Structural and Thermodynamic Studies of Antibody Binding to Blood Coagulation Factor VIII

Blood coagulation factor VIII (FVIII) is a 2332 residue glycoprotein expressed in endothelial cells and plays a significant role in the formation of blood clots. Structurally, FVIII's domains are organized as A1-A2-B-A3-C1-C2. The absence or deficiency of FVIII in the bloodstream gives rise to Hemophilia A; an X-linked bleeding disorder affecting 1 in 5000 males worldwide. To combat this deficiency, patients undergo FVIII replacement therapy which involves frequent injections of FVIII into the bloodstream in the form of blood, plasma, or protein concentrates. Although effective, this treatment commonly results in the development of anti-FVIII inhibitory antibodies in approximately 20-30% of patients. Across the domains of FVIII, antibody development is most frequently seen for the A2, C2, and A3-C1 domains. Previous crystal structures from the Spiegel lab have led to a wealth of understanding of how inhibitory antibodies interact with the C2 and C1 domains. Two of the crystal structures, those of anti-C2 inhibitors 3E6 and G99, revealed a potential positive cooperation between the two inhibitors stemming from a rigidification of the C2 domain upon G99's binding. Using isothermal titration calorimetry to investigate this, it was determined that these two inhibitors bind the C2 domain independently without any sort of cooperation. Rigidification of the C2 domain upon G99's binding however was proven using this method. Additionally, competition for FVIII binding was investigated for six anti-A2 domain inhibitors. Using bio-layer interferometry, overlap and lack of overlap was identified for five of the six antibodies, illustrating the diversity of the immune response to injected FVIII. Recent Spiegel lab Cryo-EM structures of have deepened our understanding of how inhibitors bind the C1 and A2 domains to inhibit FVIII. One of such structures is that of human/porcine chimeric construct Et3i bound to the patient-derived antibody NB2E9. In tandem with the data collection for this structure, crystallographic trials were carried out for the isolated C1 domain in complex with the NB2E9 Fab fragment. Despite successful formation of crystals, all failed to diffract.