Targeting of a mutant plasminogen activator to circulating red blood cells for prophylactic fibrinolysis.
Chemical coupling to carrier red blood cells (RBCs) converts tissue type plasminogen activator (tPA) from a problematic therapeutic into a safe agent for thromboprophylaxis. The goal of this study was to develop a more clinically relevant recombinant biotherapeutic by fusing a mutant tPA with a single-chain antibody fragment (scFv) with specificity for glycophorin A (GPA) on mouse RBCs. The fusion construct (anti-GPA scFv/PA) bound specifically to mouse but not human RBCs and activated plasminogen; this led to rapid and stable attachment of up to 30,000 copies of anti-GPA scFv/PA per mouse RBC that were thereby endowed with high fibrinolytic activity. Binding of anti-GPA scFv/PA neither caused RBC aggregation, hemolysis, uptake in capillary-rich lungs or in the reticuloendothelial system nor otherwise altered the circulation of RBCs. Over 40% of labeled anti-GPA scFv/PA injected in mice bound to RBC, which markedly prolonged its intravascular circulation and fibrinolytic activity compared with its nontargeted PA counterpart, anti-GPA scFv/PA, but not its nontargeted PA analog, prevented thrombotic occlusion in FeCl(3) models of vascular injury. These results provide proof-of-principle for the development of a recombinant PA variant that binds to circulating RBC and provides thromboprophylaxis by use of a clinically relevant approach.
Zaitsev
Sergei
S
IFEM, University of Pennsylvania School of Medicine, One John Morgan Building, 3620 Hamilton Walk, Philadelphia, PA 19104-6068, USA.
Zaitzev
Sergei
S
Spitzer
Dirk
D
Murciano
Juan-Carlos
JC
Ding
Bi-Sen
BS
Tliba
Samira
S
Kowalska
M Anna
MA
Bdeir
Khalil
K
Kuo
Alice
A
Stepanova
Victoria
V
Atkinson
John P
JP
Poncz
Mortimer
M
Cines
Douglas B
DB
Muzykantov
Vladimir R
VR
D000818:Animals; D004903:Erythrocyte Aggregation; D004912:Erythrocytes / Q000187:drug effects* / Q000502:physiology; D005343:Fibrinolytic Agents / Q000493:pharmacokinetics / Q000494:pharmacology*; D006021:Glycophorins / Q000276:immunology; D006461:Hemolysis; D006801:Humans; D066298:In Vitro Techniques; D007601:Jugular Veins; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D010960:Plasminogen Activators / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology*; D057127:Single-Chain Antibodies / Q000235:genetics / Q000494:pharmacology*; D020246:Venous Thrombosis / Q000097:blood / Q000517:prevention & control
D005343:Fibrinolytic Agents; D006021:Glycophorins; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D010960:Plasminogen Activators