[Increased susceptibility of recombinant type 2A von Willebrand factor mutant A1500E to proteolysis by ADAMTS13].
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
The A1500E mutation resulted in VWF more susceptible to ADAMTS13-dependent proteolysis, which belonged to VWD type 2A group 2 mutation.
Soochow University, Suzhou, China.
Jing-yu
JY
Zhang
Jian
J
Su
Zhen-ni
ZN
Ma
Ning-zheng
NZ
Dong
Ying-chun
YC
Wang
Chang-geng
CG
Ruan
D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism*; D000071120:ADAMTS13 Protein; D005838:Genotype; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D056728:von Willebrand Disease, Type 2 / Q000235:genetics* / Q000378:metabolism*; D014841:von Willebrand Factor / Q000235:genetics*
D011994:Recombinant Proteins; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human