Degradation of VWD Type 2B Variants by ADAMTS-13 in Simulated Circulation

von Willebrand disease (VWD) is characterized as a bleeding disorder of primary hemostasis induced by mutations of the von Willebrand factor (VWF) gene. Depending on the mutations, either quantitative (types 1 and 3) or qualitative (type 2), deficiencies of VWF can be generated. Mostly, loss-of-function mutations are causative for the bleeding phenotype. However, in the case of VWD type 2B, gain-of-function mutations in the VWF A1 domain result in spontaneous binding of VWF to platelets in the absence of injury and subsequent thrombocytopenia. Furthermore, in some type 2B patients, reduced high-molecular-weight multimers of VWF can be observed due to their enhanced proteolysis by the protease ADAMTS-13.

We strived to investigate degradation of VWF–platelet complexes by ADAMTS-13 employing a modified agglutination assay. To this end, we recombinantly expressed VWD type 2B variants, p.Pro1266Leu, p.Arg1306Trp, p.Arg1308Cys, p.Ile1309Val, p.Val1316Met, and p.Ala1461Asp, which were previously identified in VWF 2B patients. Turbidity of a solution of 300*103 washed platelets per microliter was measured using light transmission aggregometry (LTA). Changes in turbidity were first recorded after addition of VWF 2B mutants and further after adding recombinant ADAMTS-13. Both proteins were used at their normal plasma concentrations.

Our data show that most of the investigated recombinant VWF 2B mutants immediately led to agglutination when added to the platelet solution. The presence of ristocetin was necessary only to induce VWF–platelet complex formation for variant p.Pro1266Leu, but at lower doses compared to wild type (wt) VWF. While variants p.Arg1306Trp, p.Arg-1308Cys, and p.Ile1309Val were cleaved to a comparable degree as wtVWF, p.Pro1266Leu and p.Val1316Met displayed less sensitivity to ADAMTS-13 cleavage than wtVWF after binding to platelets. Interestingly, platelet complexes with p.Ala1461Asp could not be degraded by ADAMTS-13.

In conclusion, LTA is a useful tool to investigate VWF type 2B variants. Since we did not find an enhanced ADAMTS-13 sensitivity, our data indicate that increased degradation of VWF in 2B patients is most likely due to increased proteolysis of surface-bound VWF rather than cleavage of VWF–platelet complexes in circulation.

^*The first two authors contributed equally to this study.

No conflict of interest has been declared by the author(s).

Publication History

Article published online:
13 November 2020