Functional Characterization of Three VWF- A1 Domain Mutations Causing Type 2 Von Willebrand Disease.

Abstract 1305

Poster Board I-327

Through the Canadian Platelet-Type VWD Project (www.pt-vwd.org), we have identified 3 novel candidate mutations within the A1 domain of VWF gene in 5 patients that were provisionally diagnosed as type 2 VWD. These mutations are: L1460F (2 related patients), Y1363C (1 patient), E1389K (2 related patients). The VWF:Ag ranged from 0.19 – 0.54 IU/mL, VWF:RCo ranged from 0.19- 0.21 IU/mL and platelet count ranged from 206-254 × 10³/ mm³. High Molecular weight multimers of VWF were absent in the plasma of the patient with the 1460 aa change. These substitutions have not been previously reported in the literature or in the International VWF Mutation Database. All three mutations were created by site-directed mutagenesis in the full-length huVWF cDNA and transiently transfected in the HEK293T/17 cell line. The VWF protein secreted in the media was collected and quantified by ELISA before proceeding with Functional VWF-platelet binding studies. Quantification of the mutant rVWF proteins in the media and cell lysates and multimer analysis of the proteins showed normal secretion and multimer distributions. To determine the GpIb binding properties of the putative mutants, the VWF recombinant protein (0.25 ug) from each of the WT and three mutants was incubated with lyophilized platelets (1 × 10⁸) for 10 minutes at 25°C with different concentrations of ristocetin between 0-1 mg/mL. The platelets were then pelleted by centrifugation at 1200 g and the resulting supernatant was tested for the remaining unbound VWF by ELISA. Compared to the WT VWF protein, only the L1460F mutant showed increased platelet binding under lower concentration of ristocetin. Ristocetin-induced platelet agglutination (RIPA) assays performed using these recombinant proteins confirmed enhanced ristocetin responsiveness for L1460F, and that Y1363C and E1389K alternatively showed reduced responsiveness. Based on these data, a type 2B VWD diagnosis can only be made with respect the L1460F and not the other two A1 domain missense mutations. These data confirm once again that the assignment of the phenotype on clinical and laboratory basis is useful prior to genetic analysis and mutation identification. However, in some situations both will be ultimately required to understand the clinical presentation and to assign a disease subtype.