Studies on causative gene for thrombotic thrombocytopenic purpura, ADAMTS13

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1. Phenotypic analysis of mouse lacking ADAMTS13 gene We generated and characterized Adamts13-knockout mice(KO) and Adamts13-congenic mice lacking the C-terminal domains of ADAMTS13(CG) . Both KO and CG were viable and fertile. In KO, unusually large von Willbrand factor(VWF) multimers were observed in the plasma. Thrombogenesis on immobilized collagen under flow and thrombocytopenia induced by a collagen plus epinephrine infusion were significantly promoted in KO than in wild-type mice(WT). However, hematological and histological analyses failed to detect any signs of TTP in KO. CG maintained the ADAMTS13 activity and normal VWF-multimer distribution in the plasma, and did not show an enhanced thrombogenesis under flow. Thrombocytopenia induced by a collagen plus epinephrine infusion was significantly more in CG than in WT. These results suggest that ADAMTS13 deficiency alone is not sufficient to cause TTP and that the mouse lacking the C-terminal domains is prone to thromosis.2. Studies on P475S mutation of ADAMTS13 We previously identified P475S mutation in ADAMTS13 gene with the allele frequency of 0.05 in the Japanese general population. Here, we expressed the recombinant wild type ADAMTS13 protein and P475S mutant and compared their activity toward the natural substrate VWF and the synthetic substrate FRETS-VWF73. We found that mutant ADAMTS13 showed about 10% activity of wild-type using natural substrate but about 70% activity using the synthetic substrate. The difference of the activity was attributable to urea in the reaction buffer for the natural substrate. The mutant protein tended to lose its activity in the presence of 1.5 M urea.