Thrombolysis vs. bleeding from hemostatic sites by a prourokinase mutant compared with tissue plasminogen activator

Abstract

Summary. Background: A single site mutant (M5) of prourokinase (proUK) was developed to make proUK less vulnerable to spontaneous activation in plasma. This was a problem that seriously compromised proUK in clinical trials, as it precluded proUK-mediated fibrinolysis at therapeutic concentrations. Methods and results: After completing dosefinding studies, 12 anesthetized dogs with femoral artery thrombosis were given either M5 (2.0 mg kg ) by i.v. infusion over 60 min (20% administered as a bolus). Two pairs of standardized injuries were inflicted at which hemostasis was completed prior to drug administration. Blood loss was quantified by measuring the hemoglobin in blood absorbed from these sites. Thrombolysis was evaluated at 90 min and was comparably effective by both activators. Rethrombosis developed in one t-PA dog. The principal difference found was that blood loss was 10-fold higher with t-PA (mean 40 mL) than with M5 (mean 4 mL) (P ¼ 0.026) and occurred at more multiple sites (mean 2.7 vs. 1.2). This effect was postulated to be related to differences in the mechanism of plasminogen activation by t-PA and M5 in which the latter is promoted by degraded rather than intact (hemostatic) fibrin. In addition, two-chain M5 was efficiently inactivated by plasma C1 inactivator, an exceptional property which helped contain its non-specific proteolytic effect. Conclusions: Intravascular thrombolysis by M5 was accompanied by significantly less bleeding from hemostatic sites than by t-PA. This was attributed to the proUK paradigm of fibrinolysis being retained at therapeutic concentrations by the mutation.

Mutant Prourokinase with Adjunctive C1-Inhibitor Is an Effective and Safer Alternative to tPA in Rat Stroke

“…Hemostatic fibrin, being protected from degradation, contains the D but not the E-domain (see Figure 6B). Therefore, unlike tPA, M5 spares hemostatic fibrin, as previously reported [20], but this is highly dependent on the prevention of non-specific conversion to tcM5. Therefore, M5 is highly dependent on plasma inhibitors (C1I), which is not the case for tPA.…”

“…In human plasma clot lysis studies with M5, the same inhibition of tcM5 as in the dogs was observed [20], [22], and the addition of exogenous human C1I or recombinant C1I (rhC1I) further increased M5 stability without compromising clot lysis. As a result, higher M5 doses achieving optimal rates of lysis were possible without inducing fibrinogenolysis [22], [23].…”

“…The quenching of tcM5 activity was monitored over 1 h with uPA amidolytic substrate (S2244). After 1 h incubation, the plasmas were examined by zymography on plasminogen enriched casein plates as previously described [20], a method sensitive to plasminogen activator:inhibitor complexes. Male adult Sprague-Dawley rats (Harlan, Italy) weighing 250–350 g were used to compare therapeutic fibrinolysis by tPA or M5+/− C1I following middle cerebral artery (MCA) occlusion.…”

“…These led to the development of a single site-directed (Lys300→His) prouPA mutant, M5, which was significantly more stable in plasma at therapeutic doses [18], [19], but otherwise retained the basic mechanism of action of prouPA. Two thrombolytic studies with M5 in dogs with venous or arterial thrombi showed that it was as effective as tPA but caused ten-fold less bleeding from injury sites [20], [21]. In the second of these studies, an unusual plasma inhibitor of two-chain M5 (tcM5) was identified which helped explain the low incidence of bleeding complications.…”

“…This inhibitor, by inactivating tcM5, prevented non-specific plasmin generation responsible for a bleeding diathesis. The plasma inhibitor responsible was identified to be complement C1-inhibitor (C1I) [20].…”