Structure of Blood Coagulation Factor VIII in Complex With an Anti-C2 Domain Non-Classical, Pathogenic Antibody Inhibitor

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.

Structural insights into blood coagulation factor VIII: Procoagulant complexes, membrane binding, and antibody inhibition

“…More recently, the structure of the G99 inhibitor bound to the BDD FVIII construct, ET3i, highlights a modest conformational change of the C2 domain relative to the remaining FVIII structure. 64 Here, the C2 domain rotates in the opposite direction than what was previously observed for the ET3i model B structure described above C2 domain alone and in complex with the BO2C11 antibody predict differences in electrostatic hotspots that may contribute to complementary antibody binding affinity. 98…”

“…However, crystallographic B‐factor analysis suggests that simply binding the 3E6 antibody decreases the mobility of the surface loops in the opposing G99 epitope. More recently, the structure of the G99 inhibitor bound to the BDD FVIII construct, ET3i, highlights a modest conformational change of the C2 domain relative to the remaining FVIII structure 64 . Here, the C2 domain rotates in the opposite direction than what was previously observed for the ET3i model B structure described above by 8 Å (Figure 7A,B).…”

“…Further structural studies of the FVIII C2 domain in complex with antibody inhibitors that disrupt membrane binding suggest that PS headgroup binding is localized to basic clefts centered on the conserved R2320 in the C2 domain and R2163 in the C1 domain (Figure 4C). [63][64][65][66] To accommodate this binding motif, residue R2215 may change conformation toward the basic cleft and stabilize the negatively charged PS headgroup. Various hemophilia A-associated mutants to R2163/R2320 result in mild-to-severe hemophilia, depending on the mutation.…”

SAXS analysis of the intrinsic tenase complex bound to a lipid nanodisc highlights intermolecular contacts between factors VIIIa/IXa

“…6 However, recent crystal structures of fVIII have revealed a flexible C2 domain that can adopt multiple conformations while the other fVIII domains are conformationally conserved. 63,101,102 We attempted to account for alternative conformers of the Xase complex using MultiFOXS, 67 a SAXS-based tool that allows for modeling multistate conformations of macromolecular complexes, but were unsuccessful due to incompatibility with the lipid nanodisc. Future work, such as X-ray crystallography or cryoEM, will provide insight into energetically favorable conformations of the Xase complex at a higher resolution.…”

An intronic RNA element modulates Factor VIII exon-16 splicing

“…Among these, the F8 gene had the highest frequency of variants predicted to affect splicing regulatory sequences ( 23 ). The F8 gene encodes for a procoagulant called Factor VIII (FVIII), which is required for initiating the coagulation cascade to form blood clots in response to wounds ( 32 ). Through this cascade, activated FVIII must bind to another procoagulant called Factor IX in its activated form.…”