The long-term objective of this grant is to further the understanding of the genomic organization, biological roles and structural/functional relationships of platelet factor 4 (PF4) and Beta-thromboglobulin (Beta TG) in humans using recombinant DNA technologies to clone, sequence and express these genes in bacteria. PF4 and Beta TG are members of a family of related proteins (which also includes gamma IP-10, a protein synthesized by many cell lines following gamma-interferon stimulation) that appear to have significant roles in the inter-related processes of coagulation, chemotaxis, immunoregulation and tissue repair. A better understanding of the biological function of these two proteins and their structural/functional relationships may provide new insights into such pathologic states as thrombosis in atherosclerotic vessels during myocardial infarcts and cerebrovascular thromboembolisms. There are 2 specific aims to the proposed research: 1) Further characterization of PF4 and Beta TG cDNA and genomic DNA in humans and other mammalian and avian species using a PF4 cDNA probe already isolated and a Beta TG cDNA probe that I propose to isolate in a similar fashion. These studies should result in new information on protein precursors, their intracellular processing, and the evolutionary relationship between these proteins, as well as provide amino acid sequence data for deciding what modifications of these proteins may yield new biological information. 2) Studies on the relationship between protein structure and function using site- directed amino acid replacement. The natural proteins and precursor and induced mutations will be synthesized in E.coli, purified and used in biologic assays. Modification of PF4 will include a) alteration of the functionally important COOH- terminus of human PF4 to resemble Beta TG in order to study the relationships between these differences and the different biologic functions of the two proteins, and b) disruption of the secondary organization of PF4 by modification of one or more of the four cysteine residues that occupy homologous positions in PF4, Beta TG and gamma IP-10. Biologic functions to be tested include heparin affinity, platelet binding and activation, immunoregulatory activity, and chemotaxis for monocytes and fibroblasts.

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National Heart, Lung, and Blood Institute (NHLBI)
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Hematology Subcommittee 2 (HEM)
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Children's Hospital of Philadelphia
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