Results: Eighteen studies were included in the final review for a total of 45 patients.The median age was 78 years (range: 21-93).The vast majority (75.6%) were males.Presenting bleeding symptoms were widely variable; the most common were subcutaneous bleeding and muscle hematomas.In forty patients the FVIII levels at diagnosis were ≤1% or undetectable.The median inhibitor level was 40 BU/mL (1.8 to 2000 BU/mL).Kaplan-Meier analysis showed that around 56% of patients have been reported to achieve bleed control in one day after the start of treatment and almost 90% of patients had no bleeding by day 5 of starting Emicizumab (Figure 2).Side effects were infrequently reported; nevertheless, the treatment appeared to be well-tolerated.Figure: Time-to-bleed control after starting Emicizumab. Conclusion(s):Emicizumab appears to be an effective and safe hemostatic treatment for AHA.Bleeding control is rapidly achieved after initiating emicizumab.Clinical trials comparing emicizumab versus standard hemostatic agents are needed to confirm these findings.

BACKGROUND:Acquired von Willebrand syndrome (aVWS) is common in patients with mechanical circulatory support (MCS) devices. In these patients, the high shear stress in the device leads to increased shear-induced proteolysis of von Willebrand factor (VWF) by A Disintegrin And Metalloprotease with Thrombospondin type 1 repeats, number 13 (ADAMTS13). As a result, the high molecular weight (HMW) VWF multimers are lost, leading to a decreased VWF function and impaired hemostasis that could explain the bleeding complications that are frequently observed in these patients. To counteract this abnormal VWF degradation by ADAMTS13, we developed a novel targeted therapy, using an anti-ADAMTS13 monoclonal antibody (mAb) that inhibits the shear-induced proteolysis of VWF by ADAMTS13.METHODS:Human or bovine blood was circulated through in vitro MCS device systems with either inhibitory anti-ADAMTS13 mAb 3H9 or 17C7 (20 μg/ml) or control anti-ADAMTS13 mAb 5C11 or phosphate buffered saline (PBS). VWF multimers and function (collagen binding activity) were determined at different time points. Next, Impella pumps were implanted in calves and the calves were injected with PBS and subsequently treated with mAb 17C7. VWF, ADAMTS13, and blood parameters were determined.RESULTS:We demonstrated that blocking ADAMTS13 could prevent the loss of HMW VWF multimers in in vitro MCS device systems. Importantly, our antibody could reverse aVWS in a preclinical Impella-induced aVWS calf model.CONCLUSION:Hence, inhibition of ADAMTS13 could become a novel therapeutic strategy to manage aVWS in MCS device patients.

Plasma ADAMTS13 circulates in a folded conformation that is stabilized by an interaction between the central Spacer domain and the C-terminal CUB (complement components C1r and C1s, sea urchin protein Uegf, and bone morphogenetic protein-1) domains. Binding of ADAMTS13 to the VWF D4(-CK) domains or to certain activating murine monoclonal antibodies (mAbs) induces a structural change that extends ADAMTS13 into an open conformation that enhances its function. The objective was to characterize the mechanism by which conformational activation enhances ADAMTS13-mediated proteolysis of VWF. The activating effects of a novel anti-Spacer (3E4) and the anti-CUB1 (17G2) mAbs on the kinetics of proteolysis of VWF A2 domain fragments by ADAMTS13 were analyzed. mAb-induced conformational changes in ADAMTS13 were investigated by enzyme-linked immunosorbent assay. Both mAbs enhanced ADAMTS13 catalytic efficiency (kcat/Km) by ∼twofold (3E4: 2.0-fold; 17G2: 1.8-fold). Contrary to previous hypotheses, ADAMTS13 activation was not mediated through exposure of the Spacer or cysteine-rich domain exosites. Kinetic analyses revealed that mAb-induced conformational extension of ADAMTS13 enhances the proteolytic function of the metalloprotease domain (kcat), rather than augmenting substrate binding (Km). A conformational effect on the metalloprotease domain was further corroborated by the finding that incubation of ADAMTS13 with either mAb exposed a cryptic epitope in the metalloprotease domain that is normally concealed when ADAMTS13 is in a closed conformation. We show for the first time that the primary mechanism of mAb-induced conformational activation of ADAMTS13 is not a consequence of functional exosite exposure. Rather, our data are consistent with an allosteric activation mechanism on the metalloprotease domain that augments active site function.

Background In immune-mediated thrombotic thrombocytopenic purpura (iTTP), patients develop an immune response against the multidomain enzyme ADAMTS13. ADAMTS13 consists of a metalloprotease (M) and disintegrin-like (D) domain, 8 thrombospondin type 1 repeats (T1-T8), a cysteine-rich (C), a spacer (S), and 2 CUB domains (CUB1-2). Previous epitope mapping studies have used relatively large overlapping ADAMTS13 fragments. Objectives We aimed at developing small nonoverlapping ADAMTS13 fragments to fine map anti-ADAMTS13 autoantibodies in iTTP patients. Methods A library of 16 ADAMTS13 fragments, comprising several small (M, DT, C, S, T2-T5, T6-T8, CUB1, CUB2), and some larger fragments with overlapping domains (MDT, MDTC, DTC, CS, T2-T8, CUB1-2, MDTCS, T2-C2), were generated. All fragments, and ADAMTS13, were expressed as a fusion protein with albumin domain 1, and purified. The folding of the fragments was tested using 17 anti-ADAMTS13 monoclonal antibodies with known epitopes. An epitope mapping assay using small ADAMTS13 fragments was set up, and validated by analyzing 18 iTTP patient samples. Results Validation with the monoclonal antibodies demonstrated that single S and CUB1 were not correctly folded, and therefore CS and CUB1-2 fragments were selected instead of single C, S, CUB1, and CUB2 fragments. Epitope mapping of antibodies of patients with iTTP confirmed that 6 nonoverlapping ADAMTS13 fragments M, DT, CS, T2-T5, T6-T8, and CUB1-2 were sufficient to accurately determine the antibody-binding sites. Conclusion We have developed a tool to profile patients with iTTP according to their anti-ADAMTS13 antibodies for a better insight in their immune response.

Bleeding is the most frequent adverse event in patients with continuous flow mechanical circulatory support (CF-MCS) and has been linked to the occurrence of acquired von Willebrand syndrome (aVWS). MCS devices cause an increased shear-induced proteolysis of von Willebrand factor (VWF) by ADAMTS13, leading to aVWS. Hence, specifically blocking ADAMTS13 might be an efficient way to rescue the loss of HMW VWF multimers in CF-MCS patients. To investigate if blocking ADAMTS13, using an in-house developed inhibitory anti-ADAMTS13 monoclonal antibody (mAb), prevents the loss of high molecular weight (HMW) VWF multimers in in vitro CF-MCS systems and to determine the efficacy of this therapy in a CF-MCS calf model. Human blood was perfused through in vitro CF-MCS systems (Heartmate II and Impella CP, axial flow heart pumps) in the presence of the inhibitory or control mAb (20 μg/mL). Bovine blood was perfused through an in vitro Impella 5.5 system with the inhibitory mAb (20 μg/mL) or PBS. Next, Impella 5.5 pumps were implanted in calves. One dose of the inhibitory mAb (600 μg/kg) or PBS was injected eight days after Impella implantation. Plasma samples were analysed for VWF multimers, VWF antigen (VWF:Ag) and VWF collagen binding activity (VWF:CB). A time-dependent decrease in HMW VWF multimers was observed in both in vitro CF-MCS systems in the presence of the control mAb, leading to a 70% reduction of HMW VWF multimers, 180 minutes (min) after blood perfusion (p=0.01 for HM II and p=0.0003 for Impella). This was also reflected by a severely decreased VWF:CB/VWF:Ag ratio (0.59±0.11 and 0.52±0.10 at 180 min versus 1.00±0.06 and 1.07±0.09 before perfusion, for the HM II (p=0.03) and Impella (p=0.001) respectively). Interestingly, blocking ADAMTS13 using the inhibitory mAb prevented the loss of HMW VWF multimers in both systems (p=0.50 for the HM II and p=0.06 for the Impella, 180 min after the start of perfusion). The preservation of HMW VWF multimers was also reflected by normal VWF:CB/VWF:Ag ratios (0.92±0.16 and 0.97±0.11 at 180 min versus 0.93±0.09 and 1.19±0.12 before perfusion for the HM II (p=0.75) and Impella (p=0.06) respectively). Blocking bovine ADAMTS13 using the inhibitory mAb could prevent the loss of HMW VWF multimers in the in vitro Impella 5.5 system, showing that the calf is a good preclinical animal model to study the in vivo effect of this novel therapy. Impella implantation in the calves led to a decrease in HMW VWF multimers (Figure 1A and B). Hence, this animal model represents the VWF laboratory features of MCS-induced aVWS. Moreover, the loss of HMW VWF multimers after pump implantation could be rescued after injection of the inhibitory mAb (Figure 1A and B). Blocking ADAMTS13 rescues MCS-induced VWF proteolysis in calves. Hence, inhibiting ADAMTS13 function could become a promising therapeutic strategy to rescue aVWS-induced bleeding in MCS patients. Figure 1. Impella calf model Type of funding source: Foundation. Main funding source(s): Fund for Scientific Research Flanders

Recent data indicate that elite athletes are at risk of developing exercise-induced bronchoconstriction (EIB).1Bonini M. Silvers W. Exercise-induced bronchoconstriction: background, prevalence, and sport considerations.Immunol Allergy Clin North Am. 2018; 38: 205-214Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 2Boulet L.-P. O'Byrne P.M. Asthma and exercise-induced bronchoconstriction in athletes.N Engl J Med. 2015; 372: 641-648Crossref PubMed Scopus (104) Google Scholar, 3Levai I.K. Hull J.H. Loosemore M. Greenwell J. Whyte G. Dickinson J.W. Environmental influence on the prevalence and pattern of airway dysfunction in elite athletes.Respirology. 2016; 21: 1391-1396Crossref PubMed Scopus (22) Google Scholar EIB is defined as the acute narrowing of the airways during or immediately after exercise.2Boulet L.-P. O'Byrne P.M. Asthma and exercise-induced bronchoconstriction in athletes.N Engl J Med. 2015; 372: 641-648Crossref PubMed Scopus (104) Google Scholar The prevalence of EIB in athletes ranges between 3.7% and 54.8% depending on the sport discipline and the environment in which the athletes exercise.1Bonini M. Silvers W. Exercise-induced bronchoconstriction: background, prevalence, and sport considerations.Immunol Allergy Clin North Am. 2018; 38: 205-214Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar The pathophysiological mechanisms driving EIB are not fully understood. One of the hypotheses states that high ventilatory rates in combination with environmental factors such as cold dry air, air pollution, and/or chlorine by-products can lead to an increased mechanical load to the airways, resulting in epithelial shedding and damage.4Rundell K.W. Smoliga J.M. Bougault V. Exercise-induced bronchoconstriction and the air we breathe.Immunol Allergy Clin North Am. 2018; 38: 183-204Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar In line with this hypothesis, we showed that in adolescent and young adult elite swimmers, epithelial damage with increased sputum uric acid and serum club cell protein 16 (CC16) levels, is present.5Seys S.F. Hox V. Van Gerven L. Dilissen E. Marijsse G. Peeters E. et al.Damage-associated molecular pattern and innate cytokine release in the airways of competitive swimmers.Allergy. 2015; 70: 187-194Crossref PubMed Scopus (30) Google Scholar As damage-associated molecular patterns result from epithelial damage, airway innate immune cells, such as macrophages, can get activated, resulting in the release of proinflammatory cytokines such as TNF-α, IL-6, IL-8, IL-1β, and IL-1α.5Seys S.F. Hox V. Van Gerven L. Dilissen E. Marijsse G. Peeters E. et al.Damage-associated molecular pattern and innate cytokine release in the airways of competitive swimmers.Allergy. 2015; 70: 187-194Crossref PubMed Scopus (30) Google Scholar Only few studies have investigated the prevalence of EIB in early-career elite athletes. Pedersen et al6Pedersen L. Lund T.K. Barnes P.J. Kharitonov S.A. Backer V. Airway responsiveness and inflammation in adolescent elite swimmers.J Allergy Clin Immunol. 2008; 122 (327.e1): 322-327Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar did not find increased EIB or signs of airway inflammation in 13- to 16-year-old elite swimmers leading to the conclusion that elite swimmers develop EIB throughout their career. This urges for screening tools to detect EIB in young elite athletes early in and during their sports career. We hypothesize that markers of epithelial damage, which might be the first pathological event upon airway triggering, could be suitable biomarkers for the detection of EIB. Therefore, we tested 117 12- to 13-year-old early-career elite athletes performing more than 12 hours of sports per week from different Flemish prevalent sport disciplines (swimming, basketball, and football) and compared them with control subjects (see this article's Online Repository at www.jacionline.org). We investigated the prevalence of EIB with the eucapnic voluntary hyperventilation (EVH) test adapted for young elite athletes7Van der Eycken S. Schelpe A. Marijsse G. Dilissen E. Troosters T. Vanbelle V. et al.Feasibility to apply eucapnic voluntary hyperventilation in young elite athletes.Respir Med. 2016; 111: 91-93Abstract Full Text Full Text PDF PubMed Google Scholar as well as different markers of airway inflammation and epithelial damage (see this article's Online Repository at www.jacionline.org). Subjects' characteristics are presented in Table I. Interestingly, swimmers showed significantly higher FEV1 and forced vital capacity (FVC) compared with control subjects (Table I). Symptoms during exercise were investigated in the sport disciplines (see Fig E1 in this article's Online Repository at www.jacionline.org). In this study, we found that 24.5% of the tested early-career elite athletes (12-13 years old) suffer from EIB. This is slightly higher than the mean prevalence (15%) of EIB in young elite athletes (5-18 years old) as reported in a meta-analysis by de Aguiar et al.8de Aguiar K.B. Anzolin M. Zhang L. Global prevalence of exercise-induced bronchoconstriction in childhood: a meta-analysis.Pediatr Pulmonol. 2018; 53: 412-425Crossref PubMed Scopus (28) Google Scholar Besides the differences in age group, this higher prevalence can be due to the use of the EVH test instead of an exercise challenge test, such as a 6-minute running test at 85% to 90% of the maximal heart rate, which is used in most of the previous studies in athletes between 12 and 18 years old. We opted to use the EVH test because it is performed in a standardized setting similar for all sport disciplines and it is accepted by the International Olympic Committee to diagnose EIB. In our study, early-career elite swimmers (33.3%) have the highest prevalence of EIB followed by basketball (27.3%) and football players (12.8%) (Fig 1). Several studies in adult elite athletes have already shown that EIB is more common in swimmers as well as in athletes performing sports in a cold environment.3Levai I.K. Hull J.H. Loosemore M. Greenwell J. Whyte G. Dickinson J.W. Environmental influence on the prevalence and pattern of airway dysfunction in elite athletes.Respirology. 2016; 21: 1391-1396Crossref PubMed Scopus (22) Google Scholar We now show that already at the start of their sports career, swimmers have a higher EIB prevalence compared with others, probably because of the long-term exposure to chlorine by-products during intensive exercise. In addition to the presence of EIB in early-career elite athletes, signs of epithelial damage were detected in 12- to 13-year-old athletes. Indeed, in all athletes (ie, in swimmers and in basketball and football players), baseline serum CC16 level was increased compared with the level in age-matched controls (Fig 1). No significant differences were found in the percentages of sputum neutrophils and eosinophils between athletes and control subjects (see Fig E3 in this article's Online Repository at www.jacionline.org), leading to believe that at this young age epithelial damage is already present, but cellular inflammation is still missing in almost all the athletes (see Table E3 in this article's Online Repository at www.jacionline.org). Interestingly, higher serum CC16 levels correlated significantly with higher maximal fall amplitude in FEV1 after the EVH test (r = −0.1937; P = .0262; Fig 1). Sputum uric acid was increased in all athletes compared with the level in age-matched controls but was most striking in basketball players (Fig 1). In contrast, no significant differences in serum uric acid levels were found among the different athletes and control subjects (data not shown). However, EIB+ athletes have higher serum uric acid levels, but not higher CC16 levels compared with those in EIB− athletes (P = .044; Fig 1). No significant differences were observed in sputum proinflammatory cytokine mRNA levels (IL-1α, IL-1β, IL-6, IL-8, IL-17A, and TNF-α) between the 4 groups (see Fig E2 in this article's Online Repository at www.jacionline.org). However, sputum IL-8 mRNA levels (but none of the other proinflammatory cytokine levels) were significantly increased in athletes with EIB compared with those without EIB (P = .009; Fig 1).Table ISubjects' characteristicsCharacteristicBasketball playersFootball playersSwimmersControl subjectsP valueNumber (n)26444717Age (y)12.2 ± 0.613.1 ± 0.912.1 ± 0.812.9 ± 1.8<.0001Sex (M/F)14/1237/736/115/12.0001BMI (kg/m2)18.4 ± 1.719.0 ± 2.918.0 ± 1.717.7 ± 2.2.2194Atopy, n (%)6 (23.1)14 (31.8)19 (40.4)4 (23.5).3056FEV1 (L)3.2 ± 0.53.3 ± 0.63.5 ± 0.6∗P < .01 compared with control subjects.2.9 ± 0.9.0101FEV1% predicted101.9 ± 10.6110.9 ± 17.1114.1 ± 15.7†P < .05 compared with control subjects.97.5 ± 18.7.0008FVC (L)3.7 ± 0.73.8 ± 0.84.3 ± 0.8∗P < .01 compared with control subjects.3.5 ± 1.0.0007FVC% predicted100.7 ± 14.9108.5 ± 18.8117.9 ± 13.6‡P < .001 compared with control subjects.97.5 ± 20.9<.0001Hours of sports a week (median)12‡P < .001 compared with control subjects.13‡P < .001 compared with control subjects.12‡P < .001 compared with control subjects.3.5<.0001Sputum total cell count (×106)0.3-0.6-1.60.2-0.5-1.10.3-0.4-0.90.6-0.96-1.9.0764Sputum macrophages (%)69.5-93.1-9642-81.5-99.158.4-94.4-99.740-80-93.1518Sputum neutrophils (%)1.8-4-14.70.9-21.5-57.50-4-37.47-20-60.1346Sputum eosinophils (%)0-0-00-0-0.070-0-00-0-0.1601Sputum lymphocytes (%)0-0.4-1.20-0-00-0-1.10-0.8-1.8.1836Normally distributed data are represented as mean ± SD and analyzed via 1-way ANOVA. Nonparametric data are represented as median with interquartile range and analyzed with Kruskal-Wallis test.BMI, Body mass index; F, female; FVC, forced vital capacity; M, male.∗ P < .01 compared with control subjects.† P < .05 compared with control subjects.‡ P < .001 compared with control subjects. Open table in a new tab Normally distributed data are represented as mean ± SD and analyzed via 1-way ANOVA. Nonparametric data are represented as median with interquartile range and analyzed with Kruskal-Wallis test. BMI, Body mass index; F, female; FVC, forced vital capacity; M, male. Till now, an exercise challenge test or the EVH test to diagnose EIB is not included in the standard medical screening of early-career elite athletes, although EIB might seriously affect their future career. It remains very difficult to screen all early-career elite athletes yearly for EIB. Therefore, one of the goals of this research was to define risk factors and/or biomarkers that might predict EIB with high sensitivity and to screen early-career athletes from different sport disciplines who might be more susceptible to have EIB. Therefore, we looked at the atopic state (determined via skin prick test) and the AQUA score9Jonckheere A.-C. Seys S.F. Dilissen E. Marijsse G. Schelpe A.-S. Van der Eycken S. et al.AQUA© Questionnaire as prediction tool for atopy in young elite athletes.Pediatr Allergy Immunol. 2018; 29: 648-650Crossref PubMed Scopus (2) Google Scholar of our athletes and correlated these with maximal fall in FEV1 and EIB status. In our cohort of early-career elite athletes, being atopic or having an AQUA score of 6 or more on its own does not indicate the presence of EIB. Second, sputum cytokine mRNA levels and serum damage-associated molecular patterns were investigated to predict EIB. Using receiver-operating characteristic (ROC) analysis, cutoff values were determined, which were able to predict, with a high sensitivity, the presence of EIB in early-career elite athletes (see Fig E4 in this article's Online Repository at www.jacionline.org). Athletes with sputum IL-8 mRNA levels lower than 190 or serum uric acid levels below 92 μM have a very low risk of developing EIB (P = .003 and P = .044, respectively; see Table E1 in this article's Online Repository at www.jacionline.org). However, athletes with sputum IL-1β mRNA levels higher than 300 are more prone to have or develop EIB (P = .0331; positive predictive value = 0.85; Table E1). To conclude, EIB and markers of potential epithelial damage are already present in 12- to 13-year-old early-career elite athletes, in absence of overt cellular inflammation. We suggest that in early-career elite athletes, EIB can be screened by searching for "wheezing during exercise" and using the AQUA questionnaire on a first level to look for athletes who are at risk for EIB development. However, this would lead to a substantial number of nonatopic yet EIB+ athletes who would be missed in screening. On a second level, potential biomarkers such as serum CC16, serum uric acid, sputum IL-8, and IL-1β mRNA levels might be of use to identify athletes who are more prone to develop EIB, independent of their atopy status. This however needs to be validated in a larger cohort. This selected group of athletes should then be referred to specialized centers to undergo an exercise challenge test or an EVH test to confirm the presence of EIB. We thank the participating athletes for their cooperation and the "Topsportscholen" of Wilrijk and Leuven in Flanders. Forty-seven competitive swimmers, 44 football players, and 26 basketball players between the age of 12 and 13 years were recruited at different elite high schools, known in Flanders as "Topsportscholen" and among athletes of the Future Swimming Team. All athletes included performed at least 12 hours of sports a week. These represented ±95% of the available high-school elite athletes within those disciplines in Flanders. Control subjects who performed recreational sports for less than 6 hours of sports a week were recruited (n = 17). Subjects' characteristics are presented in Table I. Three of the swimmers and 1 football player were previously diagnosed with asthma by a physician. One swimmer was diagnosed with rhinitis by a physician. In contrast, none of the basketball players or controls had a physician-based asthma diagnosis. Three swimmers were using inhaled corticosteroids, 1 swimmer was using a long-acting β2 adrenergic receptor agonist, and 1 basketball player had occasionally used a bronchodilator just before intense training. The study was approved by the institutional review board and registered at clinicaltrails.gov (NCT02432183). All tests were performed outside the pollen season as well as outside a period of intense competition and/or training. The EVH test was performed according to the American Thoracic Society guidelines and adapted for young elite athletes.E1Parsons J.P. Hallstrand T.S. Mastronarde J.G. Kaminsky D.A. Rundell K.W. Hull J.H. et al.An official American Thoracic Society clinical practice guideline: exercise-induced bronchoconstriction.Am J Respir Crit Care Med. 2013; 187: 1016-1027Crossref PubMed Scopus (372) Google Scholar, E2Van der Eycken S. Schelpe A. Marijsse G. Dilissen E. Troosters T. Vanbelle V. et al.Feasibility to apply eucapnic voluntary hyperventilation in young elite athletes.Respir Med. 2016; 111: 91-93Abstract Full Text Full Text PDF PubMed Google Scholar Subjects were advised to stop regular treatment 48 hours before the test.E3Anderson S.D. Brannan J.D. Methods for "indirect" challenge tests including exercise, eucapnic voluntary hyperpnea, and hypertonic aerosols.Clin Rev Allergy Immunol. 2003; 24: 27-54Crossref PubMed Scopus (135) Google Scholar Before the start of the EVH test, the maximal voluntary ventilation (MVV) was measured with the help of a spirometer. The athlete breathed quickly and deeply for 12 to 15 seconds and afterward the data were extrapolated to a value expressed as liters per minute. The EVH test was performed for 6 minutes at a target ventilation of 85% of the MVV, and ventilation was monitored by a flow sensor (Jaeger Oxycon Mobile, Carefusion, Vilvoorde, Belgium). Spirometry (Spirolab III spirometer; MIR, Rome, Italy) was performed immediately after and 5, 10, and 15 minutes after the EVH test. Of all study subjects, 91.7% (123 of 134) performed an EVH test with lung function measurements. Four basketball players, 5 football players, and 2 swimmers did not perform the EVH test because of technical issues. All athletes obtained between 60% and 85% of MVV during the EVH test. The EVH test was considered positive if a drop in FEV1 greater than or equal to 10% compared with baseline was measured at one of the time points after the test (time point immediately after the EVH test not included). Exhaustion was excluded by the Tiffeneau index (FEV1/forced vital capacity).E1Parsons J.P. Hallstrand T.S. Mastronarde J.G. Kaminsky D.A. Rundell K.W. Hull J.H. et al.An official American Thoracic Society clinical practice guideline: exercise-induced bronchoconstriction.Am J Respir Crit Care Med. 2013; 187: 1016-1027Crossref PubMed Scopus (372) Google Scholar A higher Tiffeneau index compared with baseline could suggest a less accurate spirometry after the EVH test and might be excluded. Reversibility of FEV1 after inhalation of 400 μg of salbutamol was measured and calculated relative to the FEV1 measured 15 minutes after the EVH test.E4Van Der Eycken S. Schelpe A. Marijsse G. Dilissen E. Troosters T. Vanbelle V. et al.Feasibility to apply eucapnic voluntary hyperventilation in young elite athletes.Respir Med. 2016; 111: 91-93Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar A reversibility above 20% was used as the second criterion to consider a person EIB+ or not when only 1 drop in FEV1 greater than or equal to 10% compared with baseline was measured after the EVH test. This criterion was added because of the limited time points used to measure FEV1 after the EVH test (3, 7, and 12 minutes were not included in the analysis). The Allergy questionnaire for athletes (AQUA) was reported by Bonini et alE5Bonini M. Braido F. Baiardini I. Giacco S.D.E.L. Gramiccioni C. Manara M. et al.AQUA: allergy questionnaire for athletes. Development and validation.Med Sci Sports Exerc. 2009; 41: 1034-1041Crossref PubMed Scopus (82) Google Scholar and validated for its use in other studies.E10Fasola S. Bellafiore M. Baiardini I. Bonini M. Giacco S.D. Ferrante G. et al.Feasibility of the Allergy Questionnaire for Athletes (AQUA©) in pediatric age.Pediatr Allergy Immunol. 2019; 30: 242-245PubMed Google Scholar, E5Bonini M. Braido F. Baiardini I. Giacco S.D.E.L. Gramiccioni C. Manara M. et al.AQUA: allergy questionnaire for athletes. Development and validation.Med Sci Sports Exerc. 2009; 41: 1034-1041Crossref PubMed Scopus (82) Google Scholar, E6Teixeira R.N. Mendes F.A. Martins M.A. Mickleborough T.D. Carvalho C.R. AQUA© as predictor of allergy in elite marathon runners.World Allergy Organ J. 2014; 7: 7Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, E7Kurowski M. Jurczyk J. Krysztofiak H. Kowalski M.L. Exercise-induced respiratory symptoms and allergy in elite athletes: Allergy and Asthma in Polish Olympic Athletes (A(2)POLO) project within GA(2)LEN initiative.Clin Respir J. 2016; 10: 231-238Crossref PubMed Scopus (20) Google Scholar, E8Teixeira R. Leite G.D.S. Bonini M. Gorjão R. Agondi R. Kokron C. et al.Atopy in elite endurance athletes.Clin J Sport Med. 2018; 28: 268-271Crossref PubMed Scopus (4) Google Scholar, E9Jonckheere A.-C. Seys S.F. Dilissen E. Marijsse G. Schelpe A.-S. Van der Eycken S. et al.AQUA© Questionnaire as prediction tool for atopy in young elite athletes.Pediatr Allergy Immunol. 2018; 29: 648-650Crossref PubMed Scopus (6) Google Scholar The AQUA questionnaire was filled in by 26 of 26 basketball players, 44 of 44 football players, 46 of 47 swimmers, and 17 of 17 control subjects. Via a self-made questionnaire several symptoms of the upper and lower airways were studied. Irritated nose, itchy nose, sneezing, rhinorrhea, and nasal obstruction were questioned as symptoms for upper airway disease. For lower airway disease the following symptoms were investigated: wheezing, coughing, shortness of breath, and chest tightness. A skin prick test for 9 common aero-allergens (Dermatophagoides pteronyssinus, mixed tree pollen [hazel, birch, and alder], birch pollen, grass pollen, weed pollen, cat, dog, Alternaria Alternata, and Aspergillus fumigatus) was performed on all subjects (HAL Allergy, Leiden, The Netherlands). A person was considered "atopic" if the skin prick test result was positive (wheal diameter ≥3 mm and at least the size of the histamine control) for at least 1 of the 9 tested allergens.E11Heinzerling L. Mari A. Bergmann K.-C. Bresciani M. Burbach G. Darsow U. et al.The skin prick test—European standards.Clin Transl Allergy. 2013; 3: 3Crossref PubMed Scopus (487) Google Scholar Subjects inhaled a nebulized hypertonic salt solution (3%, 4%, and 5%) for 7 minutes and spit induced sputum into a sputum cup as described in the European Respiratory Society guidelines.E12Weiszhar Z. Horvath I. Induced sputum analysis: step by step.Breathe. 2013; 9: 300-306Crossref Scopus (42) Google Scholar Sputum samples were processed by the selected plug method as previously described.E13Seys S.F. Hox V. Van Gerven L. Dilissen E. Marijsse G. Peeters E. et al.Damage-associated molecular pattern and innate cytokine release in the airways of competitive swimmers.Allergy. 2015; 70: 187-194Crossref PubMed Scopus (43) Google Scholar, E14Seys S.F. Daenen M. Dilissen E. Van Thienen R. Bullens D.M.A. Hespel P. et al.Effects of high altitude and cold air exposure on airway inflammation in patients with asthma.Thorax. 2013; 68: 906-913Crossref PubMed Scopus (64) Google Scholar, E15Seys S.F. Grabowski M. Adriaensen W. Decraene A. Dilissen E. Vanoirbeek J.A. et al.Sputum cytokine mapping reveals an "IL-5, IL-17A, IL-25-high" pattern associated with poorly controlled asthma.Clin Exp Allergy. 2013; 43: 1009-1017Crossref PubMed Scopus (63) Google Scholar, E16Bullens D.M.A. Truyen E. Coteur L. Dilissen E. Hellings P.W. Dupont L.J. et al.IL-17 mRNA in sputum of asthmatic patients: linking T cell driven inflammation and granulocytic influx?.Respir Res. 2006; 7: 1-9Crossref PubMed Scopus (484) Google Scholar, E17Truyen E. Coteur L. Dilissen E. Overbergh L. Dupont L.J. Ceuppens J.L. et al.Evaluation of airway inflammation by quantitative Th1/Th2 cytokine mRNA measurement in sputum of asthma patients.Thorax. 2006; 61: 202-208Crossref PubMed Scopus (153) Google Scholar, E18Seys S.F. Scheers H. Van den Brande P. Marijsse G. Dilissen E. Van Den Bergh A. et al.Cluster analysis of sputum cytokine-high profiles reveals diversity in T(h)2-high asthma patients.Respir Res. 2017; 18: 1-10Crossref PubMed Scopus (53) Google Scholar Sputum supernatants were distributed into an Eppendorf tube and stored at −80°C. Total sputum cell count was determined. Blood samples were taken before a period of intense exercise and before the EVH test. Serum CC16 level was quantified by ELISA according to the manufacturer's protocol (Biovender, Brno, Czech Republic). Cytospins (Shandon cytocentifuge) were prepared from 12,500 and 25,000 sputum cells for differential cell counts, and the remaining cells were lysed for mRNA analysis at −80°C. Sputum cytokine levels (IL-1β, IL-1α, IL-8, IL-6, IL-17A, and TNF-α) were measured using real-time quantitative PCR.E13Seys S.F. Hox V. Van Gerven L. Dilissen E. Marijsse G. Peeters E. et al.Damage-associated molecular pattern and innate cytokine release in the airways of competitive swimmers.Allergy. 2015; 70: 187-194Crossref PubMed Scopus (43) Google Scholar, E14Seys S.F. Daenen M. Dilissen E. Van Thienen R. Bullens D.M.A. Hespel P. et al.Effects of high altitude and cold air exposure on airway inflammation in patients with asthma.Thorax. 2013; 68: 906-913Crossref PubMed Scopus (64) Google Scholar, E15Seys S.F. Grabowski M. Adriaensen W. Decraene A. Dilissen E. Vanoirbeek J.A. et al.Sputum cytokine mapping reveals an "IL-5, IL-17A, IL-25-high" pattern associated with poorly controlled asthma.Clin Exp Allergy. 2013; 43: 1009-1017Crossref PubMed Scopus (63) Google Scholar, E16Bullens D.M.A. Truyen E. Coteur L. Dilissen E. Hellings P.W. Dupont L.J. et al.IL-17 mRNA in sputum of asthmatic patients: linking T cell driven inflammation and granulocytic influx?.Respir Res. 2006; 7: 1-9Crossref PubMed Scopus (484) Google Scholar, E17Truyen E. Coteur L. Dilissen E. Overbergh L. Dupont L.J. Ceuppens J.L. et al.Evaluation of airway inflammation by quantitative Th1/Th2 cytokine mRNA measurement in sputum of asthma patients.Thorax. 2006; 61: 202-208Crossref PubMed Scopus (153) Google Scholar Primers and probes for IL-1β, IL-8, IL-6, IL-17A, TNF-α, and β-actin have been previously described.E13Seys S.F. Hox V. Van Gerven L. Dilissen E. Marijsse G. Peeters E. et al.Damage-associated molecular pattern and innate cytokine release in the airways of competitive swimmers.Allergy. 2015; 70: 187-194Crossref PubMed Scopus (43) Google Scholar, E14Seys S.F. Daenen M. Dilissen E. Van Thienen R. Bullens D.M.A. Hespel P. et al.Effects of high altitude and cold air exposure on airway inflammation in patients with asthma.Thorax. 2013; 68: 906-913Crossref PubMed Scopus (64) Google Scholar, E15Seys S.F. Grabowski M. Adriaensen W. Decraene A. Dilissen E. Vanoirbeek J.A. et al.Sputum cytokine mapping reveals an "IL-5, IL-17A, IL-25-high" pattern associated with poorly controlled asthma.Clin Exp Allergy. 2013; 43: 1009-1017Crossref PubMed Scopus (63) Google Scholar, E16Bullens D.M.A. Truyen E. Coteur L. Dilissen E. Hellings P.W. Dupont L.J. et al.IL-17 mRNA in sputum of asthmatic patients: linking T cell driven inflammation and granulocytic influx?.Respir Res. 2006; 7: 1-9Crossref PubMed Scopus (484) Google Scholar, E17Truyen E. Coteur L. Dilissen E. Overbergh L. Dupont L.J. Ceuppens J.L. et al.Evaluation of airway inflammation by quantitative Th1/Th2 cytokine mRNA measurement in sputum of asthma patients.Thorax. 2006; 61: 202-208Crossref PubMed Scopus (153) Google Scholar, E18Seys S.F. Scheers H. Van den Brande P. Marijsse G. Dilissen E. Van Den Bergh A. et al.Cluster analysis of sputum cytokine-high profiles reveals diversity in T(h)2-high asthma patients.Respir Res. 2017; 18: 1-10Crossref PubMed Scopus (53) Google Scholar Primers and probes for IL-1α and PPIA are listed in Table E2. Data were normalized to the geometric mean of the reference genes β-actin and PPIA. cDNA plasmid standards were used to quantify the amount of target gene in unknown samples.E19Giulietti A. Overbergh L. Valckx D. Decallonne B. Bouillon R. Mathieu C. An overview of real-time quantitative PCR: applications to quantify cytokine gene expression.Methods. 2001; 25: 386-401Crossref PubMed Scopus (1102) Google Scholar Sputum supernatant and serum uric acid levels diluted 1/5 to reduce interference of dithiothreitol in the sputum were determined with the Amplex Red uric acid/uricase assay kit from Invitrogen (Thermo Fisher Scientific, Waltham, Mass). We searched for cutoff values for the measured cytokines via a receiver-operating characteristic curve analysis (Fig E4). A total of 109 serum samples (23 basketball players, 33 football players, 44 swimmers, and 9 control subjects) and sputum samples (19 basketball players, 34 football players, 39 swimmers, and 17 control subjects) were collected and analyzed. Of the 109 sputum samples, 66 samples contained enough mRNA to analyze cytokines via quantitative PCR. Statistical analysis was performed with Graphpad prism 7 for Macintosh (Graphpad Software, Inc, San Diego, Calif). First, normal distribution was studied for each group with a D'Agostino and Pearson normality test. One-way ANOVA with Tukey post hoc test was used if the data were normally distributed, and Kruskal-Wallis test with Dunn's multiple comparison test was used for nonparametric data. Two-way ANOVA was used with Dunnett's multiple comparison tests to compare several symptoms in our different groups. Mann-Whitney test was used to compare 2 groups with nonparametric data. Paired data were analyzed with paired t test or Wilcoxon test where appropriate. A difference was considered significant when P was less than .05.Fig E2Relative expression of IL-1α (A), IL-1β (B), IL-6 (C), IL-8 (D), IL-17A (E), and TNF-α (F) on sputum mRNA level in basketball players (n = 9), football players (n = 22), swimmers (n = 23), and control subjects (n = 12). Data are represented as median with interquartile range. Kruskal-Wallis test was used to compare the different groups. Red dots represent the EIB+ athletes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Differential cell count in early-career elite athletes and control subjects. Sputum neutrophils (A) and eosinophils (B) were counted on cytospins. Data are represented as median with interquartile range. Red dots are athletes and control subjects with EIB. Kruskal-Wallis with multiple post hoc test was used to compare the different groups.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4ROC curves for sputum IL-1β, IL-8, IL-6, TNF-α mRNA levels, sputum uric acid levels, and serum CC16 levels. ROC curves were made by comparing the values of EIB+ and EIB− athletes. The dotted line with value represents the value with a sensitivity of 70% created by the ROC analysis. ROC, Receiver-operating characteristic.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Analysis of several known and unknown risk factors for EIB in early-career elite athletesBiomarkerEIB+EIB−P value∗P value, sensitivity, and specificity are determined via Fisher exact test. Cutoff value IL-8 = 190, cutoff value IL-1β = 300, cutoff value IL-6 = 6, cutoff value TNF-α = 20, cutoff value CC16 = 7 ng/mL, cutoff value sputum uric acid = 75 μM, and cutoff value serum uric acid = 92 μM. Boldface values represent significant P values with their corresponding sensitivity and specificity.Sensitivity (%)∗P value, sensitivity, and specificity are determined via Fisher exact test. Cutoff value IL-8 = 190, cutoff value IL-1β = 300, cutoff value IL-6 = 6, cutoff value TNF-α = 20, cutoff value CC16 = 7 ng/mL, cutoff value sputum uric acid = 75 μM, and cutoff value serum uric acid = 92 μM. Boldface values represent significant P values with their corresponding sensitivity and specificity.Specificity (%)∗P value, sensitivity, and specificity are determined via Fisher exact test. Cutoff value IL-8 = 190, cutoff value IL-1β = 300, cutoff value IL-6 = 6, cutoff value TNF-α = 20, cutoff value CC16 = 7 ng/mL, cutoff value sputum uric acid = 75 μM, and cutoff value serum uric acid = 92 μM. Boldface values represent significant P values with their corresponding sensitivity and specificity.AtopicNonatopic11132753.200645.8366.25AQUA score ≥ 6AQUA score < 611153247.524242.3159.49High levels IL-1βLow levels IL-1β1041423.033162.1671.43High levels IL-8Low levels IL-81311719.003592.8652.78High levels IL-6Low levels IL-6772510.19295028.57High levels TNF-αLow levels TNF-α1041819.210171.4351.35High levels sputum uric acidLow levels sputum uric acid2047012.755183.3314.63High levels serum CC16Low levels serum CC1611162351.249140.7468.92High levels serum uric acidLow levels serum uric acid1472335.042966.6760.34∗ P value, sensitivity, and specificity are determined via Fisher exact test. Cutoff value IL-8 = 190, cutoff value IL-1β = 300, cutoff value IL-6 = 6, cutoff value TNF-α = 20, cutoff value CC16 = 7 ng/mL, cutoff value sputum uric acid = 75 μM, and cutoff value serum uric acid = 92 μM. Boldface values represent significant P values with their corresponding sensitivity and specificity. Open table in a new tab Table E2Primer and probe sequences for quantitative PCRGenePrimerProbe sequencePPIAFWRVTP5′ cgc gtc tcc ttt gag ctg tt 3′5′ ctg aca cat aaa ccc tgg aat aat tc 3′5′ cag aca agg tcc caa aga cag cag aaa att t 3′IL-1αFWRVTP5′ cct gaa gaa ctg tta cag tga aaa tga 3′5′ aca gat tga tcc atg cag cct t 3′5′ tcc ttc tat cat gta agc tat ggc cca ctc c 3′FW, Forward; RV, reverse; TP, Taqman Probe. Open table in a new tab Table E3Correlation between sputum mRNA levels, sputum neutrophils, and serum CC16 levels in all subjectsIL-8IL-1αNeutrophils (%)IL-6IL-17AUric acidSerum CC16rPRPrPrPrPrPrPSputum IL-1β mRNA0.825< .00010.623< .00010.546.00040.397.0140.260.175−0.192.364−0.140.622Sputum IL-8 mRNA0.499∗Pearson correlation; P values are corrected for multiple testing via the Benjamini-Hochberg correction test. Boldface values represent significant P values..00110.510.00140.410∗Pearson correlation; P values are corrected for multiple testing via the Benjamini-Hochberg correction test. Boldface values represent significant P values..0050.322∗Pearson correlation; P values are corrected for multiple testing via the Benjamini-Hochberg correction test. Boldface values represent significant P values..066−0.333.0590.012.969Sputum IL-1α mRNA0.015.96860.523∗Pearson correlation; P values are corrected for multiple testing via the Benjamini-Hochberg correction test. Boldface values represent significant P values..0001−0.160.344−0.012.968−0.094.767Neutrophils (%)−0.048.8970.376.050−0.118.695−0.212.135Sputum IL-6 mRNA−0.095∗Pearson correlation; P values are corrected for multiple testing via the Benjamini-Hochberg correction test. Boldface values represent significant P values..615−0.097.750−0.019.969Sputum IL-17A mRNA0.01.973−0.10.757Sputum uric acid−0.003.993∗ Pearson correlation; P values are corrected for multiple testing via the Benjamini-Hochberg correction test. Boldface values represent significant P values. Open table in a new tab FW, Forward; RV, reverse; TP, Taqman Probe.

Several studies have already identified atopy as a risk factor for the development of exercise-induced bronchoconstriction (EIB) in adult elite athletes (1-3). Skin prick test or serum specific IgE to common aero-allergens is used to determine the patient's atopy status (4). In 2009, Bonini M et al. introduced a screening tool to predict atopy in elite adult athletes, namely the Allergy Questionnaire for Athletes or short the AQUA© questionnaire (5). This article is protected by copyright. All rights reserved.

IntroductionPatients suffering from congenital thrombotic thrombocytopenic purpura (cTTP) have a deficiency in ADAMTS13 due to mutations in their ADAMTS13 gene. ObjectiveThe aim of this study was to determine ADAMTS13 parameters (activity, antigen, and mutations), to investigate if the propositus suffered from child-onset cTTP, and to study the in vitro effect of the ADAMTS13 mutations. MethodsADAMTS13 activity and antigen were determined using the FRETS VWF73 assay and ELISA and ADAMTS13 mutations via sequencing of the exons. Mutant proteins were expressed in Chinese hamster ovary cells, and their expression was studied using fluorescence microscopy and ELISA. Molecular modeling was used to evaluate the effect of the mutations on ADAMTS13 structure and stability. ResultsThe propositus was diagnosed with cTTP at the age of 20. ADAMTS13 activity was below 10%, and 2 compound heterozygous mutations, the p.R498C point and the p.G259PfsX133 frameshift mutation, were identified. Expression of ADAMTS13 mutants revealed that the p.R498C and the p.G259PfsX133 mutation cause secretion and translation defects in vitro, respectively. Molecular modeling showed that the R498 intra-domain interactions are lacking in the p.R498C mutant, resulting in protein instability. ConclusionThe ADAMTS13 mutations result in a severe ADAMTS13 deficiency explaining the patient's phenotype.

Abstract Background ADAMTS13 circulates in a folded conformation, which is mediated by interactions between the C-terminal CUB domains and its central Spacer domain. Binding of ADAMTS13 to the VWF D4-CK domains disrupts the CUB-Spacer interaction, inducing a structural change that extends ADAMTS13 into an open conformation that enhances catalytic efficiency ~2-fold. This mechanism supports a model in which ADAMTS13 unfolding induces exposure of an exosite in the Spacer domain that interacts with the VWF A2 domain, increasing the affinity between the two molecules, and, therefore, the rate of proteolysis. The D4-CK-mediated conformational activation of ADAMTS13 can be mimicked in vitro with the use of antibodies that disrupt the CUB-Spacer interaction, such as the previously published anti-CUB antibody, Ab17G2. We recently generated a novel, activating antibody against the Spacer domain (Ab3E4). Aim To characterize the mechanism by which the Ab17G2 and Ab3E4 enhance the catalytic efficiency of ADAMTS13. Methods The effects of the Ab17G2 and Ab3E4 on the activity of ADAMTS13 were studied using FRETS-VWF73. The effects of the Ab17G2 and Ab3E4 on the kinetics of VWF96 (VWF G1573-R1668) proteolysis were characterized using an in-house assay. ELISA was used to investigate conformational changes in ADAMTS13 induced by the Ab17G2 and Ab3E4. Results Both Ab17G2 and Ab3E4 enhanced FRETS-VWF73 proteolysis by ~1.7-fold. This result was reproduced using the VWF96 substrate; the Ab17G2 and Ab3E4 enhanced the catalytic efficiency (kcat/Km) of ADAMTS13 by ~1.8- and ~2.0-fold, respectively. The activation was dependent on the conformational extension of ADAMTS13, since the antibodies could not enhance the activity of an ADAMTS13 variant that lacks the TSP2-CUB2 domains (MDTCS). Surprisingly, ADAMTS13 activation was not mediated through exposure of the Spacer or Cys-rich domain exosites as previously proposed, as the Ab17G2 and Ab3E4 efficiently enhanced proteolysis of VWF96 variants in which the Spacer/Cys-rich exosite binding sites were disrupted. Kinetic analysis of VWF96 proteolysis showed that the Ab17G2- and Ab3E4-induced activation of ADAMTS13 is primarily manifest through a ~1.5- to ~2-fold increase in enzyme turnover (kcat). Thus, contrary to the current model, this suggests that the conformational extension of ADAMTS13 influences the functionality of the active site, and not substrate binding affinity (Km). Incubating ADAMTS13 with either Ab17G2 or Ab3E4 exposed a cryptic epitope in the metalloprotease domain that was specifically detected by ELISA, further corroborating that the antibodies induce a conformational change in ADAMTS13 affecting the M domain. Conclusion Antibodies can be used as tools for understanding the structure/function of enzymes. Using activating antibodies against the Spacer and CUB1 domains of ADAMTS13, we show for the first time that the activation of ADAMTS13 following its unfolding is not a result of exposure of a functional exosite in Spacer/Cys-rich domain that increases affinity to VWF. Rather, our data are consistent with an allosteric activation mechanism upon the metalloprotease domain. We propose that ADAMTS13 unfolding causes a conformational change in the active site that further activates the enzyme. We are currently investigating whether the D4-CK-induced enhancement of ADAMTS13 proteolytic activity is also mediated by conformational changes in the active site. Disclosures Vanhoorelbeke: Ablynx: Consultancy; Shire: Consultancy.

Preemptive rituximab infusions prevent relapses in immune thrombotic thrombocytopenic purpura (iTTP) by maintaining normal ADAMTS13 activity. However, the long-term outcome of these patients and the potential adverse events of this strategy need to be determined. We report the long-term outcome of 92 patients with iTTP in clinical remission who received preemptive rituximab after identification of severe ADAMTS13 deficiency (activity <10%) during the follow-up. Thirty-seven patients had >1 iTTP episode, and the median cumulative relapse incidence before preemptive rituximab was 0.33 episode per year (interquartile range [IQR], 0.23-0.66). After preemptive rituximab, the median cumulative relapse incidence in the whole population decreased to 0 episodes per year (IQR, 0-1.32; P < .001). After preemptive rituximab, ADAMTS13 activity recovery was sustained in 34 patients (37%) during a follow-up of 31.5 months (IQR, 18-65), and severe ADAMTS13 deficiency recurred in 45 patients (49%) after the initial improvement. ADAMTS13 activity usually improved with additional courses of preemptive rituximab. In 13 patients (14%), ADAMTS13 activity remained undetectable after the first rituximab course, but retreatment was efficient in 6 of 10 cases. In total, 14 patients (15%) clinically relapsed, and 19 patients (20.7%) experienced benign adverse effects. Preemptive rituximab treatment was associated with a change in ADAMTS13 conformation in respondent patients. Finally, in the group of 23 historical patients with iTTP and persistently undetectable ADAMTS13 activity, 74% clinically relapsed after a 7-year follow-up (IQR, 5-11). In conclusion, persistently undetectable ADAMTS13 activity in iTTP during remission is associated with a higher relapse rate. Preemptive rituximab reduces clinical relapses by maintaining a detectable ADAMTS13 activity with an advantageous risk-benefit balance.

In autoantibody-mediated autoimmune diseases, autoantibody profiling allows patients to be stratified and links autoantibodies with disease severity and outcome. However, in immune-mediated thrombotic thrombocytopenic purpura (iTTP) patients, stratification according to antibody profiles and their clinical relevance has not been fully explored. We aimed to develop a new type of autoantibody profiling assay for iTTP based on the use of anti-idiotypic antibodies. Anti-idiotypic antibodies against 3 anti-spacer autoantibodies were generated in mice and were used to capture the respective anti-spacer idiotopes from 151 acute iTTP plasma samples. We next deciphered these anti-spacer idiotope profiles in iTTP patients and investigated whether these limited idiotope profiles could be linked with disease severity. We developed 3 anti-idiotypic antibodies that recognized particular idiotopes in the anti-spacer autoantibodies II-1, TTP73 or I-9, that are involved in ADAMTS13 binding; 35%, 24% and 42% of patients were positive for antibodies with the II-1, TTP73 and I-9 idiotopes, respectively. Stratifying patients according to the corresponding 8 anti-spacer idiotope profiles provided a new insight into the anti-spacer II-1, TTP73 and I-9 idiotope profiles in these patients. Finally, these limited idiotope profiles showed no association with disease severity. We successfully developed 3 anti-idiotypic antibodies that allowed us to determine the profiles of the anti-spacer II-1, TTP73 and I-9 idiotopes in iTTP patients. Increasing the number of patients and/or future development of additional anti-idiotypic antibodies against other anti-ADAMTS13 autoantibodies might allow idiotope profiles of clinical, prognostic value to be identified.

AbstractImmune-mediated thrombotic thrombocytopenic purpura (iTTP) is characterized by severe ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13) deficiency, the presence of anti-ADAMTS13 autoantibodies and an open ADAMTS13 conformation with a cryptic epitope in the spacer domain exposed. A detailed knowledge of anti-ADAMTS13 autoantibodies will help identifying pathogenic antibodies and elucidating the cause of ADAMTS13 deficiency. We aimed at cloning anti-ADAMTS13 autoantibodies from iTTP patients to study their epitopes and inhibitory characteristics. We sorted anti-ADAMTS13 autoantibody expressing B cells from peripheral blood mononuclear cells of 13 iTTP patients to isolate anti-ADAMTS13 autoantibody sequences. Ninety-six B cell clones producing anti-ADAMTS13 autoantibodies were identified from which 30 immunoglobulin M (IgM) and 5 IgG sequences were obtained. For this study, we only cloned, expressed and purified the five IgG antibodies. In vitro characterization revealed that three of the five cloned IgG antibodies, TTP73–1, ELH2–1 and TR8C11, indeed recognize ADAMTS13. Epitope mapping showed that antibodies TTP73–1 and TR8C11 bind to the cysteine–spacer domains, while the antibody ELH2–1 recognizes the T2–T3 domains in ADAMTS13. None of the antibodies inhibited ADAMTS13 activity. Given the recent findings regarding the open ADAMTS13 conformation during acute iTTP, we studied if the cloned antibodies could recognize cryptic epitopes in ADAMTS13. Interestingly, all three antibodies recognize cryptic epitopes. In conclusion, we cloned three anti-ADAMTS13 autoantibodies from iTTP patients that recognize cryptic epitopes. Hence, these data nicely fit our recent finding that the conformation of ADAMTS13 is open during acute iTTP.

Background. Deficient ADAMTS13 activity (TS13:act <10%) induced by anti-ADAMTS13 autoantibodies (autoAbs) causes immune-mediated thrombotic thrombocytopenic purpura (iTTP). Recently we showed that an open ADAMTS13 conformation is characteristic for acute iTTP patients, while folded ADAMTS13 was found in 78% of iTTP patients in remission with an TS13:act >50%. However, also iTTP patients in remission with a persistent (<10%) or moderately restored (10-50%) TS13:act have been described, but their ADAMTS13 conformation is unknown. Intriguingly, the factor responsible for inducing open ADAMTS13 in iTTP patients remains elusive. Identifying the cause of open ADAMTS13 in iTTP will help better understand the pathophysiology of iTTP and could help appreciate the prognosis and better manage the prevention of subsequent relapses.

In this study, we investigated a case of pregnancy-onset thrombotic thrombocytopenic purpura (TTP). The patient had severely decreased ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13) activity levels during acute phase and the presence of inhibitory anti-ADAMTS13 autoantibodies was demonstrated, which led to the diagnosis of immune-mediated TTP. However, ADAMTS13 activity was only mildly restored during remission, although inhibitory anti-ADAMTS13 antibodies were no longer detected. We hypothesized that genetic abnormalities could account for this discrepancy between ADAMTS13 activity and antigen. Genetic analysis revealed the presence of two heterozygous substitutions on the same allele: a single nucleotide polymorphism (SNP) c.2699C > T (p.A900V), located in the beginning of the T5 domain, and a mutation c.3530G > A (p.R1177Q) located in the third linker region of ADAMTS13. In vitro testing of those substitutions by expression of recombinant proteins revealed a normal secretion but a reduced ADAMTS13 activity by the novel p.R1177Q mutation, which could partially explain the subnormal activity levels found during remission. Although changes in the linker region might induce conformational changes in ADAMTS13, the p.R1177Q mutation in the third linker region of ADAMTS13 did not expose a cryptic epitope in the metalloprotease domain. In conclusion, we report on an immune-mediated pregnancy-onset TTP patient who had inhibitory anti-ADAMTS13 autoantibodies during acute phase, but not during remission. Genetic analysis confirmed the diagnosis of immune-mediated TTP and revealed the novel p.R1177Q mutation which mildly impaired ADAMTS13 activity.