FVIII/vWF concentrate on the effects of cathepsin G. Even high excess of fibrinogen added to VIII/vWF concentrate did not protect vWF multimers against proteolytic degradation by cathepsin G /Fig. 4/.

1

Fig.

4.

2

3

The effect of human fibrinogen addition to BHS on cathepsin G induced changes in vWF multimeric composition of EHS -1; BHS incubated with cathepsin G for 2 hrs at enzyratio (w/w> 1 to 100: without fibrinogen me: vWF protein -2; with fibrinogen /fbg: vWF protein ratio w/w 2.5:1/ -3.

The addition of and VIII:C against

fibrinogen cathepsin

to BHS also did not G mediated inactivation

protect RCof /Table 3/.

TABLE 3 Effect of fibrinogen/fbg/ RCof and VIII:C during

addition incubation

to BHS on susceptibility with cathepsin G f bg/vWF (w/w>

BHS BHS BHS BHS BHS

+ buffer + cathepsin + cathepsin + cathepsin + cathepsin

G G + fbg G + fbg G + fbg

2.5 5.0 10.0

of

RCof (%>

VIII:C (%>

100 3 2 2 2

100 1 1 1 1

438

FIBRINOGEN DOES NOT PROTECT vWF...

Vol. 61, No. 4

DISCUSSION In contrast to HNE (10) cathepsin G degraded large size vWF multimers and inactivated RCof and VIII:C not only in BHS, a highly purified FVIII/vWFconcentrate, but also in cryoprecipitate. Moreover fibrinogen added to HHS in quantity so excessive that it abolished completely HNE mediated alterations remained without any effect on changes in vWF and VIII:C induced by cathepsin G. This indicates a distinctly different reactivity of FVIII/vWF and fibrinogen to HNE and cathepsin G, which may be explained by the well known difference in peptide bond cleavage specificities of these two enzymes. Proteolysis of fibrinogen by HNE has been studied in detail (11, 12, 13). Fibrinogen was found highly sensitive to HNE. This enzyme complexed to alfa - macroglobulin or bound to stimulated granulocytes can degra 4 e fibrinogen in the presence of physiological concentration of plasma protease inhibitors (13). Fibrinogen degradation products produced by HNE have been characterized and found structurally? immunochemically and functionally distinct from plasmin derived cleavage products (11, 12). Recently Weitz et al (14) developed a sensitive immunoassay for the detection of the N-terminal 21 aminoacid peptide released specifically by HNE from the Ad chain of fibrinogen. This assay enables the distinction between HNE plasmin mediated fibrinogenolysis under in vivo conditions. Much less in known about the proteolysis of fibrinogen by cathepsin G. Some data however indicate that fibrinogen and fibrin are much less susceptible to proteolysis by cathepsin G than by HNE. The time required for the loss of fibrinogen clottability was found much longer and the rate of fibrin clot lysis much lower during proteolysis by cathepsin G than by HNE (15). Recently Tsai et al (16) demonstrated that human endothelial cells in culture secrete a very high molecular weight form of vWF. During incubation with granulocyte lysates it was converted into a series of multimers indistinguishable from those present in normal human plasma. This process could be prevented by serine protease inhibitors. The role of leukocytic proteases in phyvWF transformations remains to be elucidated. But a siological relative high excess of fibrinogen over vWF in plasma implies that cathepsin G contribution may be a grater importance than that of HNE. REFERENCES 1. VARAOI, K., MAROSSY, K., ASBOTH, G., ELOOI, Inactivation of human factor VIII by granulocyte Thromb. Haemostas. 43, 45-48, 1980.

P.

and ELOOI, proteases.

S.

2. KOPEC, M., BYKOWSKA, K., LOPACIUK, S., JELENSKA, M., SOPATA, I. and WOJTECKA, E. Effects of neutral KACZANOWSKA, J., proteases from human leukocytes on structure and bilogical properties of human factor VIII. Thromb. Haemostas. 43, 211-217, 1980. 3. THOMPSON, A. and HOWARD. M.A. Proteolytic cleavage von Willebrand factor induced by enzyme(s) released phonuclear cells. Blood 67, 1281-1285, 1986.

of from

human polymor-

F~BR~NOGENDOESNOTPROTECTVWF...

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439

4. BUDDE, U., DENT, J.A., BERKOWITZ, S.D., RUGGERI, Z.M. and ZIMMERMAN, T.S. Subunit composition of plasma von Willebrand factor in patients with the myeloproliferative syndrome. Blood 68, 1213-1217, 1986. 5. LOPEZ-FERNANDEZ, M.F., LOPEZ-BERGES, C., MARTIN, R., PAROO, A. RAMOS, F.J. and BATLLE, J. Abnormal structure of von Willebrand factor in myeloproliferative syndrome is associated to either thrombotic or bleeding diathesis. Thromb. Haemostas. 58, 753-757, 1987. 6. TATEWAKI, W., TAKAHASHI H., TAKAKUWA, E., WADA, K. and SHIBATA A. Plasma von Willebrand factor proteolysis in patients with chronic myeloproliferative disorders: no possibility of ex vivo degradation by calcium-dependent proteases. Thromb. Res.56, 191199, 1989. 7. FRICKE, W.A., and YU, M.W. Characterization of von Willebrand factor in factor VIII concentrates. Am.J.Haematol.31, 41-45,1989. 8. YOSHIOKA, A., SHIMA, M., NISHINO, M., YOSHIKAWA, N. and FUKUT, H. In vitro characterization of various factor VIII concentrates. Arzneim.-Forsch./Drug Res. 37, 753-756, 1987. 9. WEINSTEIN, M. and DEYKIN, D. Comparison of factor VIII-related von Willebrand factor protein prepared from human cryoprecipitate and factor VIII concentrate. Blood 53, 1095-1105, 1979. 10. JELENSKA, M., BYKOWSKA, K., KOPEC, M., VIGH. Zs., SCHARRER,I. and BREDDIN, K. Effects of human neutrophil elastase on von Willebrand factor on highly purified factor VIII concentrate and in cryoprecipitate. Thromb. Res. 59, 295-307, 1990. 11. GRAMSE, M., BINCENHEIMER, C., SCHMIDT, W., EGBRING, R. and HAVEMANN, K. Degradation products of fibrinogen by elastase-like neutradprotease from human granulocytes. J.Clin.Invest. 61, 10271032, 1978. 12. PLOW, E.F., GRAMSE, M. and HAVEMANN, K. Immunochemical discrimination of leukocyte elastase from plasmic degradation products of fibrinogen. J.Lab.Clin. Med. 102, 858-869, 1983. LANDMAN, 13. WEITZ, J.I., HUANG, A.J., and SILVESTEIN, S.C. Elastase-mediated

attractant-stimulated siologic

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S.L., NICHOLSON, fibrinogelolysis

of antiproteinases.

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the

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Med. 166, 1836 -1850, 1987. 14. WEITZ, J.I., LANOMAN, S.L., CROWLEY, K.A., BIRKEN, S. and MORGAN, F.J. Development of an assay for in vivo human neutrophil elastase activity. Increased elastase activity in patients with alfa -proteinase inhibitor deficiency. J.Clin.Invest. 78, 155-162 1986: 15. KOPEI?,M., KACZANOWSKA, J., STACHURSKA, J., TOMCZAK, Z., WOJTECKA-tUKASIK, E., BYKOWSKA, K. and LOPACIUK, S. Effect of granulocytic neutral proteases on some clotting factors. In: Progress in Fibrinolysis. J.F. Davidson, J.H. Nilson and B.Astedt (Eds.) Edinburgh. London, Melbourne and New York Churchill Living stone. 1981, 5, pp. 75-77.

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16. TSAI, H-M., NAGEL, R.L., HATCHER. V.B. and SUSSMAN, 1.1. Endothelial cell-derived high molecular weight von Willebrand factor is converted into the plasma multimer pattern by granulocyte proteases. Biochem.Biophys. Res. Commun. 158, 980-985, 1989.

Fibrinogen does not protect von Willebrand factor against proteolysis by human cathepsin G.

The susceptibility of von Willebrand factor /vWF/ to digestion by human neutrophil cathepsin G in highly purified FVIII/vWF concentrate /BHS/ and in c...
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