Characterization of the interactions of plasminogen and tissue and vampire bat plasminogen activators with fibrinogen, fibrin, and the complex of D-dimer noncovalently linked to fragment E.
Vampire bat plasminogen activator (b-PA) causes less fibrinogen (Fg) consumption than tissue-type plasminogen activator (t-PA). Herein, we demonstrate that this occurs because the complex of D-dimer noncovalently linked to fragment E ((DD)E), the most abundant degradation product of cross-linked fibrin, as well as Fg, stimulate plasminogen (Pg) activation by t-PA more than b-PA. To explain these findings, we characterized the interactions of t-PA, b-PA, Lys-Pg, and Glu-Pg with Fg and (DD)E using right angle light scattering spectroscopy. In addition, interactions with fibrin were determined by clotting Fg in the presence of various amounts of t-PA, b-PA, Lys-Pg, or Glu-Pg and quantifying unbound material in the supernatant after centrifugation. Glu-Pg and Lys-Pg bind fibrin with Kd values of 13 and 0.13 microM, respectively. t-PA binds fibrin through two classes of sites with Kd values of 0.05 and 2.6 microM, respectively. The second kringle (K2) of t-PA mediates the low affinity binding that is eliminated with epsilon-amino-n-caproic acid. In contrast, b-PA binds fibrin through a single kringle-independent site with a Kd of 0.15 microM. t-PA competes with b-PA for fibrin binding, indicating that both activators share the same finger-dependent site on fibrin. Glu-Pg binds (DD)E with a Kd of 5.4 microM. Lys-Pg binds to (DD)E and Fg with Kd values of 0.03 and 0.23 microM, respectively. t-PA binds to (DD)E and Fg with Kd values of 0.02 and 0.76 microM, respectively; interactions were eliminated with epsilon-amino-n-caproic acid, consistent with K2-dependent binding. Because it lacks a K2-domain, b-PA does not bind to either (DD)E or Fg, thereby explaining why b-PA is more fibrin-specific than t-PA.
Stewart
R J
RJ
Hamilton Civic Hospitals Research Centre and McMaster University, Hamilton, Ontario, L8V 1C3 Canada.
Fredenburgh
J C
JC
Weitz
J I
JI
D015119:Aminocaproic Acid / Q000494:pharmacology; D000818:Animals; D000933:Antifibrinolytic Agents / Q000378:metabolism*; D002685:Chiroptera; D005337:Fibrin / Q000378:metabolism*; D005338:Fibrin Fibrinogen Degradation Products / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism*; D005343:Fibrinolytic Agents / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D018082:Kringles; D010446:Peptide Fragments / Q000378:metabolism*; D010958:Plasminogen / Q000378:metabolism*; D010960:Plasminogen Activators / Q000378:metabolism*; D011485:Protein Binding; D010959:Tissue Plasminogen Activator / Q000378:metabolism*
D000933:Antifibrinolytic Agents; D005338:Fibrin Fibrinogen Degradation Products; D005343:Fibrinolytic Agents; D010446:Peptide Fragments; C036309:fibrin fragment D; C018543:fibrinogen fragment E; C018998:lysyl-plasminogen; C069588:salivary plasminogen activator alpha 1, Desmodus rotundus; D005337:Fibrin; D005340:Fibrinogen; D010958:Plasminogen; D010960:Plasminogen Activators; D010959:Tissue Plasminogen Activator; D015119:Aminocaproic Acid