Expression of Alternatively Spliced Human Tissue Factor

描述(由申请人提供)： 我，弗拉基米尔·博格丹诺夫，正在申请这个研究发展奖，以完成我向独立研究科学家的过渡。目前的目标是：1)获得新的实验室技能和产生新的研究数据，使我能够竞争常规研究项目拨款支持(RO1)；2)获得作为首席研究员的第一手经验；3)在阐明单核细胞中人类组织因子(TF)前mRNA加工的调控机制方面取得实质性进展。具体地说，我建议研究在单核细胞中产生选择性剪接的人组织因子(AsHTF)的调控机制。AsHTF是一种新近发现的分子，是一种通过选择性剪接产生的具有生物活性的可溶性人类组织因子。它存在于循环血液和血栓中，并在切变条件下与血小板相互作用。动脉血栓和体外血栓中均存在全长膜结合型Tf和asHTF。初步结果表明，循环单核细胞是人血浆中asHTF蛋白的主要来源之一。因此，单核细胞中组织因子前mRNA剪接的机制是本研究的主要研究目标。此外，我建议通过一种新的RNA干扰技术来实现单核细胞全长膜结合Tf和asHTF mRNA的选择性下调，并评估这种下调对单核细胞总凝血潜能的影响。

DESCRIPTION (provided by applicant): 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.