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

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 Tissue-type plasminogen activator (t-PA)1 is a naturally occurring serine protease that initiates fibrinolysis by converting plasminogen (Pg) to plasmin (1). Not only is fibrin the target for plasmin attack, but fibrin also stimulates t-PA-mediated Pg activation (2, 3). To accomplish this, fibrin acts as a template to which both t-PA and Pg bind (4). The fibrin-binding properties of t-PA have been ascribed to its finger and second kringle (K 2 ) domains (5, 6), although recent studies suggest that the protease domain also influences the interaction of t-PA with fibrin (4, 7, 8). The binding of both Glu-and Lys-plasminogen (Glu-Pg and Lys-Pg, respectively) to fibrin is entirely kringle-mediated, with Lys-Pg having higher affinity for fibrin than Glu-Pg (9). As a functional consequence of t-PA interaction with fibrin, the catalytic efficiency of t-PA-mediated Pg activation is 2-3 orders in magnitude higher in the presence of fibrin than in its absence (3, 10). In contrast to fibrin, fibrinogen (Fg) stimulates Pg activation by t-PA only 25-fold (3, 10). Based on these considerations, t-PA is designated a fibrin-specific plasminogen activator (11). Despite this designation, t-PA causes systemic plasminemia and fibrinogenolysis when given to patients (12). In recent studies, we have demonstrated that t-PA causes systemic plasminemia, because, like intact fibrin, soluble fibrin degradation products stimulate t-PA-mediated Pg activation (13). Furthermore, we have identified the (DD)E complex as the fibrin derivative primarily responsible for this effect (14) and have shown that the stimulatory activity of (DD)E is similar to that of fibrin.2 (DD)E, a complex of D-dimer noncovalently bound to fragment E, is the major degradation product of cross-linked fibrin (15). As a potent stimulator of t-PA-mediated activation of Pg, (DD)E generated during thrombus dissolution has the potential to induce systemic plasminemia (12,15).The limited fibrin specificity of t-PA has prompted the development of plasminogen activators with greater selectivity for fibrin (16). One such agent is the plasminogen activator isolated from the saliva of vampire bats (Desmodus rotundus) (17). Full-length vampire bat salivary plasminogen activator (designated DSPA␣ 1 ) has over 72% amino acid seque...

A Kinetic Analysis of the Tissue Plasminogen Activator and DSPAα1 Cofactor Activities of Untreated and TAFIa-treated Soluble Fibrin Degradation Products of Varying Size

“…Finally, the data show that the large decrease in the DSPA␣1 cofactor activity with the small FDPs is attributable to a large increase (ϳ43-fold) in the K A , indicating that the high affinity sites for DSPA␣1 disappear as the M w of the FDP approaches 0.48 ϫ 10 6 g/mol. This is consistent with the observations of Stewart et al (2), who showed that DSPA␣1 binds to intact fibrin with high affinity (K A ϭ 150 Ϯ 40 nM) but not to fragment DD/E (K A Ͼ Ͼ 3000 nM), the smallest possible FDP (M w ϭ 0.25 ϫ 10 6 g/mol).…”

“…Implications for the Fibrin Specificity of Plasminogen Activators-Recently, Stewart et al (2) demonstrated that plasminogen activation catalyzed by t-PA, but not DSPA␣1, is effectively stimulated by the fibrin degradation product DD/E. The authors speculated that this property may increase the fibrin selectivity of DSPA␣1, relative to t-PA, as the DD/E fragments released from the clot would continue to stimulate the t-PAcatalyzed but not the DSPA␣1-catalyzed plasminogen activation, and thus, the release of DD/E into the circulation would promote fibrinogenolysis in the presence of t-PA but not DSPA␣1.…”

“…Both t-PA and DSPA␣1 are known as fibrin-selective plasminogen activators, because the rate of plasminogen activation with both activators is increased several orders of magnitude in the presence of fibrin, as compared with fibrinogen (1). Extensive plasminogen activation in the plasma, mediated via the cofactor effect of fibrinogen, results in systemic, plasmin-mediated fibrinogenolysis and consumption of ␣ 2 -antiplasmin, severely compromising the coagulation potential of the plasma (1,2). Fibrin selectivity is thus highly desirable for systemically administered thrombolytic agents.…”

Thrombin-activable Fibrinolysis Inhibitor Attenuates (DD)E-mediated Stimulation of Plasminogen Activation by Reducing the Affinity of (DD)E for Tissue Plasminogen Activator

“…Active site blocked t-PA was prepared by incubating the activator with a 5-fold molar excess of D-phenyl-prolyl-arginine chloromethyl ketone (PPACK; Calbiochem) as described previously (5,20). The molecular weight and extinction coefficient used for t-PA were 65,000 and ⑀ 1% 280 ϭ 20.0, respectively (4).…”

“…Recently, we demonstrated that (DD)E compromises the fibrin specificity of t-PA, because this soluble fragment is as potent as fibrin at stimulating Pg activation by t-PA (10, 11). Like fibrin, (DD)E binds t-PA and Pg with high affinity (5,10,12). In contrast to its predominantly finger-dependent interaction with fibrin,…”