Abstract

Factor VIII (fVIII) is a procoagulant protein that binds to activated factor IX (fIXa) on platelet surfaces to form the intrinsic tenase complex. Due to the high immunogenicity of fVIII, generation of antibody inhibitors is a common occurrence in patients during hemophilia A treatment and spontaneously occurs in acquired hemophilia A patients. Non-classical antibody inhibitors, which block fVIII activation by thrombin and formation of the tenase complex, are the most common anti-C2 domain pathogenic inhibitors in hemophilia A murine models and have been identified in patient plasmas. In this study, we report on the X-ray crystal structure of a B domain-deleted bioengineered fVIII bound to the non-classical antibody inhibitor, G99. While binding to G99 does not disrupt the overall domain architecture of fVIII, the C2 domain undergoes an ~8 Å translocation that is concomitant with breaking multiple domain-domain interactions. Analysis of normalized B-factor values revealed several solvent-exposed loops in the C1 and C2 domains which experience a decrease in thermal motion in the presence of inhibitory antibodies. These results enhance our understanding on the structural nature of binding non-classical inhibitors and provide a structural dynamics-based rationale for cooperativity between anti-C1 and anti-C2 domain inhibitors.

Highlights

  • Hemophilia A is an X-linked recessive disorder that is caused by mutation to coagulation factor VIII

  • We report on the crystal structure of ET3i, a bioengineered factor VIII (fVIII) molecule, bound to the pathogenic, nonclassical anti-C2 domain antibody inhibitor G99

  • Considering the crystal structure of ET3i:G99 indicates binding to G99 disrupts these domain-domain contacts, we speculate how mutations to these regions influence the C2 domain conformation and inhibitor binding

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Summary

Introduction

Hemophilia A is an X-linked recessive disorder that is caused by mutation to coagulation factor VIII (fVIII). Patients with hemophilia A are prone to uncontrolled bleeding events and require regular infusions of recombinant or plasma-derived fVIII to maintain functional coagulation [1, 2]. In approximately 30% of hemophilia A treatment cases, patients will produce antibodies that inhibit infused fVIII and reduce treatment efficacy [3, 4]. Acquired hemophilia A can develop in healthy individuals through an autoimmune response, producing antibody inhibitors which bind. Structure of Factor VIII/G99 Complex to and inhibit the cofactor activity of native fVIII [5]. Inhibitory activity is detected and quantitated by the Bethesda assay of neutralization of fVIII coagulant activity in vitro [6]. Immune tolerance induction has been demonstrated to overwhelm the immune system through frequent, high-dosages of fVIII with modest success [3], but can be a physical and financial burden for the patient [7]

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