Binding of plasminogen and tissue plasminogen activator to plasmin-modulated factor X and factor Xa

Previous work in our laboratory has suggested that the fibrinolytic enzyme plasmin (Pn) inactivates coagulation factors X (FX) and Xa (FXa) in the pre sence of Ca2+ and anionic phospholipid (aPL), producing fragments which bin d plasminogen (Pg) and accelerate tissue plasminogen activator (t-PA). Our goals here were to determine if the Pn-mediated fragments of FX or FXa rema in associated, whether they directly bind t-PA, and to quantify their inter action with Pg. Binding to aPL, benzamidine-Sepharose, or the active-site i nhibitor dansyl-Glu-Gly-Arg-chloromethyl ketone demonstrated that Pn cleava ge yielded noncovalent heterodimers of a fragment containing the aPL-bindin g domain (FX gamma (47) or FXa gamma (33)) and a 13-kDa fragment (FX gamma (13) or FXa gamma (13)). Both ligand blotting and surface plasmon resonance (SPR) showed that Pn-cleaved FX and FXa bound t-PA directly when Pn-treatm ent was effected in the presence of aPL and Ca2+. Using SPR, apparent K-d v alues of 1-3 muM and 0.3-0.4 muM were measured directly and by competition for the FX gamma (47/13)-Pg and FXa gamma (33/13)-Pg interactions, respecti vely. For the first time, Pg-binding to a receptor was shown to be Ca2+ enh anced, although primarily mediated by C-terminal lysine residues. Mathemati cal modeling of kinetic data suggesting two Pg per FX gamma (47/13) or FXa gamma (33/13) was consistent with our conclusion that each subunit of FX ga mma (47/13) or FXa gamma (33/13) contains a C-terminal lysine. Earlier X-ra y structures show that these Lys residues are distal from each other and th e membrane, supporting the model where each interacts with a separate Pg. t -PA acceleration by FX gamma (47/13) or FXa gamma (33/13) may therefore inv olve simultaneous presentation of two substrate molecules.