Abstract
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.
Highlights
Curring serine protease that initiates fibrinolysis by converting plasminogen (Pg) to plasmin [1]
We have demonstrated that type plasminogen activator (t-PA) causes systemic plasminemia, because, like intact fibrin, soluble fibrin degradation products stimulate t-PA-mediated Pg activation [13]
Influence of (DD)E or Fg on t-PA- and bat plasminogen activator (b-PA)-mediated Activation of Pg—To compare the effect of (DD)E and Fg on t-PAand b-PA-mediated Pg activation, 0.4 M Glu-Pg was incubated with 1 nM t-PA or 5 nM b-PA in the absence or presence of various concentrations of (DD)E or Fg for 10 min at 37 °C, and the rate of plasmin formation was monitored (Fig. 1)
Summary
Plasminogen Activators—Wild-type recombinant t-PA was kindly provided by Dr B. Prior to use, a 1-ml volume of the plasminogen activator was dialyzed against 2 liters of 0.02 M Tris-HCl, 0.15 mM NaCl, 0.01% Tween 20, pH 7.4 (TBS) for 3 h with vigorous stirring and centrifuged at 12,000 ϫ g for 7 min at 22 °C in a microcentrifuge to remove any aggregated material. Based on these calculations of protein concentration, dialysis against TBS resulted in a.
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