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.
Highlights
Stroke is the second most common cause of death worldwide [1] of which more than 80% are thromboembolic in origin
Only an estimated 2-5% of ischemic stroke patients are currently treated with Tissue plasminogen activator (tPA) in the US [12], though the number is higher in dedicated Stroke Units
A faint complex with anti-thrombin was seen, indicating that rat plasma had little endogenous two-chain M5 (tcM5) neutralizing activity of any kind necessary to inhibit the non-specific effects of M5
Summary
Stroke is the second most common cause of death worldwide [1] of which more than 80% are thromboembolic in origin. Tissue plasminogen activator (tPA) is the only pharmacological reperfusion treatment approved for ischemic stroke [2]. The ia route was chosen because previous studies showed that at therapeutic concentrations in plasma, prouPA was vulnerable to non-specific activation to two-chain urokinase (tcuPA). Its fibrin-specificity was lost resulting in a haemorrhagic diathesis due to degradation of clotting factors like fibrinogen [15]. This activation of prouPA to tcuPA in plasma, instead of only on the fibrin clot, undermined its therapeutic exploitation and resulted in prouPA not being approved by the European Agency for the Evaluation of Medicinal Products EMEA. ProuPA had certain advantageous properties, which included a much lower rate of coronary reocclusion than tPA, no associated procoagulant haematological effects, and a low mortality [16,17]
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