D'domain Region Arg782-Cys799 of Von Willebrand Factor contributes to Factor VIII binding.

D'domain Region Arg782-Cys799 of Von Willebrand Factor contributes to Factor VIII binding. Haematologica. 2019 Sep 26;: Authors: Przeradzka MA, van Galen J, Ebberink EHTM, Hoogendijk AJ, van der Zwaan C, Mertens K, van den Biggelaar M, Meijer AB Abstract In the complex with Von Willebrand Factor, factor VIII (FVIII) is protected from rapid clearance from the circulation. Although it has been established that the FVIII binding site resides in the N-terminal D'-D3 domains of Von Willebrand Factor, detailed information about the amino acid regions that contribute to FVIII binding is still lacking. In the present study, Hydrogen- deuterium exchange mass spectrometry was employed to gain insight into the FVIII binding region on Von Willebrand Factor. To this end, time-dependent deuterium incorporation was assessed in D'-D3 and the FVIII - D'-D3 complex. Data showed reduced deuterium incorporation in D region Arg782-Cys799 in the FVIII - D'-D3 complex compared to D'- D3. This implies that this region interacts with FVIII. Site-directed mutagenesis of the six-individual charged amino acids in Arg782-Cys799 into alanine residues followed by surface plasmon resonance analysis and solid phase binding studies revealed that replacement of Asp796 affected FVIII binding. A marked decrease in FVIII binding was observed for the D'-D3 Glu787Ala variant. The same was observed for D'-D3 variants in which Asp796 and Glu787 were replaced by Asn796 and Gl...
Source: Haematologica - Category: Hematology Authors: Tags: Haematologica Source Type: research