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
M Dewerchin, HR Lijnen, JM Stassen, F De Cock, T Quertermous, MH Ginsberg, EF Plow and D Collen
Center for Thrombosis and Vascular Research, University of Leuven, Belgium.
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
IIb/IIIa) were conjugated with recombinant single-chain urokinase-type
plasminogen activator (rscu-PA) using the cross-linking reagent
N-succinimidyl 3-(2-pyridyldithio)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 immunoadsorption and gel filtration, were
obtained with recoveries of 34% to 45%, with an average stoichiometry 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 micrograms/mL rscu-PA and with 6 micrograms u-PA/mL
rscu-PA/MA-PMI-2 or 1.2 micrograms u- PA/mL rscu-PA/MA-PMI-1. In an in
vitro system composed of a 125I-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 less than .005) and fivefold to sevenfold higher than
that of the control conjugate (P less than .01).
Volume 78,
Issue 4,
pp. 1005-1018,
08/15/1991
Copyright © 1991 by The American Society of Hematology
