Abstract Number: OC 07.4
Meeting: ISTH 2022 Congress
Theme: Hemophilia and Rare Bleeding Disorders » Hemophilia - Basic
Background: The B domain of coagulation Factor VIII (FVIII) has a heavily glycosylated and highly disordered structure. Traditional structural characterization methods are unable to resolve the structure of the B domain and hence of full length FVIII (FL-FVIII).
Aims: Our study aims to structurally characterize the FVIII B domain using integrated hybrid methodology [Figure 01]. Bioanalytical techniques such as cross-linking mass spectrometry (XL-MS) are being used on purified FL-FVIII to identify interdomain (between B domain and other domains of FVIII) and intradomain (within B domain) interface residues. The interdomain and intradomain interface residues will be used as modeling restraints for modeling the structure of the B domain and of FL-FVIII. Simultaneously biophysical techniques such as AFM and cryo-EM are being carried out on the purified FL-FVIII. The structural models will be validated by matching/fitting/docking them onto biophysical maps/images generated.
Methods: Plasma concentrates and recombinant FL-FVIII products are further purified by various chromatography methods to remove all excipients. One mM of DSSO crosslinker is used to crosslink 10uM of pure FL-FVIII and the cross-linked product is further purified by SEC (size exclusion chromatography). Glycosyl residues in FL-FVIII were removed by de-glycosylation reaction using 5000 units of PNGase-F enzyme. Negative staining of the purified FL-FVIII followed by 2D classification was done towards future cryo-EM studies. Air and liquid AFM imaging was also performed on the highly pure FL-FVIII. The FL-FVIII model from the alpha-fold database was modified in silico to incorporate all post-translational modification and subjected to all-atom MD simulation.
Results: Pure FL-FVIII was successfully crosslinked with DSSO towards future XL-MS data. Negative staining and AFM of FL-FVIII showed structural heterogeneity in FL-FVIII which is also reflected in the simulation trajectory of the alpha-fold model of FL-FVIII.
Conclusion(s): FL-FVIII is structurally heterogeneous owing to the conformational variability of the B domain.
To cite this abstract in AMA style:
Ramaraje Urs S, Singh S, Javed H, Fenel D, Teulon J, Schoehn G, Pellequer J, Ma B, Ugurlar D, Imhof D, Oldenburg J, Biswas A. Exploring the conformational landscape of Factor VIII B domain in order to generate an all atom full length structure of the coagulation Factor VIII protein [abstract]. https://abstracts.isth.org/abstract/exploring-the-conformational-landscape-of-factor-viii-b-domain-in-order-to-generate-an-all-atom-full-length-structure-of-the-coagulation-factor-viii-protein/. Accessed March 21, 2024.« Back to ISTH 2022 Congress
ISTH Congress Abstracts - https://abstracts.isth.org/abstract/exploring-the-conformational-landscape-of-factor-viii-b-domain-in-order-to-generate-an-all-atom-full-length-structure-of-the-coagulation-factor-viii-protein/