34) died soon after the first rituximab infusion, and the one patient (patient no

34) died soon after the first rituximab infusion, and the one patient (patient no. national cohort study, we monitored plasma levels of ADAMTS13, clinical and laboratory findings, and outcomes. In a representative poor-responder to PE, we examined an ADAMTS13-inhibitor complex in plasma milieu, by means of a large-pore isoelectric focusing (IEF) analysis. RESULTS Of 52 aTTP patients, 20 were well-responders and 32 were poor-responders. In the latter group, plasma ADAMTS13 activity levels never increased to more than 10% of normal during 14 days after PE initiation. Mean (SD) plasma ADAMTS13 inhibitor titers (BU/ml) were 5.7 (4.5) before PE, but decreased to 1 1.4 (0.8) on 4th PE day, and then remarkably GSK-923295 increased to 14.8 (10.0) on 10th PE day, termed inhibitor boosting, and then slowly GSK-923295 decreased to undetectable level over one month. On admission, none of the routinely available clinical and laboratory markers differentiated these two groups. However, elevated pre-PE levels of ADAMTS13 inhibitor were correlated with poor-response. We visualized an ADAMTS13-inhibitor (IgG) complex in a patient plasma by an IEF analysis, and found proteolytic fragment of ADAMTS13 antigen by a 2-dimentional IEF/SDS-PAGE analysis. CONCLUSION Findings from this cohort of aTTP patients exhibited that inhibitor boosting often occurs in aTTP patients in Japan. Poor-responders could be predicted by elevated pre-PE ADAMTS13 inhibitor levels on admission, but not by routinely collected clinical or laboratory data. Keywords: primary aTTP, ADAMTS13, inhibitor boosting, IEF, ADAMTS13 inhibitor complex Introduction Thrombotic thrombocytopenic purpura (TTP), a life-threatening generalized disorder originally characterized by a pentad of thrombocytopenia, microangiopathic hemolytic anemia, renal dysfunction, neurological signs and fever,1 is now primarily defined by severe deficiency of von Willebrand factor (VWF)-cleaving protease, termed ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type 1 motifs 13) accompanied by thrombocytopenia.2C4 The ADAMTS13 specifically cleaves unusually large VWF multimer (UL-VWFM) with hyperaggregability of platelets,5 and down-regulates VWF function. Deficiency of ADAMTS13 activity (ADAMTS13:AC) is usually either caused by gene mutations in or acquired autoantibodies to this enzyme,6 occasionally without a known cause, termed primary acquired TTP (aTTP).7 Plasma exchange (PE) has long been the first-line treatment for aTTP, often with an adjunct of corticosteroid/corticosteroid pulse therapy,8 PE removes ADAMTS13 inhibitor (ADAMTS13:INH), UL-VWFM, and inflammatory cytokines that mediate UL-VWFM release from vascular endothelial cells, and replenishes ADAMTS13 and regular-sized VWFM required for normal hemostasis. Patients with aTTP frequently take several days to weeks of PE before platelet counts recover. However, one populace of aTTP patients often shows an initial increase in the platelet count that is paradoxically followed by secondary thrombocytopenia. These patients have been Fam162a categorized as having PE-refractory aTTP, but the pathogenesis of secondary thrombocytopenia is usually unclear. B?hm et al.9 reported an increase in ADAMTS13:INH titers after PE, but no systematic studies on this potentially important issue have been conducted. In addition, 30% to 50% of aTTP patients will subsequently relapse, although these relapses occur at a time-point that is much longer than the secondary thrombocytopenia described herein.6,10 Due to concerns over both short-term thrombocytopenic events and long-term aTTP relapse, the anti-CD20 antibody GSK-923295 rituximab, which removes inhibitory IgG-producing B lymphocytes from circulation,11,12has been used recently to treat aTTP patients in conjunction with PE and corticosteroid, in an effort to reduce IgG of ADAMTS13:INH with the goal of improving short-term responses as well as decreasing the rate of relapse. We have recently shown that ADAMTS13 antigen (ADAMTS13:AG) in the plasma milieu consisted of 3 groups of bands with different isoelectric points (pI); Band I (pI 4.9 to 5.6) represents ADAMTS13 not bound to VWF; Band II (pI 5.8 to 6.7) remains unaddressed, and.