Objective To investigate the mechanism of affinity down-regulation between Von Willebrand factor mutant G561S and its ligand. Methods Three molecular systems were constructed for WT-A1, G561S-A1, and R543Q-A1, respectively. G561S-A1 mutant was constructed by replacing the Gly561 with Ser561 in the wild-type A1 domain. The crystal structures of WT-A1 and R543Q-A1 were downloaded from Protein Data Base (PDB). Free molecular dynamics simulation was performed to observe the changes of conformation, alterations of flexibility, and the formation and evolution of hydrogen bond and/or salt bridge, among the three A1 domains (WT-A1, G561S-A1, and R543Q-A1). ResultsG561S mutation lowered the localized dynamic properties of α2 helix and increased the interactions between the N-terminal arm and body region in A1 domain, thus leading to the decreased binding affinity with its ligand GPIbα. However, the Gain-of-function mutation R543Q followed the pathway which was contrary to G561S. ConclusionsThe change of localized dynamic properties of α2 helix is a potential mechanism in the regulation of the binding affinity of A1, and this research finding is helpful in developing allosteric drugs against the activated A1 domain and relevant anti-thrombus drugs.