This revised proposal is directed at characterizing the molecular events of megakaryocytopoiesis, platelet production, and platelet function by analyzing the in vivo expression of platelet glycoprotein receptors. The proposed studies are devoted to improving our understanding of the GPIb-IX receptor complex, a platelet receptor for von Willebrand's factor which is essential for platelet adhesion and activation. In preliminary results, the Principle Investigator describes the use of a 6 Kb construct possessing 3 Kb of 5' upstream sequence, the two exon/ one intron coding region of alpha subunit of GpIb and 1 Kb of 3' downstream sequence to generate transgenic mice expressing the alpha subunit of human platelet GpIb associated with the beta subunit of the mouse glycoprotein. Preliminary data is also provided with regard to the characterization of mutant form of the human alpha subunit of GpIb which in the heterozygous state leads to Bernard Soulier syndrome (giant platelets with distinct changes in the microtubule network). Based upon the above preliminary results, the Principle Investigator proposes to complete work in five specific areas. First regulatory elements of the GpIb promoter will be defined by mapping the start site of the promoter, carrying out 5' deletions of the promoter and evaluating the transcriptional potency of regions by transfection into phorbal ester stimulated HEL cells, and conducting mobility shift assays with nuclear extracts of the above cells with specific domains identified as outlined above. Second, the GpIb beta and GpIX promoters will be characterized with regard to critical regulatory regions and then compared to the GpIb beta, PF4 and GpIIa promoters in order to identify common regulatory domains. Third, the GpIb alpha promoter will be evaluated in transgenic animals to ascertain whether it can be used to selectively express heterologous proteins such as GpIIb/IIIa on the surface of megakaryocytes. Fourth,the above transgenic animals will be employed to ascertain whether GpIb alpha is expressed in nonmegakaryocytic tissues such as endothelium and whether cytokines induce increased synthesis of this gene product. Fifth, transgenic animals will be generated with the above mutant form of the GpIb alpha gene product in order to ascertain whether this gene product is able to induce Bernard Soulier syndrome via abnormal interactions with cytoskeleton. The abnormalities produced with be examined by a combination of electron microscopy of intact megakaryocytes/platelets and in vitro studies of fragmentation of the abnormal megakaryocytes.
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