Binding of tissue‐type plasminogen activator to fibrinogen fragments

Abstract

In order to localize the binding site(s) for tissue-type plasminogen activator (t-PA) in the fibrin(ogen) molecule, the following binding assay was developed. Two-chain t-PA was immobilized onto microtitration plates. The t-PA-coated plates were then incubated with fibrinogen and various fibrinogen fragments. The extent of binding was quantified with enzyme-labelled antibodies against fibrin(ogen) and its fragments. Hardly any binding to t-PA was observed with fibrinogen or fragments X, Y and E; a moderate binding was observed with fragments D,,,, and DEGTA and a strong binding with the cyanogen bromide fragment FCB-2 ( K d apparent = 140 nM). The binding of fibrinogen and its fragments to immobilized Lys-plasminogen was measured by the same method as a control for the binding assay. Results were in line with literature data: virtually no binding to Lys-plasminogen with fibrinogen or fragments X and Y, a moderate binding with fragments D,,,,, DEGTA and E and a strong binding with FCB-2 (Kd apparent = 70 nM). The stimulatory capacity of the various fragments on the Lysplasminogen activation by t-PA, as studied in a spectrophotometric assay, was found to be absent for fragment E, low for fibrinogen, fragments X, Y , D,,,, and DEGTA, and high for FCB-2. It is concluded that a t-PA-binding site resides in the C-terminal globular domains of fibrinogen from which fragments D and FCB-2 originate. The site is hidden in the native fibrinogen molecule and in early fibrinogen degradation products. Binding of both Lys-plasminogen and t-PA appears to be required for a stimulator of the plasminogen activation, as illustrated by fragment E which only binds Lys-plasminogen and has no stimulatory capacity.Tissue-type plasminogen activator (t-PA) converts plasminogen, an inactive zymogen, into active plasmin and therefore plays an important role in fibrin clot dissolution. In the absence of fibrin, plasminogen is poorly activated by t-PA, but in the presence of fibrin (not fibrinogen) plasminogen activation by t-PA is greatly enhanced [l, 21. Fibrin polymerization is a decisive factor in the stimulatory effect [3, 41, and stimulation is further potentiated upon limited fibrin degradation [5 -81. Several plasmin-produced fragments of fibrinogen (e.g. fragments D,,,, and DEGTA) and a CNBrproduced fibrinogen fragment, FCB-2, also accelerate the plasminogen activation by t-PA, whereas no acceleration is seen with fragment E [9, 101. A stimulatory site is located on Kinetic studies suggest that acceleration of plasminogen activation by fibrin can be ascribed to the formation of a cyclic ternary complex of plasminogen, t-PA and fibrin [13, 141. This concept is supported by direct binding studies, showing that both t-PA and plasminogen form complexes with fibrin. The plasminogen -fibrin interaction has been studied quite extensively (for review see [15]). Plasminogen-binding sites are less well exposed in fibrinogen than in fibrin [16]. They are located both in fragments D and E [17]. Glu-plasminogen binds less strongly to fibrin than t...

Comparison of the Effects of Fibrinogen and Fibrin Products and Isolated Peptide Chains on the Fibrin-Mediated Stimulation of Plasminogen Activation by Tissue-Type Plasminogen Activator, and on the Fibrin-Dependent Enhancement of the Amidolytic Activity of One-Chain Tissue-Type Plasminogen Activator

“…Fibrinogen Ββ/γ-chains and fibrin β/γ-chains possesBrought to you by | University of California Authenticated Download Date | 6/13/15 4:37 PM sed only weak stimulatory properties, as compared with the fibrin α-chain. In contrast to observations of Dunn et alJ 26^ andVerheijen et alJ 25 \ the results of this study show that fibrinogen D-and Ε-fragments possess no stimulatory properties, in contrast to intact fibrin and FCB-2.This result confirms the mechanism of stimulation, which occurs only if both t-PA and plasminogen bind simultaneously to fibrin; it is supposed in the literature that the t-PA binding site and the plasminogen binding site to fibrin are localized in the fibrinogen D-domain [6][7][8][9][10][11][12][13][14] and fibrinogen E-domain [29] , respectively. Affinities of t-PA and plasminogen to fibrin are mediated by lysine binding sites in the kringeldomains of the proteins' 5 " 23 · 30 · 3^.…”

Biophysical Mechanisms Mediating Fibrin Fiber Lysis

“…Electron microscopy studies of plasminogen bound to fibrin also show that it binds to the peripheral “D” region, in agreement with the antibody epitope mapping (see Figure 2(a)) [37]. The binding is lysine dependent, suggesting Kringle domain involvement [38]. A lysine-independent tPA binding site has been localized to γ chain residues 312–324 that is also inaccessible to antibodies in fibrinogen, but accessible in fibrin (see Figure 2(a)) [39].…”

Molecular assembly of plasminogen and tissue‐type plasminogen activator on an evolving fibrin surface

“…Two binding sites for t-PA have been localized in the Ddomains of fibrin(ogen) [36,371. However, the kringle type-2 domain of t-PA may also interact with lysine residues in fibrin [ 181.…”