STUDY OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR BINDING-SITES ON FIBRIN USING DISTINCT FRAGMENTS OF FIBRINOGEN

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.