MOLECULAR BIOLOGY OF PLATELET ALPHAIIB-BETA3 RECEPTOR

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Platelets are circulating, highly differentiated, a nuclear cellular fragments derived from megakaryocytes. One of the tissue-specific features of these cells is their fibrinogen receptor, alphaII/beta3. This integrin receptor is densely packed on the surface of the platelet and plays a key role in platelet aggregation and thrombus formation. This application, a part of the proposed Program Project, will study the biology of this critical receptor, focusing on the regulated expression of the alphaIIb gene and on the analysis of the molecular basis of Glanzmann thrombasthenia. There are three specific aims to this application. I. Defining the Proximal Promoter Elements Regulating alphaIIb Expression. Proposed studies will concentrate on the further analysis of three regions: The first is an Sp1-based negative regulatory region centered at -135bp upstream to the transcriptional start site. We plan to define its local interactions and biological role. We also plan to examine the biological relevance of a potential PU.1-binding site at -510 bp and a potential GATA-1 interacting region between -450 to -350 bp. II. Characterizing Distal Regions of the alphaIIb Gene. Two approaches will be followed to search for such distal regulatory elements: The first will examine the alphaIIb gene locus for tissue-specific, DNase 1 hypersensitive sites (HSS). We have preliminarily defined two such sites 5 and 6 kilobasepairs (kb), respectively, upstream of the human alphaIIb gene. Using available P1 alphaIIb clones, we plan to subclone these regions, sequence them, and characterize them in vitro and also in transgenic mice models. In the second approach, we plan on creating a series of transgenic animals containing various lengths of the human alphaIIb gene locus isolated from lambda, cosmid and P1 clones that will be tested to see what is the minimal construct size required for consistent, high levels of tissue-specific human alphaIIb gene expression in mice. Once such a region is defined, we will focus on Dnase 1 HSS studies to further localize potential distal megakaryocyte-specific regulatory elements and incorporate these regions into further expression constructs. III. Functional Analysis of the alphaIIb/beta3 Receptor. These studies will focus on the analysis of thrombasthenic mutations that have already been published by us or that are under present analysis. These studies will include further analysis of the Ca2+-binding domains of alphaIIb and beta3 in intracellular receptor processing, and analysis of a new alphaIIb variant (P176 to A) and the role of this region of alphaIIb in ligand binding, and a mutation in the C-terminus of alphaIIb (Q819 to P) and the role of the C-terminal alphaIIb heavy chain in receptor processing and activation. These and related studies focus on intracellular processing and ligand binding and should provide important new insights into the biology of this important integrin receptor.