A targeted antithrombotic conjugate with antiplatelet and fibrinolytic properties which reduces in vivo thrombus formation.
OBJECTIVE
Potent therapy that could be locally delivered to inhibit blood factor-vessel wall interaction and which would remain localised to the site of damage may avoid the side effects of systemic drugs in the treatment of disorders such as subacute thrombosis of saphenous vein grafts and intravascular stents. We therefore assessed the feasibility of developing a targeted antithrombotic conjugate by covalently cross-linking urokinase to a monoclonal antibody to platelet glycoprotein IIb/IIIa (M735) and a monoclonal antibody against damaged endothelium (P14G11).


METHODS
Conjugation was carried out using N-succinimidyl-3-(2-pyridyldithio) propionate as the cross-linking reagent. The conjugate was assessed in vitro and in an in vivo model of thrombosis and local delivery.


RESULTS
The conjugate formed, ATC(3), retained specificity for damaged endothelial cells and platelets and had urokinase activity of approximately 10,000 IU.mg-1 protein. Persistence of urokinase activity on binding to intact platelets and scratch damaged endothelial monolayer preparations was confirmed. Platelet aggregation studies (using ADP and collagen) revealed complete inhibition by ATC(3) at a dose of 5 micrograms.ml-1 while an unconjugated mixture of M735 (20 micrograms.ml-1), P14G11 (20 micrograms.ml-1), and urokinase (200 IU.ml-1) failed to inhibit completely platelet aggregation induced by ADP. In an in vivo model of thrombosis and vascular injury, local delivery of ATC(3) significantly reduced the weight of thrombus formed [median 13 mg (interquartile range 9-20)] compared to an unconjugated mixture of M735, P14G11 and urokinase [35 mg (28-45)] and urokinase alone [41 mg (33-55)].


CONCLUSIONS
It is possible to produce a targeted antithrombotic conjugate which retains activity of all its individual components.
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R S
RS
Academic Department of Cardiology, University of Leicester School of Medicine, Glenfield General Hospital, United Kingdom.
Underwood
M J
MJ
Brack
M J
MJ
Pringle
S
S
de Bono
D P
DP
Gershlick
A H
AH
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000276:immunology / Q000293:injuries; D005343:Fibrinolytic Agents / Q000138:chemical synthesis* / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D010975:Platelet Aggregation Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000276:immunology; D051381:Rats; D017208:Rats, Wistar; D013388:Succinimides / Q000138:chemical synthesis* / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D005343:Fibrinolytic Agents; D010975:Platelet Aggregation Inhibitors; D010980:Platelet Membrane Glycoproteins; D013388:Succinimides; D014568:Urokinase-Type Plasminogen Activator