Recombinant ADAMTS13 in Congenital Thrombotic Thrombocytopenic Purpura.

BACKGROUND: Congenital thrombotic thrombocytopenic purpura (TTP) results from severe hereditary deficiency of ADAMTS13. The efficacy and safety of recombinant ADAMTS13 and standard therapy (plasma-derived products) administered as routine prophylaxis or on-demand treatment in patients with congenital TTP is not known. METHODS: In this phase 3, open-label, crossover trial, we randomly assigned patients in a 1:1 ratio to two 6-month periods of prophylaxis with recombinant ADAMTS13 (40 IU per kilogram of body weight, administered intravenously) or standard therapy, followed by the alternate treatment; thereafter, all the patients received recombinant ADAMTS13 for an additional 6 months. The trigger for this interim analysis was trial completion by at least 30 patients. The primary outcome was acute TTP events. Manifestations of TTP, safety, and pharmacokinetics were assessed. Patients who had an acute TTP event could receive on-demand treatment. RESULTS: A total of 48 patients underwent randomization; 32 completed the trial. No acute TTP event occurred during prophylaxis with recombinant ADAMTS13, whereas 1 patient had an acute TTP event during prophylaxis with standard therapy (mean annualized event rate, 0.05). Thrombocytopenia was the most frequent TTP manifestation (annualized event rate, 0.74 with recombinant ADAMTS13 and 1.73 with standard therapy). Adverse events occurred in 71% of the patients with recombinant ADAMTS13 and in 84% with standard therapy. Adverse events that were considered by investigators to be related to the trial drug occurred in 9% of the patients with recombinant ADAMTS13 and in 48% with standard therapy. Trial-drug interruption or discontinuation due to adverse events occurred in no patients with recombinant ADAMTS13 and in 8 patients with standard therapy. No neutralizing antibodies developed during recombinant ADAMTS13 treatment. The mean maximum ADAMTS13 activity after recombinant ADAMTS13 treatment was 101%, as compared with 19% after standard therapy. CONCLUSIONS: During prophylaxis with recombinant ADAMTS13 in patients with congenital TTP, ADAMTS13 activity reached approximately 100% of normal levels, adverse events were generally mild or moderate in severity, and TTP events and manifestations were rare. (Funded by Takeda Development Center Americas and Baxalta Innovations; ClinicalTrials.gov number, NCT03393975.).

Recombinant ADAMTS13 for Hereditary Thrombotic Thrombocytopenic Purpura.

A 27-year-old patient with a history of severe obstetrical complications and arterial thrombosis received a diagnosis of hereditary thrombotic thrombocytopenic purpura (TTP) due to severe ADAMTS13 deficiency when she presented with an acute episode in the 30th week of her second pregnancy. When the acute episode of hereditary TTP became plasma-refractory and fetal death was imminent, weekly injections of recombinant ADAMTS13 at a dose of 40 U per kilogram of body weight were initiated. The patient's platelet count normalized, and the growth of the fetus stabilized. At 37 weeks 1 day of gestation, a small-for-gestational-age boy was delivered by cesarean section. At the time of this report, the patient and her son were well, and she continued to receive injections of recombinant ADAMTS13 every 2 weeks. (Funded by the Swiss National Science Foundation.).

Successful management of refractory immune-mediated thrombotic thrombocytopenic purpura during pregnancy and delivery using the anti-VWF nanobody caplacizumab.

Pregnancy is a potential trigger of acute thrombotic thrombocytopenic purpura (TTP). The management of pregnancy-associated immune-mediated TTP (iTTP) can be challenging, especially when it is refractory to standard treatment. Caplacizumab, a nanobody to von Willebrand factor (VWF) blocking its A1 domain, is a valuable new therapeutic option. Its use is, however, not approved during pregnancy and breastfeeding. We describe the successful off-label administration of caplacizumab during pregnancy and delivery in a patient with refractory iTTP. The favourable outcome without significant thrombotic or haemorrhagic complications indicates that caplacizumab may be an effective and safe treatment option in refractory iTTP during pregnancy.

Naturally Occurring Anti-Idiotypic Antibodies Portray a Largely Private Repertoire in Immune-Mediated Thrombotic Thrombocytopenic Purpura.

Rare immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a life-threatening disease resulting from a severe autoantibody-mediated ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motifs, member 13) deficiency. Acute iTTP episodes are medical emergencies, but when treated appropriately >95% of patients survive. However, at least half of survivors will eventually experience a relapse. How remission of an initial episode is achieved and factors contributing to reemergence of anti-ADAMTS13 Abs and a relapsing course are poorly understood. In acquired hemophilia and systemic lupus erythematosus, anti-idiotypic Abs counteracting and neutralizing pathogenic autoantibodies contribute to remission. We selected and amplified the splenic anti-idiotypic IgG₁ Fab κ/λ repertoire of two relapsing iTTP patients on previously generated monoclonal inhibitory anti-ADAMTS13 Fabs by phage display to explore whether anti-idiotypic Abs have a role in iTTP. We obtained 27 single anti-idiotypic Fab clones, half of which had unique sequences, although both patients shared four H chain V region genes (V_H1-69*01, V_H3-15*01, V_H3-23*01, and V_H3-49*03). Anti-idiotypic Fab pools of both patients fully neutralized the inhibitor capacity of the monoclonal anti-ADAMTS13 Abs used for their selection. Preincubation of plasma samples of 22 unrelated iTTP patients stratified according to functional ADAMTS13 inhibitor titers (>2 Bethesda units/ml, or 1-2 Bethesda units/ml), with anti-idiotypic Fab pools neutralized functional ADAMTS13 inhibitors and restored ADAMTS13 activity in 18-45% of those cases. Taken together, we present evidence for the presence of an anti-idiotypic immune response in iTTP patients. The interindividual generalizability of this response is limited despite relatively uniform pathogenic anti-ADAMTS13 Abs recognizing a dominant epitope in the ADAMTS13 spacer domain.

Annual incidence and severity of acute episodes in hereditary thrombotic thrombocytopenic purpura.

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 <10 years of age at enrollment and were often triggered by infections. Their annual incidence of acute episodes was significantly higher than in patients aged >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.

Redefining outcomes in immune TTP: an international working group consensus report.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a potentially fatal thrombotic microangiopathy caused by autoantibody-mediated severe deficiency of ADAMTS13. Standardized definitions of response, exacerbation, remission, and relapse were initially proposed in 2003 and modified by the International Working Group for TTP in 2017. These definitions, which have been widely used in clinical practice and research, are based primarily on the platelet count and are benchmarked against the timing of discontinuation of therapeutic plasma exchange (TPE). They do not incorporate ADAMTS13 activity or the temporizing effects on the platelet count of caplacizumab, a novel anti-von Willebrand factor (VWF) nanobody. In light of these limitations, the IWG aimed to develop revised consensus outcome definitions that incorporate ADAMTS13 activity and the effects of anti-VWF therapy, by using an estimate-talk-estimate approach. The updated definitions distinguish clinical remission and clinical relapse (defined primarily by platelet count) from ADAMTS13 remission and ADAMTS13 relapse (defined by ADAMTS13 activity). The revised definitions of exacerbation and remission are benchmarked against not only the timing of discontinuation of TPE but also that of anti-VWF therapy. Retrospective validation of the revised definitions is described, although they have yet to be prospectively validated. Clinical implications of the updated outcome definitions are also discussed and an example of their application to clinical practice is provided to highlight their clinical relevance.

Caplacizumab Treatment for Acquired Thrombotic Thrombocytopenic Purpura.

BACKGROUND: In acquired thrombotic thrombocytopenic purpura (TTP), an immune-mediated deficiency of the von Willebrand factor-cleaving protease ADAMTS13 allows unrestrained adhesion of von Willebrand factor multimers to platelets and microthrombosis, which result in thrombocytopenia, hemolytic anemia, and tissue ischemia. Caplacizumab, an anti-von Willebrand factor humanized, bivalent variable-domain-only immunoglobulin fragment, inhibits interaction between von Willebrand factor multimers and platelets. METHODS: In this double-blind, controlled trial, we randomly assigned 145 patients with TTP to receive caplacizumab (10-mg intravenous loading bolus, followed by 10 mg daily subcutaneously) or placebo during plasma exchange and for 30 days thereafter. The primary outcome was the time to normalization of the platelet count, with discontinuation of daily plasma exchange within 5 days thereafter. Key secondary outcomes included a composite of TTP-related death, recurrence of TTP, or a thromboembolic event during the trial treatment period; recurrence of TTP at any time during the trial; refractory TTP; and normalization of organ-damage markers. RESULTS: The median time to normalization of the platelet count was shorter with caplacizumab than with placebo (2.69 days [95% confidence interval {CI}, 1.89 to 2.83] vs. 2.88 days [95% CI, 2.68 to 3.56], P=0.01), and patients who received caplacizumab were 1.55 times as likely to have a normalization of the platelet count as those who received placebo. The percentage of patients with a composite outcome event was 74% lower with caplacizumab than with placebo (12% vs. 49%, P<0.001). The percentage of patients who had a recurrence of TTP at any time during the trial was 67% lower with caplacizumab than with placebo (12% vs. 38%, P<0.001). Refractory disease developed in no patients in the caplacizumab group and in three patients in the placebo group. Patients who received caplacizumab needed less plasma exchange and had a shorter hospitalization than those who received placebo. The most common adverse event was mucocutaneous bleeding, which was reported in 65% of the patients in the caplacizumab group and in 48% in the placebo group. During the trial treatment period, three patients in the placebo group died. One patient in the caplacizumab group died from cerebral ischemia after the end of the treatment period. CONCLUSIONS: Among patients with TTP, treatment with caplacizumab was associated with faster normalization of the platelet count; a lower incidence of a composite of TTP-related death, recurrence of TTP, or a thromboembolic event during the treatment period; and a lower rate of recurrence of TTP during the trial than placebo. (Funded by Ablynx; HERCULES ClinicalTrials.gov number, NCT02553317 .).

Declining mortality of cerebral venous sinus thrombosis with thrombocytopenia after SARS-CoV-2 vaccination.

BACKGROUND AND PURPOSE: High mortality rates have been reported in patients with cerebral venous sinus thrombosis (CVST) due to vaccine-induced immune thrombotic thrombocytopenia (VITT) after vaccination with adenoviral vector SARS-CoV-2 vaccines. The aim of this study was to evaluate whether the mortality of patients with CVST-VITT has decreased over time. METHODS: The EudraVigilance database of the European Medicines Agency was used to identify cases of CVST with concomitant thrombocytopenia occurring within 28 days of SARS-CoV-2 vaccination. Vaccines were grouped based on vaccine type (adenoviral or mRNA). Cases with CVST onset until 28 March were compared to cases after 28 March 2021, which was the day when the first scientific paper on VITT was published. RESULTS: In total, 270 cases of CVST with thrombocytopenia were identified, of which 266 (99%) occurred after adenoviral vector SARS-CoV-2 vaccination (ChAdOx1 nCoV-19, n = 243; Ad26.COV2.S, n = 23). The reported mortality amongst adenoviral cases with onset up to 28 March 2021 was 47/99 (47%, 95% confidence interval 37%-58%) compared to 36/167 (22%, 95% confidence interval 16%-29%) in cases with onset after 28 March (p < 0.001). None of the four cases of CVST with thrombocytopenia occurring after mRNA vaccination died. CONCLUSION: The reported mortality of CVST with thrombocytopenia after vaccination with adenoviral vector-based SARS-CoV-2 vaccines has significantly decreased over time, which may indicate a beneficial effect of earlier recognition and/or improved treatment on outcome after VITT.

The potential impact of Covid-19 on the capacity of routine laboratory tests to detect heparin-induced thrombocytopenia.

In Covid-19, anticoagulation with heparin is often administered to prevent or treat thromboembolic events. Heparin-induced thrombocytopenia (HIT) is a severe complication of heparin treatment, caused by heparin-dependent, platelet activating anti-platelet factor 4 (PF4)/heparin antibodies. Diagnosis of HIT is based on the combination of clinical parameters, allowing to determine the pretest probability, and laboratory testing for anti-PF4/heparin antibodies and confirmatory functional assays, such as the heparin-induced platelet activation (HIPA) test.We report the case of a patient with severe Covid-19 pneumonia requiring ECMO treatment, who developed recurrent clotting of the ECMO filter and a drop in platelet count under heparin treatment. He was therefore suspected to have HIT and the anticoagulation was switched to argatroban. Despite high clinical probability and high titres of anti-PF4/heparin antibodies, the functional HIPA test was negative. Nevertheless, argatroban was continued rather than to reinstate anticoagulation with heparin. Reevaluation 7 days later then demonstrated a strongly positive functional HIPA test and confirmed the diagnosis of HIT. Under anticoagulation with argatroban the patient gradually improved and was finally weaned off the ECMO.In conclusion, this case highlights the critical importance of clinical judgement, exploiting the 4 T score, given that Covid-19 patients may present a different pattern of routine laboratory test results in HIT diagnostics. The possibility of a false negative HIPA test has to be considered, particularly in early phases of presentation. In cases of a discrepancy with high clinical probability of HIT and/or high titre anti-PF4/heparin antibodies despite a negative HIPA test, a reevaluation within 3 to 5 days after the initial test should be considered in order to avoid precipitant reestablishment of unfractionated heparin, with potentially fatal consequences.

Post-SARS-CoV-2-vaccination cerebral venous sinus thrombosis: an analysis of cases notified to the European Medicines Agency.

BACKGROUND AND PURPOSE: Cerebral venous sinus thrombosis (CVST) has been described after vaccination against SARS-CoV-2. The clinical characteristics of 213 post-vaccination CVST cases notified to the European Medicines Agency are reported. METHODS: Data on adverse drug reactions after SARS-CoV-2 vaccination notified until 8 April 2021 under the Medical Dictionary for Regulatory Activities Term 'Central nervous system vascular disorders' were obtained from the EudraVigilance database. Post-vaccination CVST was compared with 100 European patients with CVST from before the COVID-19 pandemic derived from the International CVST Consortium. RESULTS: In all, 213 CVST cases were identified: 187 after AstraZeneca/Oxford (ChAdOx1 nCov-19) vaccination and 26 after a messenger RNA (mRNA) vaccination (25 with Pfizer/BioNTech, BNT162b2, and one with Moderna, mRNA-1273). Thrombocytopenia was reported in 107/187 CVST cases (57%, 95% confidence interval [CI] 50%-64%) in the ChAdOx1 nCov-19 group, in none in the mRNA vaccine group (0%, 95% CI 0%-13%) and in 7/100 (7%, 95% CI 3%-14%) in the pre-COVID-19 group. In the ChAdOx1 nCov-19 group, 39 (21%) reported COVID-19 polymerase chain reaction tests were performed within 30 days of CVST symptom onset, and all were negative. Of the 117 patients with a reported outcome in the ChAdOx1 nCov-19 group, 44 (38%, 95% CI 29%-47%) had died, compared to 2/10 (20%, 95% CI 6%-51%) in the mRNA vaccine group and 3/100 (3%, 95% CI 1%-8%) in the pre-COVID-19 group. Mortality amongst patients with thrombocytopenia in the ChAdOx1 nCov-19 group was 49% (95% CI 39%-60%). CONCLUSIONS: Cerebral venous sinus thrombosis occurring after ChAdOx1 nCov-19 vaccination has a clinical profile distinct from CVST unrelated to vaccination. Only CVST after ChAdOx1 nCov-19 vaccination was associated with thrombocytopenia.

Frequency of Thrombocytopenia and Platelet Factor 4/Heparin Antibodies in Patients With Cerebral Venous Sinus Thrombosis Prior to the COVID-19 Pandemic.

Importance: Cases of cerebral venous sinus thrombosis in combination with thrombocytopenia have recently been reported within 4 to 28 days of vaccination with the ChAdOx1 nCov-19 (AstraZeneca/Oxford) and Ad.26.COV2.S (Janssen/Johnson & Johnson) COVID-19 vaccines. An immune-mediated response associated with platelet factor 4/heparin antibodies has been proposed as the underlying pathomechanism. Objective: To determine the frequencies of admission thrombocytopenia, heparin-induced thrombocytopenia, and presence of platelet factor 4/heparin antibodies in patients diagnosed with cerebral venous sinus thrombosis prior to the COVID-19 pandemic. Design, Setting, and Participants: This was a descriptive analysis of a retrospective sample of consecutive patients diagnosed with cerebral venous sinus thrombosis between January 1987 and March 2018 from 7 hospitals participating in the International Cerebral Venous Sinus Thrombosis Consortium from Finland, the Netherlands, Switzerland, Sweden, Mexico, Iran, and Costa Rica. Of 952 patients, 865 with available baseline platelet count were included. In a subset of 93 patients, frozen plasma samples collected during a previous study between September 2009 and February 2016 were analyzed for the presence of platelet factor 4/heparin antibodies. Exposures: Diagnosis of cerebral venous sinus thrombosis. Main Outcomes and Measures: Frequencies of admission thrombocytopenia (platelet count <150 ×103/µL), heparin-induced thrombocytopenia (as diagnosed by the treating physician), and platelet factor 4/heparin IgG antibodies (optical density >0.4, in a subset of patients with previously collected plasma samples). Results: Of 865 patients (median age, 40 years [interquartile range, 29-53 years], 70% women), 73 (8.4%; 95% CI, 6.8%-10.5%) had thrombocytopenia, which was mild (100-149 ×103/µL) in 52 (6.0%), moderate (50-99 ×103/µL) in 17 (2.0%), and severe (<50 ×103/µL) in 4 (0.5%). Heparin-induced thrombocytopenia with platelet factor 4/heparin antibodies was diagnosed in a single patient (0.1%; 95% CI, <0.1%-0.7%). Of the convenience sample of 93 patients with cerebral venous sinus thrombosis included in the laboratory analysis, 8 (9%) had thrombocytopenia, and none (95% CI, 0%-4%) had platelet factor 4/heparin antibodies. Conclusions and Relevance: In patients with cerebral venous sinus thrombosis prior to the COVID-19 pandemic, baseline thrombocytopenia was uncommon, and heparin-induced thrombocytopenia and platelet factor 4/heparin antibodies were rare. These findings may inform investigations of the possible association between the ChAdOx1 nCoV-19 and Ad26.COV2.S COVID-19 vaccines and cerebral venous sinus thrombosis with thrombocytopenia.

Targeting anticoagulant protein S to improve hemostasis in hemophilia.

Improved treatments are needed for hemophilia A and B, bleeding disorders affecting 400 000 people worldwide. We investigated whether targeting protein S could promote hemostasis in hemophilia by rebalancing coagulation. Protein S (PS) is an anticoagulant acting as cofactor for activated protein C and tissue factor pathway inhibitor (TFPI). This dual role makes PS a key regulator of thrombin generation. Here, we report that targeting PS rebalances coagulation in hemophilia. PS gene targeting in hemophilic mice protected them against bleeding, especially when intra-articular. Mechanistically, these mice displayed increased thrombin generation, resistance to activated protein C and TFPI, and improved fibrin network. Blocking PS in plasma of hemophilia patients normalized in vitro thrombin generation. Both PS and TFPIα were detected in hemophilic mice joints. PS and TFPI expression was stronger in the joints of hemophilia A patients than in those of hemophilia B patients when receiving on-demand therapy, for example, during a bleeding episode. In contrast, PS and TFPI expression was decreased in hemophilia A patients receiving prophylaxis with coagulation factor concentrates, comparable to osteoarthritis patients. These results establish PS inhibition as both controller of coagulation and potential therapeutic target in hemophilia. The murine PS silencing RNA approach that we successfully used in hemophilic mice might constitute a new therapeutic concept for hemophilic patients.

Caplacizumab for Acquired Thrombotic Thrombocytopenic Purpura.

BACKGROUND: Acquired thrombotic thrombocytopenic purpura (TTP) is caused by aggregation of platelets on ultralarge von Willebrand factor multimers. This microvascular thrombosis causes multiorgan ischemia with potentially life-threatening complications. Daily plasma exchange and immunosuppressive therapies induce remission, but mortality and morbidity due to microthrombosis remain high. METHODS: Caplacizumab, an anti-von Willebrand factor humanized single-variable-domain immunoglobulin (Nanobody), inhibits the interaction between ultralarge von Willebrand factor multimers and platelets. In this phase 2, controlled study, we randomly assigned patients with acquired TTP to subcutaneous caplacizumab (10 mg daily) or placebo during plasma exchange and for 30 days afterward. The primary end point was the time to a response, defined as confirmed normalization of the platelet count. Major secondary end points included exacerbations and relapses. RESULTS: Seventy-five patients underwent randomization (36 were assigned to receive caplacizumab, and 39 to receive placebo). The time to a response was significantly reduced with caplacizumab as compared with placebo (39% reduction in median time, P=0.005). Three patients in the caplacizumab group had an exacerbation, as compared with 11 patients in the placebo group. Eight patients in the caplacizumab group had a relapse in the first month after stopping the study drug, of whom 7 had ADAMTS13 activity that remained below 10%, suggesting unresolved autoimmune activity. Bleeding-related adverse events, most of which were mild to moderate in severity, were more common with caplacizumab than with placebo (54% of patients vs. 38%). The frequencies of other adverse events were similar in the two groups. Two patients in the placebo group died, as compared with none in the caplacizumab group. CONCLUSIONS: Caplacizumab induced a faster resolution of the acute TTP episode than did placebo. The platelet-protective effect of caplacizumab was maintained during the treatment period. Caplacizumab was associated with an increased tendency toward bleeding, as compared with placebo. (Funded by Ablynx; ClinicalTrials.gov number, NCT01151423.).

Recombinant ADAMTS-13: first-in-human pharmacokinetics and safety in congenital thrombotic thrombocytopenic purpura.

Safety, tolerability, and pharmacokinetics of recombinant ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; BAX 930; SHP655) were investigated in 15 patients diagnosed with severe congenital ADAMTS-13 deficiency (plasma ADAMTS-13 activity <6%) in a prospective phase 1, first-in-human, multicenter dose escalation study. BAX 930 was well tolerated, no serious adverse events occurred, and no anti-ADAMTS-13 antibodies were observed. After single-dose administration of BAX 930 at 5, 20, or 40 U/kg body weight to adolescents and adults, there was approximate dose proportionality with respect to maximum plasma concentration (Cmax [U/mL]) and area under the concentration-time curve (AUC [h∙U/mL]). Dose-related increases of individual ADAMTS-13:Ag and activity were observed and reached a maximum within 1 hour. With escalating BAX 930 doses administered, a dose-dependent persistence of ADAMTS-13-mediated von Willebrand factor (VWF) cleavage products and reduced VWF multimeric size were observed. This study demonstrated that pharmacokinetic parameters of BAX 930 were comparable to those estimated in previous plasma infusion studies and provided evidence of pharmacodynamic activity. This study was registered at www.clinicaltrials.gov as #NCT02216084.

The International Hereditary Thrombotic Thrombocytopenic Purpura Registry: key findings at enrollment until 2017.

Congenital thrombotic thrombocytopenic purpura is an autosomal recessive inherited disease with a clinically heterogeneous course and an incompletely understood genotype-phenotype correlation. In 2006, the Hereditary TTP Registry started recruitment for a study which aimed to improve the understanding of this ultra-rare disease. The objective of this study is to present characteristics of the cohort until the end of 2017 and to explore the relationship between overt disease onset and ADAMTS13 activity with emphasis on the recurring ADAMTS13 c.4143_4144dupA mutation. Diagnosis of congenital thrombotic thrombocytopenic purpura was confirmed by severely deficient ADAMTS13 activity (≤10% of normal) in the absence of a functional inhibitor and the presence of ADAMTS13 mutations on both alleles. By the end of 2017, 123 confirmed patients had been enrolled from Europe (n=55), Asia (n=52, 90% from Japan), the Americas (n=14), and Africa (n=2). First recognized disease manifestation occurred from around birth up to the age of 70 years. Of the 98 different ADAMTS13 mutations detected, c.4143_4144dupA (exon 29; p.Glu1382Argfs*6) was the most frequent mutation, present on 60 of 246 alleles. We found a larger proportion of compound heterozygous than homozygous carriers of ADAMTS13 c.4143_4144dupA with overt disease onset at < 3 months of age (50% vs 37%), despite the fact that ADAMTS13 activity was <1% in 18 of 20 homozygous, but in only 8 of 14 compound heterozygous carriers. An evaluation of overt disease onset in all patients with an available sensitive ADAMTS13 activity assay (n=97) shows that residual ADAMTS13 activity is not the only determinant of age at first disease manifestation. Registered at clinicaltrials.gov identifier NCT01257269.

Estimation of Nuwiq® (simoctocog alfa) activity using one-stage and chromogenic assays-Results from an international comparative field study.

BACKGROUND: Accurate determination of coagulation factor VIII activity (FVIII:C) is essential for effective and safe FVIII replacement therapy. FVIII: C can be measured by one-stage and chromogenic substrate assays (OSAs and CSAs, respectively); however, there is significant interlaboratory and interassay variability. AIMS: This international comparative field study characterized the behaviour of OSAs and CSAs used in routine laboratory practice to measure the activity of Nuwiq® (human-cl rhFVIII, simoctocog alfa), a fourth-generation recombinant human FVIII produced in a human cell line. METHODS: FVIII-deficient plasma was spiked with Nuwiq® or Advate® at 1, 5, 30 and 100 international units (IU)/dL. Participating laboratories analysed the samples using their routine procedures and equipment. Accuracy, inter- and intralaboratory variation, CSA:OSA ratio and the impact of different OSA and CSA reagents were assessed. RESULTS: Forty-nine laboratories from 9 countries provided results. Mean absolute FVIII:C was comparable for both products at all concentrations with both OSA and CSA, with interproduct ratios (Nuwiq® :Advate® ) of 1.02-1.13. Mean recoveries ranged from 97% to 191% for Nuwiq® , and from 93% to 172% for Advate® , with higher recoveries at lower concentrations. Subgroup analyses by OSA and CSA reagents showed minor variations depending on reagents, but no marked differences between the two products. CSA:OSA ratios based on overall means ranged from 0.99 to 1.17 for Nuwiq® and from 1.01 to 1.17 for Advate® . CONCLUSIONS: Both OSAs and CSAs are suitable for the measurement of FVIII:C of Nuwiq® in routine laboratory practice, without the need for a product-specific reference standard.

Cellular Factor XIII, a Transglutaminase in Human Corneal Keratocytes.

Cellular factor XIII (cFXIII, FXIII-A2), a transglutaminase, has been demonstrated in a few cell types. Its main function is to cross-link proteins by isopeptide bonds. Here, we investigated the presence of cFXIII in cells of human cornea. Tissue sections of the cornea were immunostained for FXIII-A in combination with staining for CD34 antigen or isopeptide cross-links. Isolated corneal keratocytes were also evaluated by immunofluorescent microscopy and flow cytometry. FXIII-A in the corneal stroma was quantified by Western blotting. FXIII-A mRNA was detected by RT-qPCR. The cornea of FXIII-A-deficient patients was evaluated by cornea topography. FXIII-A was detected in 68 ± 13% of CD34+ keratocytes. Their distribution in the corneal stroma was unequal; they were most abundant in the subepithelial tertile. cFXIII was of cytoplasmic localization. In the stroma, 3.64 ng cFXIII/mg protein was measured. The synthesis of cFXIII by keratocytes was confirmed by RT-qPCR. Isopeptide cross-links were detected above, but not within the corneal stroma. Slight abnormality of the cornea was detected in six out of nine FXIII-A-deficient patients. The presence of cFXIII in human keratocytes was established for the first time. cFXIII might be involved in maintaining the stability of the cornea and in the corneal wound healing process.