Thrombotic thrombocytopenic purpura (TTP) is associated with a severe functional deficiency of the ADAMTS13 metalloprotease resulting in accumulation of highly adhesive ultra-large von Willebrand factor (ULVWF) multimers. ULVWF promote spontaneous platelet clumping eventually leading to life-threatening widespread microvascular thrombosis in multiple organs. Inhibitory and non-inhibitory anti-ADAMTS13 autoantibodies that result in functional deficiency and/or accelerated in vivo clearance of ADAMTS13 have been detected in patients suffering from acquired idiopathic TTP. Current treatment with plasma exchange therapy is considered to remove the autoantibodies and to replenish plasma with the deficient protease. While researching therapeutic recombinant ADAMST13 (rADAMTS13) as a possible treatment modality for TTP patients, we investigated the in vitro potency of rADAMTS13 to overwhelm and neutralize circulating anti-ADAMTS13 antibodies and concurrently to reinstate plasma ADAMTS13 to normal. Mixing and incubating patient plasma with normal reagent plasma and subsequent FRETSVWF73 activity analysis was used to calculate the individual anti-ADAMTS13 inhibitor titers in 36 untreated idiopathic TTP patient plasma samples with severe ADAMTS13 deficiency (<10% of normal). The inhibitor titers (IU/ml) ranged from 1.5 to 85IU/ml (median of 3.75 IU/ml). To compare the performance of the different patient samples at equal inhibitor titers, we diluted each sample to predefined inhibitor titers (e.g. in patient samples with inhibitor titers <10IU/ml we diluted to 1, 3, 6 and 9IU/ml). Then, incrementally increasing concentrations of purified rADAMTS13 derived from stably transfected CHO cells were added to each pre-set inhibitor titer done for each of the 36 patient samples. Complex formation of rADAMTS13 and anti-ADAMTS13 patient antibody was allowed and the remaining ADAMTS13 activity was analyzed. The input of rADAMTS13 required to saturate circulating inhibitors and to elevate the activity level up to 1U/ml in the plasma was calculated by regression analysis. Our data indicate a linear correlation between supplemented rADAMTS13 and anti-ADAMTS13 antibody titer (for titers <10IU/ml) suggesting that rADAMTS13 should be further investigated as a possible treatment of acquired TTP.

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