Quantitation of membrane glycoprotein IIIa on intact human platelets using the monoclonal antibody, AP-3

P. J. Newman, Robert Allen, R. A. Kahn, T. J. Kunicki

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Abstract

A murine monoclonal antibody specific for glycoprotein (GP)IIIa was prepared by immunization with a GPIIb- and GPIIIa-enriched Triton X-114 extract of platelet membranes. This antibody, designated AP-3, was shown by indirect immunoprecipitation to react solely with GPIIIa derived from either P1(A1)-positive or -negative individuals. The epitope on GPIIIa recognized by AP-3 is expressed on dissociated GPIIIa as well as on Ca+2-dependent complexes of GPIIb and GPIIIa, as shown by crossed immunoelectrophoresis in the presence or absence of EDTA. A previously described monoclonal antibody specific for the GPIIb/IIIa complex (AP-2) inhibited platelet aggregation induced by ADP, thrombin, collagen, or arachidonic acid (Pidard et al, J Biol Chem 248:12582-12586, 1983). In contrast, AP-3 had no effect on aggregation induced by any of these reagents, a finding similar to that previously reported for the GPIIb-specific monoclonal antibody. Tab (McEver et al, J Clin Invest 66:1311-1318, 1980). At saturation, 40,200 AP-3 molecules were bound per platelet, a value similar to that obtained for AP-2 or Tab. Thus, data derived using AP-3 indicate that significant amounts of free GPIIIa are not present, thereby supporting the hypothesis that GPIIb and GPIIIa exist complexed in a 1:1 stoichiometry in the plasma membrane of intact, nonactivated platelets.

Original languageEnglish
Pages (from-to)227-232
Number of pages6
JournalBlood
Volume65
Issue number1
StatePublished - 1 Jan 1985

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Integrin beta3
Membrane Glycoproteins
Platelets
Blood Platelets
Monoclonal Antibodies
Agglomeration
Two-Dimensional Immunoelectrophoresis
Immunization
Cell membranes
Platelet Aggregation
Immunoprecipitation
Arachidonic Acid
Edetic Acid
Thrombin
Stoichiometry
Adenosine Diphosphate
Epitopes
Glycoproteins
Collagen
Cell Membrane

Cite this

Newman, P. J. ; Allen, Robert ; Kahn, R. A. ; Kunicki, T. J. / Quantitation of membrane glycoprotein IIIa on intact human platelets using the monoclonal antibody, AP-3. In: Blood. 1985 ; Vol. 65, No. 1. pp. 227-232.
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abstract = "A murine monoclonal antibody specific for glycoprotein (GP)IIIa was prepared by immunization with a GPIIb- and GPIIIa-enriched Triton X-114 extract of platelet membranes. This antibody, designated AP-3, was shown by indirect immunoprecipitation to react solely with GPIIIa derived from either P1(A1)-positive or -negative individuals. The epitope on GPIIIa recognized by AP-3 is expressed on dissociated GPIIIa as well as on Ca+2-dependent complexes of GPIIb and GPIIIa, as shown by crossed immunoelectrophoresis in the presence or absence of EDTA. A previously described monoclonal antibody specific for the GPIIb/IIIa complex (AP-2) inhibited platelet aggregation induced by ADP, thrombin, collagen, or arachidonic acid (Pidard et al, J Biol Chem 248:12582-12586, 1983). In contrast, AP-3 had no effect on aggregation induced by any of these reagents, a finding similar to that previously reported for the GPIIb-specific monoclonal antibody. Tab (McEver et al, J Clin Invest 66:1311-1318, 1980). At saturation, 40,200 AP-3 molecules were bound per platelet, a value similar to that obtained for AP-2 or Tab. Thus, data derived using AP-3 indicate that significant amounts of free GPIIIa are not present, thereby supporting the hypothesis that GPIIb and GPIIIa exist complexed in a 1:1 stoichiometry in the plasma membrane of intact, nonactivated platelets.",
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Quantitation of membrane glycoprotein IIIa on intact human platelets using the monoclonal antibody, AP-3. / Newman, P. J.; Allen, Robert; Kahn, R. A.; Kunicki, T. J.

In: Blood, Vol. 65, No. 1, 01.01.1985, p. 227-232.

Research output: Contribution to journalArticle

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AB - A murine monoclonal antibody specific for glycoprotein (GP)IIIa was prepared by immunization with a GPIIb- and GPIIIa-enriched Triton X-114 extract of platelet membranes. This antibody, designated AP-3, was shown by indirect immunoprecipitation to react solely with GPIIIa derived from either P1(A1)-positive or -negative individuals. The epitope on GPIIIa recognized by AP-3 is expressed on dissociated GPIIIa as well as on Ca+2-dependent complexes of GPIIb and GPIIIa, as shown by crossed immunoelectrophoresis in the presence or absence of EDTA. A previously described monoclonal antibody specific for the GPIIb/IIIa complex (AP-2) inhibited platelet aggregation induced by ADP, thrombin, collagen, or arachidonic acid (Pidard et al, J Biol Chem 248:12582-12586, 1983). In contrast, AP-3 had no effect on aggregation induced by any of these reagents, a finding similar to that previously reported for the GPIIb-specific monoclonal antibody. Tab (McEver et al, J Clin Invest 66:1311-1318, 1980). At saturation, 40,200 AP-3 molecules were bound per platelet, a value similar to that obtained for AP-2 or Tab. Thus, data derived using AP-3 indicate that significant amounts of free GPIIIa are not present, thereby supporting the hypothesis that GPIIb and GPIIIa exist complexed in a 1:1 stoichiometry in the plasma membrane of intact, nonactivated platelets.

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