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 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).
Dewerchin
M
M
Center for Thrombosis and Vascular Research, University of Leuven, Belgium.
Lijnen
H R
HR
Stassen
J M
JM
De Cock
F
F
Quertermous
T
T
Ginsberg
M H
MH
Plow
E F
EF
Collen
D
D
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000627:therapeutic use; D001792:Blood Platelets / Q000276:immunology* / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D006224:Cricetinae; D003432:Cross-Linking Reagents; D005337:Fibrin / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism / Q000494:pharmacology; D005342:Fibrinolysis / Q000187:drug effects*; D005343:Fibrinolytic Agents / Q000008:administration & dosage / Q000494:pharmacology* / Q000627:therapeutic use; D006801:Humans; D007700:Kinetics; D010958:Plasminogen / Q000378:metabolism; D010960:Plasminogen Activators / Q000008:administration & dosage / Q000494:pharmacology* / Q000627:therapeutic use; D010974:Platelet Aggregation / Q000187:drug effects*; D010980:Platelet Membrane Glycoproteins / Q000276:immunology; D011655:Pulmonary Embolism / Q000188:drug therapy; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000494:pharmacology / Q000627:therapeutic use; D019699:Thrombospondins; D014568:Urokinase-Type Plasminogen Activator / Q000008:administration & dosage / Q000494:pharmacology* / Q000627:therapeutic use
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D005343:Fibrinolytic Agents; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D019699:Thrombospondins; D000244:Adenosine Diphosphate; D005337:Fibrin; D010958:Plasminogen; D010960:Plasminogen Activators; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator