title: Mechanisms by which von Willebrand disease mutations destabilize the A2 domain.
abstract: von Willebrand Factor (VWF) is an ultralong, concatameric, and adhesive glycoprotein. On short time scales, adhesiveness for platelets is activated by elongation of VWF by altered hydrodynamics at sites of hemostasis. Over longer time scales, the length of VWF is regulated by ADAMTS13 (a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13), cleavage by which in the VWF A2 domain is dependent on elongational force. Patients with von Willebrand disease type 2A present with increased bleeding due to mutations within the VWF A2 domain that enhance cleavage. We tested using temperature and force the hypothesis that von Willebrand disease mutations disrupt A2 force sensing by destabilizing the folded state. Mutations R1597W, M1528V, and E1638K reduced A2 thermal stability by 10-18 °C. M1528V and E1638K showed a marked further decrease in stability upon calcium removal. In contrast, R1597W, which resides within the A2 calcium-binding loop, exhibited similar stability in the presence and absence of calcium. Using single molecule optical tweezers and R1597W, we measured the force dependence of unfolding and refolding kinetics. In the presence of calcium, the R1597W mutation slowed the rate of refolding but had no effect on unfolding. The three mutations highlight the calcium-binding loop (R1597W), the hydrophobic core around the vicinal disulfide (M1528V), and hydrogen bonds to the α4-less loop (E1638K), as structural features critically important to the function of A2 as a force sensor in regulating thrombogenic activity in the vasculature.
authors: Program in Cellular and Molecular Medicine, Children's Hospital Boston, and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors: Amy J
authors: AJ
authors: Xu
authors: Timothy A
authors: TA
authors: Springer
mesh_terms: D019943:Amino Acid Substitution; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D020125:Mutation, Missense; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014842:von Willebrand Diseases; D014841:von Willebrand Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D014841:von Willebrand Factor; D002118:Calcium