THROMBOSIS RESEARCH 36, 345-351, 1984 0049-3848/84 $3.00 + .OO Printed in the USA. Copyright (c) 1984 Pergamon Press Ltd. All rights reserved.
IMPORTANCE OF THE INTERACTION BETWEEN PLASMINOGEN AND FIBRIN FOR PLASMINOGEN ACTIVATION BY TISSUE-TYPE PLASMINOGEN ACTIVATOR. l&2 , H.R. Lijnen4, F.W. Dunnl, K. Deguchi', J. Soria3 C. Soria G. Tobeleml, J: Caenl 1. Unit6 de Soins et de Recherches d'Angiohematologie(Pr.Caen),HSpital Lariboisi&e,Units 150 INSERM, Paris, France. 2. Laboratoire Biochimie (Pr.Rousselet),H6pitalLariboisiOre,Paris,France. 3. Laboratoires Centraux de Biochimie et d'H6matologie(Pr.Fabiani,Pr.Samama) Service du Pr. Bernadou, HStel Dieu,Parvis de Notre Dame,Paris,France. 4. Department Medical Research,Universiteit Leuven, Leuven, Belgium. (Received 13.10.1983; Accepted in revised form 23.7.1984 by Editor R. Gollwitzer) ABSTRACT The potentiating effect of fibrin monomer on plasminogen activation by tissue-type plasminogen activator is much more important with lys-plasminogen than with mini-plasminogen (which lacks the high affinity lysine-binding site important for binding to fibrin). Furthermore, this potentiating effect is totally abolished when lys-plasminogen is eluted from fibrin by the addition of 1 mM c-amino caproic acid. Binding does however not seem to be the only condition required since it was found that fragment D is a much stronger potentiator of the activation of plasminogen by tissue-type plasminogen activator than fragment E although plasminogen binds to both fragment D and fragment E. Furthermore, fragment E has the same effect on the activation of lys-and mini-plasminogen by tissue-type plasminogen activator. Therefore, it is suggested that binding of plasminogen to fibrin involves a conformational change in the plasminogen molecule, facilitating its activation by tissue-type plasminogen activator. INTRODUCTION Activation of plasminogen to the active enzyme plasmin by tissue-type plasminogen activator (t-PA) is potentiated by fibrin or derivatives such as fibrin monomer (l), fragment D (2),cyanogen bromide treated fibrinogen (3). The most likely explanation for the stimulation of plasminogen activation by fibrin derivatives is an interaction between fibrin and plasminogen through its lysine binding sites and (or) between fibrin and t-PA. The present investigation was undertaken in order to examine if the potentiating effect induced by fibrin monomer on activation of plasminogen by t-PA is Key words:
Tissue-plasminogen activator -plasminogen activation-fibrinolysis - fibrinogen degradation products. 345
dependent on the binding of plasminogen to fibrin. Therefore, the potentiating effect of fibrin monomer and of fibrinogen degradation products D and E on the t-PA-mediated activation of lys-and mini-plasminogen was studied. The effect of fibrin monomer was also studied in the presence of 1 mM epsilon amino caproic acid (E-ACA) which avoids the binding of plasminogen to fibrin. MATERIAL AND METHODS Materials Soluble fibrin monomer preparation: 10 mg of fibrinogen Kabi (Stockholm Sweden) dissolved in 1 ml distilled water were clotted by addition of 0.3 ml reptilase (FTH50-Stagp,France). The clot was squeezed out and washed 4 times in 0,15 M NaCl, dissolved in 1 ml of 7 M urea, and kept at -80" until use. Fibrinogen fragments D and E were prepared according to Nilhen (4). Plasminogens: Lys-plasminogen a modified form of native Glu-plasminogen with NH2-terminal lysine, valine or methionine, MW 85,000 (Substrene) was from Choay (Paris,France). The small quantities of plasmin present in the reagent were eliminated by aprotinin agarose adsorption. Mini-plasminogen consisting of kringle 5 of plasmin A chain and the intact plasmin B chain, MW 39,000 was obtained by elastase digestion of plasminogen and separation of the fragments by gel filtration on sephadex G-75 and affinity chromatography on lysine-sepharose as previously described (5). The t-PA was isolated from human melanoma cells in culture(6). The activity was expressed in urokinase equivalent units by comparison of the fibrinolytic activity on fibrin plates. The specific activity was about 90,000 IU/mg. The chromogenic substrate S-2251 was obtained from Kabi (Stockholm, Sweden). Buffer: 0.1 M phosphate buffer. pH 7.3 containing 10 mM EDTA, O,l"/"" NaN3 and O,l"/oO Triton X 100. Methods To 0.1 ml at 85 pg/ml of plasminogen in phosphate buffer were added 0.1 ml of t-PA used at various concentrations (diluted in buffer containing o.l"/oo tween 80 to avoid unspecific adsorption) and either 10 ~1 buffer or 10 1.11fibrin monomer at 2700pg/ml followed by 0.1 ml S-2251 at 3 mM in water. Incubation was performed at 37,'Cand the absorbance at 405 nm was measured each hour in a microtiter plate using a microelisa autoreader (Dynatech. USA). The same experiment was carried out using lys-or miniplasminogen and with fibrinogen fragment D or E (10 )~l at lOOO~g/ml) instead of fibrin monomer. With fibrin monomer, the assay was also performed in the presence of 1 mM E-ACA (laboratoire Delagrange, Paris). RESULTS It appears from Fig. 1 that in the absence of fibrin monomer, the doseresponse curve of plasminogen activation (expressed as amidolytic activity on S-2251) versus increasing concentrations of t-PA is similar for lys-and mini-plasminogen. Due to the very slow activation, prolonged incubation times (22 hours) were required.
O.D.(LOS nm) 1,5 lys-plasminogen 1
095 / /
cont. of t-PA solution used (ng/ml)
FIG. 1 iunidolytic activity on S-2251 induced by t-PA-activated lys-and mini-plasminogen in the absence of fibrin monomer. (Incubation time is 22 hours at 37OC ).
As it can be seen from Fig. 2a and Fig. 2b, fibrin monomer acts as a potentiator of t-PA-induced activation of both lys-and mini-plasminogen. In the presence of fibrin Therefore, shorter incubation times can be used. monomer however the amidolytic activity induced by several doses of t-PA is higher in the presence of lys-plasminogen than that observed in the Furthermore, it appears presence of the same amount of mini-plasminogen. that in the presence of mini-plasminogen, the amidolytic activity is a linear function within time, whereas it is a parabolic function in the presence of lys-plasxinogen. Potentiating
effect of fibrin monomer
and absence of E-ACA.
The stimulating effect of fibrin monomer on the activation of lys-plasminogen b:; t-PA is completely abolished upon addition of 1 mM E-XCA (Fig. 3a). With mini-plasminogen however, the addition of the same amount of E-ACA abolish&d only partially the stimulating effect induced by fibrin monomer (Fig. 3':). When using either lys-or mini-plasminogen after 20 hours incubation wit?. t-PA in the absence of fibrin monomer, amidolytic activity was found tc be the same with or without &-ACA. Potentiating effect induced by fibrinogen activation of lys-and mini-plasminogen.
fragment D or E on t-PA induced
As shown in Fig. 4, fragment D produced a much higher potentiation than fragment E on t-PA-induced lys-plasminogen activation. In the presence of mini-plasminogen, fragment D produced a much lower potentiation than that induced in the presence of lys-plasminogen. Nevertheless, the potentiating
PLASINOGEN-FIBRIN INTERACT ON
D. (405 nm)
C ).(405 nm) 1
: 6* d
34 56 7 incubation time (hours)
12 3 4 5 6 7 incubation time (hours) b) Mini-plasminogen activation
a) Lys-plasminogen activation
FIG. 2 Activation of lys- or mini-plasminogen by t-PA. Potentiating effect induced by fibrin monomer, (0 ,0 In the presence of fibrin monomer,m , 0 in the absence of fibrin monomer, *Cont. of t-PA solution used in ng/ml).
O.D.,(I i05 nm) 1
0.D . (405 nm) 1 .fibrin monomer fibrin monomer
E-ACA + fibrin monomer
1234 5 incubation time (hours) a) Lys-plasminogen + 0,2 ng/ml t-PA
12 3 4 5 incubation time (hours)
b) Mini-plasminogen +6 ng/ml t-PA
FIG. 3 Activation of lys-and mini-plasminogen by t-PA. Effect of fibrin monomer in the presence or absence of s-ACA (1 mM final concentration).
effect of fragment D and of fragment E on mini-plasminogen activation by t-PA ;;as of the same order of magnitude as that produced by fragment E on the acti,:ation of lys-plasminogen by the same amount of t-PA. In the presence of both fragments D and E in the incubation mixture, the amidolytic activity of t-PAactivated plasminogen was found to be identical to the sum of that observed ;-ith fragment D and E respectively (either with lys- or mini-plasminogen). D+E /'
,/ / P I’ : 0’ d’ .’ . .’ . p’ ,p
/ : ; 0' u' : /.
i _0.'D +E
incubation time (hours) FIG. 4
Right panel: Assay perLeft panel: Assay performed using lys-plasminogen. Potentiating effect of D and E fragments on formed using mini-plasminogen. the activation of lys-and mini-plasminogen by (0,9 ng/ml) t-PA. The solutions of D and E added were at 1000 ~g/ml(lO~l for 310 ~1 'incubation mixture).
DISCUSSION Fibrinolysis in the blood is regulated by specific molecular interactions between fibrin, plasminogen, plasminogen activator and alpha 2-antiplasmin (7). The activation of plasminogen by t-PA is strongly increased in the presence of fibrin and it has been suggested that fibrin facilitates the interaction between t-PA and plasminogen by formation of a ternary complex on the fibrin. This study was carried out in order to analyse the importance of an interaction between plasminogen and t-PA on the fibrin surface for efficient fibrinolysis. Therefore, we have compared the enhancing effect of fibrin monomer on the activation of mini-and-lys-plasminogen by t-PA. These two types of plasminogen were tested because lys-plasminogen binds strongly to fibrin mainly via its high affinity lysine-binding site (8) whereas miniplasminogen has some affinity for fibrin which seems hcwever to be mediated by a site different from the high affinity lysine-binding site (9,lO).
Our results show that the potentiating effect induced by fibrin monomer on the activation of plasminogen by t-PA was much more important with lysplasminogen than with mini-plasminogen. The potentiation of lys-plasminogen activation by t-PA in the presence of fibrin monomer seems to be related to the binding of lys-plasminogen to fibrin through its high affinity lysinebinding site, since the potentiating effect induced by fibrin monomer is abolished upon addition of 1 mM E-ACA(ll), (it was checked that 1 mM E-ACA did not inhibit plasmin generation in the absence of fibrin monomer). These data are in good accordance with those observed by Hoylaerts et al (12) who suggested that the activation of plasminogen by t-PA in the presence of fibrin occurs through binding of an activator molecule to rhe clot surface and subsequent addition of plasminogen to form a cyclic ternary complex. The potentiating effect of fibrin monomer on the activation of mini-plasminogen by t-PA appears to be more complex. This potentiation is largely inhibited but not completely abolished in the presence of 1 mM E -ACA. It is suggested that the potentiation induced by fibrin monomer on t-PA-induced activation of mini-plasminogen may be related to a weak and thus easily disrupted binding of mini-plasminogen to fibrin independent of lysine-binding sites. In this hypothesis it has to be assumed that in the presence of E -ACA some miniplasminogen remains bound to fibrin. This is in agreement with previous observations (9,lO). We conclude that the potentiating effect induced by fibrin monomer on activation of plasminogen by t-PA is mainly related to the binding of plasminogen to fibrin. Nevertheless, binding does not seem to be the only condition required. Indeed, although plasminogen binds to both fragment D and E (the binding of fragment E is slightly more effective than that of fragment D) (13), fragment D is a stronger potentiator of plasminogen activation by t-PA than fragment E. In addition, fragment E was shown to have the same potentiating activity on mini-and on lys-plasminogen activation by t-PA. Therefore, it is suggested that binding of plasminogen to fragment D induces a structural modification of plasminogen resulting in a more efficient activation by t-PA. This is in agreement with the recent finding that the structure comprising residues 148-197 of the A dchain of fibrinogen contains a site capable of accelerating the activation of plasminogen by t-PA (14). In contrast, the binding of plasminogen to fragment E seems to play a minor role in its activation by t-PA. REFERENCES 1.
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RIJKEN, D.C., COLLEN, D. Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture. J. Biol. Chem. 256, 7035-7041, 1981.
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