Diverse Functional Implications of ADAMTS13 Gene Mutations in Patients with TTP and Congenital Deficiency.

Thrombotic thrombocytopenic purpura (TTP) is a rare disorder of small vessels that is associated with deficiency of the VWF cleaving protease ADAMTS13, causing life-threatening disseminated microvascular thrombosis. Here we report four missense mutations (V88M, G1239V, R1219W, R1123C) in three patients with congenital ADAMTS13 deficiency. The three subjects carrying the mutations had less than 10% normal ADAMTS13 plasma antigen levels (measured by ELISA), which is consistent with previously reported data showing that most ADAMTS13 mutations in TTP patients result in impaired secretion of the protein. To evaluate whether the small amount of the mutant protease present in patients’ plasma had proteolytic activity, we cloned the ADAMTS13 cDNA in the pMT/Bip/His Drosophila expression vector and introduced the respective mutations by directed mutagenesis technique using wild-type cDNA as a template. These wild-type and mutant constructs were stably transfected into S2 Drosophila cells under the influence of the Drosophila BiP protein signal sequence, which allows the protein to be secreted into the medium and overcomes impaired secretion caused by the mutations. The induction of the histidine-tagged ADAMTS13 recombinants following the addition of copper was analyzed by Western blotting using an anti-hexahistidine monoclonal antibody. Equal amounts of recombinant ADAMTS13 proteins were used to evaluate proteolytic activity of recombinant proteins by cleavage of the rVWF A1-A2-A3 substrate. The proteolytic carboxyl terminal product of about 30 kDa was visualized by Western blot with a mouse monoclonal antibody directed against an epitope contained within the A3 domain of VWF. All the four missense mutations exhibited reduced activity: V88M:40%, G1239V: 66%, R1219W: 62% and R1123C: 64% of wild type activity . The results were also confirmed by collagen binding assay. For the the V88M mutation (located in the metalloprotease domain), decreased activity was confirmed by kinetic studies. In conclusion, mutant ADAMTS13 proteins found in patients with TTP, besides defective secretion, also have reduced protease activity. The mechanisms of the deficiency will be the matter of further studies.