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Hereditary thrombotic thrombocytopenic purpura (hTTP) is a rare thrombotic microangiopathy characterized by severe congenital ADAMTS13 deficiency and recurring acute episodes causing morbidity and premature death. Information on the annual incidence and severity of acute episodes in patients with hTTP is largely lacking. This study reports prospective data on 87 patients from the Hereditary TTP Registry (clinicaltrials.gov #NCT01257269) for survival, frequency, and severity of acute episodes from enrollment until December 2019. The 87 patients, followed up for a median of 4.2 years (range, 0.01-15 years), had a median age at overt disease onset and at clinical diagnosis of 4.6 years and 18 years (range, 0.0-70 years for both), respectively. Forty-three patients received regular plasma prophylaxis, whereas 22 did not, and treatment changed over time or was unknown in the remaining 22. Forty-three patients experienced 131 acute episodes, of which 91 (69%) occurred in patients receiving regular prophylaxis. This resulted in an annual incidence of acute episodes of 0.36 (95% confidence interval [CI], 0.29-0.44) with regular plasma treatment and of 0.41 (95% CI, 0.30-0.56) without regular plasma treatment. More than one-third of acute episodes (n = 51) were documented in children 40 years (1.18 [95% CI, 0.88-1.55] vs 0.14 [95% CI, 0.08-0.23]). The prophylactic plasma infusion regimens used were insufficient to prevent acute episodes in many patients. Such regimens are burdensome, and caregivers, patients, and their guardians are reluctant to start regular plasma infusions, from which children particularly would benefit.

ObjectivesUncontrolled thromboinflammation plays an important role in the pathogenesis of coronavirus disease (COVID-19) caused by SARS-CoV-2 virus. Complement was implicated as key contributor to this process, therefore we hypothesized that markers of the complement profile, indicative for the activation state of the system, may be related to the severity and mortality of COVID-19.MethodsIn this prospective cohort study samples of 102 hospitalized and 26 outpatients with PCR-confirmed COVID-19 were analyzed. Primary outcome was in-hospital, COVID-19 related mortality, and secondary outcome was COVID-19 severity as assessed by the WHO ordinal scale. Complement activity of alternative and classical pathways, its factors, regulators, and activation products were measured by hemolytic titration, turbidimetry, or enzyme-immunoassays. Clinical covariates and markers of inflammation were extracted from hospital records.ResultsIncreased complement activation was characteristic for hospitalized COVID-19 patients. Complement activation was significantly associated with markers of inflammation, such as interleukin-6, C-reactive protein, and ferritin. Twenty-five patients died during hospital stay due to COVID-19 related illness. Patients with uncontrolled complement activation leading to consumption of C3 and decrease of complement activity were more likely to die, than those who had complement activation without consumption. Cox models identified anaphylatoxin C3a, and C3 overactivation and consumption (ratio of C3a/C3) as predictors of in-hospital mortality [HR of 3.63 (1.55–8.45, 95% CI) and 6.1 (2.1–17.8), respectively].ConclusionIncreased complement activation is associated with advanced disease severity of COVID-19. Patients with SARS-CoV-2 infection are more likely to die when the disease is accompanied by overactivation and consumption of C3. These results may provide observational evidence and further support to studies on complement inhibitory drugs for the treatment of COVID-19.

Background: Type 2 diabetes mellitus (T2DM) and obesity are risk factors of COVID-19 disease course. Retrospective observational analyses reported improvement in COVID-19 severe outcomes and mortality in T2DM patients using a DPP4 inhibitor. However, little is known about the prognostic value of circulating soluble DPP4 activity in patients with SARS-CoV-2 infection. Methods: We conducted an observational, retrospective cohort study with 184 Hungarian adults. Hospitalized patients with acute COVID-19 disease (n=102) and plasma donors (n=43) recovered from prior SARS-CoV-2 infection were enrolled from April to July, 2020 at two institutions in Budapest. “Non-COVID-19 controls” (n=39) were employed with serum sDPP4 activity determined from samples taken before September, 2019. Duplicate serum sDPP4 activity determinations by microplate base enzyme kinetic assay were successful in 182 individuals from different COVID-19 disease severity groups (acute COVID-19 patients were classified into 4 and plasma donors into 2 categories based on peak severity). Findings: A significant difference in sDPP4 activity was found among study groups (p

Anti-A Disintegrin and Metalloproteinase with a ThromboSpondin type 1 motif, member 13 (ADAMTS13) autoantibodies cause a severe ADAMTS13 deficiency in immune-mediated thrombotic thrombocytopenic purpura (iTTP). ADAMTS13 consists of a metalloprotease (M), a disintegrin-like (D) domain, 8 thrombospondin type 1 repeats (T1-T8), a cysteine-rich (C), a spacer (S), and 2 CUB domains (CUB1-2). We recently developed a high-throughput epitope mapping assay based on small, nonoverlapping ADAMTS13 fragments (M, DT, CS, T2-T5, T6-T8, CUB1-2). With this assay, we performed a comprehensive epitope mapping using 131 acute-phase samples and for the first time a large group of remission samples (n = 50). Next, samples were stratified according to their immunoprofiles, a field that is largely unexplored in iTTP. Three dominant immunoprofiles were found in acute-phase samples: profile 1: only anti-CS autoantibodies (26.7%); profile 2: both anti-CS and anti-CUB1-2 autoantibodies (12.2%); and profile 3: anti-DT, anti-CS, anti-T2-T5, anti-T6-T8, and anti-CUB1-2 autoantibodies (8.4%). Interestingly, profile 1 was the only dominant immunoprofile in remission samples (52.0%). Clinical data were available for a relatively small number of patients with acute iTTP (>68), and no correlation was found between immunoprofiles and disease severity. Nevertheless, profile 1 was linked with younger and anti-T2-T5 autoantibodies with older age and the absence of anti-CUB1-2 autoantibodies with cerebral involvement. In conclusion, identifying acute phase and remission immunoprofiles in iTTP revealed that anti-CS autoantibodies seem to persist or reappear during remission providing further support for the clinical development of a targeted anti-CS autoantibody therapy. A large cohort study with acute iTTP samples will validate possible links between immunoprofiles or anti-domain autoantibodies and clinical data. ispartof: BLOOD ADVANCES vol:5 issue:17 pages:3427-3435 ispartof: location:United States status: published

Hematopoietic stem cell transplantation (HSCT)-associated thrombotic microangiopathy (TA-TMA) is a multifactorial complication. Complement dysregulation may play an important role in the pathogenesis of TA-TMA. Our previous observations suggested that early increase of soluble C5b-9 (sC5b-9), before the development of other complications, can predict the development of later TA-TMA. The present study aims to validate our earlier findings in an independent cohort enrolling 67 pediatric patients who underwent allogeneic HSCT during the study period (October 2015–January 2019). Five different TA-TMA diagnostic criteria were applied, and all important clinical and laboratory parameters of TA-TMA activity were registered. Complement pathway activities, components and sC5b-9 levels were systematically measured before transplantation and on days 28, 56, and 100 after HSCT. A strong and remarkable association still have been found between early increase of sC5b-9 (10 of 10 patients with TA-TMA vs. 27 of 57 without TA-TMA; P = 0.002) and the development of TA-TMA during 100 days post-transplantation. An increase in sC5b-9 concentration had 100% sensitivity and 53% specificity for TA-TMA in the cohort. All TA-TMA cases have been observed during cyclosporine immunosuppression, no TA-TMA was diagnosed during tacrolimus or mycophenolat mofetil therapy. In the majority of patients TA-TMA was mild and self-limiting, without any signs of organ damage. No additional complement parameters were closely associated with the development of TA-TMA. Early raise of the sC5b-9 activation marker was predictive for later development of TA-TMA throughout the whole study period. In patients with a marked increase, early and frequent monitoring of TA-TMA activity markers should be attempted, to facilitate subsequent therapy decisions in time. However, patients with TA-TMA were only identified during or after cyclosporine immunosuppression. Further studies enrolling higher number of patients are necessary to determine the role of immunosuppression in the pathogenesis of TA-TMA.

Hematopoietic stem cell transplantation (HSCT)–associated thrombotic microangiopathy (TA-TMA) is a multifactorial complication, and its prediction is largely unresolved. Our aim was to analyze changes of complement profile after HSCT to identify potential markers of TA-TMA development. Thirty-three consecutive pediatric patients (9.6 ± 4.4 years old) who underwent allogeneic HSCT due to malignant (n = 17) or nonmalignant (n = 16) indications were included in this study. Graft-versus-host disease (GVHD) was diagnosed using Glucksberg criteria, viral reactivation was monitored, 5 different TA-TMA diagnostic criteria were applied, and all important clinical and laboratory parameters of TA-TMA activity were registered. Complement pathway activities, components and terminal pathway activation marker (sC5b-9) levels were systematically measured before transplantation and on days 28, 56, and 100 after HSCT. During the first 100 days after HSCT, 1 of 33 patients died (day 50, multiple organ failure), whereas 10 subjects met the criteria for TA-TMA, typically on day 61 (range, 16 to 98 days). TA-TMA was preceded by acute GVHD in 3 of 10 patients, by viral reactivation in 2 of 10, or by both in 4 of 10 cases. Baseline sC5b-9 levels did not differ in patients without (200 [interquartile range, 144 to 266] ng/mL), or with (208 [interquartile range, 166 to 271] ng/mL) subsequent TA-TMA; however, on day 28 significant differences were observed (201 [interquartile range, 185 to 290] ng/mL versus 411 [interquartile range, 337 to 471] ng/mL; P = .004). Importantly, all 10 patients with TMA showed increase in sC5b-9 level from baseline level to day 28, whereas in patients without TMA the same tendency was observed for only 9 of 23 patients (P = .031). No additional complement parameters were closely associated with the development of TA-TMA. Development of TA-TMA occurred in 30% of our patients, typically after GVHD and/or viral reactivation. However, early raise of sC5b-9 activation marker was predictive for later development of TA-TMA, and should therefore be considered as an alarming sign necessitating a careful monitoring of all TA-TMA activity markers. Further studies enrolling a higher number of patients are necessary to determine if terminal pathway activation is an independent predictor of TA-TMA.

Thrombotic microangiopathies (TMAs) are rare but life-threatening disorders characterized by microvascular hemolytic anemia and acute thrombocytopenia with or without organ damage. The term TMA covers various subgroups of diseases, the pathogenesis of which is briefly summarized in this review. As highlighted here, complement activation may represent an important amalgamating process in all of these conditions, since it is able to link together activation and damage of multiple involved cell types, such as endothelial cells, platelets, and neutrophils.

The acquired form of idiopathic thrombotic thrombocytopenic purpura (TTP) is an autoimmune disease, in which the underlying ADAMTS13-deficiency is caused by inhibitory autoantibodies against the protease. Human leukocyte antigens (HLA), responsible for antigen presentation, play an important role in the development of antibodies. The loci coding HLA DR and DQ molecules are inherited in linkage as haplotypes. The c.1858C>T polymorphism of the PTPN22 gene, which codes a protein tyrosine phosphatase important in lymphocyte activation, predisposes to a number of autoimmune diseases. We determined the HLA-DRB1-DQB1 haplotypes and the PTPN22 c.1858C>T genotypes in 75 patients with acquired idiopathic TTP and in healthy controls, in order to assess the role of these genetic factors and their interactions in the susceptibility to TTP. We found that the carrier frequencies of the DRB1∗11-DQB1∗03 and DRB1∗15-DQB1∗06 haplotypes were higher, while those of the DRB1∗07-DQB1∗02 and DRB1∗13-DQB1∗06 haplotypes were lower in TTP patients. There was no difference in the overall frequency of the PTPN22 c.1858T allele between TTP patients and controls. In conclusion, we identified four HLA-DRB1-DQB1 haplotypes associated with an increased (DRB1∗11-DQB1∗03 and DRB1∗15-DQB1∗06) or a decreased (DRB1∗07-DQB1∗02 and DRB1∗13-DQB1∗06) susceptibility to acquired idiopathic TTP.

Recently, we showed that ADAMTS13 circulates in an open conformation during the acute phase of immune-mediated thrombotic thrombocytopenic purpura (iTTP). Although the cause of this conformational change remains elusive, ADAMTS13 is primarily closed in iTTP patients in remission with ADAMTS13 activity >50% and undetectable anti-ADAMTS13 autoantibodies, as well as after rituximab treatment, suggesting a role for anti-ADAMTS13 autoantibodies. Therefore, immunoglobulin G from 18 acute iTTP patients was purified and added to closed ADAMTS13 in healthy donor plasma. This resulted in open ADAMTS13 in 14 of 18 (78%) samples, proving that anti-ADAMTS13 autoantibodies can induce an open ADAMTS13 conformation. To further elucidate the conformation of ADAMTS13 in iTTP patients, we studied a novel iTTP patient cohort (n = 197) that also included plasma samples from iTTP patients in remission in whom ADAMTS13 activity was 50%, although free anti-ADAMTS13 autoantibodies were not always detected. Thus, open ADAMTS13 is a hallmark of acute iTTP, as well as a novel biomarker that can be used to detect subclinical iTTP in patients in remission. Finally, a long-term follow-up study in 1 iTTP patient showed that the open conformation precedes a substantial drop in ADAMTS13 activity. In conclusion, we have shown that anti-ADAMTS13 autoantibodies from iTTP patients induce an open ADAMTS13 conformation. Most importantly, an open ADAMTS13 conformation is a biomarker for subclinical iTTP and could become an important tool in TTP management. ispartof: BLOOD vol:136 issue:3 pages:353-361 ispartof: location:United States status: published

Pentraxin-3 (PTX3) and C-reactive protein (CRP) have been shown to regulate complement activation in vitro, but their role has not been investigated in complement consumption in vivo. Thrombotic microangiopathies (TMA) are often accompanied by complement overactivation and consumption, therefore we analyzed the relation of the systemic pentraxin levels to the complement profile, laboratory parameters and clinical outcome of TMA patients. We determined the PTX3 and CRP levels, complement factor and activation product concentrations in blood samples of 171 subjects with the diagnosis of typical hemolytic uremic syndrome (STEC-HUS) (N = 34), atypical HUS (aHUS) (N = 44), secondary TMA (N = 63), thrombotic thrombocytopenic purpura (TTP) (N = 30) and 69 age-matched healthy individuals. Clinical data, blood count and chemistry were collected from medical records. To determine the in vitro effect of PTX3 on alternative pathway (AP) activation, sheep red blood cell-based hemolytic assay and AP activity ELISA were used. We found that PTX3 levels were elevated in the acute phase of STEC-HUS, aHUS and secondary TMA, whereas PTX3 elevation was exceptional is TTP. Conversely, a significantly higher median CRP was present in all patient groups compared to controls. PTX3, but not CRP was associated with signs of complement consumption in vivo, and PTX3 significantly decreased the AP hemolytic activity in vitro. Our results provide a detailed description of acute phase-TMA patients' complement profile linked to changes in the systemic pentraxin levels that may support further molecular studies on the function of PTX3 in disease pathogenesis and add to the laboratory assessment of complement consumption in TMA.

Background: The acquired form of idiopathic thrombotic thrombocytopenic purpura (TTP) is an autoimmune disease, in which the underlying deficiency of the ADAMTS13 protease is caused by autoantibodies, predominantly of the IgG isotype. Certain HLA-DR-DQ haplotypes were associated with the risk of developing TTP. Objectives: To investigate the development of the ADAMTS13-specific antibody response during the course of the disease, we analyzed the concentration, subclass distribution, and inhibitory potential of anti-ADAMTS13 IgG autoantibodies in samples of TTP patients drawn during the first acute phase, in remission, and during relapse. Additionally, we compared the anti-ADAMTS13 IgG levels between patients carrying and not carrying risk and protective HLA-DR-DQ haplotypes. Patients and methods: We determined the anti-ADAMTS13 IgG concentration and subclass distribution in 101 antibody-positive samples of 81 acquired TTP patients by ELISA methods. The presence and semi-quantitative amount of anti-ADAMTS13 inhibitors were determined in 97 of 100 deficient samples, and the specific inhibitory potential of anti-ADAMTS13 autoantibodies were determined in 49 selected samples, by mixing ADAMTS13 activity assays. HLA-DR-DQ typing and haplotype prediction were performed in 70 of the above patients. Results: We found that IgG1 and IgG4 were the predominant subclasses, present in almost all samples. While IgG1 was the dominant subclass in almost half of the samples taken during the first acute episode, IgG4 was dominant in all samples taken during or following a relapse. The inhibitory potential of the samples correlated with levels of the IgG4 subclass. Anti-ADAMTS13 antibodies of IgG4-dominant samples had higher specific inhibitory potentials than IgG1-dominant samples, independently of disease stage. Interestingly, we found that patients carrying the protective DR7-DQ2 and DR13-DQ6 haplotypes had higher anti-ADAMTS13 IgG levels. Conclusion: Our results indicate that IgG4 becomes the dominant subtype at some point of the disease course, apparently before the first relapse, parallel to the increase in inhibitory potential of the anti-ADAMTS13 autoantibodies. Furthermore, we found an association between the genetic background and the antibody response in TTP.

SummaryThrombotic thrombocytopenic purpura (TTP) is characterised by the deficiency of the von Willebrand factor (VWF) cleaving protease (ADAMTS-13). Although several observations indicate an important role of endothelial activation in the pathogenesis of TTP, no reliable endothelial activation markers are available in the clinical management of TTP. Our aim was to investigate the presence of endothelial activation in TTP and to determine its connections with disease activity, therapy and complement activation. We enrolled 54 patients (median age 40.5; 44 females) and 57 healthy controls (median age 34; 30 females),VWF antigen, carboxiterminal-pro-endothelin-1 (CT-proET-1), complement Factor H and complement activation products (C3bBbP and SC5b-9) were measured. In both the acute and remission phase of TTP we found increased CT-proET-1 and VWF levels, while Factor H levels decreased compared with healthy controls. In remission, however, the elevated CT-proET-1 levels showed 22 % decrease when compared with the acute phase in paired samples (p=0.0031), whereas no changes for VWF and Factor H levels were observed. We also found positive correlations between CT-proET-1 levels and alternative pathway activation markers (C3bBbP; p=0.0360; r=0.4299). The data we present here demonstrate a role of endothelium activation in patients with acute TTP. The finding that CT-proET-1 levels decreased in remission compared with the acute phase further supports endothelial involvement. In addition, we show that endothelial activation also correlated with the activation of the alternative complement pathway. The data suggest that complement and endothelium activation jointly contribute to the development of TTP episodes in patients with predisposition to TTP.Supplementary Material to this article is available online at www.thrombosis-online.com.

Distant metastasis is a major cause of colorectal cancer–related death, but the mechanism of tumour progression is not fully understood. There is growing evidence of an interaction between tumour cells and platelets which may influence tumour progression and metastasis formation. Quality and quantity of von Willebrand factor may regulate the interaction between tumour cells and platelets. Our aim was to measure the platelet count, von Willebrand factor antigen (VWF:Ag) levels and ADAMTS13 activity in a large (n = 232) cohort of colorectal cancer patients and to examine their relationships with the stage of the disease and 5-year survival without thrombotic complications using multivariable models. Significantly higher platelet counts (p = 0.005), VWF:Ag levels (p = 0.008) and decreased ADAMTS13 activity (p = 0.006) were observed in patients with metastatic disease. Results of the Kaplan–Meier analysis showed that lower platelet counts (p 1.7, p

Background. Deficient ADAMTS13 activity (TS13:act 50%. However, also iTTP patients in remission with a persistent ( Aim. Determine ADAMTS13 conformation in plasma of iTTP patients during acute TTP and remission when TS13:act is 50% and investigate if anti-ADAMTS13 autoAbs induce conformational changes in ADAMTS13. Methods. TS13:act was determined in 120 iTTP plasma samples from 4 different centers (Marseille, Milan, Budapest, Utrecht). Samples were categorized according to the presence of clinical symptoms (acute versusremission) and their TS13:act in remission (>50%, 10-50%, Results. Of the 120 iTTP plasma samples, 46 were obtained during the acute (clinical signs present) and 74 during the remission phase (clinical signs absent). Further subdividing remission samples showed that TS13:act was >50% in 41, 10-50% in 14 and 50%, confirming our previous results. Interestingly, ADAMTS13 was open in 93% and 89% of remission samples with TS13:act 10-50% and Conclusion. We show that ADAMTS13 is not only in the open conformation in iTTP patient plasma during the acute phase but also in remission when TS13:act is Disclosures Peyvandi: Octapharma US: Honoraria; Kedrion: Consultancy; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Grifols: Speakers Bureau; Sobi: Speakers Bureau; Shire: Speakers Bureau; Novo Nordisk: Speakers Bureau; Octapharma US: Honoraria; Octapharma US: Honoraria; Novo Nordisk: Speakers Bureau; Kedrion: Consultancy; Roche: Speakers Bureau; Novo Nordisk: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Shire: Speakers Bureau; Sobi: Speakers Bureau; Roche: Speakers Bureau; Roche: Speakers Bureau; Shire: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Octapharma US: Honoraria; Roche: Speakers Bureau; Roche: Speakers Bureau; Grifols: Speakers Bureau; Sobi: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Sobi: Speakers Bureau; Shire: Speakers Bureau; Sobi: Speakers Bureau; Novo Nordisk: Speakers Bureau; Shire: Speakers Bureau; Kedrion: Consultancy; Novo Nordisk: Speakers Bureau; Grifols: Speakers Bureau; Grifols: Speakers Bureau; Kedrion: Consultancy; Kedrion: Consultancy; Grifols: Speakers Bureau; Octapharma US: Honoraria. Coppo:Ablynx: Consultancy. Veyradier:LFB: Other: Investigator. Vanhoorelbeke:Shire: Consultancy; Ablynx: Consultancy.

Introduction Genetic and autoimmune risk factors contribute to the development of thrombotic thrombocytopenic purpura (TTP) but triggers are needed to bring about acute disease. The aim of the study was to investigate the association of neutrophil activation with acute TTP, to assess whether neutrophil activation changes during plasma exchange therapy and to show if complement- and neutrophil activation are parallel, characteristic processes in acute TTP. Materials and Methods Altogether 49 EDTA-plasma samples of 21 TTP patients with acute disease and 17 in remission were investigated along with 20 healthy controls. A stable complex of PMNE-proteinase-inhibitor was measured by ELISA (Calbiochem, Merck-Millipore, Darmstadt, Germany). Results Acute disease was associated with significantly increased PMNE levels, the group medians were similarly low in TTP patients in remission and in healthy controls. Increased PMNE levels were characteristic for hematologically active and ADAMTS13 deficient form of TTP. PMNE concentration inversely correlated to disease activity markers platelet count (r = − 0.349, p = 0.032) and hemoglobin levels (p = − 0.382 p = 0.018). Achievement of remission was associated with significant reduction of plasma PMNE levels (p = 0.031, Wilcoxon test). There was positive correlation between PMNE levels and complement activation markers C3a and Bb. Conclusions We report increased PMNE levels in acute TTP and showed its association to activity markers of acute TTP and complement activation. Effective treatment of an acute TTP episode resulted in marked decrease in PMNE levels. Our data support and extend previous observations that neutrophil extracellular traps may be released in acute TTP and potentially contribute to the pathophysiology of this disease.

Background: Neutrophil extracellular traps (NETs) can stimulate thrombosis, and their degradation is decreased in several autoimmune disorders. It was recently reported that some patients with haemolytic uraemic syndrome (HUS) also fail to degrade NETs and that neutrophils from Shiga toxin-associated HUS are primed to form NETs. Method: We used a well-characterized cohort of 74 thrombotic microangiopathy (TMA) patients, with a subset also providing follow-up samples, and 112 age-matched controls to investigate NET degradation and serum nuclease activity in TMA before, during and after treatment. Results: We identified that in the cohort of TMA patients, 50% of patients with Shiga toxin-associated HUS displayed a decreased ability to degrade NETs. NET degradation correlated with serum nuclease activity, but not with autoantibodies against double-stranded DNA, which has been previously observed in some autoimmune disorders. Further, NET degradation negatively correlated with serum creatinine levels, suggesting that kidney function was negatively impacted by the low NET degradation ability. Conclusions: We revealed that decreased NET degradation is a common feature of Shiga toxin-associated HUS and that it is associated with decreased kidney function in these patients. It remains to be clarified whether improving NET degradation would be beneficial for the patient.