Previous work in our laboratory has suggested that the fibrinolytic enzyme plasmin (Pn) inactivates coagulation factors X (FX) and Xa (FXa) in the presence of Ca(2+) and anionic phospholipid (aPL), producing fragments which bind plasminogen (Pg) and accelerate tissue plasminogen activator (t-PA). Our goals here were to determine if the Pn-mediated fragments of FX or FXa remain associated, whether they directly bind t-PA, and to quantify their interaction with Pg. Binding to aPL, benzamidine-Sepharose, or the active-site inhibitor dansyl-Glu-Gly-Arg-chloromethyl ketone demonstrated that Pn cleavage yielded noncovalent heterodimers of a fragment containing the aPL-binding domain (FXgamma(47) or FXagamma(33)) and a 13-kDa fragment (FXgamma(13) or FXagamma(13)). Both ligand blotting and surface plasmon resonance (SPR) showed that Pn-cleaved FX and FXa bound t-PA directly when Pn-treatment was effected in the presence of aPL and Ca(2+). Using SPR, apparent K(d) values of 1-3 microM and 0.3-0.4 microM were measured directly and by competition for the FXgamma(47/13)-Pg and FXagamma(33/13)-Pg interactions, respectively. For the first time, Pg-binding to a receptor was shown to be Ca(2+) enhanced, although primarily mediated by C-terminal lysine residues. Mathematical modeling of kinetic data suggesting two Pg per FXgamma(47/13) or FXagamma(33/13) was consistent with our conclusion that each subunit of FXgamma(47/13) or FXagamma(33/13) contains a C-terminal lysine. Earlier X-ray structures show that these Lys residues are distal from each other and the membrane, supporting the model where each interacts with a separate Pg. t-PA acceleration by FXgamma(47/13) or FXagamma(33/13) may therefore involve simultaneous presentation of two substrate molecules.