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Differences in effects of fibrin(ogen) fragments on the activation of 1-glu-plasminogen and 442-VAL-plasminogen by tissue-type plasminogen activator
THROMBOSISRESEARCH 32; 87-92, 1983
0049-3848/83 $3.00 + .OO Printed in the USA. Copyright (c) 1983 Pergamon Press Ltd. All rights reserved.
DIFFERENCES IN EFFECTS OF FIBRIN(OGEN) FRAGMENTS ON THE ACTIVATION OF I-GLU-PLASMINOGEN AND 442-VAL-PLASMINOGEN BY TISSUE-TYPE PLASMINOGEN ACTIVATOR
J.H. Verheijen, W. Nieuwenhuizen, D.Y. Traas, G.T.G. Chang, E. Hoegee Gaubius Institute, Health Research Division TNO, Herenstraat Sd, 2313 AD Leiden, The Netherlands
(Received 28.3.83; Accepted in revised form 12.7.83 by Editor T. Astrup)
ABSTRACT The activation rate of plasminogen by tissue-type plasminogen activator can be increased by fibrin(ogen) fragments. There is a remarkable difference in the effect of these fragments on the stimulation of l-glu-plasminogen activation and 442-val-plasminogen (mini-plasminogen) activation. Fibrin monomer as well as plasmic fragments Y, D EGTA and D-dimer have a stimulating effect on both 1-glu-plasminogen and 442-val-plasminogen activation, whereas cyanogen bromide fragment FCB2 stimulates only the activation of 1-glu-plasminogen. Results indicate that two types of sites may be operational in fibrin and fibrin(ogen) fragments Y, D EGTA and D-dimer. One type of site (FCBZ-related) interacts probably with plasminogen and may be dependent on the kringle 1-4 region; the other type of site probably interacts either with plasminogen in a non-kringle 1-4 region-dependent manner or with tissue-type plasminogen activator.
INTRODUCTION Plasmin is a major enzyme responsible for fibrin degradation in vivo. It is present in 'blood as an inactive zymogen, plasminogen. The conversion of plasminogen to plasmin occurs by the hydrolysis of a single (560-arg-561-val) peptide bond, catalyzed by specific proteolytic enzymes called plasminogen activators. The tissue-type plasminogen activator (t-PA or extrinsic plasminogen activator) is probably the most important enzyme for plasminoqen activation in vivo (for a review, see ref. 1).
Key words: Plasminogen, fibrin fragments, plasminogen activator, plasmin formation.
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Fibrin plays an important role in plasminogen activation by t-PA. It is not merely the substrate for plasmin but it also accelerates the activation process (2). We have recently shown that these stimulating properties are retained in some defined soluble fibrin(ogen) fragments (3,4,5). Fibrin monomer and the enzymatically prepared fragments Y, D EGTA and D-dimer (3) and also the cyanogen bromide fragment FCB2 (identical with Hol-DSK (8)) are all strongly stimulatory (5). However, it has not been shown until now, whether the stimulating properties of fibrin(ogen) fragments are due to an effect on plasminogen, on activator or on both. In this paper results are presented indicating that two different types of site present in fibrin contribute to the stimulating properties of fibrin, and that the kringle 1-4 region of plasminogen is involved in stimulation by one of these sites, but is not essential for stimulation by the other site.
MATERIALS AND METHODS Fibrin monomer was prepared by dissolving a fibrin clot in 3 mol/l NaBr pH 5.5 as described (3). For some experiments fibrin monomer was prepared from fibrinogen in the presence of antipolymerizing Dcate fragments (3). Fibrin and fibrinogen fragments Y, Dcate, D EGTA, D-dimer and E were prepared by limited plasmic digestion of fibrin(ogen) and purified according to published procedures (6,7). Cyanogen bromide digestion of fibrinogen was carried out as described (4). The dialysed mixture of fragments is called CNBr-digest. Fragment FCB2 with a molecular weight of 43,000 was purified from the CNBr-digest as described (5). Tissue-type plasminogen activator (t-PA) was purified from Bowes melanoma cell culture medium, in its two chain form, using the procedure of Rijken (10). l-Glu-plasminogen was prepared from fresh human blood plasma using the lysine-sepharose adsorption-desorption method (11). C-Aminohexanoic acid present in the desorbed plasminogen solution was removed by precipitating plasminogen with (NHI,)~SOI, at 50% saturation at 4°C. The precipitate was then redissolved in 0.05 mol/l Tris.HCl, 0.1 mol/l NaCl, pH 7.5 and a stock solution in Tris buffer/glycerol l/l (v/v) was prepared and kept at -2O.C. This plasminogen has glutamic acid as the only N-terminal amino acid residue as established by a determination according to Gray (12). 442-Val-plasminogen (mini-plasmincgen) was prepared essentially as described by Sottrup-Jensen et al. (13). The starting material was I-glu-plasminogen. The final product has valine as the N-terminal amino acid residue, is homogeneous on SDS gel electrophoresis and has a molecular weight of approximately 37,000. As with l-glu-plasminogen, a stock solution in Tris-buffer-glycerol was prepared. The effect of fibrin(ogen) fragments on plasminogen activation by t-PA was measured in a system analogous to that described by Drapier et al. (14) which contained t-PA (0.05 Iv/ml), plasminogen (0.13 pmol/l), S-2251 (0.30 mmol/l) in 0.1 mol/l Tris.HCl pH 7.5, 0.1% (v/v) Tween 80, the total volume was 0.25 ml (3,4). Assays were carried out in 96 well polystyrene microtitration plates at 25-C and measured with an eight channel photometer (Titertek, multiscan). Fibrinogen (grade L) and H-D-Valyl-L-Leucyl-L-Lysine-p-nitroanilide.dihydrochloride (S-2251) were obtained from Kabi Diagnostica, Stockholm, Sweden.
RESULTS AND DISCUSSION In earlier studies from this laboratory it has been shown that the activation of a mixture of I-glu-plasminogen and 77-lys-plasminogen by t-PA was acce-
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lerated in the presence of certain fibrin(ogen) fragments. Especially fragment D-dimer (3) and cyanogen bromide fragment FCB2 (5) were found to have a pronounced stimulating effect, comparable to the effect of fibrin monomer (15). Experiments with pure 1-glu-plasminogen gave essentially the same results (Figs. lA, C). For this reason no further experiments with purified 77-lys-plasminogen were carried out. Fibrin monomer, prepared either by dissolving fibrin with NaBr or generated from fibrinogen with thrombin in the presence of antipolymerizing Dcate fragments to prevent gelation (not shown), a whole fibrinogen CNBr digest (4) as well as pure FCB2 (S), fragments Y and D-dimer were all stimulatory. Fibrinogen, fragment Dcate, fragment X and fragment E did not act as stimulants. Fragment D EGTA showed a slight stimulatory effect (Figs. lA, C). In a previous study (3) we have shown that some plasmic fibrin(ogen) fragments have an inhibitory rather than a stimulatory effect on plasmin-catalyzed S-2251 hydrolysis, recently we have shown that CNBr-fragments behave very much like fragment D-dimer in this respect (not shown). The measured net stimulating effect must therefore be ascribed to an effect on plasminogen activation. Different mechanisms for this stimulating effect can be proposed: 1. An interaction of fibrin(ogen) fragment with only t-PA, making the latter a better activator for plasminogen. 2. An interaction of intact fibrin or specific fibrin(ogen) fragment with plasminogen, making the latter a better substrate for t-PA. 3. An interaction of fibrin or fragments with both t-PA and plasminogen. In this study we have tried to get more insight in the interactions involved in the stimulation of t-PA catalyzed plasminogen activation by fibrin(ogen) fragments. Wa carried out experiments with 442-val-plasminogen (mini-plasminogen), a product of limited proteolysis of 1-glu-plasminogen with elastase; this plasminogen has a fully activatable active site, but lacks four of the five kringle structures present in 1-glu-plasminogen. These kringle structures are believed to play a role in the interaction of plasminogen with fibrin and of plasmin with o2-antiplasmin (16). The possibility exists that these kringle structures are also involved in the stimulating effect of plasminogen activation. The results of the experiments with mini-plasminogen are shown in Figs. lB, D. The activation of mini-plasminogen by t-PA is stimulated by fibrin monomer, D-dimer, D EGTA and fragment Y, fragments which also stimulate the activation of glu-plasminogen. A cyanogen bromide digest of fibrinogen stimulates activation of glu-plasminogen. It does not, however, stimulate the activation of mini-plasminogen. The same result was obtained with the pure cyanogen bromide fragment FCBZ, the fragment from the cyanogen bromide fragments mixture, which contains a stimulatory site (5). The results are summarized in Table I. From these results the following conclusions can be drawn. Mechanism 1, an interaction of a fibrin(ogen) fragment only with t-PA, seems a very unlikely possibility, since mini-plasminogen activation is not, in contrast with glu-plasminogen activation, enhanced by fibrinogen fragment FCB2. If t-PA should become a more suitable enzyme, also an accelerated mini-plasminogen activation would be expected. Mechanism 2, an interaction of intact fibrin or a fragment with plasminogen, or 3, an interaction of fibrin or fragments with both t-PA and plasminogen seem much more likely. The interaction of plasminogen with fibrin(ogen) fragments leading to stimulation of plasminogen activation could depend on the integrity of the kringle regions of plasminogen. This view appears to be supported by the fact that glu-plasminogen but not mini-plasminogen activation is accelerated in the presence of FCBZ. However, mini-plasmincgen activation is enhanced in the Presence of fibrin monomers, D-dimer, D EGTA and fragment Y, i.e., fragments which also accelerate the activation of glu-plasminogen.
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CNBr-digest
/
-
-
-
Fbg
0’4
04
Ed80d:E,
OF:
0 FlBRIN(OGEN)
OR FRAGMENT
04 (m/l
0.8
)
Fig. 1. Effect of fibrfn(ogen) and fragments on t-PA catalyzed activation of 1-glu-plasminogen and 442-val-plasminogen. Tissue plasminogen activator (0.012 IU) was incubated at 25'C in a system described in materials and methods. Various amounts of fibrin monomer fibrinogen and fragments as indicated were added. The absorbance change at 405 runwas recorded at different incubation times and plotted against the square of the incubation time, The slopes of the straight lines obtained in the AA versus t2 graphs were used as a measure of the rate of plasminogen activation. A and C with 0.13 pmol/l 1 glu-plasmfnogen; B and D with 0.13 umol/l 442-val-plasminogen (mini-plasminogen).
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TABLE I Stimulation of 1-glu-plasminogen and 442-val-plasminogen activation by fibrin(ogen) and some of their fragments mini-plasminogen glu-plasminogen + + Fibrin monomer Fibrinogen + + Fragment Y Fragment Dcate + + Fragment D EGTA + + Fragment D-dimer Fragment E + CNBr-digest + Fragment FCB2 Results are obtained from Fig. 1; a(+) indicates that stimulation occurs, a(-) indicates that no stimulation is observed.
From these results it would appear that different types of site exist which contribute to the rate-enhancing capacity. One type of site is operational in cyanogen bromide fragment FCB2 and only capable of enhancing I-glu- and 77-lys-plasminogen activation (f), while another type of site appears to be present in e.g. D BGTA, D-dimer and Y. This site is capable of enhancing the rate of activation of both glu-plasminogen and mini-plasminogen. Stimulation by the site operational in FCB2 seems dependent on the kringle l-4 region of plasminogen while stimulation by the additional site, operational in e.g. D EGTA and D-dimet, is independent of the kringle l-4 region. Kringle l-4 independent plasminogen binding to fibrin has recently been observed by other investigators (9,17). It it also conceivable that the non-FCB2-related site is involved in an interaction with t-PA, which has also been observed (18). Both possibilities 2. and 3. imply the presence of an interaction between plasminogen and FCB2. The role of fibrin and some of its fragments in plasminogen activation is complex. More direct binding studies of fibrin and fibrinogen fragments with various forms of plasminogen and with t-PA will be needed to fully clarify this role.
REFBRENCES 1.
COLLEN, D. On the regulation and control of fibrinolysis. Thrombos. Haemostas. 43. 77-89, 1980.
2.
WALLEN, P. Activation of plasminogen with urokinase and tissue activator. In: Thrombosis and Urokinase, Paoletti, R. and Sherry, S. eds., Academic Press, New York, pp. 91-102, 1977.
3.
VERBEIJEN, J.H., NIEDWENBUIZEN, W. and WIJNGAARDS, G. Activation of plasminogen by tissue activator is increased specifically in the presence of certain soluble fibrin(ogen) fragments. Thromb. Res. 27, 377-385, 1982.
4.
VEZHEIJEN, J.R., MULLAART, E., CHANG, G.T.G., KLUET, C. and WIJNGAARDS, G. A simple sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurements in plasma. Thrombos. Haemostas. 48, 266-269, 1982.
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5. NIEUWENHUIZEN,W., VERHEIJEN,J.H., VBRMOND,A. and CHANG, G.T.G. Plasminogen activation by tissue activator is accelerated in the presence of fibrin(ogen) cyanogen bromide fragment FCB2. Biochim.Biophys.Acta 755,
531-533, 1983. 6. NIEUWENHUIZEN,W. and GRAVESEN,M. Anticoagulant and calcium binding properties of high molecular weight derivatives of human fibrinogen, produced by plasmin (fragments X). Biochim. Biophys. Acta 668, 81-88, 1981.
7. VAN RUIJVEN-VERMEER,I.A.M.,NIEUWENHUIZEN,W., HAVERKATE,F. and TIMAN, T. A novel method for the rapid purification of human and rat fibrin(ogen) degradation products in high yields. Hoppe-Seyler's Z. physiol. Chem, 360, 633-637, 1979. 8.
G&DLUND, B., HBSSEL, B., MARGUERIE, G., MURANO, G. and BLOMBACK, B. Primary structure of human fibrinogen: Characterization of disulfide-containing cyanogen-bromide fragments. Eur J. Biochem. 77, 595-610, 1977.
9.
THORSEN, S., CLBMMENSEN, L., SDTTRUP-JENSEN, S. and MAGNUSSON, S. Adsorption to fibrin of native fragments of known primary structure from human plasminogen. Biochim. Biophys. Acta 668, 377-387, 1981.
10.
RIJKEN, D.C. and COLLEN, D. Purificationand characterization of the plasminogen activatorsecretedby human melanomacells in culture.J. Biol. Chem. 256, 7035-7041, 1981.
11.
ROBBINS,K.C. and SUMMARIA, L. In: Methods in EnzymologyVol. XLV, Part 8. L. Lorand (Ed.)New York: Academic Press, 1976, chapter 22.
12.
GRAY, W.R. In: Methods in EnzymologyVol. XI. C.H.W. Hirs (Pd.) New York: r Academic Press, 1967, pp. 139-151.
13.
SOTTRUP-JENSEN,L., CLAEYS, ?I.,ZAJDEL, M.,
PETERSEN, T.E. and MAGNDSSON, S. The primary structure of human plasminogen: I. Isolation of two lysine-binding fragments and one "mini"-plasminogen (MW 38,000) by elastase-catalyzed specific limited proteolysis. In: Progress in Chemical Ffbrinolysis and Thrombolysis. J.F. Davidson, R. M. Rowan, M.M. SlllllLMcl, P.C. Desnoyers (Rds.) New York: Raven Press, 1978, pp. 191-209.
14.
DRAPIER, I.C., TENU, I.P., LEMAIRE, G. and PETIT, I.F. Regulationof plasminogen activatorsecretion in mouse peritoneal macrophages. Biochimie 61, 463-477, 1979.
15.
&BY, M., NORRMAN, B. and WALLiN, P. A sensitive assay for tissue plasminogen activator. Thrcinb.Res. 27, 743-749, 1982.
16.
WIMAN, B., LIJNEN, H.R. and COLLEN, D. On the specific interactions between the lysine-binding sites in plasmin and complementary sites in a2-antiplasmin and in fibrinogen. Biochim. Biophys. Acta 579, 142-154, 1979.
17.
LIJNEN, H.R., VAN HOEF,.B. and COLLEN, D. On the role of the carbohydrate side chains of human plasminogen in its interaction with o2-antiplasmin and fibrin. Bur. J. Biochem. 120, 149-154, 1981.
18.
RIJKEN, D.C., HOYLAERTS, M. and COLLEN, D. Fibrinolytic properties of one-chain and two-chain human extrinsic (tissue-type) plasminogen activator. J. Biol. Chem. 257, 2920-2925, 1982.
0049-3848/83 $3.00 + .OO Printed in the USA. Copyright (c) 1983 Pergamon Press Ltd. All rights reserved.
DIFFERENCES IN EFFECTS OF FIBRIN(OGEN) FRAGMENTS ON THE ACTIVATION OF I-GLU-PLASMINOGEN AND 442-VAL-PLASMINOGEN BY TISSUE-TYPE PLASMINOGEN ACTIVATOR
J.H. Verheijen, W. Nieuwenhuizen, D.Y. Traas, G.T.G. Chang, E. Hoegee Gaubius Institute, Health Research Division TNO, Herenstraat Sd, 2313 AD Leiden, The Netherlands
(Received 28.3.83; Accepted in revised form 12.7.83 by Editor T. Astrup)
ABSTRACT The activation rate of plasminogen by tissue-type plasminogen activator can be increased by fibrin(ogen) fragments. There is a remarkable difference in the effect of these fragments on the stimulation of l-glu-plasminogen activation and 442-val-plasminogen (mini-plasminogen) activation. Fibrin monomer as well as plasmic fragments Y, D EGTA and D-dimer have a stimulating effect on both 1-glu-plasminogen and 442-val-plasminogen activation, whereas cyanogen bromide fragment FCB2 stimulates only the activation of 1-glu-plasminogen. Results indicate that two types of sites may be operational in fibrin and fibrin(ogen) fragments Y, D EGTA and D-dimer. One type of site (FCBZ-related) interacts probably with plasminogen and may be dependent on the kringle 1-4 region; the other type of site probably interacts either with plasminogen in a non-kringle 1-4 region-dependent manner or with tissue-type plasminogen activator.
INTRODUCTION Plasmin is a major enzyme responsible for fibrin degradation in vivo. It is present in 'blood as an inactive zymogen, plasminogen. The conversion of plasminogen to plasmin occurs by the hydrolysis of a single (560-arg-561-val) peptide bond, catalyzed by specific proteolytic enzymes called plasminogen activators. The tissue-type plasminogen activator (t-PA or extrinsic plasminogen activator) is probably the most important enzyme for plasminoqen activation in vivo (for a review, see ref. 1).
Key words: Plasminogen, fibrin fragments, plasminogen activator, plasmin formation.
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Fibrin plays an important role in plasminogen activation by t-PA. It is not merely the substrate for plasmin but it also accelerates the activation process (2). We have recently shown that these stimulating properties are retained in some defined soluble fibrin(ogen) fragments (3,4,5). Fibrin monomer and the enzymatically prepared fragments Y, D EGTA and D-dimer (3) and also the cyanogen bromide fragment FCB2 (identical with Hol-DSK (8)) are all strongly stimulatory (5). However, it has not been shown until now, whether the stimulating properties of fibrin(ogen) fragments are due to an effect on plasminogen, on activator or on both. In this paper results are presented indicating that two different types of site present in fibrin contribute to the stimulating properties of fibrin, and that the kringle 1-4 region of plasminogen is involved in stimulation by one of these sites, but is not essential for stimulation by the other site.
MATERIALS AND METHODS Fibrin monomer was prepared by dissolving a fibrin clot in 3 mol/l NaBr pH 5.5 as described (3). For some experiments fibrin monomer was prepared from fibrinogen in the presence of antipolymerizing Dcate fragments (3). Fibrin and fibrinogen fragments Y, Dcate, D EGTA, D-dimer and E were prepared by limited plasmic digestion of fibrin(ogen) and purified according to published procedures (6,7). Cyanogen bromide digestion of fibrinogen was carried out as described (4). The dialysed mixture of fragments is called CNBr-digest. Fragment FCB2 with a molecular weight of 43,000 was purified from the CNBr-digest as described (5). Tissue-type plasminogen activator (t-PA) was purified from Bowes melanoma cell culture medium, in its two chain form, using the procedure of Rijken (10). l-Glu-plasminogen was prepared from fresh human blood plasma using the lysine-sepharose adsorption-desorption method (11). C-Aminohexanoic acid present in the desorbed plasminogen solution was removed by precipitating plasminogen with (NHI,)~SOI, at 50% saturation at 4°C. The precipitate was then redissolved in 0.05 mol/l Tris.HCl, 0.1 mol/l NaCl, pH 7.5 and a stock solution in Tris buffer/glycerol l/l (v/v) was prepared and kept at -2O.C. This plasminogen has glutamic acid as the only N-terminal amino acid residue as established by a determination according to Gray (12). 442-Val-plasminogen (mini-plasmincgen) was prepared essentially as described by Sottrup-Jensen et al. (13). The starting material was I-glu-plasminogen. The final product has valine as the N-terminal amino acid residue, is homogeneous on SDS gel electrophoresis and has a molecular weight of approximately 37,000. As with l-glu-plasminogen, a stock solution in Tris-buffer-glycerol was prepared. The effect of fibrin(ogen) fragments on plasminogen activation by t-PA was measured in a system analogous to that described by Drapier et al. (14) which contained t-PA (0.05 Iv/ml), plasminogen (0.13 pmol/l), S-2251 (0.30 mmol/l) in 0.1 mol/l Tris.HCl pH 7.5, 0.1% (v/v) Tween 80, the total volume was 0.25 ml (3,4). Assays were carried out in 96 well polystyrene microtitration plates at 25-C and measured with an eight channel photometer (Titertek, multiscan). Fibrinogen (grade L) and H-D-Valyl-L-Leucyl-L-Lysine-p-nitroanilide.dihydrochloride (S-2251) were obtained from Kabi Diagnostica, Stockholm, Sweden.
RESULTS AND DISCUSSION In earlier studies from this laboratory it has been shown that the activation of a mixture of I-glu-plasminogen and 77-lys-plasminogen by t-PA was acce-
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lerated in the presence of certain fibrin(ogen) fragments. Especially fragment D-dimer (3) and cyanogen bromide fragment FCB2 (5) were found to have a pronounced stimulating effect, comparable to the effect of fibrin monomer (15). Experiments with pure 1-glu-plasminogen gave essentially the same results (Figs. lA, C). For this reason no further experiments with purified 77-lys-plasminogen were carried out. Fibrin monomer, prepared either by dissolving fibrin with NaBr or generated from fibrinogen with thrombin in the presence of antipolymerizing Dcate fragments to prevent gelation (not shown), a whole fibrinogen CNBr digest (4) as well as pure FCB2 (S), fragments Y and D-dimer were all stimulatory. Fibrinogen, fragment Dcate, fragment X and fragment E did not act as stimulants. Fragment D EGTA showed a slight stimulatory effect (Figs. lA, C). In a previous study (3) we have shown that some plasmic fibrin(ogen) fragments have an inhibitory rather than a stimulatory effect on plasmin-catalyzed S-2251 hydrolysis, recently we have shown that CNBr-fragments behave very much like fragment D-dimer in this respect (not shown). The measured net stimulating effect must therefore be ascribed to an effect on plasminogen activation. Different mechanisms for this stimulating effect can be proposed: 1. An interaction of fibrin(ogen) fragment with only t-PA, making the latter a better activator for plasminogen. 2. An interaction of intact fibrin or specific fibrin(ogen) fragment with plasminogen, making the latter a better substrate for t-PA. 3. An interaction of fibrin or fragments with both t-PA and plasminogen. In this study we have tried to get more insight in the interactions involved in the stimulation of t-PA catalyzed plasminogen activation by fibrin(ogen) fragments. Wa carried out experiments with 442-val-plasminogen (mini-plasminogen), a product of limited proteolysis of 1-glu-plasminogen with elastase; this plasminogen has a fully activatable active site, but lacks four of the five kringle structures present in 1-glu-plasminogen. These kringle structures are believed to play a role in the interaction of plasminogen with fibrin and of plasmin with o2-antiplasmin (16). The possibility exists that these kringle structures are also involved in the stimulating effect of plasminogen activation. The results of the experiments with mini-plasminogen are shown in Figs. lB, D. The activation of mini-plasminogen by t-PA is stimulated by fibrin monomer, D-dimer, D EGTA and fragment Y, fragments which also stimulate the activation of glu-plasminogen. A cyanogen bromide digest of fibrinogen stimulates activation of glu-plasminogen. It does not, however, stimulate the activation of mini-plasminogen. The same result was obtained with the pure cyanogen bromide fragment FCBZ, the fragment from the cyanogen bromide fragments mixture, which contains a stimulatory site (5). The results are summarized in Table I. From these results the following conclusions can be drawn. Mechanism 1, an interaction of a fibrin(ogen) fragment only with t-PA, seems a very unlikely possibility, since mini-plasminogen activation is not, in contrast with glu-plasminogen activation, enhanced by fibrinogen fragment FCB2. If t-PA should become a more suitable enzyme, also an accelerated mini-plasminogen activation would be expected. Mechanism 2, an interaction of intact fibrin or a fragment with plasminogen, or 3, an interaction of fibrin or fragments with both t-PA and plasminogen seem much more likely. The interaction of plasminogen with fibrin(ogen) fragments leading to stimulation of plasminogen activation could depend on the integrity of the kringle regions of plasminogen. This view appears to be supported by the fact that glu-plasminogen but not mini-plasminogen activation is accelerated in the presence of FCBZ. However, mini-plasmincgen activation is enhanced in the Presence of fibrin monomers, D-dimer, D EGTA and fragment Y, i.e., fragments which also accelerate the activation of glu-plasminogen.
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CNBr-digest
/
-
-
-
Fbg
0’4
04
Ed80d:E,
OF:
0 FlBRIN(OGEN)
OR FRAGMENT
04 (m/l
0.8
)
Fig. 1. Effect of fibrfn(ogen) and fragments on t-PA catalyzed activation of 1-glu-plasminogen and 442-val-plasminogen. Tissue plasminogen activator (0.012 IU) was incubated at 25'C in a system described in materials and methods. Various amounts of fibrin monomer fibrinogen and fragments as indicated were added. The absorbance change at 405 runwas recorded at different incubation times and plotted against the square of the incubation time, The slopes of the straight lines obtained in the AA versus t2 graphs were used as a measure of the rate of plasminogen activation. A and C with 0.13 pmol/l 1 glu-plasmfnogen; B and D with 0.13 umol/l 442-val-plasminogen (mini-plasminogen).
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TABLE I Stimulation of 1-glu-plasminogen and 442-val-plasminogen activation by fibrin(ogen) and some of their fragments mini-plasminogen glu-plasminogen + + Fibrin monomer Fibrinogen + + Fragment Y Fragment Dcate + + Fragment D EGTA + + Fragment D-dimer Fragment E + CNBr-digest + Fragment FCB2 Results are obtained from Fig. 1; a(+) indicates that stimulation occurs, a(-) indicates that no stimulation is observed.
From these results it would appear that different types of site exist which contribute to the rate-enhancing capacity. One type of site is operational in cyanogen bromide fragment FCB2 and only capable of enhancing I-glu- and 77-lys-plasminogen activation (f), while another type of site appears to be present in e.g. D BGTA, D-dimer and Y. This site is capable of enhancing the rate of activation of both glu-plasminogen and mini-plasminogen. Stimulation by the site operational in FCB2 seems dependent on the kringle l-4 region of plasminogen while stimulation by the additional site, operational in e.g. D EGTA and D-dimet, is independent of the kringle l-4 region. Kringle l-4 independent plasminogen binding to fibrin has recently been observed by other investigators (9,17). It it also conceivable that the non-FCB2-related site is involved in an interaction with t-PA, which has also been observed (18). Both possibilities 2. and 3. imply the presence of an interaction between plasminogen and FCB2. The role of fibrin and some of its fragments in plasminogen activation is complex. More direct binding studies of fibrin and fibrinogen fragments with various forms of plasminogen and with t-PA will be needed to fully clarify this role.
REFBRENCES 1.
COLLEN, D. On the regulation and control of fibrinolysis. Thrombos. Haemostas. 43. 77-89, 1980.
2.
WALLEN, P. Activation of plasminogen with urokinase and tissue activator. In: Thrombosis and Urokinase, Paoletti, R. and Sherry, S. eds., Academic Press, New York, pp. 91-102, 1977.
3.
VERBEIJEN, J.H., NIEDWENBUIZEN, W. and WIJNGAARDS, G. Activation of plasminogen by tissue activator is increased specifically in the presence of certain soluble fibrin(ogen) fragments. Thromb. Res. 27, 377-385, 1982.
4.
VEZHEIJEN, J.R., MULLAART, E., CHANG, G.T.G., KLUET, C. and WIJNGAARDS, G. A simple sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurements in plasma. Thrombos. Haemostas. 48, 266-269, 1982.
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