Design, expression, and initial characterization of von Willebrand Factor A2 domain and its mutants

Abstract The von Willebrand Factor (VWF) is an essential clotting factor in hemostasis. It is secreted into blood plasma as an ultralarge multimer to mediate platelet adhesion at vascular injured sites. The VWF can be cleaved to smaller multimers by the metalloprotease ADAMTS13, which is the feedback control to maintain the proper thrombogenic potential of the VWF. The A2 domain of the VWF contains the only cleavage site for ADAMTS13. Mutations in the A2 domain may lead to type-2M von Willebrand Disease (VWD), which is characterized by a bleeding phenotype and a lack of large VWF multimers in blood plasma.This project focuses on the design, expression, and characterization of three A2 mutants: Q1541R, N1515Q, and N1574Q. Q1541R is a newly identified, type-2M VWD mutation that causes a bleeding phenotype. N1515Q and N1574Q are artificial mutants with N-linked glycans removed at sites N1515 and N1574, respectively. Genes encoding wild-type and mutant A2 domains were PCR-amplified and constructed in the pHLsec vector. N-terminal SpyTag and C-terminal Avi- and His(6)- tags were added to the constructs. Wild-type and mutant A2 proteins were expressed in HEK 293T cells, and purified from a culture supernatant using Ni-NTA chromatography, followed by size exclusion chromatography. The proteins were biotinylated using an Avi-tag biotinylation kit. The purified A2 and mutants were characterized by the ADAMTS13 cleavage assay and optical-tweezer pulling assay. Our data suggest that the Q1541R and N1574Q mutants are more susceptible to forced unfolding and ADAMTS13 cleavage compared to the wild-type A2.