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Date Permissions Signed
Date of Award
Department or Program Affiliation
Master of Science (MS)
Spiegel, P. Clint
Anthony-Cahill, Spencer J.
Factor VIII (FVIII) is a 2332 amino acid glycoprotein with domain organization of A1-A2-B-A3-C1-C2 which is a crucial component of the blood coagulation cascade. After secretion, FVIII circulates in the bloodstream at a concentration of one nanomolar bound to von Willebrand Factor (vWF) which protects FVIII from clearance. In the event of an injury to the bloodstream, FVIII is proteolytically cleaved, releases from vWF and binds to both activated platelet surfaces and activated Factor IX with nanomolar affinity. These interactions increase the rate of blood clot formation 100,000 fold. Hemophilia A is an x-linked recessive disease affecting 1 in 5000 males which is due to a deficiency of functional FVIII. Treatment involves prophylactically injecting FVIII into the bloodstream. Although this treatment works, the short half-life of FVIII demands multiple injections per week and nearly one-third of patients receiving treatment develop antibody inhibitors which target injected FVIII. This results in dramatically worse quality of life for hemophilia A patients. Greater understanding of the physiological role of FVIII in its interactions with vWF and inhibitor antibodies can lead to improved therapy by extending the half-life of engineered FVIII therapeutics and reducing immunogenicity. Towards this end, FVIII chimeric variant Et3i was co-crystallized in two different complexes. Et3i is a B-domain deleted chimera consisting of porcine A1 and A3 domains and human A2, C1 and C2 domains. The first structure was with the antigen binding fragment of murine inhibitory antibody 2A9 which targets the C1 domain of FVIII and inhibits platelet surface binding as well as vWF binding. The crystals grown resulted in a structure with a resolution of 3.9 Å, r-free of 0.34 and r-work of 0.27. This structure is the first crystal structure to date showing complete FVIII bound to an inhibitory antibody fragment and sheds new light on the physiological function of FVIII with respect to its binding interactions. The second structure is of Et3i bound to Til’E’, the domains of vWF which give rise to the high-affinity interaction with FVIII. This structure consists of two copies of FVIII at a resolution of 2.8 Å. Unfortunately, the model is not complete and appears to not contain the Til’E’ domains. However, the data is the highest resolution ever collected for FVIII and, when completed, the structure will allow for improved insight into the function of FVIII.
Hemophilia A, Coagulation, Inhibitor Antibody, 2A9, Til'E', C1 Domain, ET3i
Western Washington University
Subject – LCSH
Blood coagulation factor VIII; Glycoproteins--Structure; Blood proteins--Structure; Blood coagulation factors; Hemophilia--Treatment; Von Villebrand disease--Treatment
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Gish, Joseph S., "Structural Studies of Complexes of Blood Coagulation Factor VIII" (2019). WWU Graduate School Collection. 902.