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Platelet glycoprotein (Gp) IX associates with GpIb alpha in the platelet
membrane GpIb complex
G Wu, FJ Meloni and SS Shapiro
Department of Medicine, Jefferson Medical College of Thomas Jefferson
University, Philadelphia, PA, USA.
The platelet membrane glycoprotein (Gp) Ib complex consists of four
polypeptides: the disulfide-linked GpIb alpha and GpIb beta subunits; GpIX,
tightly, but noncovalently associated with GpIb alpha-beta; and the more
weakly associated GpV. It is not certain whether the association of GpIX to
Gplb alpha-beta is via GpIB alpha, GpIb beta, or both subunits, although
recently published evidence implicates an interaction with GpIb beta. We
have investigated the interaction of GpIX with GpIb alpha-beta using
polyclonal rabbit antibodies to GpIb alpha and GpIb beta raised by
immunization with purified glycocalicin and with synthetic peptide
sequences from GpIb beta, respectively, as well as monoclonal antibodies
directed against GpIX (FMC-25) and against GpIb alpha (AP-1). We performed
two types of experiments, using either purified GpIb complex or platelets.
(1) When wells were coated with nonreduced GpIb complex, the binding of
FMC-25 was inhibited 73% by GpIb alpha antibody, but only 30% by the GpIb
beta antibody; when wells were coated with reduced complex, FMC-25 binding
was inhibited by the same two antibodies by 86% and 13%, respectively. When
wells were coated with polyclonal GpIb alpha or GpIb beta antibodies and
then incubated with reduced GpIb complex, only wells coated with GpIb alpha
antibodies captured GpIX reactivity. When wells were coated with FMC-25 and
then incubated with nonreduced GpIb complex, both the GpIb alpha and GpIb
beta polyclonal antibodies reacted strongly; in contrast, only GpIb alpha
reactivity was retained when wells coated with FMC-25 were incubated with
reduced GpIb complex. In the reciprocal experiment, AP-1- coated wells
incubated with either nonreduced or reduced GpIb complex bound radiolabeled
FMC-25. (2) The ability of polyclonal GpIb alpha and GpIb beta antibodies
to inhibit binding of FMC-25 to platelets was studied by ELISA and by flow
cytometry. In both systems, FMC-25 binding was inhibited by the GpIb alpha
antibody, but not significantly by the GpIb beta antibody. We conclude that
GpIX is strongly associated with GpIb alpha in the purified platelet GpIb
complex and in the platelet membrane.
Volume 87,
Issue 7,
pp. 2782-2787,
04/01/1996
Copyright © 1996 by The American Society of Hematology
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