In Vitro Expression Studies of Two Mutations on the Metalloprotease and First Cub Domains of the ADAMTS-13 Gene Leading to Severe ADAMTS-13 Deficiency and Chronic Recurrent TTP.

Thrombotic thrombocytopenic purpura (TTP) is associated with a severe deficiency of ADAMTS-13, a metalloprotease that processes physiologically the hyperactive ultralarge multimers of von Willebrand factor (VWF). We report the molecular characterization of a congenital case of TTP with < 6% ADAMTS-13 activity and no ADAMTS-13 inhibitor in plasma. Mutational analysis showed heterozygosity for 2 missense mutations: a G to A substitution at nucleotide 262 in exon 3 (Val88Met), in the metalloprotease domain of ADAMTS-13 and a substitution of G to T at nucleotide 3707 in exon 27 (Gly1239Val), in the first CUB domain. To explore the mechanism of the defect, the wild type protease (ADAMTS-13-WT) and each mutant (ADAMTS-13-Val88Met, ADAMTS-13-Gly1239Val) were transiently expressed in HEK 293 and COS-7 cells. WT and mutant recombinant proteins (rADAMTS-13) was analysed in conditioned medium and cell lysates by western blot analysis using anti-ADAMTS-13 monoclonal and polyclonal antibodies. The enzymatic activity of the expressed rADAMTS-13 forms was evaluated by measuring the extent of VWF multimer degradation with a quantitative immunoblot assay. Immunohistochemistry experiments were performed to study the transport of the recombinant protein between the ER and the Golgi complex. A secondary structure prediction (SSP) was also carried out to predict the effect of these mutations on the ADAMTS-13 structure. Western blots showed a dense band in the medium with a molecular weight of ~190 Kda, corresponding to the secreted rADAMTS-13-WT protein. The medium of the cells transfected with ADAMTS-13-Val88Met showed a fainter band roughly estimated to be 40–50% of the WT band. rADAMTS-13-Gly1239Val was not detectable in the medium. Both mutants were detected in cell lysates in amounts similar to ADAMTS-13-WT. rADAMTS-13 WT was able to degrade of HMW multimers, whereas rADAMTS-13 Val88Met as the well as media of cells cotrasfected by both mutant constructs lacked enzymatic activity, recapitulating the patient’s plasma pattern. By immunohistochemistry rADAMTS-13-WT was mainly localized in the perinuclear area, whereas rADAMTS-13-Gly1239Val was diffused throughout the cytoplasm with no perinuclear enhancement. rADAMTS-13-Val88Met showed increased staining in the cytoplasm but also a perinuclear enhancement. SSP analysis showed no change in the helical region including Gly1239 following replacement with Val. We hypothesize that Gly1239Val may directly alter molecular recognition, leading to a secretion pathway alteration and to retention of the mutant protein in the ER compartment. SSP analysis also showed that the Val88Met mutation, located in the metalloprotease domain, may enhance the conformational instability of the 80–87 region containing Glu83 involved in a Ca++ ion coordination. This effect may be responsible for both the observed partial secretion alteration and enzymatic activity defect of the mutant. These expression studies explain how these two gene mutations together lead to the absence of ADAMTS-13 enzymatic activity in patient’s plasma.