Structural Features Mediating Fibrin Selectivity of Vampire Bat Plasminogen Activators

Abstract

The distinguishing characteristic of vampire bat (Desmodus rotundus) salivary plasminogen activators (DSPAs) is their strict requirement for fibrin as a cofactor. DSPAs consist of structural modules known from urokinase (u-PA) and tissue-type plasminogen activator (t-PA) such as finger (F), epidermal growth factor (E), kringle (K), and protease (P), combining to four genetically and biochemically distinct isoenzymes, exhibiting the formulas FEKP (DSPA␣1 and ␣2) and EKP and KP (DSPA␤ and DSPA␥). Only DSPA␣1 and ␣2 bind to fibrin. All DSPAs are single-chain molecules, displaying substantial amidolytic activity. In a plasminogen activation assay, all four DSPAs are almost inactive in the absence of fibrin but strongly stimulated by fibrin addition. The catalytic efficiency (k cat /K m ) of DSPA␣1 increases 10 5 -fold, whereas the corresponding value of t-PA is only 550. The ratio of the bimolecular rate constants of plasminogen activation in the presence of fibrin versus fibrinogen (fibrin selectivity) of DSPA␣1, ␣2, ␤, ␥, and t-PA was found to be 13,000, 6500, 250, 90, and 72, respectively. Whereas all DSPAs are therefore more fibrin dependent and fibrin selective than t-PA, the extent depends on the respective presence of the various domains. The introduction of a plasmin-sensitive cleavage site in a position akin to the one in t-PA partially obliterates fibrin cofactor requirement. Fibrin dependence and fibrin selectivity of DSPAs are accordingly mediated by fibrin binding, which involves the F domain, as yet undefined determinants within the K and P domains, and by the absence of a plasmin-sensitive activation site. These findings transcend the current understanding of fibrin-mediated stimulation of plasminogen activation: in addition to fibrin binding, specific protein-protein interactions come into play, which stabilize the enzyme in its active conformation.

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

“…Fibrin selectivity is thus highly desirable for systemically administered thrombolytic agents. DSPA␣1 is considerably more fibrin-selective than t-PA, as the catalytic efficiency of DSPA␣1 is stimulated 13,000-fold, compared with only 820-fold for t-PA, when fibrin is the cofactor instead of fibrinogen (1). Furthermore, DSPA␣1 is intrinsically less fibrinogenolytic than t-PA because the catalytic efficiency of DSPA␣1 is 13-fold lower than t-PA when fibrinogen is the cofactor (50 versus 640 M Ϫ1 s Ϫ1 ) (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.…”

“…Fibrinolysis, the breakdown of the fibrin clot, is achieved primarily by the activation of plasminogen to the serine protease plasmin, which catalyzes degradation of the insoluble fibrin clot to soluble fibrin degradation products (FDPs). 1 The activation of plasminogen can be catalyzed by both endogenous activators such as tissue-type plasminogen activator (t-PA) and urokinase or exogenous activators such as streptokinase, staphylokinase, and Desmodus rotundus salivary plasminogen activators (DSPAs). These enzymes have all been used as thrombolytic agents for the dissolution of pathological thrombi, which can cause both myocardial infarction and stroke.…”

The evolution of recombinant thrombolytics: Current status and future directions

“…Further, it exists in the single chain form due to the absence of any plasmin cleavage site in the protease domain. 113 The molecule is almost inactive in the absence of fibrin. However, the presence of fibrin and fibrinogen enhances its activity by 12,900 fold, which is much higher than that for t-PA. 113 Furthermore, bat-PA is inhibited by tranexamic acid, a synthetic lysine analog, that can be used to control any bleeding complications upon its administration.…”

“…113 The molecule is almost inactive in the absence of fibrin. However, the presence of fibrin and fibrinogen enhances its activity by 12,900 fold, which is much higher than that for t-PA. 113 Furthermore, bat-PA is inhibited by tranexamic acid, a synthetic lysine analog, that can be used to control any bleeding complications upon its administration. 114 In a canine model of arterial thrombosis, the bat-PA promoted effective, rapid and sustained reperfusion without systemic fibrinogenolysis in comparison with t-PA. 115 The use of bat-PA in the treatment of acute ischemic stroke does not promote excitotoxic injury which has been implicated during the administration of t-PA. 116 Desmoteplase administered after 3-9 h of symptom onset during the treatment of acute ischemic stroke showed a higher rate of reperfusion and better clinical outcomes compared with placebo.…”