P. Grailhe et al., STUDY OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR BINDING-SITES ON FIBRIN USING DISTINCT FRAGMENTS OF FIBRINOGEN, European journal of biochemistry, 219(3), 1994, pp. 961-967
It is well established that tissue-type plasminogen activator (t-PA) b
inds to the D region of fibrin(ogen) and that two distinct CNBr fragme
nts of fibrinogen (FCB), FCB-2 and FCB-5, comprising parts of this reg
ion, stimulate plasminogen activation by t-PA. In the present work, li
gand-binding studies were performed to characterize the interactions b
etween t-PA and the corresponding fibrin regions using a well defined
model of a fibrin surface and both FCB-2 and FCB-5 in liquid and solid
phase. Binding isotherms showed a characteristic Langmuir adsorption
saturation profile. The dissociation constants determined for the bind
ing of t-PA to immobilized FCB-2 (K-d = 0.70 +/- 0.10 nM) and FCB-5 (K
-d = 0.47 +/- 0.08 nM) were of the same order of magnitude as the K, f
or fibrin binding (K-d = 1 +/- 0.2 nM). The specificity of the binding
was demonstrated by the ability of soluble FCB-2 and FCB-5 to inhibit
t-PA binding to solid-phase fibrin (K-i = 3.3 mu M and 6.4 mu M, resp
ectively). The binding of t-PA to fibrin and to immobilized FCB-2 was
partially inhibited by the lysine analogue 6-aminohexanoic acid (K-i =
123 +/- 47 mu M and 364 mu M, respectively) but was not modified by c
arboxypeptidase B, thus indicating involvement of internal lysine resi
dues. Removal of lysine residues by treatment with, successively, plas
min and carboxypeptidase B, produced only a partial inhibition of t-PA
binding, thus confirming the existence of both a lysine-dependent and
a lysine-independent mechanism of binding of t-PA to both fibrin and
FCB-2. In contrast, the binding of t-PA to FCB-5 was not significantly
affected by 6-aminohexanoic acid. Altogether, these data indicate tha
t the mechanism of binding of t-PA to fibrin involves mainly a lysine-
independent interaction with the D region which is contributed by sequ
ences present in FCB-5 and FCB-2; contribution to binding by a lysine-
dependent interaction was detected only in FCB-2 and is probably of mi
nor relevance as suggested by the limited effect of 6-aminohexanoic ac
id.