Pre-mRNA Processing and Function of Alternatively Spliced Isoforms of TFPI

止血是促凝血因子和抗凝血因子、血小板和血管系统之间持续的平衡行为。 这是防止过度出血或病理性凝血所必需的。抗凝剂--组织因子途径 抑制物(TFPI)是这种平衡中的一个重要因素，调节一系列出血和凝血障碍 通过抑制Tf-FVIIa、FXA和凝血酶原酶(FXA-FVA)。TFPI基因在进化上是保守的 由于选择性剪接，不同的TFPI亚型在不同的池中占主导地位。而具体的 每种TFPI异构体的抑制功能已经被表征，关于异构体的差异知之甚少。 在血栓前疾病条件下的特殊贡献，如因子V莱顿(FVL)和 胚胎发育。此外，决定表达的前mRNA剪接和处理机制 每种异构体都是未知的。由于Fv和TFPI异构体的异常剪接之间存在因果关系- 在人类出血性疾病中的特定功能，这些机制由针对 维持止血平衡，是高度相关的。因此，这项提议的长期目标是 在分子水平上区分每一种TFPI异构体的生理性、位点特异性的产生，并定义其 抗凝血在胚胎发育和疾病中的作用。组织因子抑制物α是存在于血小板中的唯一亚型 凝血启动过程中唯一能抑制凝血酶原酶的异构体。此外，全球TFPI 缺乏导致FVL小鼠血栓前围产期死亡，以及TFP Iα凝血酶原酶抑制活性 在FVL存在的情况下会降低。为此，K99期研究探讨了tfpiα作为一种 FV/FVL的调节，特别是在发育过程中血小板表面凝血酶原酶的组装(目标1) 并鉴定了在小鼠和人类中发现的新的血小板特异性组织因子抑制物α剪接变异体的生物学活性 (目标2)。候选人将获得技术专业知识，以确定组织因子抑制α抗凝功能，在体内使用 两个独特的异构体和位点特异性TFP Iα突变小鼠模型和体外使用人和小鼠血小板。 在AIM 3(R00阶段)中，候选人将利用替代TFP I的进化保守性 拼接形式和拼接信号嵌入高度保守的序列中以确定顺式RNA元件和 反式剪接因子相互作用调节TFPI在小鼠和人类中的异构体多样性。破译 调节位点特异性TFPI异构体表达的前mRNA处理机制将描绘如何 选择性剪接有助于胚胎期间的生理和病理生理止血平衡 发展阶段和成年阶段。由于有许多患者患有不明原因的出血和凝血障碍 原因，异常剪接与这些疾病的关系代表了一个相对新的和未被探索的领域 开启成功的独立职业生涯的巨大潜力。这项建议还概述了一项强化培训 计划课程、研讨会和实践培训，将候选人转变为装备精良的独立人士 具有独特的研究技能和极具前景的基础研究管道的调查人员。

Hemostasis is a constant balancing act between pro- and anticoagulant factors, platelets, and the vasculature that is required to prevent excessive bleeding or pathological clotting. The anticoagulant, Tissue Factor Pathway Inhibitor (TFPI), is a vital factor in this balance and modulates a broad range of bleeding and clotting disorders through inhibition of TF-FVIIa, FXa, and prothrombinase (FXa-FVa). The TFPI gene is evolutionarily conserved and due to alternative splicing, different TFPI isoforms are predominant within distinct pools. While the specific inhibitory function of each TFPI isoform has been characterized, little is known regarding differences in isoform- specific contributions under prothrombotic disease conditions such as Factor V Leiden (FVL) and during embryonic development. Further, the pre-mRNA splicing and processing mechanisms dictating expression of each isoform are unknown. As a causal relationship exists between aberrant splicing of FV and TFPI isoform- specific function in human bleeding disorders, these mechanisms, coordinated by precise cues directed at maintaining the hemostatic balance, are highly relevant. Thus, the long-term objective of this proposal is to differentiate the physiological, site-specific production of each TFPI isoform at a molecular level and define their anticoagulant function in embryonic development and disease. TFPIα is the only isoform present in platelets and the only isoform that inhibits prothrombinase during the initiation of blood coagulation. Additionally, global TFPI deficiency results in prothrombotic perinatal lethality in FVL mice, and TFPIα prothrombinase inhibitory activity is reduced in the presence of FVL. To this end, K99 phase studies probe the physiological role of TFPIα as a regulator of FV/FVL, particularly in prothrombinase assembly on platelet surfaces during development (AIM 1) and characterizes biological activity of new platelet-specific TFPIα splice variants identified in mice and humans (AIM 2). The candidate will acquire technical expertise to define TFPIα anticoagulant function both in vivo using two unique isoform- and site-specific TFPIα mutant mouse models and ex vivo using human and mouse platelets. In AIM 3 (R00 phase), the candidate will take advantage of the evolutionary conservation of alternative TFPI splice forms and splicing signals embedded in highly conserved sequences to determine cis-RNA element and trans-acting splicing factor interactions regulating TFPI isoform diversity in mice and humans. Deciphering the pre-mRNA processing mechanisms that regulate site-specific TFPI isoform expression will delineate how alternative splicing contributes to the physiological and pathophysiological hemostatic balance during embryonic development and in adulthood. As there are many patients with bleeding and clotting disorders of unknown cause, the relation of aberrant splicing to these diseases represents a relatively new and unexplored area with great potential for launching a successful independent career. This proposal also outlines an intensive training plan of courses, seminars, and hands-on training for transitioning the candidate into a well-equipped independent investigator with a unique combination of research skills and a highly promising basic research pipeline.