MOLECULAR & BIOCHEMICAL ANALYSIS OF MUTANT VWF RECEPTORS

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The P.I. has been focused upon developing a career in academic medicine since her early years as a part-time research student in her native Puerto Rico. For the past few years, the P.I. has done research with the sponsor, Dr. Jonathan Miller. The P.I. has studied patients with defective platelet glycoprotein (GP) Ib/IX receptor complexes for von Willebrand factor (vWF). This receptor consists of GPIb-alpha, GPIb-beta, and GPIX. In the course of her research training, the P.I. has learned numerous molecular biology techniques, including: isolation and purification of DNA and RNA, Southern and Northern blot hybridization, RNA dot blot, reverse transcription and PCR techniques, competitive oligonucleotide priming, DNA cloning and sequencing, site-directed mutagenesis, and in vitro expression of human glycoproteins in insect cells. The P.I. will apply these and other newer techniques to accomplish the proposed research work. One of the goals of the proposed work is to express mutant GPIb-alpha on the surface membrane of stable mammalian cell lines expressing GPIb-beta and GPIX. Initially, the work will be focused in the expression of receptor complexes containing the mutant valine GPIb-alpha or the mutant phenylalanine GPIb-beta described in our patients with platelet-type von Willebrand disease and variant Bernard-Soulier disease, respectively. The P.I.'s research approach will be to express homozygous (containing mutant GPIb-beta alone) or heterozygous (containing mutant GPIb-alpha and wild-type GPIb-alpha) mutant vWF receptors. The P.I. will then study the functional activity of each complex. To accomplish this goal, the P.I. will study the binding of vWF to these mutant receptors in the presence of ristocetin or of other modulators. It is anticipated that these studies will reveal the roles of particular amino acid sequences within the mature GPIb-alpha that are important in modulating vWF binding. Finally, it is the P.I.'s intent to identify the molecular abnormality responsible for the classic Bernard-Soulier disease phenotype in patients with normal GPIb-alpha genes. This work should yield relevant information about the interaction among the components of the GPIb/IX receptor, as well as between this receptor and its ligand, vWF. To accomplish these goals, the institution, through the P.I.'s chairman, is committed to providing adequate laboratory space and the necessary resources to create a productive research environment for the P.I.