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Effect of Chemical Conjugation of Recombinant Single-Chain Urokinase-Type Plasminogen Activator With Monoclonal Antiplatelet Antibodies on Platelet Aggregation and on Plasma Clot Lysis In Vitro and In Vivo

The murine monoclonal antiplatelet antibodies MA-TSPI-1 (directed against human thrombospondin) and MA-PMI-2, MA-PMI-1, and MA-LIBS-1 (directed against ligand-induced binding sites [LIBS] on human platelet glycoprotein llb/llla) were conjugated with recombinant single-chain urokinase-type plasminoge...

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Bibliographic Details
Published in:Blood 1991-08, Vol.78 (4), p.1005-1018
Main Authors: Dewerchin, M., Lijnen, H.R., Stassen, J.M., De Cock, F., Quertermous, T., Ginsberg, M.H., Plow, E.F., Collen, D.
Format: Article
Language:English
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Summary:The murine monoclonal antiplatelet antibodies MA-TSPI-1 (directed against human thrombospondin) and MA-PMI-2, MA-PMI-1, and MA-LIBS-1 (directed against ligand-induced binding sites [LIBS] on human platelet glycoprotein llb/llla) were conjugated with recombinant single-chain urokinase-type plasminogen activator (rscu-PA) using the cross-linking reagent N-succinimidγl 3-(2-pγridyldithio)propionate (SPDP). The conjugates (rscu-PA/MA-TSPI-1, rscu-PA/MA-PMI-2, rscu-PA/MA-PMI-1, and rscu-PA/MA-LIBS-1), purified by immu-noadsorption and gel filtration, were obtained with recoveries of 34% to 45%, with an average stoichiomβtry of 1.6 to 1.8 IgG molecules per rscu-PA molecule, and with unaltered specific activities and affinities. Preincubation of human platelet-rich plasma with rscu-PA/MA-PMI-2, rscu-PA/MA-PMI-1, or unconjugated rscu-PA resulted in partial inhibition of ADP-induced aggregation; 25% inhibition was obtained with 63 μg/mL rscu-PA and with 6 μg u-PA/mL rscu-PA/MA-PMI-2 or 1.2 μg u-PA/mL rscu-PA/MA-PMI-1. In an in vitro system composed of a 125l-fibrin-labeled platelet-rich human plasma clot immersed in normal human plasma, the conjugates had threefold to greater than 15-fold less fibrinolytic potency than unconjugated rscu-PA. The thrombolytic potency of rscu-PA/MA-PMI-1 and rscu-PA/MA-LIBS-1 was compared with that of rscu-PA and that of a control conjugate rscu-PA/MA-1C8 in a pulmonary embolism model in the hamster, using clots prepared from platelet-poor or platelet-rich human plasma. Lysis was measured 30 minutes after the end of a 60-minute intravenous infusion of the thrombolytic agents. rscu-PA, rscu-PA/MA-PMI-1, rscu-PA/MA-LIBS-1, as well as rscu-PA/MA-1C8 had comparable thrombolytic potencies (percent lysis per dose administered) towards platelet-poor human plasma clots. In contrast, the thrombolytic potency of rscu-PA/MA-PMI-1 and of rscu-PA/MA-LIBS-1 towards platelet-rich clots was 2.3- to 3-fold higher than that of rscu-PA (P ≪ .005) and fivefold to sevenfold higher than that of the control conjugate [P ≪ .01).
ISSN:0006-4971
1528-0020
DOI:10.1182/blood.V78.4.1005.1005