The inherited deficiency of the von Willebrand factor-cleaving protease, ADAMTS13, is usually associated with severe forms of thrombotic thrombocytopenic purpura (TTP). Among the reported 53 mutations on ADAMTS13 gene, none has been described on TSP1-6 repeat domain. We investigated an Iranian man with history of chronic recurrent TTP and very low plasma levels of ADAMTS13 activity (2.3%). He had his first TTP episode at the age of 21 years and 6 subsequent episodes occurred without precipitating events. Genetic analysis revealed a homozygous deletion of nucleotides 2930–2935 (GTGCCC), in exon 23 of ADAMTS13 gene, leading to the replacement of Cys977 residue by a Trp and the deletion of Ala978 and Arg979, in the TSP1-6 repeat domain. The patient’s brother, homozygous for the same mutation, has no clinical manifestation of TTP so far, despite the same degree of ADAMTS13 deficiency. The patient’s parents resulted to be heterozygous. To explore the mechanism of ADAMTS13 deficiency, wild type (ADAMT13WT) and mutant (ADAMTS13del6bp) expression vectors were transiently transfected in HEK293 and COS-7 cells. The enzymatic activity of the expressed rADAMTS13 proteins was evaluated by measuring the extent of VWF multimer degradation, using a quantitative immunoblotting assay. rADAMT13WT was able to degrade multimers completely (100% of activity), whereas rADAMTS13del6bp had enzymatic activity reduced to ~10% of WT. Both WT and mutant proteases present in conditioned media and cell lysates, were analyzed by Western blot analysis, using anti-V5 monoclonal antibody against the C-terminal tag of rADAMTS13. Blots showed a dense band of ~190 KDa in the medium, corresponding to the secreted rADAMT13WT protein. Cells transfected with ADAMTS13del6bp showed a fainter band roughly estimated to be 5% of the WT obtained by densiometric analysis. Therefore, pulse-chase labelling experiments were performed to evaluate the presence of a secretion pathway alteration. After 60 min pulse with [35S] methionine the maximum level of rADAMT13WT in conditioned media was found at 24 hours. rADAMTS13del6bp was minimally present after 3 hours only, no band being detected after 7 hours of chase. Differential immunofluorescence studies were performed using an anti-V5 monoclonal antibody against ADAMTS13 proteins and monoclonal antibodies recognizing markers of Cis-Golgi and ER. The merging studies in WT and mutant transfected cells showed that rADAMTS13WT was mostly localized in the perinuclear area, suggesting a primary localization in the Golgi apparatus, whereas rADAMTS13del6bp showed less intense staining diffusely throughout the cytoplasm, only a minimal amount of the mutant protein being localized in the Cis-Golgi and ER. The structural consequences of the deletion mutation in the TSP1-6 domain may cause a loss of the known antiparallel, three-stranded fold characterizing the architecture of the TSP-like domains. In these domains, each strand is capped by disulfide bonds on each end. This study suggests that the residue Cys977 could be involved in the formation of disulphide bonds responsible for a correct folding of one of the TSP1-like domains of ADAMTS13. Therefore the deletion could lead to uncorrected folding which reflects an intracellular degradation and secretion defect of the mutant protease without any intracellular accumulation. These results reflect the severe deficiency of ADAMTS13 in the patient’s plasma.

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