Nonfamilial thrombotic thrombocytopenic purpura is due to an inhibitor of von Willebrand factor-cleaving protease, whereas the familial form seems to be caused by a constitutional deficiency of the protease. Patients with the hemolyticuremic syndrome do not have a deficiency of von Willebrand factor-cleaving protease or a defect in von Willebrand factor that leads to its resistance to protease.

In both atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G) complement plays a primary role in disease pathogenesis. Herein we report the outcome of a 2015 Kidney Disease: Improving Global Outcomes (KDIGO) Controversies Conference where key issues in the management of these 2 diseases were considered by a global panel of experts. Areas addressed included renal pathology, clinical phenotype and assessment, genetic drivers of disease, acquired drivers of disease, and treatment strategies. In order to help guide clinicians who are caring for such patients, recommendations for best treatment strategies were discussed at length, providing the evidence base underpinning current treatment options. Knowledge gaps were identified and a prioritized research agenda was proposed to resolve outstanding controversial issues.

Patients with end-stage renal disease suffer from complex hemostatic disorders. Uremic patients show a bleeding diathesis that is mainly due to abnormalities of primary hemostasis; in particular, platelet dysfunction and impaired platelet-vessel wall interaction. However, despite decreased platelet function, these patients have a high prevalence of cardiovascular and thrombotic complications. Platelet dysfunction in uremic patients is partially due to uremic toxins present in circulating blood. Dialysis improves platelet abnormalities and reduces, but does not eliminate, the risk of hemorrhage. Hemodialysis can even contribute to the bleeding through the continuous platelet activation induced by the interaction between blood and artificial surfaces. Thrombocytopenia, glomerular thrombosis, and thrombi in small arteries and glomerular capillaries are common pathological features in many renal diseases. Platelets are also involved directly in the pathogenesis of glomerular diseases through a variety of mechanisms, including release of active molecules, by enhancing immune complex deposition, and by altering glomerular permeability.

Key Points• Endothelial-restricted complement activation occurs in aHUS, and clinical remission relies on efficient endothelial complement inhibition.• Ex vivo serum-induced endothelial C5b-9 deposits are a sensitive tool to monitor complement activation and eculizumab effectiveness in aHUS.Atypical hemolytic-uremic syndrome (aHUS) is associated with genetic complement abnormalities/anti-complement factor H antibodies, which paved the way to treatment with eculizumab. We studied 44 aHUS patients and their relatives to (1) test new assays of complement activation, (2) verify whether such abnormality occurs also in unaffected mutation carriers, and (3) search for a tool for eculizumab titration. An abnormal circulating complement profile (low C3, high C5a, or SC5b-9) was found in 47% to 64% of patients, irrespective of disease phase. Acute aHUS serum, but not serum from remission, caused wider C3 and C5b-9 deposits than control serum on unstimulated human microvascular endothelial cells (HMEC-1). In adenosine 59-diphosphate-activated HMEC-1, also sera from 84% and 100% of patients in remission, and from all unaffected mutation carriers, induced excessive C3 and C5b-9 deposits. At variance, in most patients with C3 glomerulopathies/immune complex-associated membranoproliferative glomerulonephritis, serum-induced endothelial C5b-9 deposits were normal. In 8 eculizumab-treated aHUS patients, C3/SC5b-9 circulating levels did not change posteculizumab, whereas serum-induced endothelial C5b-9 deposits normalized after treatment, paralleled or even preceded remission, and guided drug dosing and timing. These results point to efficient complement inhibition on endothelium for aHUS treatment. C5b-9 endothelial deposits might help monitor eculizumab effectiveness, avoid drug overexposure, and save money considering the extremely high cost of the drug. (Blood. 2014;124(11):1715-1726

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 ...

Shiga toxin (Stx)-producing E.coli O157:H7 has become a global threat to public health; it is a primary cause of diarrhea-associated hemolytic uremic syndrome (HUS), a disorder of thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure with thrombi occluding renal microcirculation. In this study, we explored whether Stx triggers complement-dependent microvascular thrombosis in in vitro and in vivo experimental settings of HUS. Stx induced on human microvascular endothelial cell surface the expression of P-selectin, which bound and activated C3 via the alternative pathway, leading to thrombus formation under flow. In the search for mechanisms linking complement activation and thrombosis, we found that exuberant complement activation in response to Stx generated an increased amount of C3a that caused further endothelial P-selectin expression, thrombomodulin (TM) loss, and thrombus formation. In a murine model of HUS obtained by coinjection of Stx2 and LPS and characterized by thrombocytopenia and renal dysfunction, upregulation of glomerular endothelial P-selectin was associated with C3 and fibrin(ogen) deposits, platelet clumps, and reduced TM expression. Treatment with anti–P-selectin Ab limited glomerular C3 accumulation. Factor B-deficient mice after Stx2/LPS exhibited less thrombocytopenia and were protected against glomerular abnormalities and renal function impairment, indicating the involvement of complement activation via the alternative pathway in the glomerular thrombotic process in HUS mice. The functional role of C3a was documented by data showing that glomerular fibrin(ogen), platelet clumps, and TM loss were markedly decreased in HUS mice receiving C3aR antagonist. These results identify Stx-induced complement activation, via P-selectin, as a key mechanism of C3a-dependent microvascular thrombosis in diarrhea-associated HUS.

In this report we demonstrate that proteolytic cleavage of the constituent subunit is one of the causes determining the heterogeneous size distribution of plasma von Willebrand factor (vWf) multimers. As shown by two-dimensional nonreduced/ reduced agarose/polyacrylamide gel electrophoresis, the structure of circulating vWf molecules may deviate from that represented by assemblage of a variable number of identical subunits. Indeed, even though the largest multimers in normal plasma appear to be composed predominantly of intact 225-kD subunits, those of intermediate and smaller size contain also 189-, 176-, and 140-kD proteolytic fragments. Different subunit composition patterns are repeated regularly in multimers ofincreasing molecular mass, yielding series of bands with similar structure. One of these series consists of molecules without evidence of proteolytic fragmentation, and its smallest member appears to be a dimer of 225-kD subunits. Type IIA von Willebrand disease, characterized by absence of the largest multimers, displays a pattern wherein the fragments of 176 and 140 kD are relatively increased, that of 189 kD is markedly decreased or absent, but the composition of individual multimers is otherwise similar to that of species seen also in normal plasma. In contrast to those in the circulation, all normal platelet vWf multimers contain only intact subunit. These results suggest that proteolytic cleavage of plasma vWf subunits occurs after release from cellular sites, whereas platelet vWf stored in a-granules is protected from proteolysis. These findings provide information that may be relevant for understanding the normal processing of vWf multimers and for elucidating the pathogenesis of some of the congenital and acquired structural abnormalities of this molecule. (J. Clin. Invest. 1991.

for the Italian Registry of Recurrent and Familial HUS/TTP Whether measurement of ADAMTS13 activity may enable physicians to distinguish thrombotic thrombocytopenic purpura (TTP) from hemolytic uremic syndrome (HUS) is still a controversial issue. Our aim was to clarify whether patients with normal or deficient ADAMTS13 activity could be distinguished in terms of disease manifestations and multimeric patterns of plasma von Willebrand factor (VWF). ADAMTS13 activity, VWF antigen, and multimeric pattern were evaluated in patients with recurrent and familial TTP (n ‫؍‬ 20) and HUS (n ‫؍‬ 29). Results of the collagen-binding assay of ADAMTS13 activity were confirmed in selected samples by testing the capacity of plasma to cleave recombinant VWF A1-A2-A3. Most patients with TTP had complete or partial deficiency of ADAMTS13 activity during the acute phase, and in some the defect persisted at remission. However, complete ADAMTS13 deficiency was also found in 5 of 9 patients with HUS during the acute phase and in 5 patients during remission. HUS patients with ADAMTS13 deficiency could not be distinguished clinically from those with normal ADAMTS13. In a subgroup of patients with TTP or HUS, the ADAMTS13 defect was inherited, as documented by half-normal levels of ADAMTS13 in their asymptomatic parents, consistent with the heterozygous carrier state. In patients with TTP and HUS there was indirect evidence of increased VWF fragmentation, and this occurred also in patients with ADAMTS13 deficiency. In conclusion, deficient ADAMTS13 activity does not distinguish TTP from HUS, at least in the recurrent and familial forms, and it is not the only determinant of VWF abnormalities in these conditions. (Blood. 2002;100:778-785)