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 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.
Grundy
J E
JE
R&D Department, Canadian Blood Services, 1800 Alta Vista Drive, Ottawa, Ontario, Canada K1G 4J5.
Lavigne
N
N
Hirama
T
T
MacKenzie
C R
CR
Pryzdial
E L
EL
D000838:Anions; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D019281:Dimerization; D005170:Factor X / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D010958:Plasminogen / Q000378:metabolism*; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D010959:Tissue Plasminogen Activator / Q000378:metabolism*
D000838:Anions; D010446:Peptide Fragments; D010743:Phospholipids; D005170:Factor X; D010958:Plasminogen; D015951:Factor Xa; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin; D002118:Calcium