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

Abstract

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.

Why so little progress in therapeutic thrombolysis? The current state of the art and prospects for improvement

“…These fractional doses were almost identical to those of tPA and proUK in the clinical PATENT trial, which were 5 and 40 %, as described above. This similarity between the findings from this in vitro study to those found clinically provided evidence for the clinical relevance of the clot lysis model [ 56 ].…”

An activated-platelet-sensitive nanocarrier enables targeted delivery of tissue plasminogen activator for effective thrombolytic therapy

“…However, the difference between tPA-PEG-cRGD-lip and free tPA was not considerable (approximately 15%). This is probably because the clot lysis induced by tPA is a gradual process, as reported by other researchers [57,58]. However, after 75 min of treatment when majority of tPA was released from tPA-PEG-cRGD-lip ( Figure 4B), the liposomal tPAs, like free tPA, caused almost the complete blood clot lysis.…”

Thrombolysis: A Critical First-Line Therapy with an Unfulfilled Potential

“…Because proUK's instability was unrelated to its fibrinolytic mechanism of action, this problem was addressed by site-directed mutagenesis, which improved its plasma stability fivefold without interfering with its mechanism of action. [22][23][24] By contrast, the bleeding side effects with tPA are primarily due to its lysis of hemostatic fibrin at vascular repair sites, 25,26 which is due to its therapeutic fibrinolytic mechanism of action.…”