Intersection of Mechanisms of Type 2A Von Willebrand Disease through Defects in Von Willebrand Factor Multimerization, Secretion, ADAMTS-13 Susceptibility, and Regulated Storage

Type 2A von Willebrand disease (VWD) is characterized by the absence of large von Willebrand factor (VWF) multimers and decreased platelet binding function. These variants are classified as either group 1 (2A-1) with impaired assembly and secretion of VWF multimers, or group 2 (2A-2) with increased susceptibility to proteolysis by ADAMTS13. However, laboratory parameters in individual patients may not discriminate between 2A-1 and 2A-2; this must be established through expression studies. Type 2A VWD patients recruited through the TS Zimmerman Program For The Molecular And Clinical Biology Of VWD (ZPMCB VWD) were phenotypically identified based upon laboratory parameters. By sequencing genomic DNA we identified 13 potentially causative sequence variations in the VWF D2, D3, A1, and A2 domains of type 2A VWD patients. Expression studies were performed to examine the effect of these mutations on

1. VWF processing and proteolysis, and

2. VWF regulated storage. The VWF variants L1503R, S1506L, and V1607D demonstrated severely impaired secretion and lacked mid- and high-molecular weight multimers, consistent with a group 1 classification (2A – 1).

Some variants including I1568N, G1579R, and G1631D were normally multimerized and secreted, but had an increased susceptibility to cleavage by ADAMTS13 cleavage, indicative of group 2 classification (2A – 2). Five variants involving cysteine residues (C1190S, C1099P, C1272R/S/Y) had multimer abnormalities, a modest reduction in secretion, and variably enhanced ADAMTS-13 cleavage. These variants did not easily fit the criteria for either 2A subgroup. We also identified sequence variations (M740I and I1380V) that had no effect on VWF secretion, structure, or proteolysis that are likely innocuous polymorphisms but are present in patients with a type 2A phenotype. VWF is stored for regulated release in endothelial cell Weibel-Palade bodies and in platelet alpha-granules. The effect of mutations on VWF regulated storage has been documented for few VWF variants. The 2A variants were expressed in HEK293 cells, immunostained, and examined by confocal microscopy. Several variants including C1272S/Y, L1503R, S1506L, and V1607D did not form storage granules. A loss or reduction in endothelial cell Weibel-Palade bodies may explain the diminished desmopressin response observed in many type 2A VWD patients. In sum, our data indicate that 2A-2 variants are associated with VWF A2 domain mutations and normal regulated storage, while 2A-1 mutations are not restricted to a particular domain and result in loss of regulated VWF storage. Mutations involving cysteines may not affect VWF secretion, but are likely to cause abnormalities in multimer structure. In summary, type 2A VWD appears to result from at least three intersecting mechanisms: intracellular retention, defective multimerization, or increased plasma proteolysis.