Characterisation of von Willebrand factor A1 domain mutants I1416N and I1416T: correlation of clinical phenotype with flow‐based platelet adhesion

Background: Type 2M von Willebrand disease (VWD) results from mutations in the A1 domain of von Willebrand factor (VWF) that reduce its platelet‐binding function. However, currently employed VWF functional static assays may not distinguish between clinical phenotype. Methods: Fifteen individuals from five kindreds with VWF‐A1 domain mutations I1416T or I1416N, correlated with mild and moderate clinical phenotypes, respectively, were investigated. The mutations were reproduced by site‐directed mutagenesis and expressed in HEK293T cells; functional studies of the recombinant mutants, including GPIbα binding using a flow‐based assay, were performed. Results: Plasma from all individuals demonstrated discordant reductions in VWF antigen and platelet‐binding function in the presence of high‐molecular‐weight VWF multimers consistent with VWD type 2M. There was lowered expression and secretion of both mutants compared with wild type (WT) recombinant (r)VWF as well as a significant reduction in GPIbα binding. Binding to collagen was normal and electrophoretic analysis demonstrated a similar multimer distribution between the mutant proteins and wt‐rVWF. GPIbα binding under flow was also significantly reduced for I1416N and I1416T rVWF. Impairment of GPIbα binding was more marked for I1416N rVWF than I1416T under both static and flow conditions: this was in spite of similar VWF:Ristocetin cofactor (RCo) activities in patient plasma and is consistent with a respective clinical phenotype. Conclusions: Our findings have established for the first time that I1416N and I1416T are responsible for a type 2M VWD phenotype and demonstrate that quantification of VWF function under shear stress may provide a more accurate measure of clinical severity than the static functional measurements in current diagnostic use.