Page 241 - Haematologica - Vol. 105 n. 6 - June 2020

The FVIII binding site on VWF
required to reduce FVIII binding to immobilized VWF by 50%. For the Arg782Ala, Glu784Ala, and Glu798Ala vari- ants of D’-D3 about 100 nM was required to reach the same effect. A markedly reduced competition efficiency was observed for the Asp796Ala variant as more than 800 nM was required to reduce the binding to 50%. Almost no competition was observed for D’-D3 Glu787Ala. The data further reveal a biphasic competition curve for the Lys790Ala variant. This implies that D’-D3 Lys790Ala may exist in two conformations that differentially inter- fere with complex formation between FVIII and VWF. We
therefore cannot make any reliable conclusions about the putative role of Lys790 for FVIII binding. Based on the results, we also constructed two new D’D3 variants i.e. Glu787Gln and Asp796Asn and assessed their FVIII bind- ing efficiency using SPR analysis. Results showed that changing the charged amino acids with their neutral coun- terpart also affected FVIII binding (Online Supplementary Figure S2). Changing Glu787 for a Gln in full-length VWF also revealed a major impact on FVIII using a solid phase binding assay (Online Supplementary Figure S3). The data together confirm the observation that amino acid residues
AB
CD
EF
GH
Figure 3. Surface plasmon resonance analysis of D’-D3 variants in interaction with FVIII. (A-G) Multiple concentrations (0-200 nM) of the indicated D’-D3 variants were passed over the coagulation factor VIII (FVIII) that was immobilized via antibody EL14 to the surface of a CM5 sensor chip. The binding response is represented in response units (RU) and was assessed in 20 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM CaCl2, 0.05% (v/v) Tween 20 at a flow rate of 30 μL/min at 25oC. (H) Shows the maximum binding response in RU of the D’-D3 variants at a function of the employed concentration.
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