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Updated: May 20, 2026
Investigating von Willebrand Factor Pathophysiology Using a Flow Chamber Model of von Willebrand Factor-platelet String Formation
Published on: August 14, 2017
[Increased susceptibility of recombinant type 2A von Willebrand factor mutant A1500E to proteolysis by ADAMTS13]
Jing-yu Zhang1, Jian Su, Zhen-ni Ma
1Soochow University, Suzhou, China.
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.
Conclusion:
The A1500E mutation resulted in VWF more susceptible to ADAMTS13-dependent proteolysis, which belonged to VWD type 2A group 2 mutation.
Insights
The von Willebrand factor (VWF) A1500E mutation increases susceptibility to ADAMTS13 proteolysis, confirming its role in von Willebrand disease (VWD) type 2A. This finding clarifies VWD pathogenesis.
Area of Science:
- Hematology
- Molecular Biology
- Genetics
Background:
- Von Willebrand disease (VWD) is a bleeding disorder caused by defects in von Willebrand factor (VWF).
- VWF type 2A mutations are characterized by reduced high molecular weight multimers, leading to impaired platelet adhesion.
- The specific role of the A1500E mutation in VWF type 2A pathogenesis requires further elucidation.
Purpose of the Study:
- To investigate the proteolysis susceptibility of the VWF type 2A mutant A1500E by ADAMTS13.
- To provide direct evidence for the pathogenesis of the VWF A1500E mutation in VWD type 2A.
Main Methods:
- Recombinant wild-type VWF (WT-VWF) and A1500E mutant VWF were expressed in HeLa cells.
- Expression media were analyzed for VWF antigen (VWF:Ag) levels.
- Proteolysis by recombinant ADAMTS13 (rADAMTS13) was assessed using SDS-agarose gel VWF multimer analysis.
Main Results:
- A1500E mutant VWF showed reduced VWF:Ag levels in supernatant compared to WT-VWF.
- A1500E mutant VWF was efficiently cleaved by ADAMTS13 under static conditions, unlike WT-VWF.
- VWF multimer analysis revealed a decrease in high and intermediate molecular weight multimers and an increase in low molecular weight multimers for A1500E mutant VWF after ADAMTS13 treatment.
Conclusions:
- The A1500E mutation confers increased susceptibility to ADAMTS13-dependent proteolysis.
- This heightened proteolysis explains the characteristic VWF multimer deficiency in VWD type 2A.
- The A1500E mutation is confirmed as a VWD type 2A group 2 mutation.
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