Clot uptake of labeled active and inhibited tissue plasminogen activator.
The clot uptake of labeled active and inhibited t-PA was compared. The most efficient inhibition was obtained with diisopropyl fluorophosphate (DFP) after 4 h incubation at room temperature. Enzyme activity was followed by fibrin-plate assay, radioactivity-release technique and proton magnetic resonance (PMR). The novel PMR method developed by us is sensitive to the effect of as low as nanogram amounts of t-PA on the interaction between the fibrin and the compartmentalized water trapped in the clot. Binding of labeled enzyme to fibrin-coated plates showed that the deactivation by DFP did not impair the affinity of t-PA for fibrin. A rapid binding of 125I-labeled t-PA to the clot occurred, which reached a maximum in 30 min and declined with time. This pattern was explained by consecutive clot binding and lysis. The binding of DFP-t-PA to the clot differed markedly from that of the active protein; 2 h post-incubation the uptake of DFP-t-PA was more than double that of the untreated t-PA. Parallel measurements in clots prepared from human blood showed a qualitatively similar trend. The biodistribution of radiolabeled t-PA in mice was similar for the active and inhibited forms. Blood activity reached 10% of the injected dose within 10 min. DFP-t-PA may prove to be a useful reagent for in-vivo localization of thrombi.
Lavie
E
E
Radiopharmaceuticals Department, Soreq Nuclear Research Center, Yavne, Israel.
Bitton
M
M
Shalev
J
J
Weininger
J
J
Abrashkin
S
S
Varga
E
E
Lubin
E
E
Azoury
R
R
D000818:Animals; D001682:Biological Availability; D001777:Blood Coagulation / Q000187:drug effects*; D005260:Female; D005337:Fibrin / Q000378:metabolism*; D005338:Fibrin Fibrinogen Degradation Products / Q000378:metabolism; D005342:Fibrinolysis / Q000187:drug effects; D006207:Half-Life; D006801:Humans; D007531:Isoflurophate / Q000097:blood / Q000494:pharmacology; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D051379:Mice; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D013927:Thrombosis / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors / Q000097:blood / Q000378:metabolism*
D005338:Fibrin Fibrinogen Degradation Products; D011994:Recombinant Proteins; D007531:Isoflurophate; D010664:Phenylmethylsulfonyl Fluoride; D005337:Fibrin; D010959:Tissue Plasminogen Activator