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428 Cryoem Structures and Conformational Landscapes of Activated Blood Coagulation Factor VIII and the Intrinsic Tenase Complex

Program: Oral and Poster Abstracts
Type: Oral
Session: 321. Coagulation and Fibrinolysis: Basic and Translational. A Coagulation Grab Bag
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Bleeding and Clotting, Bleeding disorders, Hemophilia, Diseases, Thrombotic disorders
Sunday, December 8, 2024: 9:45 AM

Kenneth C Childers1*, Omar Davulcu2*, Carmen Helena Coxon, PhD3*, Pete Lollar, MD4, Christopher B. Doering, PhD5* and P. Clint Spiegel Jr., PhD1

1Chemistry Department, Western Washington University, Bellingham, WA
2Pacific Northwest Center For Cryo-EM, Portland, OR
3National Institute of Biological Standards and Control, Potters Bar, ENG, GBR
4Emory University, Children's Center, Atlanta, GA
5Aflac Cancer and Blood Disorders Center, Emory University and Children's Healthcare of Atlanta, Atlanta, GA

Blood coagulation factor VIII (FVIII) is a key regulator of fibrin clot formation. Once activated by thrombin, activated FVIII (FVIIIa) binds to activated platelet surfaces and forms a complex with activated factor IX (FIXa) termed the intrinsic tenase (Xase) complex which sustains fibrin clot development. Excessive Xase complex activity is clinically linked with venous thromboembolism. Conversely, dysfunctional or deficient levels of FVIII or FIX is associated with hemophilia A or B, respectively. Despite decades of biochemical and structural research, the structural determinants for Xase complex assembly remain elusive. Here, we describe the structures of FVIIIa and the Xase complex, providing the first structural glimpse into thrombin-catalyzed activation of FVIII and assembly of the Xase complex.

We utilized single-particle cryogenic electron microscopy (cryoEM) to determine the structures of FVIIIa and the Xase complex using ET3i, a bioengineered FVIII human/porcine chimera with enhanced stability suitable for cryoEM experiments, and wildtype human FIXa treated with a protease inhibitor to prevent degradation of the Xase complex. CryoEM maps of FVIIIa and the Xase complex were determined to nominal resolutions of 3.56 Å and 3.64 Å, respectively. Leveraging recent advancements in heterogeneous cryoEM data analysis, our results reveal continuous conformational changes to isolated FVIIIa that are concomitant with thrombin-catalyzed activation. One of the most pronounced structural rearrangements is a novel ~20° bending centered at the interface between the A domains and C domains. This tilted conformer is only present in the FVIIIa structure with no FIXa bound, suggesting that this conformation of FVIIIa is not conducive for forming the active tenase complex. We are also able to visualize continuous dissociation of the A2 domain, allowing for structural investigation into how mutations at the A2 interface with the A1 and A3 domains may stabilize or destabilize FVIII/FVIIIa.

Furthermore, we identified a separate set of particles consisting of intact FVIIIa bound to FIXa. Our structure of the Xase complex reveals for the first time how the FIXa catalytic domain binds to the FVIIIa A2 domain, centered on FVIIIa residues 558-565. This structural arrangement places FIXa residue p.Arg384 (legacy numbering) docked onto several hydrophilic FVIIIa residues, neighboring a hydrophobic patch, on the A2 domain, providing a structure-based rationale for FIXa variants p.Arg384Leu (Padua) and p.Arg384Gln (Shanghai) which have been identified in patients with thrombosis. The Xase structure also suggests that the acidic C-terminal tail of the A2 domain anchors FVIIIa to FIXa through electrostatic interactions with FIXa exosite II. Further inspection of the cryoEM map of the Xase complex reveals how the FIXa light chain wraps around FVIIIa A3-C1 domains for optimal binding to lipid membranes. Correlating these results with the CDC Hemophilia Mutation Projects (CHAMP and CHBMP) databases, we are able to hypothesize how hemophilia A or B missense mutations disrupt Xase complex assembly and/or activity.

Disclosures: Lollar: Expression Therapeutics: Current equity holder in private company. Doering: Expression Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Spiegel: Uniqure: Consultancy.

*signifies non-member of ASH