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.
Highlights
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
This study shows the feasibility of producing chimeric plasminogen activators in which annexin V provides the thrombus-targeting component; not yet tested in vivo, such chimeras may have advantages over antibody-based targeting agents
This study shows the feasibility of producing hybrid thrombolytic agents in which annexin V provides the thrombustargeting component
Summary
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. 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. We expressed a chimera lacking the first 143 amino acids of prourokinase, which encode the epidermal growth factor-like and kringle domains of prourokinase [24]; this molecule, while retaining the proenzyme character of full-length prourokinase, might have certain advantages in production or clinical use due to its smaller size We show that these proteins can be expressed and purified in active form; they fully retain the component enzymatic and membrane-binding activities of the parent molecules; and they are active in assays of in vitro clot lysis
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
