The 3.2 angstrom structure of a bioengineered variant of blood coagulation factor VIII indicates two conformations of the C2 domain

• Type of Material: Article
• Authors: Ian W. Smith, Western Washington University; Anne E. d'Aquino, Western Washington University; Christopher W. Coyle, Emory University; Andrew Fedanov, Emory University; Ernest T. Parker, Emory University; Gabriela Denning, Expression Therapeutics, LLC.; H Trent Spencer, Emory University; John Lollar, Emory University; Christopher Doering, Emory University; P. Clint Spiegel. Jr., Western Washington University
• Language: English
• Date: 2019-09-08
• Publisher: WILEY
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: 2019
• Final Published Version (URL): http://dx.doi.org/10.1111/jth.14621
• Title of Journal or Parent Work: JOURNAL OF THROMBOSIS AND HAEMOSTASIS
• Volume: 18
• Issue: 1
• Start Page: 57
• End Page: 69
• Grant/Funding Information: The authors thank Betty Shen from the Fred Hutchinson Cancer Research Center for assistance with data processing and the SIBYLS beamline 12.3.1 at the Advanced Light Source for data collection access. This work was supported by the Dreyfus Foundation [Henry Dreyfus Teacher-Scholar Award], National Science Foundation [MRI 1429164], The Arlan Norman Award for Excellence in Student Mentoring and National Institutes of Health/National Heart, Lung and Blood Institute [award numbers R15 HL103518 and U54HL141981] to PCS and National Institutes of Health/National Heart, Lung and Blood Institute [award numbers R44HL117511, R44HL110448, U54HL112309 and U54HL141981 to CBD, HTS and PL].
• Keywords: Cardiovascular System & Cardiology; HIGH-LEVEL EXPRESSION; GENE-THERAPY; CRYSTAL-STRUCTURE; Science & Technology; Peripheral Vascular Disease; IMMUNE-RESPONSE; x-ray crystallography; HEMOPHILIA-A; ACTIVATED FACTOR-VIII; RECOMBINANT FACTOR-VIII; MEMBRANE-BINDING; Hematology; blood coagulation; COFACTOR ACTIVITY; VON-WILLEBRAND-FACTOR; factor VIII; Life Sciences & Biomedicine
• Research Categories: Biology, Genetics; Biology, General

Abstract

Background: Coagulation factor VIII represents one of the oldest protein-based therapeutics, serving as an effective hemophilia A treatment for half a century. Optimal treatment consists of repeated intravenous infusions of blood coagulation factor VIII (FVIII) per week for life. Despite overall treatment success, significant limitations remain, including treatment invasiveness, duration, immunogenicity, and cost. These issues have inspired research into the development of bioengineered FVIII products and gene therapies. Objectives: To structurally characterize a bioengineered construct of FVIII, termed ET3i, which is a human/porcine chimeric B domain-deleted heterodimer with improved expression and slower A2 domain dissociation following proteolytic activation by thrombin. Methods: The structure of ET3i was characterized with X-ray crystallography and tandem mass spectrometry-based glycoproteomics. Results: Here, we report the 3.2 Å crystal structure of ET3i and characterize the distribution of N-linked glycans with LC-MS/MS glycoproteomics. This structure shows remarkable conservation with the human FVIII protein and provides a detailed view of the interface between the A2 domain and the remaining FVIII structure. With two FVIII molecules in the crystal, we observe two conformations of the C2 domain relative to the remaining FVIII structure. The improved model and stereochemistry of ET3i served as a scaffold to generate an improved, refined structure of human FVIII. With the original datasets at 3.7 Å and 4.0 Å resolution, this new structure resulted in improved refinement statistics. Conclusions: These improved structures yield a more confident model for next-generation engineering efforts to develop FVIII therapeutics with longer half-lives, higher expression levels, and lower immunogenicity.