Dialyzer Performance During Hemodialysis Without Systemic Anticoagulation Using a Heparin-Grafted Dialyzer Combined With a Citrate-Enriched Dialysate: Results of the Randomized Crossover Noninferiority EvoCit Study.

RATIONALE & OBJECTIVE: The EvoCit study was designed to evaluate performance of a heparin-grafted dialyzer during hemodialysis with and without systemic anticoagulation. STUDY DESIGN: Randomized, crossover, noninferiority trial. Noninferiority was defined as a difference of≤10% for the primary outcome. SETTING & PARTICIPANTS: Single hemodialysis center; 26 prevalent patients treated with 617 hemodialysis sessions. INTERVENTIONS: Hemodialysis using a heparin-grafted dialyzer combined with a 1.0mmol/L citrate-enriched dialysate ("EvoCit") without systemic anticoagulation compared with hemodialysis performed with a heparin-grafted dialyzer with systemic heparin ("EvoHep"). Patients were randomly allocated to a first period of 4 weeks and crossed over to the alternative strategy for a second period of 4 weeks. OUTCOMES: The primary end point was the difference in Kt/Vurea between EvoCit and EvoHep. Secondary end points were urea reduction ratio, middle molecule removal, treatment time, thrombin generation, and reduction in dialyzer blood compartment volume. RESULTS: The estimated difference in Kt/Vurea between EvoCit and EvoHep was-0.03 (95% CI, -0.06 to-0.007), establishing noninferiority with mean Kt/Vurea of 1.47±0.05 (SE) for EvoCit and 1.50±0.05 for EvoHep. Noninferiority was also established for reduction ratios of urea and ß2-microglobulin. Premature discontinuation of dialysis was required for 4.2% of sessions among 6 patients during EvoCit and no sessions during EvoHep. Effective treatment time was 236±5 minutes for EvoCit and 238±1 minutes for EvoHep. Thrombin generation was increased and there was greater reduction in dialyzer blood compartment volume after treatments with EvoCit compared with EvoHep. LIMITATIONS: The effects of avoiding systemic anticoagulation on clinical outcomes were not evaluated. CONCLUSIONS: EvoCit is noninferior to EvoHep with respect to solute clearance but results in a greater number of shortened treatments, more membrane clotting, and greater thrombin generation TRIAL REGISTRATION: Registered at ClinicalTrials.gov with study number NCT03887468.

Incidence and features of thrombosis in children with inherited antithrombin deficiency.

Pediatric thromboembolism (≤18 years) is very rare (0.07-0.14/10,000/year) but may be more prevalent in children with severe thrombophilia (protein C, protein S or antithrombin deficiency). The aim of this study was to define the prevalence and clinical characteristics of pediatric thrombosis in subjects with inherited antithrombin deficiency. Our observational retrospective multicentric study from two countries recruited 968 patients of any age from 441 unrelated families with genetically, biochemically and functionally characterized antithrombin deficiency. Seventy-three subjects (7.5%) developed thrombosis before 19 years of age. Two high-risk periods for thrombosis were identified: adolescence (12-18 years, n=49) with thrombus localization (lower limb deep venous thrombosis or pulmonary embolism) and triggering factors common to adults (oral contraceptives, surgery or pregnancy); and the neonatal period (<30 days, n=15) with idiopathic thrombosis at unusual sites. The clinical evaluation of pediatric thrombosis in subjects with antithrombin deficiency revealed: i) a high prevalence of cerebral sinovenous thrombosis (n=13, 17.8%), mainly at young age (8 neonates and 4 children <6 years); ii) severe outcome with fatality in six cases (3 neonates, two of them homozygous for p.Leu131Phe). The majority of subjects (76.7%) carried quantitative type I deficiency. This retrospective analysis includes the largest cohort of subjects with inherited antithrombin deficiency so far and provides strong evidence for an increased risk of pediatric thrombosis associated with this thrombophilia (300-fold compared with the general population: 0.41%/year vs 0.0014%/year, respectively). Our results support testing for antithrombin deficiency in children of affected families, particularly in case of type I deficiency.

Child-onset thrombotic thrombocytopenic purpura caused by p.R498C and p.G259PfsX133 mutations in ADAMTS13.

INTRODUCTION: Patients suffering from congenital thrombotic thrombocytopenic purpura (cTTP) have a deficiency in ADAMTS13 due to mutations in their ADAMTS13 gene. OBJECTIVE: The 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. METHODS: ADAMTS13 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. RESULTS: The 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. CONCLUSION: The ADAMTS13 mutations result in a severe ADAMTS13 deficiency explaining the patient's phenotype.

Homozygous antithrombin deficiency in adolescents presenting with lower extremity thrombosis and renal complications: two case reports from Turkey.

We present 2 cases of lower extremity deep venous thrombosis in 2 gypsy adolescents from related families. The patients had low antithrombin activity levels and inherited homozygous antithrombin deficiency was confirmed by molecular analysis (Leu131Phe mutation). One patient had a history of nephrectomy at the age of 9 due to nonfunctioning kidney and 2 siblings died at 4 months of age. His mother had 3 fetal losses in the third trimester. The other propositus had an elder sister who suffered from postpartum deep vein thrombosis and pulmonary embolism. Heterozygous mutation was demonstrated in both parents.

The use of direct oral anticoagulants in the secondary prevention of venous thromboembolism in patients with severe thrombophilia: communication from the ISTH SSC Subcommittee on Physiological Anticoagulants and Thrombophilia.

Direct oral anticoagulants (DOACs) are the first-line anticoagulants for the secondary prevention of venous thromboembolism (VTE). However, patients with severe inherited thrombophilias represent a group in whom the efficiency and safety of DOACs is poorly studied. In this communication, we focus on the utility of DOACs in the secondary prevention of VTE in patients with severe thrombophilia. Current evidence is based only on cohort or single-center studies, and poor data are available on compliance of the patients in the studies. Analysis of the studies suggested that full-dose DOACs and vitamin K antagonists have a similar efficacy and bleeding risk in the secondary prevention of VTE in patients with thrombophilia, with a low hazard ratio for recurrent VTE calculated from cohort studies for DOAC vs warfarin, ranging from 0.3 to 0.75. We wish to highlight that treatment failure is greater in those with severe forms of protein S deficiency (below 20%) and possibly in antithrombin deficiency type II heparin-binding site homozygous Budapest 3. In summary, the current approach to using DOACs in patients with severe thrombophilia is dependent on clinical judgment and experience. Limited evidence suggests that for those with severe thrombophilias, full-dose DOACs have similar utility as vitamin K antagonists. We recommend caution in using low-dose DOACs due to lack of evidence. Ideally, large randomized multicenter studies are required to develop a reliable treatment algorithm.

The 2023 Belgian clinical guidance on anticoagulation management in hospitalized and ambulatory COVID-19 patients.

COVID-19 is associated with an increased risk for thrombotic complications. The trials investigating the optimal thromboprophylactic dose are performed in challenging times and seemingly produce conflicting evidence. The burdensome circumstances, divergent endpoints, and different analytical approaches hamper comparison and extrapolation of available evidence. Most importantly, clinicians should provide thromboprophylaxis in hospitalized COVID-19 patients while (re)assessing bleeding and thrombotic risk frequently. The COVID-19 Thromboprophylaxis Working Group of the BSTH updated its guidance document. It aims to summarize the available evidence critically and to guide clinicians in providing the best possible thromboprophylaxis.

Belgian clinical guidance on anticoagulation management in hospitalised and ambulatory patients with COVID-19.

OBJECTIVES: COVID-19 predisposes patients to thrombotic disease. The aim of this guidance document is to provide Belgian health-care workers with recommendations on anticoagulation management in COVID-19 positive patients. METHODS: These recommendations were based on current knowledge and a limited level of evidence. RESULTS: We formulated recommendations for the prophylaxis and treatment of COVID-related venous thromboembolism in ambulatory and hospitalised patients, as well as recommendations for the use of antithrombotic drugs in patients with prior indication for anticoagulation who develop COVID-19. CONCLUSIONS: These recommendations represent an easy-to-use practical guidance that can be implemented in every Belgian hospital and be used by primary care physicians and gynaecologists. Of note, they are likely to evolve with increased knowledge of the disease and availability of data from ongoing clinical trials.

Molecular Dissection of Structural Variations Involved in Antithrombin Deficiency.

Inherited antithrombin deficiency, the most severe form of thrombophilia, is predominantly caused by variants in SERPINC1. Few causal structural variants have been described, usually detected by multiplex ligation-dependent probe amplification or cytogenetic arrays, which only define the gain or loss and the approximate size and location. This study has done a complete dissection of the structural variants affecting SERPINC1 of 39 unrelated patients with antithrombin deficiency using multiplex ligation-dependent probe amplification, comparative genome hybridization array, long-range PCR, and whole genome nanopore sequencing. Structural variants, in all cases only affecting one allele, were deleterious and caused a severe type I deficiency. Most defects were deletions affecting exons of SERPINC1 (82.1%), but the whole cohort was heterogeneous, as tandem duplications, deletion of introns, or retrotransposon insertions were also detected. Their size was also variable, ranging from 193 bp to 8 Mb, and in 54% of the cases involved neighboring genes. All but two structural variants had repetitive elements and/or microhomologies in their breakpoints, suggesting a common mechanism of formation. This study also suggested regions recurrently involved in structural variants causing antithrombin deficiency and found three structural variants with a founder effect: the insertion of a retrotransposon, duplication of exon 6, and a 20-gene deletion. Finally, nanopore sequencing was determined to be the most appropriate method to identify and characterize all structural variants at nucleotide level, independently of their size or type.

Antithrombin p.Thr147Ala: The First Founder Mutation in People of African Origin Responsible for Inherited Antithrombin Deficiency.

BACKGROUND: Hereditary antithrombin deficiency is a rare autosomal-dominant disorder predisposing to recurrent venous thromboembolism (VTE). To date, only two founder mutations have been described. OBJECTIVES: We investigated the antithrombin p.Thr147Ala variant, found in 12 patients of African origin. This variant is known as rs2227606 with minor allele frequency of 0.5% in Africans and absent in Europeans. A possible founder effect was investigated. METHODS: Phenotypical characterization was established through immunological and functional methods, both under basal and stress conditions. Recombinant antithrombin molecules were constructed by site-directed mutagenesis and expressed in HEK-293T cells. Secreted antithrombin was purified and functionally characterized. Structural modeling was performed to predict the impact of the mutation on protein structure. A novel nanopore sequencing approach was used for haplotype investigation. RESULTS: Ten patients experienced VTE, stroke, or obstetric complications. Antithrombin antigen levels and anti-IIa activity were normal or slightly reduced while anti-Xa activity was reduced with only one commercial assay. On crossed immunoelectrophoresis, an increase of antithrombin fractions with reduced heparin affinity was observed under high ionic strength conditions but not under physiological conditions. The recombinant p.Thr147Ala protein displayed a reduced anti-Xa activity. Structural modeling revealed that residue Thr147 forms three hydrogen bonds that are abolished when mutated to alanine. The investigated patients shared a common haplotype involving 13 SERPINC1 intragenic single nucleotide polymorphisms. CONCLUSION: Antithrombin p.Thr147Ala, responsible for antithrombin type II heparin binding site deficiency, is the first founder mutation reported in people of African ancestry. This study further emphasizes the limitations of commercial methods to diagnose this specific subtype.

Hemodialysis Does Not Induce Detectable Activation of the Contact System of Coagulation.

INTRODUCTION: Systemic anticoagulation is administered during hemodialysis to prevent clotting of the extracorporeal circuit. The role of contact system activation in thrombin generation during hemodialysis using current era dialyzer membranes is unknown. METHODS: We performed a single-center randomized crossover study. Ten patients treated with hemodialysis underwent 3 standardized hemodialysis sessions. For every patient, each session was performed with a different type of dialyzer membrane (polyphenylene [PP], polymethylmetacrylate [PMMA], polyethylenimine-coated polyacrylonitrile [AN69ST]). Blood samples were collected before and 5, 15, 30, 90, and 240 minutes after blood pump start to evaluate coagulation activation (thrombin-antithrombin complex [TAT], prothrombin fragment 1+2 [PF1+2], activated factor XII [FXIIa], kallikrein, activated factor XI [FXIa]). Plasma of healthy volunteers (n = 20) was used as a reference. RESULTS: Baseline TAT and PF1+2 levels were higher in hemodialysis patients compared to healthy controls (median [interquartile range] for TAT: 3.3 [2.9-4.2] vs. 2.4 [2.3-2.5] µg/l [P = 0.0002] and for PF1+2: 647 [478-737] vs. 138 [125-254] pmol/l [P < 0.0002]). Despite the use of systemic anticoagulation, TAT further increased during treatment, with the increase starting after 30 minutes (median TAT at t240: 9.0 µg/l (PP), 5.5 µg/l (PMMA), and 7.2 µg/l (AN69ST), all P < 0.05 vs. baseline). Contact system markers FXIIa and kallikrein did not differ significantly between dialysis patients and healthy controls, whereas baseline FXIa levels were significantly lower in dialysis patients compared to healthy controls (P = 0.001). Levels of all contact system markers remained unchanged during hemodialysis with all types of dialyzer membranes. CONCLUSION: Routine hemodialysis using systemic heparin anticoagulation induces coagulation activation without measurable contact system activation.

Recommendations for clinical laboratory testing for antithrombin deficiency; Communication from the SSC of the ISTH.

Hereditary deficiency of antithrombin, a natural anticoagulant, causes a thrombophilia with a high risk for venous thromboembolism. Guidance for laboratory testing to diagnose antithrombin deficiency include the use of an activity assay for initial testing, performing an antigen test and activity-to-antigen ratio when the activity level is low, using pediatric reference ranges until the age of 6 months, excluding acquired causes of low antithrombin (e.g. liver dysfunction, proteinuria, heparin, disseminated intravascular coagulation, thrombosis, surgery) or falsely normal/elevated results (e.g. argatroban, bivalirudin, dabigatran in factor IIa-based assays; rivaroxaban, apixaban, edoxaban, but not betrixaban in Xa-based assays). Molecular testing, if available, may help determine the risk for thrombosis as this might vary among the different mutations. Moreover, it will identify mutations that can be missed by traditional activity assays. Strategies for interpreting laboratory test results are provided.

Heterozygous FGA p.Asp473Ter (fibrinogen Nieuwegein) presenting as antepartum cerebral thrombosis.

INTRODUCTION: The propositus - a two-week-old boy - was transferred to our university hospital for investigation of increased head circumference and full fontanel. On ultrasound, thrombosis of the right internal cerebral vein and intraventricular haemorrhage was diagnosed, confirmed by MRI. Family history revealed a bleeding history in the mother. A haemostatic work-up in both mother and child was performed in order to rule out congenital coagulopathy. AIM: We document a clinical case of congenital dysfibrinogenemia, caused by heterozygosity for the mutation FGA p.Asp473Ter, previously reported as fibrinogen Nieuwegein in homozygosity in an asymptomatic patient. METHODS: Fibrinogen activity in plasma was determined by functional Clauss assay, and immunological fibrinogen concentration by nephelometry. In vitro fibrin clot investigations and genetic analysis of the fibrinogen gene were performed. Complete haemostatic work-up was done by conventional methods. RESULTS AND DISCUSSION: After full laboratory work-up, dysfibrinogenemia was diagnosed, based on fibrinogen activity:antigen ratio, thrombin time, and reptilase time. Molecular analysis showed a frameshift mutation in exon 5 of FGA: c.1415_1416 insC, leading to a termination codon immediately after the insertion (CCT GAT>CCC TGA) and resulting in a truncated αC-domain. This mutation has been reported earlier as fibrinogen Nieuwegein. Further in vitro investigations revealed an abnormally tight clot structure, prolonged clot lysis time and affected polymerization, suggesting a thrombotic phenotype. Cerebral imaging revealed thrombosis, most likely developed in the antenatal period, leading to extensive intraventricular haemorrhage and posthaemorrhagic ventricular dilatation. CONCLUSION: We highlight the combined thrombotic and haemorrhagic phenotype linked to heterozygous fibrinogen Nieuwegein, in contrast to the previously reported asymptomatic homozygous case.

Antithrombin heparin binding site deficiency: A challenging diagnosis of a not so benign thrombophilia.

BACKGROUND: Hereditary antithrombin (AT) deficiency is a rare autosomal dominant disorder characterised by decreased AT activity in plasma and predisposition to recurrent venous thromboembolism (VTE). Thrombotic risk is thought to vary according to the subtype of deficiency, with Heparin Binding Site (HBS) deficiencies being the less thrombogenic. OBJECTIVES AND METHODS: The study population consisted of 82 genetically confirmed HBS deficient patients sharing six different mutations. Plasma samples of 35 of them, including one homozygous patient, were used for the evaluation of 4 commercial activity assays in their ability to diagnose HBS deficiency. We assessed mutation-specific prevalence of venous and arterial thrombosis and the contribution of additional thrombophilic risk factors. RESULTS AND CONCLUSIONS: Only one assay showed 100% sensitivity for all HBS mutations. The other ones failed mainly in the cases with p.Pro73Leu and p.Arg79His mutations. Shortening of incubation time resulted in an increase in sensitivity. In one patient, a novel HBS mutation, p.Asn77His, was identified, a quite exceptional and important finding given the restricted number of causal mutations reported so far in AT HBS deficiency. The overall prevalence of VTE in our study population (35%) was higher than previously reported (6-8%) in these patients. The presence of additional thrombophilic risk factors such as Factor V Leiden or prothrombin gene mutation G20210A contributed to a higher risk of VTE. Interestingly, the p.Pro73Leu and p.Arg79His mutations were associated with a high prevalence of arterial thrombosis. Our data suggest that AT HBS deficiencies are probably more prevalent and less benign than previously assumed.