Binding of tissue‐type plasminogen activator to fibrinogen fragments

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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 Dcate and DEGTA and a strong binding with the cyanogen bromide fragment FCB‐2 (Kd 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 Dcate, DEGTA and E and a strong binding with FCB‐2 (Kd apparent = 70 nM). The stimulatory capacity of the various fragments on the Lys‐plasminogen activation by t‐PA, as studied in a spectrophotometric assay, was found to be absent for fragment E, low for fibrinogen, fragments X, Y, Dcate 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.

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