Increased susceptibility of recombinant type 2A von Willebrand factor mutant A1500E to proteolysis by ADAMTS13 / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the susceptibility of von Willebrand factor (VWF) type 2A mutant A1500E to proteolysis by metalloprotease ADAMTS13 and to provide the direct supports for the pathogenesis of VWF mutation A1500E responsible for von Willebrand disease (VWD) type 2A.</p><p><b>METHODS</b>Recombinant wild-type VWF (WT-VWF) and A1500E mutant VWF transiently expressed on transfected HeLa cell lines. Expression media were collected and concentrated, then cleaved directly by recombinant ADAMTS13 (rADAMTS13). Compared with WT-VWF, the susceptibility of A1500E mutant VWF to proteolysis by ADAMTS13 was analyzed using SDS-agarose gel VWF multimers analysis.</p><p><b>RESULTS</b>In vitro the expression of VWF:Ag in the supernatants of WT-VWF and A1500E mutant VWF were 1.10 U/ml and 0.78 U/ml, respectively, while VWF:Ag in cells lysates of A1500E mutant VWF was 90.6% of that of WT-VWF. The SDS-agarose gel VWF multimers analysis showed that there were no differences between WT-VWF and A1500E mutant VWF. The A1500E mutant VWF could be efficiently cleaved by ADAMTS13 under static condition without denaturants such as urea and guanidine HCl. VWF multimeric analysis showed that high and intermediate molecular weight multimers dramatically decreased while low molecular weight multimers obviously increased. Conversely, WT-VWF could not be cleaved by ADAMTS13 under the same condition.</p><p><b>CONCLUSION</b>The A1500E mutation resulted in VWF more susceptible to ADAMTS13-dependent proteolysis, which belonged to VWD type 2A group 2 mutation.</p>

Gene mutation analysis of one case with von willebrand disease type 2A / 中国实验血液学杂志

Objective of this study was to identify gene mutation involved in a patient with type 2A von Willebrand disease (vWD). The bleeding time, vWF:Ag, FVIII:C, RIPA and multimeric assay were used for phenotypic diagnosis. All of the 52 exons and the exon-intron boundaries of vWF gene were amplified by polymerase chain reaction (PCR) and direct sequencing was carried out. The results indicated that the levels of vWF:Ag, FVIII:C and RIPA decreased in this patient, the vWF multimer with high and intermediate molecular weight was absent in plasma. The sequencing of genomic DNA revealed a C4738G (L1580V) missense mutation in the vWF gene from the patient. In conclusion, the C4738G (L1580V) missense mutation effecting the form of vWF multimer was responsible to molecular mechanism in this patient with vWD.

Detection of Novel C4517G (Ser743Trp) Mutation in a Family with Type 2A von Willebrand Disease / 대한혈액학회지

Quantitative von Willebrand disease (VWD) are divided into partial deficiency (type 1) and total deficiency (type 3). Qualitative VWD are devided further into four subcategories (2A, 2B, 2M, 2N) based upon the major mechanism by which von Willebrand factor (VWF) function is impaired. Type 2A is characterized by the absence of large molecular weight VWF multimers and a number of mutations have been identified in the region encoding the A2 domain of VWF where a normal cleavage site is situated. Here, we report a case of type 2A VWD in a 5 year-old girl with a novel C4517G (Ser743Trp) mutation, which was also detected in her mother.