In-vitro and in-vivo consequences of mutations in the von Willebrand factor cleaving protease ADAMTS13 in thrombotic thrombocytopenic purpura

Abstract

SummaryThrombotic thrombocytopenic purpura (TTP) is a disease characterized by microvascular thrombosis, often associated with deficiency of the von Willebrand factor (VWF) cleaving protease ADAMTS13.We investigated the spectrum of ADAMTS13 gene mutations in patients with TTP and congenital ADAMTS13 deficiency to establish the consequences on ADAMTS13 processing and activity. We describe five missense (V88M, G1239V, R1060W, R1123C and R1219W), 1 nonsense (W1016Stop) and 1 insertion (82_83insT) mutations. In two patients no mutation was identified despite undetectable protease activity. Expression in HEK293 mammalian cells (V88M, G1239V, R1123C and R1219W) documented that three missense mutants were not secreted, whereas theV88M was secreted at low levels and with reduced activity. We also provide evidence that impaired secretion of ADAMTS13 mutants observed in vitro translates into severely reduced ADAMTS13 antigen levels in patients in vivo. To evaluate whether the small amounts of mutant protease present in the circulation of patients had VWF cleaving activity, WT and mutant rADAMTS13 were stably expressed in Drosophila S2 cells under the influence of the Drosophila BiP protein signal sequence, which allows protein secretion. Drosophila expression system showed a 40–60% protease activity in the mutants. Several single nucleotide polymorphisms (SNPs) within exons and intron boundaries were found in patients, suggesting that the interplay of SNPs could at least in part account for ADAMTS13 functional abnormalities in patients without mutations. In conclusion, defective secretion and impaired activity of the mutants concur to determine an almost complete deficiency of ADAMTS13 activity in patients with a homozygous or two heterozygous ADAMTS13 mutations.

ADAMTS, Thrombotic Thrombocytopenic Purpura and Pregnancy

“…In 24 patients [27][28][29][30][31][32][33][34][35][36], ADAMTS13 was documented (ADAMTS13 activity lower than 10% in the absence of auto-antibodies) including 15 cases in whom ADAMTS13 genotype could also be characterized (Supplementary Table 2) [30,32,34,36]. Interestingly, all 6 cases reported from the Western World [30,32,36] carried the R1060W mutation (either heterozygous or homozygous), reported to be a marker for adult TTP [32], and they were indeed associated with an adult-onset USS initially recognized with the first pregnancy (Supplementary Table 2). In contrast, the largest series of 9 pregnancy-associated USS reported in Japan by Fujimura et al [34], showed miscellaneous mutations not including the R1060W ADAMTS13 variant: interestingly, 6 of these 9 women had episodes of severe to mild thrombocytopenia during childhood that had been incorrectly diagnosed as ITP while 3 patients had an adult-onset TTP initially recognized with pregnancy.…”

Thrombotic Thrombocytopenic Purpura: A Thrombotic Disorder Caused by ADAMTS13 Deficiency

“…More than 65 mutations, including nonsense, missense, frame-shifting insertion or deletion and splicing mutations, have been detected in patients with hereditary TTP [99][100][101][102][103]. The mutations affect the process of protease synthesis, activity, or more commonly, secretion.…”

The D173G mutation in ADAMTS-13 causes a severe form of congenital thrombotic thrombocytopenic purpura

“…Inherited TTP is a rare autosomal recessive disorder due to homozygous or double heterozygous mutations in the ADAMTS-13 gene, causing a severe decrease of ADAMTS-13 level and activity (10)(11)(12)(13)(14)(15)(16). About 100 mutations causing inherited ADAMTS-13 deficiency have been identified so far in regions of the gene encoding different domains (10)(11)(12)(13)(14)(15)(16)(17) with only a few of them characterized by in vitro expression studies (12,15,(18)(19)(20)(21)(22). In this manuscript, we report cases of congenital TTP, due to the homozygous mutation in ADAMTS-13 gene in two young brothers born from two consanguineous parents of Romanian origin.…”