6C). patients harbored Exendin-4 Acetate inhibitors that blocked cleavage of VWF by normal human plasma (NHP) [13]. The likelihood of detecting an anti-ADAMTS-13 autoantibody decreases to 31%C48% in prospective studies in less selective patient populations [20,22]. This low-detection rate may reflect false-negatives in activity-based assays, due to very low autoantibody concentration, presence of denaturing reagents in the assay system or prolonged incubation of the reaction. Alternatively, some patients may harbor autoantibodies that bind ADAMTS-13, but do not inhibit its activity [27]; therefore, they are not detected by the functional assays. Our previous longitudinal study has shown that plasma exchange therapy does not quickly normalize plasma ADAMTS-13 activity as expected in some patients with undetectable autoantibodies. Rather, 2C7 days of plasma exchange were necessary to raise the plasma ADAMTS-13 activity [20], suggesting that the autoantibodies may be present, but undetectable by the functional assays. To determine the prevalence of the inhibitory and non-inhibitory autoantibodies, we used functional assays (collagen binding, GST-VWF73, and FRETS-VWF73) to identify the inhibitory autoantibodies and immunological assays [enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation plus Western blot] to identify both inhibitory and non-inhibitory autoantibodies in patients with TTP. In addition, we determined ADAMTS-13 antigen levels to assess whether the binding of the inhibitory and non-inhibitory IgG autoantibodies to ADAMTS-13 protease can accelerate its clearance = 21 patients) is defined as TTP occurring in patients with no apparent pre-existing or concurrent illness; non-idiopathic TTP (= 19 patients) is defined as TTP occurring in patients after various obvious etiologies including hematopoietic stem cell transplantation, disseminated cancer/chemotherapy, use of certain medications, and pregnancy [20,22,28]. Some may consider this group as thrombotic microangiopathy (TMA) due to other causes. Table 1 Summary of laboratory data in patients with thrombotic thrombocytopenic purpura (TTP) = 21)= 19)assays. Inhibitory anti-ADAMTS-13 IgG was defined as the immunoglobulin G that binds ADAMTS-13 [detected by immunological assays (see below)] and blocks ADAMTS-13 proteolytic activity (detected by FRET-VWF73 assay). Non-inhibitory anti-ADAMTS-13 IgG was defined as the immunoglobulin G that merely binds ADAMTS-13 protease, but does not block ADAMTS-13 activity in the functional assay (Table 2). Table 2 Definition of autoantibodies in patients with thrombotic thrombocytopenic purpura (TTP) for 10 min, collected and stored at ?80 C. Pooled normal human plasma Exendin-4 Acetate Rabbit polyclonal to KCNV2 from 20 healthy donors was used for a reference. Collagen-binding assay This assay using purified human plasma VWF as substrate was described previously [20,29]. Briefly, patient plasma was diluted 1:10 with 1.5 M urea in 5 mM TrisCHCl, pH 8.0 and activated with 10 mM BaCl2 for 5 min. It was then mixed with purified VWF (10 g mL?1) in presence of 0.1% protease inhibitor cocktail (Sigma, St Louis, MO, USA) and incubated at 37 C overnight. The reaction was stopped with 10 mM of Na2SO4 and centrifuged at 1100 for 3 min at room temperature. The supernatant was diluted 1:5 in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA), 0.05% Tween 20, and then added to a MaxiSorb microtiter plate (Nunc, Rochester, NY, USA) that had been precoated with human collagen type III (Southern Biotech, Birmingham, AL, USA). The plate was incubated at 37 C for 1 h and then washed three times with PBS. Peroxidase-conjugated antihuman VWF antibody (P0226; DakoCytomation, Carpinteria, CA, USA) was diluted 1:3000 in PBS containing 0.5% BSA, 0.05% Tween 20 and incubated at 37 C for 1 h. After three washes with PBS, the peroxidase substrate BL21 cells and purified by HiTrap Ni-chelating column and glutathione-agarose (BD Biosciences, San Jose, Exendin-4 Acetate CA, USA) as previously described [31C33]. It consists of 73 amino acids derived from the central A2 domain of VWF and is flanked by a glutathione S-transferase protein (GST) at its N-terminus and a 6xHis epitope at its C-terminus [31C33]. The molecular weight of uncleaved Exendin-4 Acetate substrate is ~38 kDa. Specific cleavage of GST-VWF73 at the TyrCMet bond by plasma ADAMTS-13 protease yielded a N-terminal fragment (~34 kDa) that was detected with anti-GST antibody [31C33] on Western blot. The quantification of the chemiluminescent signal of the cleaved band was performed with densitometry.