I, Vladimir Bogdanov, am applying for this research development award in order to finalize my transition into an independent research scientist. The immediate goals are: 1) to acquire new laboratory skills and generate novel research data that would enable me to compete for regular research project grant support (RO1), 2) to acquire first-hand experience in being a principal investigator, and 3) to make substantial progress in elucidating the mechanisms involved in regulation of human tissue factor (TF) pre-mRNA processing in monocytic cells. Specifically, I propose to study regulatory mechanisms responsible for generation of alternatively spliced human tissue factor (asHTF) in monocytes. asHTF, a recently identified molecule, is a soluble, biologically active form of human tissue factor generated by alternative splicing. It is present in circulating blood and thrombi and interacts with platelets under shear conditions. Full-length membrane-bound TF and asHTF are both present in arterial and ex-vivo thrombi. Initial results show that circulating monocytes serve as one of the major sources of asHTF protein in human plasma. Thus, mechanics of tissue factor pre-mRNA splicing in monocytic cells is the primary research objective of the proposed study. In addition, I propose to achieve selective down-regulation of full-length membrane bound TF and asHTF mRNA in monocytic cells by a novel technique of RNA interference and assess the impact of this down-regulation on the total coagulant potential of monocytic cells.
