Binding of tissue-type plasminogen activator to fibrinogen fragments.
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.
D001665:Binding Sites; D005338:Fibrin Fibrinogen Degradation Products / Q000378:metabolism*; D006801:Humans; D007124:Immunoenzyme Techniques; D010446:Peptide Fragments / Q000378:metabolism; D010958:Plasminogen / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000378:metabolism*
D005338:Fibrin Fibrinogen Degradation Products; D010446:Peptide Fragments; C018998:lysyl-plasminogen; D010958:Plasminogen; D010959:Tissue Plasminogen Activator