Characterization of activity and cleavage of von Willebrand disease type 2B variants

Objective Von Willebrand disease type 2B (VWD2B) is a bleeding disorder of primary hemostasis, characterized by variable lack of von Willebrand factor (VWF) high molecular weight multimers (HMWM) and varying degree of thrombocytopenia. VWD2B is caused by gain-of-function (GOF) mutations in the VWF A1 domain inducing spontaneous binding to platelets. Objective of this study was the identification of the underlying mutations in patients with suspected VWD2B and functional characterization of the respective variants.

Material and Methods VWF exon 28 was sequenced in patient DNA samples. To circumvent the requirement of fresh platelet-rich plasma for the Ristocetin Induced Platelet Aggregation (RIPA), we used a modified VWF:GPIbα binding ELISA employing a recombinant GPIbα peptide as capture component to determine GPIbα binding of plasmatic and recombinant variants. Degradation of the variants by ADAMTS13 was measured employing a modified light transmission aggregometry (LTA) assay.

Results By genetic analysis of 113 patients with suspected VWD2B, we found 15 different mutations (Figure 1A). p.Arg1315Cys could be excluded from the investigation of GPIbα binding as it has previously been characterized as VWD2 M. All variants, except p.Val1279Ile, exhibited significantly increased GPIbα binding (Figure 1B). Thereby, diagnosis of VWD2B was confirmed for 13 mutations. We further characterized those sixteen patients, in more detail, for whom complete clinical data and VWF multimers were available and found a correlation between degree of increased GPIbα binding and loss of HMWM for some mutations. Since the latter could be caused by increased cleavage of the variants in vivo, we employed a modified LTA assay to investigate degradation of 2BVWF-platelet-complexes by ADAMTS13. Counterintuitively, we found that some variants show decreased sensitivity for proteolytic cleavage under flow conditions.

Conclusion Summarizing, we characterized VWD2B variants found in a VWD2B patient cohort. The used ELISA proved to be applicable to differentiate 2B variants from other types of VWD and the absence of patient platelets prevents false positive results due to platelet type-VWD. Additionally, our data indicate that increased proteolysis of some variants does not arise from enhanced degradation of circulating 2BVWF-platelet-complexes. Our data could increase understanding of VWD2B disease phenotypes.

Fig. 1 (A) Distribution of mutations identified in suspected VWD2B patients and (B) GPIbα binding of the recombinant variants.

Publication History

Article published online:
18 June 2021