Vascular endothelial cells harbor specific storage granules called Weibel-Palade bodies. These organelles contain two adhesion proteins involved in platelet and leukocyte binding"""""""" von Willebrand factor (vWf) and P-selectin. As a consequence, the granules play a role many and pathological processes that involve recruitment of platelets, monocytes or neutrophils. Vwf is a multimeric soluble glycoprotein that is synthesized with a large prosequence whose function after secretion is unknown. P-selectin is a transmembrane receptor that is directed to the storage granules by its cytoplasmic domain. The mechanisms involved in the Weibel- Palade bodies' formation and the function of the soluble granular components after secretion are the main topics of this application. The following are the specific aims of the proposed research: 1) to study the mechanisms involved in Vwf multimerization and storage. We will study the function of the prosequence and of the prosequence cleaving enzymes in these processes. 2) to investigate the role of Vwf prosequence after secretion. This will be done both in vitro and by overproduction of the prosequence in a transgenic animal model. 3) to produce an animal model of Vwf deficiency. We will prepare pro-Vwf deficient mouse by gene targeting to study the role of pro-Vwf in hemostasis, atherosclerosis, cancer metastasis and inflammation. 4) to define the role of the cytoplasmic domain of P-selectin in intracellular targeting. We will identify amino acid sequences involved in endocytosis and targeting to storage granules. 50 to use P-selectin cytoplasmic and transmembrane region as a tool for delivery of proteins to specific storage granules. We will express CDNA constructs endoding a P- selectin/beta-galactosidase chimera under the mekakaryocyte specific Platelet Factor 4 promoter in cells and then in mice to study the delivery of the reporter protein to alpha-granules. 60 to identify and characterize new molecular components of Weibel-Palade bodies. The research in this proposal will combine a variety of techniques including protein chemistry, cell culture, cell fractionation, site-specific mutagenesis and protein expression, gene cloning, genetic engineering of animals deficient in a particular gene (gene targeting by homologous recombination), engineering transgenic animals expressing a foreign gene and animal experimentation such as diet induced atherosclerosis. Understanding of the general processes involved in Weibel-Palade body formation should allow delivery of drugs or new gene products to specific storage granules in the future. Understanding of Weibel-Palade body function may be relevant not only for treatment of patients with von Willebrand disease but also with other disorders where this organelle and its contents are likely to take part, such as thrombosis, cancer metastasis, inflammation and atherosclerosis.
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