Identification of fibrin contact regions in the A2 domain of VWF. (A) Crystal structure of the A2 domain. Magenta, green, and blue colors show regions containing putative binding sites for fibrin. The amino acid residue E1567 (green) analyzed in this study is depicted as a point of reference; red shows the location of the ADAMTS-13 cleavage site. (B) Increasing concentrations of either the WT A2 protein or A2 mutant were incubated with immobilized thrombin-generated fibrin in microtiter wells. The A2 mutant had significantly lower binding activity for fibrin (half-maximal binding, 1.03 ± 0.079 µM) than the WT A2 protein (half-maximal binding, 0.06 ± 0.004 µM). Each point in the graph represents the mean ± standard error of the mean of 3 determinations. Use of monoclonal antibody (C) and circular dichroism thermal unfolding (D) shows that the overall structure of the A2 protein was not altered by the mutation E1567A. (D) Assessment of 2 different batches of the WT A2 protein. *P < .05. Abs, absorbance; n.s., not significant.