# Translating Mechanistic Insights into Intrinsic Xase Function

> **NIH NIH P01** · CHILDREN'S HOSP OF PHILADELPHIA · 2024 · $605,200

## Abstract

Project 4—Abstract
The intrinsic Xase enzyme complex, composed of the substrate factor (F) X, cofactor FVIIIa, and protease
FIXa, catalyzes the rate-limiting step of sustained coagulation. Deficiencies or dysfunction in these clotting
factors leads to the bleeding disorders FX-deficiency, hemophilia A (HA), and hemophilia B (HB), respectively.
The molecular details of the structure and function of FVIIIa, FIXa, and FX within the intrinsic Xase are
incompletely understood. Current treatments for these diseases have limited worldwide availability and are
highly burdensome. Our objectives in this proposal are to develop new therapeutic approaches for these
bleeding disorders and clarify the mechanisms that govern the assembly and function of the intrinsic Xase. To
this end, we have preliminary data indicating that FVIII-mimetics (a class of drugs approved for HA treatment)
can be repurposed for select HB genotypes. We have identified >40 HB-causing loss-of-function FIX variants
whose procoagulant activity is enhanced by FVIII-mimetics. We hypothesize that their loss-of-function is due
to disrupted interactions with FVIIIa or FX; these variants also provide a new experimental system where the
role of intrinsic Xase assembly of specific regions of FIXa can be biochemically and structurally interrogated.
We further hypothesize that there are disease-causing loss-of-function FX variants with disrupted FVIIIa
interactions whose activation could be rescued by the alternative FVIIImimetic/FIXa Xase complex.
Conversely, we have also identified a class of gain-of-function FVIII variants with high specific activity, which
we hypothesize is due to enhanced FVIIIa/FIXa interactions. Our experimental approach aims to 1) determine
the translation potential of FVIII-mimetics for select HB and FX-deficiency genotypes as well as the potential
for high-specific activity FVIII variants to address current limitations of HA gene therapy and 2) to clarify the
molecular assembly and function of the intrinsic Xase complex utilizing these clinically-relevant loss-of-function
and gain-of-function variants. In aim 1, using biochemical and structural approaches, we will determine the
mechanism of loss-of-function of FVIII-mimetic rescuable HB-causing FIX variants with amino acid
substitutions throughout the protein. In aim 2, we will develop a FVIII-mimetic adopted HB and FX-deficient
mouse model and determine the in vivo hemostatic potency of FVIII-mimetics for these disorders. In aim 3,
using biochemical and structural approaches, we will determine the mechanism of the high-specific activity of
our new gain-of-function FVIII variants. Further, we will determine their hemostatic potency over a range of
injury models in HA mice as well as their ability to enhanced gene therapy for HA. Together, these studies will
provide mechanistic insights into the regulation and activity of the intrinsic Xase as well as developing
alternative therapeutic approaches for these bleeding disord...

## Key facts

- **NIH application ID:** 10768832
- **Project number:** 2P01HL139420-06
- **Recipient organization:** CHILDREN'S HOSP OF PHILADELPHIA
- **Principal Investigator:** Ben J Samelson-Jones
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $605,200
- **Award type:** 2
- **Project period:** 2018-09-01 → 2029-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10768832

## Citation

> US National Institutes of Health, RePORTER application 10768832, Translating Mechanistic Insights into Intrinsic Xase Function (2P01HL139420-06). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10768832. Licensed CC0.

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