Binding of Plasminogen and Tissue Plasminogen Activator to Plasmin-Modulated Factor X and Factor Xa

Abstract

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.

Thrombin and plasmin generation in patients with plasminogen or plasminogen activator inhibitor type 1 deficiency

“…23 These results are comparable to results of others, who reported shortened lag time with afibrinogenemia and with FXIII deficiency as well. [27][28][29][30][31][32] The cumulative list of potential coagulation proteins cleaved by plasmin may result in a shortened lag time. For instance, plasmin influences coagulation by (in)activation of factor VII, factor VIII, factor IX, factor X and factor XII.…”

Does plasmin have anticoagulant activity?

“…Plasmin-mediated cleavage of FXa exposes a Plg binding site16,32 and inhibits coagulation. In the presence of anionic phospholipid, FX and FXa enhance Plg activation by tPA33,34 to plasmin. Thus, plasmin cleavage of FX not only inhibits coagulation but also enhances fibrinolysis.…”

Coagulation Factor VIIa

“…This also contributes to the formation of the Na 1 and factor Va binding sites [57], and appears to cause the transition from zymogen to active protease. A second cleavage, plasmin-mediated or autocatalytic, at the Lys435kSer436 bond yields factor Xaβ [39]. The procoagulant activity of both forms of factor Xa is similar.…”

“…Membrane-bound factor X/Xa can be inactivated by plasmin. Cleavage of the Lys435kSer436 bond generates factor Xaβ and further cleavage at the Lys330kGly331 peptide bond generates factor Xa33/13, which destroys clotting activity [39]. These cleavages expose plasminogen binding sites that augment the generation of plasmin and thus seem to switch factor X/Xa from a procoagulant to an anticoagulant role by stimulating fibrinolysis [40].…”