Investigation of VWD Type 2B Variants by Light Transmission Aggregometry

Scientific Research Question: Von Willebrand disease (VWD) type 2B is characterized as a bleeding disorder of primary hemostasis with reduced high molecular weight multimers of von Willebrand factor (VWF) due to their enhanced proteolysis by the protease ADAMTS13 and reduction of free plasma VWF caused by vessel damage-independent binding to platelets. The result is formation of platelet-VWF-conglomerates leading to diagnosis of thrombocytopenia. The molecular pathomechanism is based on VWF gain-of-function mutations in its A1 domain, which increase binding to platelet GPIbα. We strived to investigate ADAMTS13 degradation of these conglomerates by Light Transmission Aggregometry (LTA).

Methodology: We recombinantly expressed VWD type 2B variants previously identified in patients. Turbidity of a solution of 300*10³ washed platelets per µl was measured employing LTA. Changes in turbidity were first recorded after addition of VWF 2B variants and further after addition of recombinant ADAMTS13.

Findings: Most of the investigated recombinant VWF 2B variants immediately led to formation of aggregates when added to the platelet solution. For a few mutants presence of Ristocetin was necessary to induce aggregate formation but in lower doses compared with wildtype (wt) VWF. All variants displayed less sensitivity to ADAMTS13 cleavage than wtVWF after binding to platelets.

Conclusions: LTA is a useful tool to investigate VWF type 2B variants. The decrease in ADAMTS13 sensitivity indicates that enhanced degradation of VWF in 2B patients in most likely due to increased proteolysis of surface-bound VWF rather than cleavage of VWF-platelet aggregates in circulation.

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