Severe Thrombophilia Research Articles

Targeted management of coexistent severe thrombophilias-A case report of a successful pregnancy despite paroxysmal nocturnal hemoglobinuria and hereditary protein C deficiency.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematological disorder characterized by the absence of complement regulatory proteins on the surface of erythrocytes, leading to intravascular hemolysis and thrombosis. Managing PNH during pregnancy poses significant challenges due to increased risks of morbidity and mortality. This case report describes the detailed obstetric course of a 44-year-old woman with PNH and additional hereditary protein C deficiency who had previously experienced multiple thrombotic events and adverse pregnancy outcomes (two early miscarriages and one stillbirth at 25 weeks gestation [WG]), treated with eculizumab (terminal C5 inhibitor) and optimal anticoagulation management. Close monitoring of hemolysis and hemostasis parameters was conducted throughout the gestation period together with increased obstetrical surveillance. The pregnancy progressed without thrombotic complications or breakthrough hemolysis, and the patient delivered a healthy newborn at 36 WG after induction of labor due to restricted fetal growth. To the best of our knowledge, this is the first reported case of a positive pregnancy outcome despite PNH in conjunction with hereditary thrombophilia. This case report highlights the importance of a multidisciplinary approach involving hematologists and obstetricians in the management of pregnant women with PNH. Tailored therapy, close monitoring, and comprehensive care are crucial to minimize risks and optimize outcomes.

Antithrombin testing and treatment in pregnancy: Their real-world relationship to clinical outcomes

BackgroundAntithrombin (AT) deficiency is a severe thrombophilia associated with increased rates of maternal morbidity, mortality, and greater healthcare resource utilization during pregnancy and postpartum. MethodsTwo large U.S. healthcare databases were queried for women aged 15–44 with delivery-related encounters: Cerner Real-World Data (CRWD, 01/01/2000–12/31/2021) and Premier Healthcare Database (PHD, 01/01/2016–01/01/2019). Individuals receiving cardiopulmonary bypass were excluded. Three cohorts were created: 1) Individuals who had AT levels tested any time between 9-months pre- through 3-months post-delivery (CRWD Test Cohort); 2) individuals prescribed AT concentrate (ATc) within 1-year pre- or 1-year post-delivery in CRWD (CRWD Medication Cohort); and 3) the same criteria as 2) applied to PHD (PHD Medication Cohort). ResultsThere were 5411 individuals in the CRWD Test Cohort, 13 in the CRWD Medication Cohort and 38 in the PHD Medication Cohort. Demographic and baseline clinical characteristics were similar across cohorts. AT level testing occurred pre-delivery in 47.9 % of the CRWD Test Cohort and 23.1 % of the CRWD Medication Cohort. ATc was administered during the delivery hospitalization to 0.1 %, 23.1 % and 50.0 % of the CRWD Test, CRWD Medication, and PHD Medication Cohorts, respectively. Across cohorts, 5.4–7.9 % of individuals experienced thrombosis during the delivery-related encounter. Mean (SD) total costs for delivery through 1-year post-delivery were $190,894 ($276,893) with $123,763 ($177,122) of total costs related to abnormal coagulation. ConclusionOpportunities exist to enhance the care of pregnant individuals with low AT levels throughout pregnancy, aiming for optimal maternal outcomes.

Clinical and functional characterization of rare compound heterozygous mutations in the SERPINC1 gene causing severe thrombophilia

IntroductionHereditary antithrombin (AT) deficiency is a rare autosomal dominant disorder with significant clinical heterogeneity. In the study, we identified a patient with AT deficiency caused by compound heterozygous mutations in the SERPINC1 gene. MethodsA total of 9 individuals from three generations were investigated. The mutations were identified by direct sequencing of SERPINC1. Multiple in silico tools were programmed to predict the conservation of mutations and the effect on the AT structure. The coagulation state was evaluated by the thrombin generation assay. Recombinant AT was overexpressed in HEK293T cells; the mRNA level was determined using RT-qPCR. Western blotting, ELISA, and immunocytofluorescence were applied to characterize the recombinant AT protein. ResultsThe proband was a 26-year-old male who experienced recurrent venous thrombosis. He presented the type I deficiency with 33 % AT activity and a synchronized decrease in AT antigen. Genetic screening revealed that he carried a heterozygous c.318_319insT (p.Asn107*) in exon 2 and a heterozygous c.922G > T (p.Gly308Cys) in exon 5, both of which were completely conserved in homologous species and resulted in enhanced thrombin generation capability. Hydrophobicity analysis suggested that the p.Gly308Cys mutation may interfere with the hydrophobic state of residues 307–313. In vitro expression studies indicated that the levels of the recombinant protein AT-G308C decreased to 46.98 % ± 2.94 % and 41.35 % ± 1.48 % in transfected cell lysates and media, respectively. After treatment with a proteasome inhibitor (MG132), the quantity of AT-G308C protein in the cytoplasm was replenished to a level comparable to that of the wild type. The mRNA level of AT-N107* was significantly reduced and the recombinant protein AT-N107* was not detected in either the lysate or the culture media. ConclusionThese two mutations were responsible for the AT defects and clinical phenotypes of the proband. The p.Gly308Cys mutation could lead to proteasome-dependent degradation of the AT protein in the cytoplasm by altering local residue hydrophobicity. The c.318_319insT could eliminate aberrant transcripts by triggering nonsense-mediated mRNA degradation. Both mutations resulted in type I AT deficiency.

Risk Factors, Clinical Manifestations, Anticoagulation Duration, and Outcomes in Carriers of Severe Inherited Thrombophilia

Severe Inherited thrombophilia comprises deficiencies of antithrombin (AT), protein C (PC), and protein S (PS), while gain-of-function (GoF) variants include homozygosity for factor V Leiden (FVL) or prothrombin gene G20210A mutation (PGM), or double-heterozygosity. Persons with AT, Protein C, or S deficiencies have a heightened risk of developing thrombosis from a young age. Several family members are usually affected, and thrombosis may occur in unusual locations.Homozygote or double heterozygous of GoF variants may not have a family history of thrombosis, and the first thrombotic event may present later in life. The optimal duration and type of anticoagulation and their long-term outcomes for persons with severe thrombophilia require further investigation. This study aimed to compare risk factors, clinical manifestations, type and duration of anticoagulation and clinical outcomes between persons with AT, protein C, and S deficiencies and homozygote or double heterozygous persons with GoF variants. Retrospective evaluation of electronic medical records of persons with severe inherited thrombophilia referred to the Center for Blood Disorders at Weill Cornell Medicine-New York Presbyterian Hospital between January 2009 and December 2022. Severe deficiencies of AT, PC and PS were defined as (AT ≤ 60%, PC ≤ 50% and PS ≤ 40%). Results needed to be confimed in second testing in one month interval. Statistical analysis was performed using descriptive statistics, and chi-square test and Fisher's exact test were applied to compare variables between persons with AC deficiencies and GoF variants. 795 persons with inherited thrombophilia were identified, of those 671 were excluded as they had mild thrombophilia. Of the remaining 124 persons, 17 were eliminated due to absence of confirmatory results. A total of 107 persons were analyzed. There were 47 (44%) persons with anticoagulant deficiencies (AT, PC, and PS) and 60 (56%) homozygotes for FVL, PGM or double heterozygote. Mean age (SD) was 48 (13.6) and 49 (12.2) for anticoagulation deficiency and GoF mutation, respectively (Table 1). Overall risk factors for thrombosis were similar in both groups. A positive family history of thrombosis in a first-degree family member was higher in the anticoagulant deficient group (68%) compared to the GoF mutation (42%) (p=0.008). In addition, persons with anticoagulant deficiency had higher rates of thrombosis before 40 years of age compared to the GoF mutation group, 68% and 26%, respectively (p=0.031). The most frequent anticoagulant prescribed was a DOAC in both groups, with 38% and 30% used for the anticoagulation deficient group and GoF mutation group, respectively. The duration of anticoagulation was similar in both groups with most patients remaining on anticoagulation for more than one year (79% anticoagulant deficient group, 65% GoF mutation group) (p=0.3). Patients with AC deficiency had more recurrent thrombotic events while on anticoagulation compared to GoF variants (17% vs 6.7%) (Table 2). Our results indicate that patients with severe inherited thrombophilia either due to anticoagulant deficiencies or GoF mutations are likely to remain on anticoagulation for long durations and may have recurrent thrombosis despite therapeutic anticoagulation.

PB0989 The Efficacy and Safety of Direct Oral Anticoagulants in 50 Patients with Venous Thromboembolism and Severe Thrombophilia: A Multicenter Study

PB0989 The Efficacy and Safety of Direct Oral Anticoagulants in 50 Patients with Venous Thromboembolism and Severe Thrombophilia: A Multicenter Study

Systematic search for the UBA1 mutation in men after a first venous thrombotic episode

VEXAS syndrome (acronym for Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) is an autoinflammatory disease, described in adults in 2020, caused by a somatic mutation of the Ubiquitin like modifier activating enzyme 1 (UBA1) gene located on the X chromosome. VEXAS syndrome is mainly described in men after 50 years of age with a systemic inflammatory disease associated with febrile episodes, but also hematological, pulmonary, cutaneous, vascular, cartilaginous and articular involvement. Thrombotic complications have been reported in approximately 40% of VEXAS patients, with a predominance of venous thromboembolic events (VTE) rather than arterial thromboembolic events. Among the VTEs, deep vein thrombosis (DVT) was more frequent than pulmonary embolism (PE). The pathophysiology of VTE in VEXAS remains unknown. The objective of the study was to determine the frequency of UBA1 mutations in a cohort of men over 50 years of age with a first VTE. Our study included male patients aged > 50 years included in our center between January 2003 and January 2009, as part of the multicenter FARIVE study. The FARIVE case-control study included consecutive patients treated as inpatients or outpatients for a first episode of proximal DVT and/or PE. All patients were sampled for genetic study. In this study, exon 3 of UBA1 was sequenced. A total of 97 patients were included with a median age of 65 years and a median body mass index of 25.5. Of the patient cohort, 49 (50.5%) presented with an unprovoked VTE. Thrombophilia testing was available for 56 (57.7%) of the 97 patients and no severe thrombophilia was diagnosed among the patients tested. In the end, no UBA1 mutations were detected among the 97 patients (Table 1). VEXAS syndrome is associated with a high incidence of VTE but our study does not encourage its investigation in all men over 50 years of age with a first VTE without considering other clinical symptoms related to VEXAS. Routine screening for UBA1 exon 3 mutations should not be recommended after a first VTE.

Usefulness and Limitations of Multiple Ligation-Dependent Probe Amplification in Antithrombin Deficiency

Multiplex ligation-dependent probe amplification (MLPA) identifies genetic structural variants in SERPINC1 in 5% of cases with antithrombin deficiency (ATD), the most severe congenital thrombophilia. Our aim was to unravel the utility and limitations of MLPA in a large cohort of unrelated patients with ATD (N = 341). MLPA identified 22 structural variants (SVs) causing ATD (6.5%). MLPA did not detect SVs affecting introns (four cases), and the diagnosis was inaccurate in two cases according to long-range PCR or nanopore sequencing. MLPA was used to detect possible hidden SVs in 61 cases with type I deficiency with single nucleotide variations (SNVs) or small insertion/deletion (INDEL). One case had a false deletion of exon 7, as the 29-bp deletion affected an MLPA probe. We evaluated 32 variants affecting MLPA probes: 27 SNVs and 5 small INDELs. In three cases, MLPA gave false-positive results, all diagnosed as deletions of the affected exon: a small INDEL complex, and two SNVs affecting MLPA probes. Our study confirms the utility of MLPA to detect SVs in ATD, but also shows some limitations in detecting intronic SVs. MLPA renders imprecise and false-positive results for genetic defects which affect MLPA probes. Our results encourage the validation of MLPA results.

Hereditary Antithrombin Deficiency Pilot Project Registry from the American Thrombosis and Hemostasis Network (ATHN)

Background: Hereditary antithrombin deficiency (HAD) is a severe rare thrombophilia with an estimated prevalence of 0.02% to 0.2% in the general population. Patients with HAD have a high risk of developing arterial and venous thromboembolism (VTE). The purpose of ATHN 12: HAD Pilot Project is to collect data on participants diagnosed with HAD across the United States to better understand this rare thrombophilia and to develop guidelines regarding timing of screening for HAD, type of antithrombotic therapy, and indications for using antithrombin concentrate. The ATHN 12 study is sponsored by the American Thrombosis and Hemostasis Network (ATHN). ATHN will leverage its existing data collection infrastructure and its ATHN-affiliated network of 146 hemostasis and thrombosis treatment centers in the United States to identify a cohort of approximately 100 patients with HAD who are receiving care in these centers. Methods: We designed a national HAD registry to collect clinical, laboratory, and treatment data for participants with HAD with the goal of optimizing their management. The ongoing prospective HAD registry project (ATHN 12) was created in 2020 to identify a cohort of 100 adult and pediatric participants with HAD receiving care at thrombosis centers affiliated with ATHN. We collected information on demographics, laboratory data, thrombotic events, risk factors, antithrombotic therapy, and use of antithrombin (AT) concentrate during pregnancy and procedures. Results: 71 participants have been enrolled, 47 (66.2%) are female; mean age is 35 years (1-82); the majority 58 (82%) are White and 31 (43.6%) have type 1 HAD; 62 (87.32%) had a known diagnosis of HAD; 45 (63.38%) had a history of VTE (Table 1). The most common VTE locations included: lower extremity deep vein thrombosis 51 events (57%) and pulmonary embolisms 23 events (26%). 23 participants (51%) had recurrent VTEs. 4 participants (5.5%) had history of arterial thrombosis (AT) and 1 (25%) had a recurrent event. Of 45 participants, 17 (38%) had no other known risk factors for VTE; 21 (46.5%) were estrogen-related; 11 (24.4%) occurred after surgery, prolonged immobilization, hospitalization, travel, or trauma (Table 1). A total of 173 anticoagulants were prescribed on 45 (63.38%) participants (Table 2) There were 87 pregnancies in 37 women. Low molecular weight heparin (LMWH) was administered in 46% of the pregnancies and in 3 women LMWH was switched to UFH between 36 to 38 weeks of gestation. AT concentrate was administered in 22 (25%) of the pregnancies before delivery and in 14 pregnancies 24 hours post-delivery. 31 women (36%) restarted anticoagulation post-delivery. The earliest initiation of anticoagulation was 6 hours after delivery. Overall, there were 34 (39%) pregnancy complications: 23 pregnancy losses mainly in first trimester, 4 thrombosis, 4 pre-eclampsia, and 6 other complications (Table 2). 59 participants (83%) had a total of 171 surgeries or procedures. The most frequent surgeries were gastrointestinal, and the most common procedures were vascular (Table 2). Overall, 28 (39.4%) have received AT concentrate without adverse events. Conclusions: VTE was the most frequent clinical manifestation with DVT accounting for 57% of events and PE 26%. Oral factor Xa inhibitors were the most common anticoagulants prescribed. LMWH was administered in almost half of the pregnancies and AT concentrates in 25% of the pregnancies before delivery, and in 39.4% overall in pregnancies and procedures without reported adverse events. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prolonged Combinatorial Pharmacological Therapy Targeting the Epigenome Increases Fetal Hemoglobin to Clinically Relevant Levels in Normal, Nonanemic Baboons

Elevated levels of Fetal Hemoglobin (HbF; α2γ2) reduce the symptoms associated with Sickle Cell Disease (SCD) and increase the lifespan of patients. An effective pharmacological therapy that increases HbF would advance the treatment of SCD. HbF levels are developmentally regulated by transcriptional repression of the γ-globin gene in adult erythroid cells. This is achieved by a co-repressor complex containing multiple enzymes that catalyze repressive epigenetic modifications of chromatin targeted to the γ-globin promoter by site-specific DNA binding proteins. Pharmacological inhibitors of repressive epigenetic enzymes within the complex including DNMT1, LSD1 (KDM1A), G9A, and HDACs increase HbF in cell line and animal models and SCD patients. Many of these drugs have been studied in Phase 1 trials, but only the DNMT inhibitor decitabine (DAC) has advanced to Phase 3 trials. The successful development of a pharmacological therapy to increase HbF for SCD has thus far been hindered by dose-limiting side effects including neutropenia, thrombocytopenia, and/or thrombophilia. The goal of this current work is to develop a pharmacological therapy employing a combination of drugs that target different repressive epigenetic enzymes within the co-repressor complex based on the hypothesis that administration of these drugs in combination will reduce the dose and/or time of exposure to any single agent to minimize adverse effects while still stimulating clinically meaningful HbF elevations. To test this hypothesis, baboons, long considered the best animal model to test the effect of HbF-inducing agents due to conservation of the structure and developmental regulation of the β-globin gene locus between baboons and humans, were treated with a combination of two drugs, DAC and RN-1, that inhibit DNMT1 and LSD1 respectively. Analysis of seven baboons used in our studies showed that they comprised two phenotypic groups differentiated by baseline pre-treatment HbF expression (Figure 1). Two baboons of each phenotypic group were chosen to study the effect of DAC and RN-1, alone and in combination, on HbF. Either RN-1 (0.25mg/kg/d) or DAC (0.5mg/kg/d), were administered subcutaneously individually or in combination to normal, non-anemic animals for 2 days. The effect on γ-globin expression was measured by flow cytometry analysis of F-retics and RT-PCR of γ-globin mRNA in peripheral blood at least 3X per week post-drug treatments. Each drug induced a significant increase in F-retics and peripheral blood γ-globin mRNA in both phenotypic groups that peaked on d8 (Figure 1) while differences in F-retics and γ-globin mRNA between the two groups were maintained. Combined administration (RN-1 (0.25mg/kg/d; DAC (0.5mg/kg/d); 2d) increased levels of F-retics and γ-globin mRNA to greater than additive levels (Figure 1) while the difference in γ-globin mRNA (p<0.02), but not F-retics, between the two groups was still observed. To assess the effect of sustained administration of the two drug combination on HbF expression and evaluate possible hematological toxicity, two normal baboons, one of the low HbF group (PA 9254) and one of the high HbF group (PA 9194), were treated weekly with 12 total courses of the DAC-RN-1 combination (DAC, 0.5mg/kg/d; RN-1, 0.25mg/kg/d; 2d/wk). F cells increased approximately 6 fold to 60.5% in the high HbF animal and >10 fold to 42% in the low HbF baboon (Table 1). HPLC analysis of globin chain expression showed that HbF (γ/γ+β) increased tenfold in each animal (Table 1). The calculated % HbF/ F cell values were 33% for PA 9194 (High HbF group) and 23.5% for PA 9254 (Low HbF group). No instances of moderate or severe neutropenia, thrombocytopenia, or thrombophilia were observed during the treatment period (Table 1). Decreased Hct, Hb, and RBCs were apparent in the high HbF group baboon (PA 9194) but not the low HbF group baboon (PA 9254). In conclusion, combinatorial administration of DAC and RN-1 increased HbF and F cell numbers in a greater than additive manner. Prolonged treatment of two normal, nonanemic baboons increased F cells and HbF to levels predicted to be clinically significant in the absence of neutropenia, thrombocytopenia and/or thrombophilia throughout the treatment period although a reduction in Hct, RBCs, and Hb was observed in one animal. These results strongly suggest additional drug combinations, dosing, and schedules to increase HbF for the treatment of SCD should be pursued. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Systematic search for the UBA1 mutation in men after a first episode of venous thromboembolism: A monocentric study

Systematic search for the UBA1 mutation in men after a first episode of venous thromboembolism: A monocentric study

Evolving Knowledge on Primary and Secondary Prevention of Venous Thromboembolism in Carriers of Hereditary Thrombophilia: A Narrative Review

The association between heritability of venous thromboembolism (VTE) and thrombophilia was first reported clinically in 1956, later followed by the first description of a congenital cause of hypercoagulability-antithrombin deficiency-in 1965. Since then, our knowledge of hereditary causes of hypercoagulability, which may predispose carriers to VTE has improved greatly. Novel genetic defects responsible for severe thrombophilia have been recently identified and we have learned that a wide range of interactions between thrombophilia and other genetic and acquired risk factors are important determinants of the overall individual risk of developing VTE. Furthermore, therapeutic strategies in thrombophilic patients have benefited significantly from the introduction of direct oral anticoagulants. The present review is an overview of the current knowledge on the mechanisms underlying inherited thrombophilia, with a particular focus on the latest achievements in anticoagulation protocols and prevention strategies for thrombosis in carriers of this prothrombotic condition.

The Vascular Endothelium and Coagulation: Homeostasis, Disease, and Treatment, with a Focus on the Von Willebrand Factor and Factors VIII and V.

The vascular endothelium has several important functions, including hemostasis. The homeostasis of hemostasis is based on a fine balance between procoagulant and anticoagulant proteins and between fibrinolytic and antifibrinolytic ones. Coagulopathies are characterized by a mutation-induced alteration of the function of certain coagulation factors or by a disturbed balance between the mechanisms responsible for regulating coagulation. Homeostatic therapies consist in replacement and nonreplacement treatments or in the administration of antifibrinolytic agents. Rebalancing products reestablish hemostasis by inhibiting natural anticoagulant pathways. These agents include monoclonal antibodies, such as concizumab and marstacimab, which target the tissue factor pathway inhibitor; interfering RNA therapies, such as fitusiran, which targets antithrombin III; and protease inhibitors, such as serpinPC, which targets active protein C. In cases of thrombophilia (deficiency of protein C, protein S, or factor V Leiden), treatment may consist in direct oral anticoagulants, replacement therapy (plasma or recombinant ADAMTS13) in cases of a congenital deficiency of ADAMTS13, or immunomodulators (prednisone) if the thrombophilia is autoimmune. Monoclonal-antibody-based anti-vWF immunotherapy (caplacizumab) is used in the context of severe thrombophilia, regardless of the cause of the disorder. In cases of disseminated intravascular coagulation, the treatment of choice consists in administration of antifibrinolytics, all-trans-retinoic acid, and recombinant soluble human thrombomodulin.

An Integrated Approach on the Diagnosis of Cerebral Veins and Dural Sinuses Thrombosis (a Narrative Review).

(1) Objective: This review paper aims to discuss multiple aspects of cerebral venous thrombosis (CVT), including epidemiology, etiology, pathophysiology, and clinical presentation. Different neuroimaging methods for diagnosis of CVT, such as computer tomography CT/CT Venography (CTV), and Magnetic Resonance Imaging (MRI)/MR Venography (MRV) will be presented. (2) Methods: A literature analysis using PubMed and the MEDLINE sub-engine was done using the terms: cerebral venous thrombosis, thrombophilia, and imaging. Different studies concerning risk factors, clinical picture, and imaging signs of patients with CVT were examined. (3) Results: At least one risk factor can be identified in 85% of CVT cases. Searching for a thrombophilic state should be realized for patients with CVT who present a high pretest probability of severe thrombophilia. Two pathophysiological mechanisms contribute to their highly variable clinical presentation: augmentation of venular and capillary pressure, and diminution of cerebrospinal fluid absorption. The clinical spectrum of CVT is frequently non-specific and presents a high level of clinical suspicion. Four major syndromes have been described: isolated intracranial hypertension, seizures, focal neurological abnormalities, and encephalopathy. Cavernous sinus thrombosis is the single CVT that presents a characteristic clinical syndrome. Non-enhanced CT (NECT) of the Head is the most frequently performed imaging study in the emergency department. Features of CVT on NECT can be divided into direct signs (demonstration of dense venous clot within a cerebral vein or a cerebral venous sinus), and more frequently indirect signs (such as cerebral edema, or cerebral venous infarct). CVT diagnosis is confirmed with CTV, directly detecting the venous clot as a filling defect, or MRI/MRV, which also realizes a better description of parenchymal abnormalities. (4) Conclusions: CVT is a relatively rare disorder in the general population and is frequently misdiagnosed upon initial examination. The knowledge of wide clinical aspects and imaging signs will be essential in providing a timely diagnosis.

Two SERPINC1 variants affecting N-glycosylation of Asn224 cause severe thrombophilia not detected by functional assays

AbstractAntithrombin deficiency, the most severe congenital thrombophilia, might be underestimated, as some pathogenic variants are not detected by routine functional methods. We have identified 2 new SERPINC1 variants, p.Glu227Lys and p.Asn224His, in 4 unrelated thrombophilic patients with early and recurrent thrombosis that had normal antithrombin activity. In one case, the mutation was identified by whole genome sequencing, while in the 3 remaining cases, the mutation was identified by sequencing SERPINC1 based on a single functional positive finding supporting deficiency. The 2 variants shared a common functional defect, an impaired or null N-glycosylation of Asn224 according to a eukaryotic expression model. Carriers had normal anti-FXa or anti-FIIa activities but impaired anti-FVIIa activity and a detectable loss of inhibitory function when incubating the plasma for 1 hour at 41°C. Moreover, the β glycoform of the variants, lacking 2 N-glycans, had reduced secretion, increased heparin affinity, no inhibitory activity, and a potential dominant–negative effect. These results explain the increased thrombin generation observed in carriers. Mutation experiments reflected the role that Lysine residues close to the N-glycosylation sequon have in impairing the efficacy of N-glycosylation. Our study shows new elements involved in the regulation of N-glycosylation, a key posttranslational modification that, according to our results, affects folding, secretion, and function, providing new evidence of the pathogenic consequence of an incorrect N-glycosylation of antithrombin. This study supports that antithrombin deficiency is underestimated and encourages the development of new functional and genetic tests to diagnose this severe thrombophilia.

Molecular and clinical characterization of transient antithrombin deficiency: A new concept in congenital thrombophilia.

Antithrombin deficiency, the most severe thrombophilia, might be underestimated, since it is only investigated in cases with consistent functional deficiency or family history. We have analyzed 444 consecutive, unrelated cases, from 1998 to 2021, with functional results supporting antithrombin deficiency in at least one sample. Plasma antithrombin was evaluated by functional and biochemical methods in at least two samples. SERPINC1 gene was analyzed by sequencing and MPLA. Hypoglycosylation was studied by electrophoresis and high-performance liquid chromatography (HPLC). In 260 of 305 cases (85.2%) with constitutive deficiency (activity < 80% in all samples), a SERPINC1 (N= 250), or N-glycosylation defect (N= 10) was observed, while 45 remained undetermined. The other 139 cases had normal antithrombin activity (≥ 80%) in at least one sample, what we called transient deficiency. Sixty-one of these cases (43.9%) had molecular defects: 48 had SERPINC1 variants, with two recurrent mutations (p.Ala416Ser[Cambridge II], N= 15; p.Val30Glu[Dublin], N= 12), and 13 hypoglycosylation. Thrombotic complications occurred in transient deficiency, but were less frequent, latter-onset, and had a higher proportion of arterial events than in constitutive deficiency. Two mechanisms explained transient deficiency: The limitation of functional methods to detect some variants and the influence of external factors on the pathogenic consequences of these mutations. Our study reveals a molecular defect in a significant proportion of cases with transient antithrombin deficiency, and changes the paradigm of thrombophilia, as the pathogenic effect of some mutations might depend on external factors and be present only at certain timepoints. Antithrombin deficiency is underestimated, and molecular screening might be appropriate in cases with fluctuating laboratory findings.

Incidential Findings of Mutations in the PIGA Gene Are Highly Specific for the Presence of PNH Clones

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a hemolytic anemia associated with severe thrombophilia and characterized by complement-mediated lysis of erythrocytes lacking glycosylphosphatidylinositol (GPI)-anchored proteins. In the majority of cases, GPI deficiency is caused by somatic mutations in the PIGA gene. Presence of PNH clones is associated with acquired aplastic anemia (AA) and can be found in patients with myelodysplastic syndrome (MDS) or rarely other myeloid neoplasms (MN). Flow cytometric analysis for deficiency of GPI-anchored proteins on multiple cell lineages detects PNH clones, and PIGA mutational analysis is not mandatory to establish the diagnosis. In contrast, molecular genetic analysis of targeted gene panels is widely used in the diagnostic workup of MN. We hypothesized that the inclusion of PIGA into the myeloid gene panel could identify obscure cases with PNH clones irrespective of the initial clinical suspicion.Aim: To assess the significance of incidental findings of mutations in PIGA in the diagnostic workup of MN.Methods: 20,320 consecutive patients undergoing sequencing analysis for a confirmed or suspected MN were analyzed for the presence of mutations in the PIGA gene. Patients with confirmed PNH analyzed only for PIGA were not included, and cases with previously known PIGA mutations were excluded from further analysis. DNA was isolated from peripheral blood (PB) or bone marrow, and sequencing was performed on NovaSeq after Illumina DNA Prep for Enrichment library preparation (Illumina, San Diego, CA) and hybrid capture of a 41 gene panel including the complete coding sequence of PIGA (IDT Inc., Coralville, IA); data was analyzed with Pisces and Pindel (BaseSpace, Illumina). Flow cytometry was performed on granulocytes, monocytes, and erythrocytes in PB using antibodies against GPI-anchored proteins (CD14, CD24, CD55, and CD59), fluorescein-labeled proaerolysin (FLAER) staining, and Navios cytometers; analysis was done using Kaluza software (both Beckman Coulter, Miami, FL).Results: PIGA mutations were newly identified in 67 patients (0.3%) undergoing targeted sequencing within the diagnostic workup of MN. 30 patients were excluded from further analysis as the gene panel had been requested for a MN associated with previously diagnosed PNH. From the remaining patients, PB for flow cytometry analysis could be obtained from 20 patients. Flow cytometry confirmed the presence of a PNH clone in 17 (85%) of these patients (median clone size: 41% for granulocytes, 54.5% for monocytes, and 12% for erythrocytes). In 3 patients (15%) with unexpected PIGA mutations, flow cytometry detected no PNH clone. The type of PIGA mutations differed significantly in those cases: Patients in whom a PNH clone was confirmed, showed protein-truncating frame-shift (41%) or nonsense (6%) mutations, splice site mutations (18%), or multiple mutations (35%) including at least one protein-truncating mutation at a median variant allele frequency (VAF) of 15.4% (range 2.0% to 50.1%). In contrast, patients without a PNH clone showed only singular missense mutations of PIGA with a VAF of 3.6% to 5.3% (Figure 1). Final diagnoses in patients with confirmed clones were sole PNH (n=9), or PNH clone associated with MDS (n=4), AA (n=3), and AML (n=1), and additional mutations in other genes were observed in 9 cases. While the initial clinical presentation included the differential diagnosis of PNH in some of the patients, flow cytometry was requested as a direct result of the PIGA mutation in 4 cases with an accompanying MN and in 3 patients without - the later showing a median latency of 6.5 years from the initial clinical presentation to the diagnosis.Conclusions: The inclusion of PIGA into a standardized targeted sequencing panel for MN helps to identify patients with PNH clones irrespective of the initial clinical suspicion but is not sufficient to rule out PNH. Protein-truncating PIGA mutations are highly specific for PNH clones whereas singular missense mutations may not necessarily effect GPI biosynthesis. Our data indicate that the incidental finding of a PIGA mutation in sequencing analysis shall entail flow cytometry of GPI-anchored proteins in PB. The potential clinical sequelae and the availability of specific treatment options such as complement inhibitors warrant the thorough exclusion of PNH in the diagnostic workup of suspected MN. [Display omitted] DisclosuresHoermann: Novartis: Honoraria. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership.

High penetrance of inferior vena cava system atresia in severe thrombophilia caused by homozygous antithrombin Budapest 3 variant: Description of a new syndrome

Atresia of inferior vena cava (IVC) is a rare congenital malformation associated with high risk of venous thrombosis that still has unknown etiology, although intrauterine IVC thrombosis has been suggested to be involved. The identification of IVC atresia in a case with early idiopathic venous thrombosis and antithrombin deficiency caused by the homozygous SERPINC1 c.391C > T variant (p.Leu131Phe; antithrombin Budapest 3) encouraged us to evaluate the role of this severe thrombophilia in this vascular abnormality. We have done a cross-sectional study in previously identified cohorts of patients homozygous for the Budapest 3 variant (N=61) selected from 1118 patients with congenital antithrombin deficiency identified in two different populations: Spain (N=692) and Hungary (N=426). Image analysis included computed tomography and phlebography. Atresia of the IVC system was observed in 17/24 cases (70.8%, 95% confidence interval [CI]: 48.9%-87.3%) homozygous for antithrombin Budapest 3 with available computed tomography (5/8 and 12/16 in the Spanish and Hungarian cohorts, respectively), 16 had an absence of infrarenal IVC and one had atresia of the left common iliac vein. All cases with vascular defects had compensatory mechanisms, azygos-hemiazygos continuation or double IVC, and seven also had other congenital anomalies. Short tandem repeat analysis supported the specific association of the IVC system atresia with SERPINC1. We show the first evidence of the association of a severe thrombophilia with IVC system atresia, supporting the possibility that a thrombosis in the developing fetal vessels is the reason for this anomaly. Our hypothesis-generating results encourage further studies to investigate severe thrombophilic states in patients with atresia of IVC.

How we('ll) treat paroxysmal nocturnal haemoglobinuria: diving into the future.

SummaryParoxysmal nocturnal haemoglobinuria (PNH) is characterized by complement‐mediated intravascular haemolysis, severe thrombophilia and bone marrow failure. While for patients with bone marrow failure the treatment follows that of immune‐mediated aplastic anaemia, that of classic, haemolytic PNH is based on anti‐complement medication. The anti‐C5 monoclonal antibody eculizumab has revolutionized treatment, resulting in control of intravascular haemolysis and thromboembolic risk, with improved long‐term survival. Novel strategies of complement inhibition are emerging. New anti‐C5 agents reproduce the safety and efficacy of eculizumab, with improved patient convenience. Proximal complement inhibitors have been developed to address C3‐mediated extra‐vascular haemolysis and seem to improve haematological response.

Lead thrombus under standard-dose edoxaban in a patient with normal to high creatinine clearance and protein S deficiency

BackgroundNon-vitamin K antagonist oral anticoagulants (NOACs) are as effective and safe as warfarin for thromboembolic prevention and treatment. The efficacy of NOACs lacks evidence from large and randomized studies in patients with inherited severe thrombophilia, including protein S deficiency. Further, some concerns still exist regarding the relative efficacy of edoxaban in preventing arterial thromboembolism in patients with normal to high creatinine clearance (CrCl). We present a case of a rare complication of lead thrombus under standard-dose edoxaban in a patient with protein S deficiency and supernormal renal function.Case presentationA 65-year-old man experienced persistent chest tightness and a high level of D-dimer. Chest computed tomography (CT) showed a lead thrombus at the superior vena cava. He had a medical history including, paroxysmal atrial fibrillation (PAf), sick sinus syndrome after permanent pacemaker implantation, and transient ischemic attack. He received standard-dose edoxaban (60 mg daily) after PAf was diagnosed. His estimated CrCl was 98.6–102.1 mL/min. However, protein S deficiency (22.8%; normal range: 55–130%) was diagnosed. After switching to dabigatran (150 mg twice daily) for 3 months, the chest CT showed lead thrombus resolution and no symptoms were seen during the follow-up period.ConclusionsThis case was a rare complication of lead thrombus in a protein S deficient patient with normal renal function receiving standard-dose edoxaban. Edoxaban efficacy is uncertain in patients with protein S deficiency, and intracardiac devices also increase the risk of thromboembolic events.

Partial F8 gene duplication (factor VIII Padua) associated with high factor VIII levels and familial thrombophilia

AbstractHigh coagulation factor VIII (FVIII) levels comprise a common risk factor for venous thromboembolism (VTE), but the underlying genetic determinants are largely unknown. We investigated the molecular bases of high FVIII levels in 2 Italian families with severe thrombophilia. The proband of the first family had a history of recurrent VTE before age 50 years, with extremely and persistently elevated FVIII antigen and activity levels (>400%) as the only thrombophilic defects. Genetic analysis revealed a 23.4-kb tandem duplication of the proximal portion of the F8 gene (promoter, exon 1, and a large part of intron 1), which cosegregated with high FVIII levels in the family and was absent in 103 normal controls. Targeted screening of 50 unrelated VTE patients with FVIII levels ≥250% identified a second thrombophilic family with the same F8 rearrangement on the same genetic background, suggesting a founder effect. Carriers of the duplication from both families showed a twofold or greater upregulation of F8 messenger RNA, consistent with the presence of open chromatin signatures and enhancer elements within the duplicated region. Testing of these sequences in a luciferase reporter assay pinpointed a 927-bp region of F8 intron 1 associated with >45-fold increased reporter activity in endothelial cells, potentially mediating the F8 transcriptional enhancement observed in carriers of the duplication. In summary, we report the first thrombophilic defect in the F8 gene (designated FVIII Padua) associated with markedly elevated FVIII levels and severe thrombophilia in 2 Italian families.