Pre-mRNA Processing and Function of Alternatively Spliced Isoforms of TFPI - 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-FVlla, 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. TFPla 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 TFPla prothrombinase inhibitory activity is reduced in the presence of FVL. To this end, K99 phase studies revealed that TFPla and its inhibition of FVL-containing prothrombinase play an important role in placental angiogenesis and embryonic survival and further characterized the biological activity of new platelet-specific TFPla splice variants identified in mice and humans. The R00 phase studies will leverage the evolutionary conservation of alternative TFPI splice forms and splicing signals embedded in highly conserved sequences to determine cisRNA 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 work will establish an innovative research program at the intersection of RNA biology and coagulation, enabling the Pl to lead a uniquely interdisciplinary investigation into how splicing regulation influences platelet function and thrombotic risk. The R00 phase will provide the foundation for future R01-level studies and sustained contributions to the fields of thrombosis and hemostasis and RNA regulation.
