Tissue factor pathway inhibitor (TFPI) is an essential anticoagulant protein. Decreased plasma TFPI is associated with venous and arterial thrombosis, and pharmaceutical agents that block TFPI activity are being developed to treat patients with hemophilia. TFPI is an alternatively spliced protein and all isoforms are capable of inhibiting tissue factor (TF)-initiated blood coagulation. Our laboratory identified TFPI? as the primary isoform on endothelium, while TFPI? is the primary isoform within platelets. Using murine model systems, we demonstrated that platelet TFPI? limits thrombus growth following vascular injury and weakens the hemostatic response in hemophilia. These results suggested that TFPI? has a specific anticoagulant activity that is not performed by TFPI?. We recognized that the basic C-terminal region of TFPI? and the basic region of the FV B-domain have striking homology, and sought to define how TFPI? may specifically interact with FV/FVa. Our biochemical studies described how TFPI? effectively inhibits prothrombinase assembled with forms of FVa that retain the acidic region of the FV B-domain, such as that activated by FXa or found within platelets, but not with forms of FVa that have the entire B-domain removed, such as that activated by thrombin. During the previous funding period, we have worked to further characterize the TFPI?-FV interaction, finding that TFPI? has reduced ability to inhibit prothrombinase assembled with FV Leiden (FVL) and that the LIKT amino acids within the TFPI? C-terminus are required for inhibitory activity, but do not contribute to the affinity of TFPI? for FVa. We also generated several new murine models to investigate the physiological role of the TFPI?-FV interaction in vivo. Preliminary findings indicate that altering the TFPI?-FV interaction by modulating expression of TFPI? or FV in platelets dramatically alters embryonic survival of TFPI-K1 null mice. Additionally, we found that a transgene over-expressing hyperactivatable mouse protein C alters embryonic survival of TFPI-K1 null mice. Some of the surviving mice live to adulthood, but then often develop severe hydrocephalus. The proposed aims are designed to further pursue these intriguing preliminary findings to define TFPI- associated biology during embryonic development.
Aim 1 will examine how altering expression of TFPI? and FV, in general or specifically within platelets, modulates embryonic development.
Aim 2 will characterize murine brain and how TFPI deficiency modulates its development. Inhibition of prothrombinase by TFPI? is a recently recognized anticoagulant mechanism not performed by any other human protein. Therefore, it is anticipated that the results of the proposed experiments will describe the pathophysiology of coagulation and the coagulation-mediated cellular signaling developmental processes modulated by TFPI.
Tissue factor pathway inhibitor (TFPI) is a protein that prevents blood from clotting. This proposal will examine how basic biochemical interactions between TFPI and other blood clotting proteins modulate survival in animal model systems. The results of these studies will be important for understanding and developing new treatments for thrombotic diseases, such as how estrogen-containing drugs impact embryonic survival.
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