Sustained thromboprophylaxis mediated by an RBC-targeted pro-urokinase zymogen activated at the site of clot formation.
Plasminogen activators (PAs) are used to treat life-threatening thrombosis, but not for thromboprophylaxis because of rapid clearance, risk of bleeding, and central nervous system (CNS) toxicity. We describe a novel strategy that may help to overcome these limitations by targeting a thrombin-activated PA pro-drug to circulating red blood cells (RBCs). We fused a single chain antibody (scFv Ter-119) that binds to mouse glycophorin A (GPA) with a variant human single-chain low molecular weight urokinase construct that can be activated selectively by thrombin (scFv/uPA-T). scFv/uPA-T bound specifically to mouse RBCs without altering their biocompatibility and retained its zymogenic properties until converted by thrombin into an active 2-chain molecule. As a result, RBC-bound scFv/uPA-T caused thrombin-induced fibrinolysis. One hour and 48 hours after intravenous (IV) injection in mice, approximately 70% and approximately 35% of scFv/uPA-T was retained in the blood, respectively, and approximately 95% of the circulating scFv/uPA-T remained bound to RBCs. A single IV injection of scFv/uPA-T provided effective prophylaxis against arterial and venous thrombosis for up to 24 hours. Thus, prophylactic delivery of RBC-targeted PA pro-drugs activated selectively at the site of clot formation represents a new approach to prevent thrombosis in clinical settings where the risk of clotting is high.
Zaitsev
Sergei
S
Program in Targeted Therapeutics, Institute for Translational Medicine and Therapeutics and Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104-6068, USA.
Spitzer
Dirk
D
Murciano
Juan-Carlos
JC
Ding
Bi-Sen
BS
Tliba
Samira
S
Kowalska
M Anna
MA
Marcos-Contreras
Oscar A
OA
Kuo
Alice
A
Stepanova
Victoria
V
Atkinson
John P
JP
Poncz
Mortimer
M
Cines
Douglas B
DB
Muzykantov
Vladimir R
VR
D000818:Animals; D016503:Drug Delivery Systems; D004792:Enzyme Precursors / Q000494:pharmacology*; D004912:Erythrocytes; D005343:Fibrinolytic Agents / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D011355:Prodrugs / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000494:pharmacology; D057127:Single-Chain Antibodies / Q000494:pharmacology*; D013927:Thrombosis / Q000517:prevention & control*; D013997:Time Factors; D014568:Urokinase-Type Plasminogen Activator / Q000494:pharmacology*
D004792:Enzyme Precursors; D005343:Fibrinolytic Agents; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D014568:Urokinase-Type Plasminogen Activator; C067551:saruplase
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