To study both the pathophysiologic and the prognostic value of ADAMTS13 in thrombotic microangiopathies (TMAs), we enrolled a cohort of 35 adult patients combining a first acute episode of TMA, an undetectable (below 5%) ADAMTS13 activity in plasma, and no clinical background such as sepsis, cancer, HIV, and transplantation. All patients were treated by steroids and plasma exchange, and an 18-month follow-up was scheduled. Remission was obtained in 32 patients (91.4%), and 3 patients died (8.6%) after the first attack. At presentation, ADAMTS13 antigen was decreased in 32 patients (91.4%), an ADAMTS13 inhibitor was detectable in 31 patients (89%), and an anti-ADAMTS13 IgG/IgM/IgA was present in 33 patients (94%). The 3 decedent patients were characterized by the association of several anti-ADAMTS13 Ig isotypes, including very high IgA titers, while mortality was independent of the ADAMTS13 inhibitor titer. In survivors, ADAMTS13 activity in remission increased to levels above 15% in 19 patients (59%) but remained undetectable in 13 patients (41%). Six patients relapsed either once or twice (19%) during the follow-up. High levels of inhibitory anti-ADAMTS13 IgG at presentation were associated with the persistence of an undetectable ADAMTS13 activity in remission, the latter being predictive for relapses within an 18-month delay. IntroductionThrombotic microangiopathies (TMAs) are defined by the association of acute mechanical hemolytic anemia, thrombocytopenia, and visceral ischemic manifestations related to the formation of platelet thrombi in the microcirculation. 1 Clinically, TMA includes mainly the thrombotic thrombocytopenic purpura (TTP) and the hemolytic uremic syndrome (HUS) characterized by a multivisceral ischemia and a renal ischemia, respectively. 2 Although mechanisms for HUS remain very heterogeneous, pathophysiology for most forms of TTP is related to a severe deficiency of a plasma metalloprotease, ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats). [3][4][5][6] Physiologically, ADAMTS13 is the specific cleaving protease for von Willebrand factor (VWF), a large multimeric glycoprotein crucial for both platelet adhesion and aggregation in the high stress-associated hemodynamic conditions of the microcirculation. 7 A severe enzymatic deficiency of ADAMTS13 causes highly adhesive unusually large multimers of VWF to accumulate in plasma, which may spontaneously bind to platelets and thus induce the formation of platelet thrombi in the microcirculation. In rare cases, clinically relevant ADAMTS13 severe deficiency is related to compound heterozygous or homozygous mutations of the ADAMTS13 gene (Upshaw-Schulman syndrome). [8][9][10] In most cases, severe ADAMTS13 deficiency is secondary to the development of anti-ADAMTS13 autoantibodies (auto-Abs). [11][12] Anti-ADAMTS13 auto-Abs can be detected in vitro either functionally because of their inhibitory effect on ADAMTS13 enzymatic activity [13][14] or, more recently, physically as immunoglobulin G (IgG) or IgM by enzyme-lin...

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Autoantibodies neutralizing human ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type 1 motif), the metalloprotease that physiologically cleaves von Willebrand factor, are a major cause of severe deficiency of the protease and of acquired thrombotic thrombocytopenic purpura (TTP). We evaluated prevalence of anti-ADAMTS13 antibodies in 59 patients with thrombotic microangiopathies (TMAs) and in 160 patients with immunologic or thrombocytopenic diseases different from TTP, using an enzyme-linked immunosorbent assay (ELISA). Immunoglobulin G (IgG) antibodies directed against ADAMTS13 were found in 97% of untreated patients with acute acquired TMA who had plasma levels of ADAMTS13 activity below 10%. The corresponding prevalence of IgM antibodies was 11%. In contrast, anti-ADAMTS13 antibodies of G or M isotypes were detected in 20% of patients with TMA with ADAMTS13 activity above 10%. The ELISA was more sensitive than the standard functional inhibitor assay for detecting antibodies against ADAMTS13. Patients with thrombocytopenia from various causes (n ‫؍‬ 50), systemic lupus erythematosus (SLE; n ‫؍‬ 40), and the antiphospholipid antibody syndrome (APS; n ‫؍‬ 55) had prevalences of IgG antibodies of 8%, 13%, and 5% respectively, only slightly higher than the prevalence in 111 healthy donors (4%). A rather high prevalence of anti-ADAMTS13 IgM antibodies was found in patients with SLE and APS (18% each). The clinical significance of IgM antibodies in these groups is unclear. In conclusion, the ELISA method detected anti-ADAMTS13 IgG antibodies in a very large proportion of patients with acquired TMA associated with severe ADAMTS13 deficiency, and was more sensitive than the inhibitor assay. IntroductionThrombotic microangiopathies (TMAs) are a heterogeneous group of diseases characterized by microangiopathic hemolytic anemia and thrombocytopenia due to platelet clumping in the microcirculation leading to ischemic organ dysfunction with neurologic symptoms and renal impairment. The 2 principal forms of TMA are the hemolytic-uremic syndrome (HUS), in which severe renal failure prevails, and thrombotic thrombocytopenic purpura (TTP), characterized by the systemic occlusion of the microcirculation that frequently affects the central nervous system. [1][2][3][4] von Willebrand factor (VWF) plays an important mechanistic role in TTP. Upon stimulation of endothelial cells, this adhesive glycoprotein is released from storage organelles (Weibel-Palade bodies) as unusually large multimers (ULVWF) that are physiologically cleaved by the plasma metalloprotease ADAMTS13 and degraded into smaller multimers ranging in size from 500 kDa to approximately 20 000 kDa. 5 On the basis of in vitro studies using artificial flow systems, the following pathogenetic mechanisms are postulated in TTP. Upon release, ULVWF multimers remain anchored to the endothelial-cell surface in a P-selectin-dependent manner, and form extraordinary long strings. 6,7 ADAMTS13 docks to the endothelial cells through binding to both the ...

Acquired thrombotic thrombocytopenic purpura (TTP) has been linked to severe deficiency of ADAMTS-13 activity caused by autoantibodies inhibitory to ADAMTS-13. We report data on a patient with confirmed TTP who had severely reduced ADAMTS-13 activity but showed no ADAMTS-13 inhibition in a widely used fluid phase activity assay. With a newly developed enzyme-linked immunosorbent assay, using immobilized recombinant ADAMTS-13, we found high titers of IgM and IgG antibodies that bound to ADAMTS-13, but did not neutralize protease activity. These autoantibodies probably influenced the half-life of ADAMTS-13 or its binding to the endothelial cell surface, thereby compromising ADAMTS-13 activity in vivo. Given that ADAMTS-13 may interact physiologically with various receptors or ligands, the occurrence, distribution, and the epitope mapping of nonneutralizing antibodies will be an important area for future research. (Blood. 2003;102:3241-3243)

Summary.  Background: ADAMTS13‐neutralizing IgG autoantibodies are the major cause of acquired thrombotic thrombocytopenic purpura (TTP). Objective: To analyze the IgG subclass distribution of anti‐ADAMTS13 antibodies and a potential relationship between subclass distribution and disease prognosis. Methodology: An enzyme‐linked immunosorbent assay‐based method was used to quantify the relative amounts of IgG subclasses of anti‐ADAMTS13 antibodies in acquired TTP plasma. Results: IgG4 (52/58, 90%) was the most prevalent IgG subclass in patients with acquired TTP, followed by IgG1 (52%), IgG2 (50%), and IgG3 (33%). IgG4 was found either alone (17/52) or with other IgG subclasses (35/52). IgG4 was not detected in 10% of the patients. There was an inverse correlation between the frequency and abundance of IgG4 and IgG1 antibodies (P < 0.01). Patients with high IgG4 levels and undetectable IgG1 are more prone to relapse than patients with low IgG4 levels and detectable IgG1. Conclusions: All IgG subclasses of anti‐ADAMTS13 antibodies were detected in patients with acquired TTP, with IgG4, followed by IgG1, antibodies dominating the anti‐ADAMTS13 immune response. Levels of IgG4 could be useful for the identification of patients at risk of disease recurrence.

Severe ADAMTS13 deficiency in thrombotic thrombocytopenic purpura (TTP) is either constitutional and caused by ADAMTS13 mutations, or acquired and most often due to ADAMTS13 inhibitory autoantibodies. In strongly hemolytic serum of a pediatric patient, diagnosed with TTP postmortem, ADAMTS13 activity was less than 3%. Both parents had an AD-AMTS13 activity of approximately 50%. Sequencing of the ADAMTS13 gene revealed an intronic 687-2A>G substitution affecting exon 7, homozygous in the propositus and heterozygous in both parents, confirming constitutional AD-AMTS13 deficiency. ADAMTS13 activity of normal plasma was inhibited by incubation with the propositus' serum, suggesting alloantibody formation to ADAMTS13. However, immunoglobulin purified from serum had no ADAMTS13 inhibitory effect, whereas the immunoglobulin-

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine and counterregulator of glucocorticoids, is a potential therapeutic target. MIF is markedly different from other cytokines because it is constitutively expressed, stored in the cytoplasm, and present in the circulation of healthy subjects. Thus, the concept of targeting MIF for therapeutic intervention is challenging because of the need to neutralize a ubiquitous protein. In this article, we report that MIF occurs in two redox-dependent conformational isoforms. We show that one of the two isoforms of MIF, that is, oxidized MIF (oxMIF), is specifically recognized by three mAbs directed against MIF. Surprisingly, oxMIF is selectively expressed in the plasma and on the cell surface of immune cells of patients with different inflammatory diseases. In patients with acute infections or chronic inflammation, oxMIF expression correlated with inflammatory flare-ups. In addition, anti-oxMIF mAbs alleviated disease severity in mouse models of acute and chronic enterocolitis and improved, in synergy with glucocorticoids, renal function in a rat model of crescentic glomerulonephritis. We conclude that oxMIF represents the disease-related isoform of MIF; oxMIF is therefore a new diagnostic marker for inflammation and a relevant target for anti-inflammatory therapy.

Sequence analysis of the ADAMTS13 locus of 2 patients with hereditary thrombotic thrombocytopenic purpura (TTP) revealed the homozygous presence of 4 single nucleotide polymorphisms (SNPs) (R7W, Q448E, P618A, A732V) and a rare missense mutation (R1336W). Analysis of the individual effect of any amino acid exchanges showed that several sequence variations can interact with each other, thereby altering the phenotype of ADAMTS13 deficiency. Introduction of polymorphisms R7W, Q448E, and A732V had no or only minor effects on ADAMTS13 secretion. In contrast, P618A, R1336W, and the A732V-P618A combination strongly reduced ADAMTS13-specific activity and antigen levels. Surprisingly, R7W and Q448E were positive modifiers of ADAMTS13 secretion in the context of P618A and A732V but neither could rescue the severely reduced specific activity conferred by P618A. However, in the context of R1336W, polymorphisms R7W and Q448E enhanced the detrimental effect of the missense mutation and led to undetectable enzyme activity. We show that dependent on the sequence context, the same polymorphisms might be either positive or negative modifiers of gene expression. Our results might therefore be widely relevant to understanding the influence of polymorphisms on the phenotypic expression of complex diseases. Introductionvon Willebrand factor (VWF) is crucial for primary hemostasis by mediating extracellular matrix-platelet interactions and delivering factor VIII to the site of vascular injury. The large and heterogeneous glycoprotein circulates as a multimer composed of identical units, ranging in molecular weight from 500 kDa to more than 20 000 kDa. 1 Plasma VWF is predominately secreted as high molecular weight, "unusually large," VWF (ULVWF) multimers. Because ULVWF binds more avidly to components of the extracellular matrix 2 and to various platelet receptors 3,4 than low molecular weight VWF, the hemostatic activity of VWF is size dependent and much more pronounced for high molecular weight VWF than for smaller multimers. This intrinsic property of ULVWF allows for platelet attachment to sites of vascular injury under high shear stress conditions of fastflowing blood. 5 However, immediately downstream from the site of injury, the multimeric size of VWF needs to be physiologically regulated to prevent the formation of platelet and VWF-rich thrombi.A VWF-cleaving protease, now called ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type-1 motifs) has been identified. [6][7][8][9][10][11][12][13] ADAMTS13 cleaves VWF between Tyr1605 and Met1606 in the A2 domain of the VWF monomer, 6,7,14,15 yielding typical fragments of 176 kDa and 140 kDa and smaller VWF multimers. Severe deficiency of ADAMTS13 may result in ULVWF spontaneously interacting with platelet receptors 5 and cause the life-threatening disorder thrombotic thrombocytopenic purpura (TTP). 16,17 Acquired TTP has been associated with the inhibition of ADAMTS13 activity by autoimmune antibodies, [18][19][20][21] whereas mutations in the ADAMTS13 gene have been ...