Characterization of an isoform specific anticoagulant function of TFPI-alpha

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Abstract 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 TFPIβ as the primary isoform on endothelium, while TFPIα is the primary isoform within platelets. Using murine model systems, we demonstrated that platelet TFPIα limits thrombus growth following vascular injury and weakens the hemostatic response in hemophilia. These results suggested that TFPIα has a specific anticoagulant activity that is not performed by TFPIβ. We recognized that the basic C-terminal region of TFPIα and the basic region of the FV B-domain have striking homology, and sought to define how TFPIα may specifically interact with FV/FVa. Our biochemical studies described how TFPIα 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 TFPIα-FV interaction, finding that TFPIα has reduced ability to inhibit prothrombinase assembled with FV Leiden (FVL) and that the LIKT amino acids within the TFPIα C-terminus are required for inhibitory activity, but do not contribute to the affinity of TFPIα for FVa. We also generated several new murine models to investigate the physiological role of the TFPIα-FV interaction in vivo. Preliminary findings indicate that altering the TFPIα-FV interaction by modulating expression of TFPIα 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 TFPIα 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 TFPIα 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.