Mutations and common polymorphisms in ADAMTS13 gene responsible for von Willebrand factor-cleaving protease activity

Abstract

von Willebrand factor (VWF) is synthesized primarily in vascular endothelial cells and secreted into the plasma as unusually large VWF multimers. Normally, these multimers are quickly degraded into smaller forms by a plasma metalloproteinase, VWF-cleaving protease (VWF-CP). Decreases in the activity of this enzyme result in congenital and acquired thrombotic thrombocytopenic purpura (TTP). The human VWF-CP has recently been purified. Cloning of the corresponding cDNA revealed that the 1,427-aa polypeptide is a member of the ADAMTS gene family, termed ADAMTS13. Twelve rare mutations in this gene have been identified in patients with congenital TTP. Here, we report missense and nonsense mutations in two Japanese families with Upshaw-Schulman syndrome, congenital TTP with neonatal onset and frequent relapses. The comparison of individual ADAMTS13 genotypes and plasma VWF-CP activities indicated that the R268P, Q449stop, and C508Y mutations abrogated activity of the enzyme, whereas the P475S mutant retained low but significant activity. The effects of these mutations were further confirmed by expression analysis in HeLa cells. Recombinant VWF-CP containing either the R268P or C508Y mutations was not secreted from cells. In contrast, Q449stop and P475S mutants were normally secreted but demonstrated minimal activity. Genotype analysis of 364 Japanese subjects revealed that P475S is heterozygous in 9.6% of individuals, suggesting that approximately 10% of the Japanese population possesses reduced VWF-CP activity. We report on a single-nucleotide polymorphism associated with alterations in VWF-CP activity; it will be important to assess this single-nucleotide polymorphism as a risk factor for thrombotic disorders.

Figure 1

Pedigrees and haplotypes of patient families. (A) Family A. (B) Family B. Squares and circles indicate male and female, respectively, and arrows indicate the probands. The VWF-CP activities determined by multimer analysis, as reported (5), are shown as a percentage of the normal control. Missense and nonsense mutations found in the ADAMTS13 gene are shown in one-letter amino acid residues numbering from the initiation methionine. (C) Schematic domain structure of VWF-CP. S, signal peptide; P, propeptide; D, disintegrin-like; T, TSP1; Cys, Cys-rich.

Figure 2

Expression of recombinant VWF-CP. The human WT VWF-CP and mutants with C-terminal FLAG-tag were transiently expressed in HeLa cells. The culture media (Upper) and cell lysates (Lower) were analyzed by Western blot with an anti-FLAG antibody. The representative of three repetitive experiments is shown. The sizes of the protein standards are indicated at the left.

Figure 3

N-glycosidase treatment of recombinant VWF-CP. FLAG-tagged WT VWF-CP derived from culture media and cell lysates were treated with PNGase F and analyzed by Western blot with an anti-FLAG antibody. The sizes of the protein standards are indicated at the left.

Figure 4

Cleavage of VWF multimers. The culture media of HeLa cells either untransfected or transfected with VWF-CP expression plasmids were incubated with purified VWF. A portion of each reaction mixture was then separated by SDS-agarose gel electrophoresis. The multimeric states of VWF were visualized by Western blot analysis with an anti-VWF antibody. The representative of three repetitive experiments is shown. HMW, high molecular weight; LMW, low molecular weight.