Impact of mutations in the von Willebrand factor A2 domain on ADAMTS13-dependent proteolysis

Abstract

Classical von Willebrand disease (VWD) type 2A, the most common qualitative defect of VWD, is caused by loss of highmolecular-weight multimers (HMWMs) of von Willebrand factor (VWF). Underlying mutations cluster in the A2 domain of VWF around its cleavage site for ADAMTS13. We investigated the impact of mutations commonly found in patients with VWD type 2A on ADAMTS13-dependent proteolysis of VWF. We used recombinant human ADAMTS13 (rhuADAMTS13) to digest recombinant full-length VWF and a VWF fragment spanning the VWF A1 through A3 domains, harboring 13 different VWD type 2A mutations (C1272S, G1505E, G1505R, S1506L, M1528V, R1569del, R1597W, V1607D, G1609R, I1628T, G1629E, G1631D, and E1638K). With the exception of G1505E and I1628T, all mutations in the VWF A2 domain increased specific proteolysis of VWF independent of the expression level. Proteolytic susceptibility of mutant VWF in vitro closely correlated with the in vivo phenotype in patients. The results imply that increased VWF susceptibility for ADAMTS13 is a constitutive property of classical VWD type 2A, thus explaining the pronounced proteolytic fragments and loss of HMWM seen in multimer analysis in patients.

Mutation G1629E Increases von Willebrand Factor Cleavage via a Cooperative Destabilization Mechanism

“…Note, that even in the absence of ADAMTS13 the VWF mutant was degraded to some extent just after dialysis in the 5 mM Tris-HCl. Hence, our recombinant proteins (wild-type and mutated) reproduced the same cleavage response as in previous studies (17).…”

“…C-terminal domain. Full-length ADAMTS13 cDNA was cloned into the expression vector pIRES neo2 for stable transfection of HEK 293 cells by liposomal transfer under selective pressure 48 h after transfection (17). Wild-type rhuADAMTS13, secreted to the medium, was used for cleavage experiments.…”

“…In a broad study, Hassenpflug et al investigated the impact of 13 different clinically relevant VWD type 2A mutations on ADAMTS13-dependent proteolysis (17). They found that 11 of the studied mutants showed increased specific proteolysis compared with wild-type VWF.…”

“…Although the effect of the Gly1629Glu (G1629E) mutation on the static multimer size distribution has been determined (17), quantification of its impact on the cleavage kinetics is lacking so far. Furthermore, the molecular mechanism by which this mutation drastically enhances proteolytic cleavage remains to be fully resolved.…”

Mechanisms by which von Willebrand Disease Mutations Destabilize the A2 Domain*

“…Patients with type 2A VWD present with increased bleeding due to the decreased thrombogenicity of smaller VWF multimers (9). Certain mutations have been described to impair VWF biosynthesis (group 1) (10), whereas other mutations increase VWF proteolysis despite normal VWF multimer assembly and secretion (group 2) (11,12). Group 2A mutations cluster within the A2 domain and are thought to enhance cleav-age by disrupting the stability of the folded A2 structure.…”