Mutant prourokinase with adjunctive C1-inhibitor is an effective and safer alternative to tPA in rat stroke

Abstract

A single-site mutant (M5) of native urokinase plasminogen activator (prouPA) induces effective thrombolysis in dogs with venous or arterial thrombosis with a reduction in bleeding complications compared to tPA. This effect, related to inhibition of two-chain M5 (tcM5) by plasma C1-inhibitor (C1I), thereby preventing non-specific plasmin generation, was augmented by the addition of exogenous C1I to plasma in vitro. In the present study, tPA, M5 or placebo +/- C1I were administered in two rat stroke models. In Part-I, permanent MCA occlusion was used to evaluate intracranial hemorrhage (ICH) by the thrombolytic regimens. In Part II, thromboembolic occlusion was used with thrombolysis administered 2 h later. Infarct and edema volumes, and ICH were determined at 24 h, and neuroscore pre (2 h) and post (24 h) treatment. In Part I, fatal ICH occurred in 57% of tPA and 75% of M5 rats. Adjunctive C1I reduced this to 25% and 17% respectively. Similarly, semiquantitation of ICH by neuropathological examination showed significantly less ICH in rats given adjunctive C1I compared with tPA or M5 alone. In Part-II, tPA, M5, and M5+C1I induced comparable ischemic volume reductions (>55%) compared with the saline or C1I controls, indicating the three treatments had a similar fibrinolytic effect. ICH was seen in 40% of tPA and 50% of M5 rats, with 1 death in the latter. Only 17% of the M5+C1I rats showed ICH, and the bleeding score in this group was significantly less than that in either the tPA or M5 group. The M5+C1I group had the best Benefit Index, calculated by dividing percent brain salvaged by the ICH visual score in each group. In conclusion, adjunctive C1I inhibited bleeding by M5, induced significant neuroscore improvement and had the best Benefit Index. The C1I did not compromise fibrinolysis by M5 in contrast with tPA, consistent with previous in vitro findings.

Conflict of interest statement

Competing Interests: Victor Gurewich, PhD, is the scientific officer of Thrombolytic Science, LLC, which is developing M5. PS is the scientific officer of Primm s.r.l., which supplied M5. Pharming Technologies B.V. supplied rhC1I for the study. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1. Quenching of tcM5 activity by inhibitors in plasmas from different species.

A. tcM5 (10 µg/mL) incubated (37°C, 1 h) in plasma and activity monitored with uPA enzymatic substrate (S2244). Rat plasma induced little tcM5 inhibition, in contrast to the other plasmas. B. After 1 h of incubation, each plasma sample was examined by zymography. No tcM5 complex with C1I, and only a faint complex with AT formed in rat plasma, in contrast to the other species.

Figure 2. (Part I): representative brain sections from Groups 1, 2,4.

A–C: Rat which died from tPA showing extensive hemorrhagic infiltration with edema compression (arrows) of the ischemic hemisphere (enlarged in C). D: Diffuse hemorrhagic transformation of ischemic cortex with edema compression in a Group 2 rat which died of ICH in a few hours. E–F: Sections 24 h after tPA+C1I infusion showing blood within the ischemic core (arrowheads), but no infiltration below the cortical surface (details in F). G–I: Thick surface hematoma above ischemic area, but no major blood infiltration in a M5+C1I rat. This Group showed the least bleeding with no alteration of brain architecture. L–M: ischemic sections from saline group, showing no blood infiltration within the infarct areas (details in N). Scale bars: 2 mm in A–B–D–E–G–H–L–M and 500 µm in C–F–I–N. Group 3 (M5 alone) rats not shown due to extensive disruption of brain by ICH.

Figure 3. (Part II) Neuroscores (A) and Ischemic and Edema volumes (B).

A: Neuroscore pre-(2 h) (black) and post-treatment (24 h) (gray) of thromboembolic stroke. Only Group 4 (M5+C1I) showed significant (*p

Figure 4. (Part II): Brain infarct zones from representative rats in each group.

Five antero-posterior TTC stained 2 mm-thick coronal sections from a representative animal from each group. Infarct areas in cerebral cortex, hippocampus and striatum appear white. The least amounts of infarction were with tPA, M5, and M5+rhC1I. Scale bar: 5 mm.

Figure 5. Semiquantitative ICH from Part I (A) and Benefit Index from Part II (B).

A. ICH (%) by anatomical region in Groups 1, 2 (tPA) and 4 (M5+C1I). Group 3 could not be examined microscopically due to extensive hemorrhage. Extent of ICH in Group 4 was significantly (*p

Figure 6. Illustration of ProuPA vs M5 at therapeutic concentrations in plasma and of the lysis of hemostatic vs occlusive fibrin by tPA and M5.

A. At therapeutic concentrations of prouPA or M5, their intrinsic activities can activate plasminogen in plasma and the plasmin generated will then activate these proenzymes to their respective enzymes uPA and tcM5. The latter is irreversibly inactivated by C1I, which prevents the positive feedback, thereby preventing non-specific plasmin generation responsible for hemorrhagic side effects. B. Hemostatic fibrin, being protected from degradation physiologically, contains only the plasminogen binding site on the fibrin fragment-D domain of intact fibrin. At therapeutic concentrations, tPA, being free of its inhibitor PAI-1, will bind to an adjacent site to plasminogen resulting in its activation and bleeding from the degraded hemostatic site. Occlusive thrombus triggers the release of tPA from the vessel wall which initiates its degradation physiologically. This exposes new plasminogen binding site, particularly the high affinity site on fibrin fragment E domain with its three C-terminal lysine. Plasminogen binding to this site undergoes a conformational shape change which permits its activation by M5 causing lysis. TPA will also lyse the occlusive clot since other plasminogen binding sites remain. The two plasminogen activators are complementary and synergistic in their fibrinolytic mechanisms.