Page 239 - Haematologica - Vol. 105 n. 6 - June 2020

The FVIII binding site on VWF
D’ region Arg782-Cys799 shows reduced deuterium incorporation in the presence of FVIII. HDX-MS was employed on the FVIII-D’-D3 complex
The complex was transferred to D2O-containing buffer and the incorporation of deuterium was assessed at three
different time points. The obtained results were compared to the time-dependent deuterium incorporation in D’-D3 in the absence of FVIII. Most peptides originating from the FVIII-D’-D3 complex did not show a change in deuterium uptake compared to isolated D’-D3 (Figure 2 and Online
A
B
Figure 1. Hydrogen-deuterium exchange mass spectrometry analysis of the D’-D3 fragment. D’-D3 was incubated for 10 sec, 100 sec and 1000 sec in a deuterium buffer consisting of 20 mM HEPES (pH 7.4), 150 mM NaCl and 5 mM CaCl2. D’-D3 was processed for hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis as described in the methods. (A) Shows the identified peptides as blue lines underneath the primary sequence of D’-D3. (B) Shows the percentage of deu- terium incorporation for the individual identified peptides for the different incubation times with deuterium buffer. The sequence of the peptide’s numbers, shown on the x-axis, is displayed in the Online Supplementary Table S1.
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