title: Prourokinase-annexin V chimeras. Construction, expression, and characterization of recombinant proteins.
abstract: Annexin V is a human protein that binds with high affinity to the abundant phosphatidylserine molecules exposed on activated platelets and accumulates selectively in thrombi after intravenous administration in animal models of arterial thrombosis. We designed two chimeras that use annexin V as a means to target thrombolytic agents to platelet-containing thrombi: prourokinase (1-411)-annexin V (1-320); and prourokinase (144-411)-annexin V (1-320) (amino acid numbers of parent proteins given in parentheses). Chimeras were produced by cytoplasmic expression in Escherichia coli, refolded, and purified in single-chain form. Both chimeras had the same specific activity as annexin V in binding to cell membranes containing exposed phosphatidylserine. After activation with plasmin, both chimeras had specific amidolytic activity similar to that of urokinase. Both chimeras activated plasminogen in vitro with kinetic parameters similar to those for urokinase, and both showed full activity compared to urokinase in an assay of clot lysis in vitro. This study shows the feasibility of producing chimeric plasminogen activators in which annexin V provides the thrombus-targeting component; although not yet tested in vivo, such chimeras may have advantages over antibody-based targeting agents.
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle 98195, USA.
authors.forename: J F
authors.initials: JF
authors.lastname: Tait
authors.forename: S
authors.initials: S
authors.lastname: Engelhardt
authors.forename: C
authors.initials: C
authors.lastname: Smith
authors.forename: K
authors.initials: K
authors.lastname: Fujikawa
mesh_terms: D000595:Amino Acid Sequence; D017304:Annexin A5 / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005342:Fibrinolysis; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010958:Plasminogen / Q000378:metabolism; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D017304:Annexin A5; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D010958:Plasminogen; D014568:Urokinase-Type Plasminogen Activator; C067551:saruplase