Highly Effective Fibrinolysis by a Sequential Synergistic Combination of Mini-Dose tPA plus Low-Dose Mutant proUK

Results of thrombolysis by monotherapy with either tPA or proUK have not lived up to expectations. Since these natural activators are inherently complementary, this property can be utilized to a synergistic advantage; and yet, this has undergone little evaluation. ProUK is no longer available because at pharmacological concentrations it converts to UK in plasma. Therefore, a single site proUK mutant, M5, was developed to address this problem and was used in this study. Fibrinolysis was measured using preformed fluoresceinated 24 h old clots in a plasma milieu rather than by the standard automated method, because proUK/M5 is sensitive to inactivation by thrombin and activation by plasmin. The shortest 50% clot lysis time that could be achieved by tPA or M5 alone was determined: mean times were 55 and 48 minutes respectively. These bench marks were matched by 6% of the tPA monotherapy dose combined with 40% that of M5: mean lysis time 47 minutes with less associated fibrinogenolysis. Results showed that the tPA effect was limited to initiating fibrinolysis which was completed by M5 and then tcM5. Plasma C1-inhibitor inhibited fibrinogenolysis by M5, providing protection from side effects not available for proUK. In conclusion, by utilizing the complementary properties and sequential modes of action of each activator, more efficient fibrinolysis with less non-specific effects can be achieved than with traditional monotherapy. In vivo validation is needed, but in a previous clinical trial using a similar combination of tPA and proUK (5% and 50% monotherapy doses) very promising results have already been obtained

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

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

Experiences with pro-urokinase and potentiation of its fibrinolytic effect by urokinase and by tissue plasminogen activator

Pro-urokinase is a single chain, precursor form of two chain, 54,000 Mrurokinase. Although originally isolated from urine, pro-urokinase is also found in blood, where it is believed to participate in natural fibrinolysis alongside tissue plasminogen activator (t-PA). These two plasminogen activators share the property of inducing fibrin-selective plasminogen activation, but many of their other properties, including their modes of action, are dissimilar. A comparison of some of the clinically relevant properties of pro-urokinase and t-PA is provided.A multicenter, dose-finding clinical trial of native prourokinase is underway in the United States and in West Germany. At the time of this writing, 110 patients with angiographically proved acute coronary thrombosis have been treated. The findings from one center are summarized in some detail and the overall experience is reviewed. Preliminary evidence for a potentiating effect on pro-urokinase-induced thrombolysis by urokinase is presented. The findings suggest that a bolus of urokinase (200,000 IU) at the outset increases the reperfusion rate from 60 to >80% and shortens the lysis time from about 50 to 30 minutes. A modest (19%) but significant (p < 0.05) decrease in fibrinogen accompanied thrombolysis by urokinase/pro-urokinase. Nonetheless, significant bleeding complications in the multicenter study have, to date, not been encountered and it is suggested that this may be related to pro-urokinase's sensitivity to inactivation by thrombin and lack of potentiation by heparin.Some in vitro data are summarized that provide an explanation for the potentiating effect of urokinase, the mechanism for which is believed to be similar to that by which synergism between t-PA and pro-urokinase has been postulated to occur. Evidence is reviewed that indicates that pro-urokinase and t-PA induce thrombolysis far more efficiently when they are combined than when each is used alone. The findings suggest that a bolus of t-PA, in place of urokinase, followed by an infusion of pro-urokinase may be optimal because t-PA is more effective in triggering lysis and pro-urokinase is more reactive after fibrinolysis has started

Fibrinolytic therapy with tPA failed because it was based on a flawed concept

Fibrinolytic therapy has become synonymous with tissue plasminogen activator (tPA) based on the belief that tPA alone was responsible for natural fibrinolysis. Although this assumption was belied from the outset by disappointing clinical results, it persisted, eventually causing fibrinolysis to be discredited and replaced by an endovascular procedure. Since time to reperfusion is the critical determinant of outcome, which in acute myocardial infarction (AMI) means within two hours, a time-consuming hospital procedure is ill-suited as first line treatment. For this purpose, fibrinolysis is more fitting. The assumption that tPA is responsible for fibrinolysis is contradicted by published findings. Instead, tPA &lsquo;s function is limited to the initiation of fibrinolysis, which is continued by urokinase plasminogen activator (uPA) and that has the dominant effect. tPA and uPA gene deletion and clot lysis studies showed the activators have complementary functions, requiring both for a full effect at fibrin-specific doses. They are also synergistic in combination thereby requiring lower doses for efficacy. A clinical proof of concept study in 101 AMI patients who were treated with a 5 mg bolus of tPA followed by a 90 minute infusion of prouPA, the native form of uPA. A near doubling of the 24 h TIMI-3 infarct artery patency rate was obtained compared to that in the best of the tPA trials (GUSTO). In further contrast to tPA, there were no reocclusions and the mortality was only 1% [1]. A sequential combination of both activators, mimicking natural fibrinolysis, holds promise to significantly improve the efficacy and safety of therapeutic fibrinolysis

Thrombolysis, the only Optimally Rapid Reperfusion Treatment

Thrombolysis with tissue plasminogen activator (tPA) has been plagued by inadequate efficacy and a high risk of intracranial hemorrhage (ICH), which led to its replacement by procedures like percutaneous coronary intervention (PCI) whenever possible. Since this requires hospitalization, it is time-consuming, and compromising salvage of brain tissue and myocardium. Thrombolysis is the only first-line treatment that can provide sufficiently timely treatment for optimal recovery of organ function. However, for this potential to be realized, its efficacy and safety must be significantly improved over the current method. By adopting the sequential, synergistic fibrinolytic paradigm of the endogenous system, already verified by a clinical trial, this becomes possible. The endogenous system&rsquo;s function is evidenced by the fibrinolytic product D-dimer that is invariably present in blood, and which increases &gt;20-fold in the presence of thromboembolism. This system uses tPA to initiate lysis, which is then completed by the other fibrin-specific activator prourokinase (proUK). Since tPA and proUK in combination are synergistic in fibrinolysis, it helps explain their efficacy at their low endogenous concentrations