Calcium ions and the conformation of glycoprotein IIIa that is essential fibrinogen binding to platelets: analysis by a new monoclonal anti-GP IIIa antibody, TM83.
Using a newly developed murine monoclonal antibody (MoAb), TM83, against glycoprotein IIIa (GPIIIa) of human platelets, we have analyzed the relationship between platelet fibrinogen binding and conformational changes in GPIIIa under EDTA treatment. Crossed radioimmunoelectrophoresis demonstrated that TM83 reacted with only the GPIIb/IIIa complex but also with GPIIIa alone. TM83 dose-dependently inhibited both thrombin-induced aggregation and fibrinogen binding to activated platelets. 125I-TM83 bound to an average of 20,890 +/- 1,600 (mean +/- SE, n = 12) sites on a resting platelet, with Kd = 2.06 nmol/L in the presence of Ca2+. When platelets were incubated in 2 nmol/L EDTA-containing medium, pH 7.4, at 22 degrees C for 30 minutes, binding of TM83 decreased to 70% of the control level. The decreased binding was fully recovered to the control level when the platelets were resuspended in Ca2+-containing medium. These platelets retained their aggregability. In contrast, when platelets were incubated in 2 mmol/L EDTA-containing medium, pH 7.4, at 37 degrees C for 30 minutes, TM83 binding to the platelets markedly decreased to 7% of the control, which could only be recovered to 40% of the control by replacing the medium with calcium-containing medium; these platelets lacked thrombin-induced aggregability. These findings suggest that the epitope for TM83 may be located near the fibrinogen binding site on GPIIIa and that its conformation is dependent on Ca2+ ions.
Yamamoto
N
N
Department of Cardiovascular Research, Tokyo Metropolitan Institute of Medical Science, Japan.
Kitagawa
H
H
Yamamoto
K
K
Tanoue
K
K
Yamazaki
H
H
D000911:Antibodies, Monoclonal / Q000276:immunology*; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000502:physiology*; D004492:Edetic Acid / Q000494:pharmacology; D000939:Epitopes; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007123:Immunoelectrophoresis, Two-Dimensional; D010980:Platelet Membrane Glycoproteins / Q000502:physiology* / Q000648:ultrastructure; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D013696:Temperature
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010980:Platelet Membrane Glycoproteins; D005340:Fibrinogen; D004492:Edetic Acid; D002118:Calcium