Study of tissue‐type plasminogen activator binding sites on fibrin using distinct fragments of fibrinogen

Abstract

It is well established that tissue-type plasminogen activator (t-PA) binds to the D region of fibrin(ogen) and that two distinct CNBr fragments of fibrinogen (FCB), FCB-2 and FCB-5, comprising parts of this region, stimulate plasminogen activation by t-PA. In the present work, ligandbinding studies were performed to characterize the interactions between 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 binding of t-PA to immobilized FCB-2 (& = 0.70 2 0.10 nM) and FCB-5 (& = 0.47 * 0.08 nM) were of the same order of magnitude as the Kd for fibrin binding (Kd = 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, = 3.3 pM and 6.4 pM, respectively). The binding of t-PA to fibrin and to immobilized FCB-2 was partially inhibited by the lysine analogue 6-aminohexanoic acid (K, = 123 * 47 pM and 364 pM, respectively) but was not modified by carboxypeptidase B, thus indicating involvement of internal lysine residues. Removal of lysine residues by treatment with, successively, plasmin 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 F C B J was not significantly affected by 6-aminohexanoic acid. Altogether, these data indicate that the mechanism of binding of t-PA to fibrin involves mainly a lysine-independent interaction with the D region which is contributed by sequences 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 minor relevance as suggested by the limited effect of 6-aminohexanoic acid.Fibrinolysis, the plasmin-induced degradation of a fibrin clot that preserves blood vessel flow, is finely regulated at several levels. Among them, fibrin itself triggers its own degradation by offering specific binding sites for tissue-type plasminogen activator (t-PA) and plasminogen on its surface. The formation of this ternary complex [l] affects the K,,, of the plasminogen activation by t-PA. In the absence of fibrin, In contrast to fibrin, fibrinogen is a soluble protein that does not stimulate plasminogen activation significantly [5]. However, plasmin-generated fibrin degradation products [6] and CNBr fragments of fibrinogen (FCB) stimulate plasminogen activation by t-PA [7]. Based on these observations, it has been suggested [8] that stimulatory site(s) buried on fibrinogen are unmasked by conformational changes induced either by thrombin, by plasmin or by CNBr cleavage of fibrinogen. Indeed, with the use of the monoclonal antibodies anti-(Aal48 -160) and anti-(y312-324), raised against amino-acid sequences derived from FCB-2 and FC...

Tissue-type plasminogen activator is a target of the tumor suppressor gene maspin

“…sctPA is secreted as an active enzyme and is further strongly activated by specific interactions, primarily through its second kringle domain, with a wide variety of extracellular matrix components including fibrin(ogen) (19,20), laminin (21), fibronectin (22), and lysinerich molecules as shown here. Because sctPA activated by poly-D-lysine or fibrinogen͞gelatin is inhibited by rMaspin(i), it is likely that the second kringle domain of sctPA plays a critical role in the fine modulation of its activity by both stimulators and inhibitors.…”

Macromolecular Ensembles of Internal and External Fibrinolysis: the Resources for Enhancement of Thrombolysis Efficacy

“…The binding of tPA to Fb is mediated by the finger-like and kringle 2 domains [4]. It has been suggested that these domains are involved in two different types of binding: lysin-dependent (kringle 2 and Ddomain of Fb) and lysin-independent (finger-like domain and D-domain of Fb) [29]. The latter domains contribute to a greater extent to the total binding which becomes highly affinity due to cooperative binding of tPA by the two abovementioned low-affinity centres and accelerates Pmg activation [30].…”

Biophysical Mechanisms Mediating Fibrin Fiber Lysis

“…The finger domain and the Kringle-2 domain serve as the primary fibrin binding sites [86]. The Kringle-2 domain plays a role in C-terminal lysine binding, while the finger domain can bind to a region in the fibrin γ -nodule in a lysine-independent mechanism [91] or to amyloid-like cross-beta structures [44], which have been hypothesized to form in fibrin α -polymers [14, 92]. Recent work has shown that the tPA finger domain plays the predominant role in binding to fibrin during fibrinolysis [93, 94], even in proteolytically degraded fibrin.…”