# Structural and Biochemical Studies of Blood Coagulation Factor VIII to Overcome the Immune Response

> **NIH NIH R15** · WESTERN WASHINGTON UNIVERSITY · 2021 · $389,770

## Abstract

Summary
Blood coagulation factor VIII is a protein cofactor that is essential for the proper regulation of the clotting
cascade. Deficiencies in factor VIII cause hemophilia A, the most common severe genetic bleeding
disorder, affecting 1 in 5,000 males worldwide. Treatment for hemophilia A consists of therapeutic
infusions of a functional form of factor VIII, termed “replacement therapy.” Complications to factor VIII
replacement therapy consist of an inhibitory immune response to the infusion in 25-30% of hemophilia
patients receiving treatment. Moreover, autoimmune disorders arise in healthy individuals against native
factor VIII, causing acquired hemophilia. As factor VIII has been repeatedly described as highly
immunogenic, understanding the structural nature of this immune response, how factor VIII forms
complexes in circulation with von Willebrand factor (VWF), and how activated factor VIII (fVIIIa) binds
activated platelet surfaces, may lead to more effective therapies for hemophilia A patients.
In our previous studies, we have: developed and tested a working model of membrane binding by the C-
terminal (C2) domain of factor VIII, determined the first high resolution structure of B domain-deleted
factor VIII, and structurally characterized inhibitory antibodies that target the factor VIII C1 and C2
domains, which have resulted in further understanding of the pathogenic anti-factor VIII immune
response. To further examine the structural nature of factor VIII protein complexes that exist in
circulation, this proposal will accomplish three specific aims. First, we will examine the structural basis of
activated platelet binding by: determining the X-ray crystal structure of an activated form of
bioengineered fVIIIa; characterizing the relative membrane binding affinity of isolated C1 and C2
domains, a tandem C1-C2 domain construct, and fVIIIa; generating a series of hemophilia A-associated
point mutants in the C1 and C2 domains that we will subject to protein stability studies and membrane
binding assays; and developing a quantitative assay to measure the binding of the fVIIIa/factor IXa
tenase complex to lipid nanodiscs (Specific Aim 1). Second, we will elucidate the molecular basis for the
factor VIII/VWF circulatory complex by determining the X-ray crystal structure of B domain-deleted factor
VIII bound to the VWF D’ and/or D’D3 domains (Specific Aim 2). Third, we will continue to characterize
the immune response to factor VIII replacement therapy by determining the X-ray crystal structures of B
domain-deleted factor VIII in complex with (1) anti-A2 domain inhibitory antibodies, and (2) hemophilia A
patient-derived inhibitory antibodies (Specific Aim 3). By examining the factor VIII immune response at
atomic resolution, paired with structural characterization of factor VIII circulatory complexes, our results
will illustrate in detail the life cycle of factor VIII and its procoagulant activity. The structural data that
result from this study...

## Key facts

- **NIH application ID:** 10114099
- **Project number:** 2R15HL135658-02
- **Recipient organization:** WESTERN WASHINGTON UNIVERSITY
- **Principal Investigator:** Paul Clinton Spiegel
- **Activity code:** R15 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $389,770
- **Award type:** 2
- **Project period:** 2017-04-01 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10114099

## Citation

> US National Institutes of Health, RePORTER application 10114099, Structural and Biochemical Studies of Blood Coagulation Factor VIII to Overcome the Immune Response (2R15HL135658-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10114099. Licensed CC0.

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