# Characterization of an isoform specific anticoagulant function of TFPI-alpha

> **NIH NIH R01** · VERSITI BLOOD HEALTH, INC. · 2022 · $745,219

## Abstract

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.

## Key facts

- **NIH application ID:** 10380837
- **Project number:** 5R01HL068835-16
- **Recipient organization:** VERSITI BLOOD HEALTH, INC.
- **Principal Investigator:** Alan E Mast
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $745,219
- **Award type:** 5
- **Project period:** 2002-08-01 → 2024-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10380837

## Citation

> US National Institutes of Health, RePORTER application 10380837, Characterization of an isoform specific anticoagulant function of TFPI-alpha (5R01HL068835-16). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10380837. Licensed CC0.

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