PROUROKINASE-ANNEXIN-V CHIMERAS - CONSTRUCTION, EXPRESSION, AND CHARACTERIZATION OF RECOMBINANT PROTEINS

Annexin V is a human protein that binds with high affinity to the abun dant phosphatidylserine molecules exposed on activated platelets and a ccumulates selectively in thrombi after intravenous administration in animal models of arterial thrombosis. We designed two chimeras that us e annexin V as a means to target thrombolytic agents to platelet-conta ining thrombi: prourokinase (1-411)-annexin V (1-320); and prourokinas e (144-411)-annexin V (1-320) (amino acid numbers of parent proteins g iven in parentheses). Chimeras were produced by cytoplasmic expression in Escherichia coil, refolded, and purified in single-chain form. Bot h chimeras had the same specific activity as annexin V in binding to c ell membranes containing exposed phosphatidylserine. After activation with plasmin, both chimeras had specific amidolytic activity similar t o that of urokinase. Both chimeras activated plasminogen in vitro with kinetic parameters similar to those for urokinase, and both showed fu ll activity compared to urokinase in an assay of clot lysis in vitro. This study shows the feasibility of producing chimeric plasminogen act ivators in which annexin V provides the thrombus-targeting component; although not yet tested in vivo, such chimeras may have advantages ove r antibody-based targeting agents.