Independent modulation of von Willebrand factor and fibrinogen binding to the platelet membrane glycoprotein IIb/IIIa complex as demonstrated by monoclonal antibody.
In this study we have used two new monoclonal antibodies, designated LJP5 and LJP9, as well as a previously described one, AP2, all specific for the platelet membrane glycoprotein (GP)IIb/IIIa complex. None of them reacted with dissociated GPIIb or GPIIIa. The monovalent Fab fragment of both LJP5 and LJP9 bound to unstimulated platelets in a saturable manner, but binding was markedly decreased after platelets had been incubated at 37 degrees C in the absence of added extracellular calcium. The binding of LJP9 was not affected by AP2, but was blocked by excess LJP5. On the contrary, the binding of LJP5 was blocked in the presence of both AP2 and LJP9. Thus, these antibodies bound to distinct epitopes of GPIIb/IIIa. At saturation, the binding to unstimulated platelets was between 2.41 and 10.9 X 10(4) molecules/platelet for LJP5 and between 3.47 and 9.1 X 10(4) molecules/platelet for LJP9 (range of 11 and 10 experiments, respectively). Binding increased up to 50% after thrombin stimulation. The estimated association constant, Ka, was 2.7 X 10(7) M-1 for LJP5 and 3.85 X 10(7) M-1 for LJP9. Both LJP5 and LJP9 partially inhibited the association of 45Ca2+ with the surface of unstimulated platelets. Moreover, both antibodies blocked the binding of von Willebrand factor (vWF) to stimulated platelets, whereas only LJP9, but not LJP5, blocked fibrinogen binding. LJP9 was also a potent inhibitor of platelet aggregation, whereas LJP5 was without effect in this regard. The results of the present study demonstrate that independent modulation of vWF and fibrinogen binding to stimulated platelets can be attained with monoclonal antibodies directed against distinct epitopes of GPIIb/IIIa.
V T
VT
Lombardo
E
E
Hodson
J R
JR
Roberts
T J
TJ
Kunicki
T S
TS
Zimmerman
Z M
ZM
Ruggeri
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D001792:Blood Platelets / Q000276:immunology / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D000939:Epitopes; D005340:Fibrinogen / Q000378:metabolism*; D006023:Glycoproteins / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins; D013696:Temperature; D013917:Thrombin / Q000494:pharmacology; D014841:von Willebrand Factor / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006023:Glycoproteins; D007140:Immunoglobulin Fab Fragments; D008565:Membrane Proteins; D010980:Platelet Membrane Glycoproteins; D014841:von Willebrand Factor; D000244:Adenosine Diphosphate; D005340:Fibrinogen; D013917:Thrombin; D002118:Calcium