Platelet factor 4 (PF4) and Beta-thromboglobulin (BetaTG) are platelet- specific proteins stored within alpha granules and released following platelet activation. These proteins belong to a family of proteins encoded by the small inducible genes (SIG). SIG proteins are important in the overlapping biological functions of inflammation, coagulation and tissue repair. This grant proposes to use molecular biology techniques to study PF4 and BetaTG from two aspects: (1) Defining protein structural/functional relationships: The emphasis of these studies is to better define the biological function of PF4 and betaTG as well as providing insight into the biology of SIG proteins in general. Biological studies using our as well as providing insight into the biology of SIG proteins in general. Biological studies using our recombinant PF4 (rPF4) have documented that this protein has neutrophil chemotactic and negative megakaryocytopoietic proteins. Further studies concerning its immunologic and angiogenic properties are proposed. Vectors to express site-directed mutations of RPF4 have been constructed and initial protein expression accomplished. These proteins will be used to further define the relationship between the body of the protein and the Lys-rich C-terminus. rBeta and recombinant forms of platelet basic protein (PBP), connective tissue activating protein III (CTAP III) and neutrophil activating protein-2 (NA)-2), which are different cleavage forms of the same full-length precursor, have been constructed and initial expression accomplished. These constructs will be used to study the molecular basis for the >10(3) greater potency of NAP-2 compared to the other BetaT-like proteins and to PF4. (2) Megakaryocyte- specific gene expression: Both PF4 and BetaTG are expressed in a megakaryocyte-specific fashion. The emphasis of the proposed studies will be to better understand this tissue-specific expression utilizing two separate approaches. The first will focus on tissue-specific promoter sequences in the 5'-flanking region sequences of the cloned human and rat BetaTG genes utilizing a transient expression rat marrow system. These studies will be supplemented by mobility shift and footprinting gels to more fully define the cis-acting regulatory sites and to begin to characterize trans-acting nuclear proteins. The long-term objective will be to isolate and clone tissue-specific nuclear factors. In addition, the genomic organization of the human PF4/BetaTG complex on chromosome 4 is being defined by pulse-field gel electrophoresis and genomic library cloning. Using unique genomic DNA fragments from these clones, tissue- specific Dnase 1 hypersensitivity sites will then be determined with the ultimate goal of defining tissue-specific enhancers that are involved in regulating expression within the PF4/BetaTG complex. Such studies may provide insights into the regulation of megakaryocyte differentiation and may ultimately allow modulation of megakaryocyte-specific gene expression, thereby altering the thrombogenic tendency of platelets.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Hematology Subcommittee 2 (HEM)
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Children's Hospital of Philadelphia
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