Structure-resolved dynamics of type 2M von Willebrand disease

Genetically determined amino acid substitutions in the platelet adhesive A1 domain alter von Willebrand factor's (VWF) platelet agglutination competence, resulting in both gain- (type 2B) and loss-of-function (type 2M) phenotypes of von Willebrand disease. Prior studies of variants in both phenotypes revealed defects in secondary structure that altered stability and folding of the domain. An intriguing observation was that loss of function arose from both misfolding of A1 and, in a few cases, hyperstabilization of the native structure.

To fully understand the 2M phenotype, we thoroughly investigated the structure/function relationships of 15 additional type 2M variants and 2 polymorphisms in the A1 domain.

These variants were characterized using circular dichroism, fluorescence, calorimetry, hydrogen-deuterium exchange mass spectrometry, surface plasmon resonance, and platelet adhesion under shear flow.

Six variants were natively folded, with 4 being hyperstabilized. Nine variants disordered A1, causing a loss in α-helical structure and unfolding enthalpy. GPIbα binding affinity and platelet adhesion dynamics were highly correlated to helical structure. Hydrogen-deuterium exchange resolved specific C-terminal secondary structure elements that differentially diminish the GPIbα binding affinity of A1. These localized structural perturbations were highly correlated to GPIbα binding affinity and shear-dependent platelet adhesion.

While hyperstabilized dynamics in A1 do impair stable platelet attachment to VWF under flow, variant-induced localized disorder in specific regions of the domain misfolds A1 and abrogates platelet adhesion. These 2 opposing conformational properties represent 2 structural classes of VWF that drive the loss-of-function phenotype that is type 2M von Willebrand disease.