The dissociation constants and stoichiometries of the interactions of Lys-plasminogen and chloromethyl ketone derivatives of tissue plasminogen activator and the variant delta FEIX with intact fibrin.
Active-site-blocked, fluorescent derivatives of tPA (Activase) and a variant (delta FEIX) which lacks the finger and epidermal growth factor-like domains and possesses Asn to Gln and Val to Met mutations at residues 117 and 245, respectively, were prepared. The binding of these to fibrin was studied by adding them at systematically varying concentrations to fibrinogen, at a fixed concentration, inducing clotting with thrombin, separating free and bound tPA or delta FEIX by centrifugation, and measuring the concentration of unbound material by extrinsic fluorescence. Similar studies were performed with Glu and Lys-plasminogen, using intrinsic fluorescence. epsilon-amino caproic acid (EACA) was utilized to distinguish kringle-dependent from finger-dependent binding. In the absence of EACA, delta FEIX-bound fibrin through a single class of sites with Kd = 0.69 microM and n = 1.34 delta FEIX/fibrin. The binding of delta FEIX was completely inhibited by EACA and 50% displacement occurred at [EACA] = 300 microM. Fibrin-bound tPA was only partially displaced with EACA. In the presence of 30 mM EACA, tPA binding reflected a single class of sites with Kd = 0.26 microM and n = 0.60 tPA/fibrin. In the absence of EACA, tPA binding was complex, typified by downwardly curved Scatchard plots, and was consistent with interactions of the two classes of sites, characterized by Kd = 0.13 microM, n = 0.60 and Kd = 0.61 microM, n = 1.23. These were attributed to finger and kringle-dependent interactions, respectively. Under the experimental conditions employed, Glu-plasminogen exhibited no binding to fibrin, whereas Lys-plasminogen bound to a single class of sites with Kd = 0.25 microM and n = 1.02 plasminogen/fibrin. This binding was completely inhibited by EACA and 50% displacement occurred at [EACA] = 28 microM. Competition experiments indicated that Lys-plasminogen does not displace either tPA or delta FEIX from fibrin. From these results the conclusions are drawn that tPA can interact with intact fibrin by two different and independent modes, involving, respectively, the finger and kringle 2 domains, and neither of these modes are competitive with the kringle-dependent binding of Lys-plasminogen.
Nesheim
M
M
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Fredenburgh
J C
JC
Larsen
G R
GR
D015119:Aminocaproic Acid / Q000494:pharmacology; D001665:Binding Sites; D005337:Fibrin / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D010958:Plasminogen / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000378:metabolism* / Q000648:ultrastructure
D005337:Fibrin; D010958:Plasminogen; D013917:Thrombin; D010959:Tissue Plasminogen Activator; D015119:Aminocaproic Acid