Binding of Tissue-Type Plasminogen Activator and Plasminogen to Fibrinogen and Its Degradation Products

Abstract

In order to localize the binding site(s) for tissue-type plasminogen activator (t-PA) in the fibrin(ogen) molecule, two-chain t-PA was immobilized onto microtitration plates and incubated with fibrinogen and various fibrinogen fragments. The extent of binding was quantified with an enzyme immunoassay. Hardly any binding to t-PA was observed with fibrinogen, fragments X, Y and E. A moderate binding was observed with fragments D(cate) and D(EGTA) and a strong binding with the cyanogen bromide fragment FCB-2 (Kd apparent = 65 nM). Results of control experiments, in which the binding of fibrinogen and its fragments to immobilized Lys-plasminogen was measured, using the same assay, were in line with literature data: hardly any binding was found with fibrinogen, fragments X and Y. A moderate binding was observed with fragments D and E and a strong binding with FCB-2 (Kd apparent = 100 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 and D, and high for FCB-2. It is concluded that the t-PA binding site in the fibrin (ogen) molecule resides in the distal domains from which fragments D and FCB-2 are derived. The site is apparently hidden in fibrinogen and early fibrinogen degradation products. Binding of both plasminogen and t-PA is required for stimulation of the plasminogen activation, as illustrated by fragment E which binds plasminogen and no t-PA, and has no stimulatory capacity.