Molecular characterization of four ADAMTS13 mutations responsible for congenital thrombotic thrombocytopenic purpura (Upshaw-Schulman syndrome).

ADAMTS13 mutations S203P, R268P, R507Q and A596V were previously identified in French patients with hereditary thrombotic thrombocytopenic purpura (TTP) (Upshaw-Schulman syndrome). Mutated recombinant (r) ADAMTS13 were transiently expressed in COS-7 cells and characterized in comparison with wild-type (WT) rADAMTS13. ADAMTS13 antigen was qualitatively and quantitatively estimated by electrophoretic analysis and ELISA. Enzymatic activity was qualitatively and quantitatively estimated using GST-VWF73, FRETS-VWF73 fragments and full-length rVWF-WT as substrates. The four mutants and rADAMTS13-WT were present within the cells. Secretion level of rADAMTS13-WT reached 1,200 ng/ml. The four mutations strongly altered the secretion and biological activity of rADAMTS13. The percentage secretion was 21, 38 and 17% for rADAMTS13-S203P, -R268P and -A596V compared with rADAMTS13-WT. rADAMTS13-R507Q concentration was under the detection limit of the assay. In the four cases, no enzymatic activity was detected. After concentration, we confirmed that mutations S203P and R268P totally abolished the proteolytic activity of ADAMTS13. Due to the very low protease concentration, activity of rADAMTS13-R507Q was below the threshold of the assays. rADAMTS13-A596V had no proteolytic activity towards the full-length rVWF-WT whereas it exhibited a decreased specific activity of about 30% of that of rADAMTS13-WT towards FRETS-VWF73 fragment. Binding study of mutated rADAMTS13-S203P, -R268P and -A596V showed that the three mutations strongly decreased the interaction of ADAMTS13 with VWF. In conclusion, the four mutations, which led to a secretion defect, a loss of enzymatic activity and a decreased binding to the substrate, are responsible for the hereditary TTP in patients.

Impaired dimerization of von Willebrand factor subunit due to mutation A2801D in the CK domain results in a recessive type 2A subtype IID von Willebrand disease.

The CK domain of von Willebrand factor (VWF) is involved in the dimerization of the protein. We identified the homozygous substitution A2801D of the CK domain in two siblings. Patients had low levels of VWF in plasma, abnormal ristocetin-induced binding to platelets and abnormal multimeric pattern with a lack of high molecular weight (HMW) forms and the presence of intervening bands between normal multimers. Accordingly, they were classified in type 2A, subtype IID, von Willebrand disease (VWD). Both asymptomatic parents carried the mutation at the heterozygous state. Their plasmaVWF exhibited the full range of multimers found in normal plasma. When analyzed by high resolution gel electrophoresis, very faint bands corresponding to the position of intervening bands of the propositus can be observed. The mutated recombinant (r)VWF-D2801, the hybrid rVWF-A/D2801 and the mutated C-terminal VWF fragment rSPII-D2801 were expressed in COS-7 cells. rVWF-D2801 showed an abnormal multimeric distribution similar to that of the propositus'VWF with intervening bands and a lack of HMW species. rVWF-A/D2801 exhibited the full range of multimers and the aberrant sized forms observed both in propositus'VWF and in rVWF-D2801. rSPII-WT assembled correctly into a dimer of 220 kDa. rSPII-D2801 appeared as a mixture of monomeric and dimeric forms which may be related to the abnormal multimeric pattern of the propositus and both mutated rVWF. We concluded that mutation A2801D disturbs the folding of the CK domain, which may result in a mixture of monomers and dimers of VWF. Multimers containing either an odd or even number of mature subunits are produced, and the presence of monomers appears to limit the degree of multimerization. In the heterozygousVWF, the presence of normal dimers improves the multimerization process. In conclusion, the mutation A2801D appears to be responsible for a recessive type 2A, subtype IID, VWD.