Project: Research project

Project Details


Platelet aggregation and platelet adhesion to components of the
extracellular matrix are control events in hemostasis and thrombosis and
are regulated through the binding of a set of adhesive proteins including
fibrinogen, fibronectin and von Willebrand factor all which contain Arg-
Gly-Asp (RGD) sequences. GPIIb-IIIa is a component of a common receptor
for these adhesive proteins and binds to peptides containing the RGD
sequence. Moreover, GPIIb-IIIa is a member of the Integrin adhesion
receptor superfamily and shares the RGD recognition function with certain
other integrins. The various alpha and beta subunits are homologous
proteins and possess several highly conserved sequences. The binding of
adhesive proteins to these receptors is divalent cation dependent, the
amino acid sequences of the putative divalent cation binding sites in the
Integrin alpha subunits is highly conserved, and the binding RGD containing
ligands to GPIIb-IIA causes the expression of divalent cation regulated
antigenic sites on GPIIb and GPIIIa. This proposal will test the
hypothesis that one or more of these putative calcium binding sites
participate in RGD binding. In addition, we will test the hypothesis that
the highly conserved region of GPIIIa to which bound RGD peptides may be
chemically crosslinked also contributes to RGD binding. We will first
analyze the sequence of these regions in GPIIb and GPIIIa cDNAs derived
from a natural mutant in which the GPIIb-IIIa hetrodimer lacks RGD binding
function. Secondly, we will asses the effects of recombinant GPIIb-IIIa in
a transient expression system utilizing a novel cytometric assay, affinity
chromatography, and chemical crosslinking. Third, in those regions whose
deletion abolishes RGD binding,we will identify those individual amino
acids essential for the RGD recognition by saturation mutagenesis. Finally,
in addition to those specific regions we will identify those regions of
GPIIb-IIIa whose structural integrity is essential for normal adhesive
protein binding function through random nucleotide insertion mutations
throughout both GPIIb and GPIIIa. These studies will provide fundamental
information about those regions of GPIIb-IIIa which are essential for the
platelet adhesion and aggregation.
StatusNot started


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