Embryo implantation involves the initial interaction of the external surface of the trophectoderm of the blastocyst and the apical cell surface of uterine epithelial cells (UEC). A number of studies indicate that heparan sulfate (HS) proteoglycans expressed on embryonic cell surfaces participate in early stages of embryo attachment to UEC in both rodent and human model systems. HS, and the related molecule, heparin (Hp), are structurally diverse polysaccharides that appear to play important roles in normal and malignant cell function, including mediation of cell adhesion and modulation of thrombin activity through their action as a required cofactor for anti-thrombin-3. A prediction from these studies is that UEC surfaces display corresponding HS/Hp binding sites. This prediction has been borne out by the identification of such sites on the surfaces of primary cultures of mouse UEC and human UEC lines. Ultimately, these studies have lead to the identification of a novel, cell surface heparin sulfate/heparin interacting protein (HIP) from human UEC, and other epithelial cells and cell lines. Furthermore, a 17 amino acid synthetic peptide (HIP-peptide-1) of this protein is sufficient to support specific, high affinity binding of Hp/HS in vitro as well as cell adhesion of a variety of human and non-human HS-expressing cell lines. Moreover, HIP and HIP-peptide-1 recognize the subset of Hp that binds to anti-thrombin-3 with highest affinity. As a result, HIP competes with anti-thrombin-3 for Hp binding and effectively activates thrombin and related proteases. Consequently, HIP may directly promote HS-dependent cell adhesion and indirectly promote thrombin-stimulated proteolytic cascades, induction of additional cell adhesion systems and cell proliferation. HIP is expressed in an appropriate locale to participate in HS-dependent processes of UEC. In the proposed studies, the investigators will use site-directed mutagenesis and biochemical approaches to define the structural requirements for HIP activity. Definition of these structural requirements will provide peptide targets and reagents to selectively perturb associated activities. These reagents then may be used to interrupt or enhance these activities, not only in the context of implantation, but in a variety of other contexts in which HS or thrombin is known to play a critical role, e.g., blot clotting. In addition, they will specifically examine HIP expression and function in murine embryo attachment in vitro and in vivo.
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