Delivery system for targeted thrombolysis without the risk of hemorrhage

Cardiovascular diseases that result from thrombosis of critically situated blood vessels remain the leading cause of death in industrialized countries. One primary clinical treatment is dissolution of the thrombus with thrombolytic agents, plasminogen activators (PA). Activation of plasminogen by a PA agent produces plasmin that degrades fibrin. However, plasmin also degrades other circulating clotting factors. Therefore, thrombolytic therapy, which introduces systemic generation of excess plasmin, carries the risk of hemorrhage. We propose a novel approach that could lead to targeted thrombolysis without bleeding risk. The system is comprised of a protein conjugate made of two parts: a fibrin-targeting antibody (Ab) linked with anionic heparin; and a PA derivatized with cationic species. These two parts are linked via an electrostatic interaction. Because the cationic species are relatively small, the derivatized PA would retain its thrombolytic activity, but this activity would be inhibited after binding with the Ab-heparin counterpart because of the blockage of the PA's active site by these appended macromolecules. Because protamine is a clinical heparin antagonist with a much stronger affinity for heparin than the incorporated cations, it can be used safely to dissociate the modified PA from the Ab-heparin counterpart. Therefore, this approach would permit administration of a fibrin-targeting but inactive thrombolytic, and subsequently, a triggered release of the active modified PA drug in close proximity to the fibrin deposit. These features would enhance the potency and the specificity of the thrombolytic agent and alleviate the bleeding risk by avoiding systemic generation of excess plasmin. In this report, we present preliminary results demonstrating the feasibility of the approach. A cationic octapeptide, (Arg)7-Cys, was linked to urokinase (UK) using the N-succinimidyl-3-[2-pyridylidithio]propionate activation method. This UK peptide retained a significant amount of its catalytic activity, as measured by the S-2251 chromogenic assay. However, this activity was almost completely inhibited (approximately 99%) after the addition of heparin, but was fully reversed (100%) after the addition of protamine.