Antithrombin Deficiency Research Articles

Novel factors affecting fibrin clot formation and their clinical implications.

Fibrin formation is pivotal in hemostasis, serving as a temporary barrier to blood loss following vascular injury, while in thrombosis this process is involved in thrombus progression, stability, and recurrence. Growing evidence shows an exceptional complexity of processes that determine fibrin clot structure and function, especially lysability, both in health and disease, which might be relevant in the pathogenesis of arterial and venous thromboembolic diseases. In this overview we summarized available data on novel factors that in recent years have been suggested to contribute to prothrombotic fibrin clot properties, involving formation of compact fibrin networks (reduced clot permeability) displaying impaired susceptibility to lysis (prolonged clot lysis time). The factors discussed in this review encompass elevated levels of factor (F)XI, and its activated form (FXIa), protein carbonylation as the most common type of post-translational modification, neutrophil extracellular traps (NETs) formation, increased levels of circulating lipopolysaccharide (LPS) and zonulin, a marker of gut permeability, along with antithrombin deficiency. These factors have been shown to be not only associated with ischemic stroke, myocardial infarction, pulmonary embolism, and cardiovascular death, but also with unfavorably altered fibrin clot characteristics, which underscores clinical relevance of fibrin clot properties. Given preclinical or ongoing studies aimed at modifying some of these factors, in particular FXI / FXIa inhibitors, recent findings might expand our knowledge on fibrin-related mechanisms of emerging therapeutic agents tested and stimulate further research into new targets for future therapeutic interventions to prevent thromboembolic events.

A novel SERPINC1 c.119G>A (p.Cys40Tyr) mutation with variable clinical expression in an Indian family.

Hereditary antithrombin (AT) deficiency due to mutations in SERPINC1 is known to be the most severe form of thrombophilia. We report three members in a family with hereditary AT deficiency with a novel mutation in exon 2 of SERPINC1, that is c.119 G>A (p.Cys40Tyr). Two brothers presented with acute pulmonary thromboembolism (PTE) at 18 and 21 years of age, whereas their 58-year-old father did not have any thrombotic episode till date. The in-silico prediction of the variant was found to be highly damaging by PolyPhen-2, SIFT and MutationTaster. Clinical exome sequencing did not show any strong coinherited thrombophilia genes, except SERPINE1 -844 G>A variant in homozygous state in the two affected brothers as compared to the father who was heterozygous for this variant. The additive effect of SERPINE1 variant in the clinical expression in two siblings cannot be ruled out, in the absence of any other known environmental triggering factors.

Novel insights into antithrombin deficiency enabled by mass spectrometry-based precision diagnostics

BackgroundAlthough P5 (preventive, personalized, predictive, participatory, psychocognitive) medicine and patient-focused healthcare are gaining ground in various healthcare areas, the diagnosis of antithrombin deficiency (ATD) is still based on crude diagnostic tests, clustering patients into clinically heterogeneous subgroups whereby relevant thrombophilia phenotypes may go unnoticed. Clinical pathways and the majority of evidence are based on these tests, therefore, generic treatment is still the norm. ObjectivesTo unravel the heterogeneity of ATD, a mass spectrometry (liquid chromatography coupled to multiple-reaction-monitoring mass spectrometry [LC-MRM-MS])-based test for antithrombin was developed allowing molecular characterization of the antithrombin proteoforms in patient plasma. This study provides the first insight into the tests’ clinical performance. MethodsPlasma from 91 unrelated ATD patients and 41 patients with a congenital disorder of glycosylation affecting antithrombin glycosylation were characterized functionally, genetically, and analyzed by LC-MRM-MS. An established data analysis strategy was applied for quantitation and molecular characterization of antithrombin proteoforms. ResultsThe test recognized patients with a quantitative defect, discriminated between type I and type II ATD, and identified variant proteoforms. Overall, the diagnostic sensitivity for ATD was 100% for LC-MRM-MS compared with 81.1% by the functional test. Type II ATD, a subtype prone to misdiagnosis, revealed an even larger difference of 100% identification by LC-MRM-MS vs 56.8% by functional test. ConclusionThe qualitative and quantitative mass spectrometry–based AT-test can serve as a platform for investigating the molecular basis of the clinical heterogeneity of ATD. This “precision diagnostics” approach for ATD can lower diagnostic uncertainty and modernize the ATD diagnostic and clinical pathways.

Novel SERPINC1 Mutation in a Brazilian Patient with Sudden-onset Sagittal Sinus Thrombosis Caused by Congenital Antithrombin Deficiency

A 19-year-old Brazilian man visited the emergency department of our hospital complaining of nausea and fever. He presented with left hemispatial neglect. Magnetic resonance imaging led to a diagnosis of sagittal sinus thrombosis. Blood tests showed decreased antithrombin (ATIII) activity, and ATIII deficiency was diagnosed. He was treated with heparin sodium and warfarin. Genetic counseling through medical interpreters was recommended, and the patient and his mother consented to genetic testing, which revealed a novel mutation variant of the SERPINC1 gene. Genetic counseling is important to connect patients to genetic testing, even if language barriers are present.

Genetic analysis of antithrombin deficiency among Thai patients with thrombosis

Hereditary antithrombin (AT) deficiency, primarily caused by mutations of SERPINC1 mutations, is a strong risk factor for occurrences and recurrences of venous thrombosis. While AT activity level measurements may be interfered by several factors, genetic analysis is more definitive for diagnosis. This study aims to address the knowledge gap of genetic basis of AT deficiency among Thai patients. Samples were collected from patients with thrombosis and low AT activities. Cases with acquired causes of low AT were excluded. All 7 exons of the SERPINC1 gene were PCR amplified and subjected to Sanger sequencing. The study included 15 patients and 6 controls with normal AT activity levels. The results revealed the presence of 6 benign single nucleotide polymorphisms (SNPs) located in intron 1, 2, exon 5 and intron 5. Two of these SNPs in intron 2 were first described here. Notably, a novel SNP in exon 3, SERPINC1 c.570 C>G (p.Y190X), was discovered. This is likely pathogenic because the premature stop codon led to a non-functional protein. The results suggest that pathogenic SERPINC1 variants detectable by DNA sequencing are uncommon among Thai patients with low AT activity levels underscoring the role of genetic analysis for definitive diagnosis. Further studies involving larger numbers of patients are required.

Heparin-Bead Extraction Enhanced Fluorogenic Aptamer-Thrombin Composite Reporter Enables Sensitive and Rapid Detection of Functional Antithrombin.

Antithrombin (AT) deficiency in the extracorporeal circulation during cardiac surgery leads to uncontrolled inflammation and vascular damage in patients. AT levels decrease in sepsis, major trauma, extracorporeal membrane oxygenation, and eclampsia. Monitoring plasma AT levels facilitates the accurate restoration of AT to baseline values through precise supplementation. Traditional methods of chromogenic assay and enzyme-linked immunosorbent assay (ELISA) kits encounter challenges, such as interference, inconsistency, and delayed response times, making real-time, reliable antithrombin monitoring a clinical gap. To address this critical need, we develop a heparin-bead extraction enhanced fluoroGenic aptamer-thrombin composite reporter (HExGATOR) for the rapid, sensitive, and precise detection of functional AT in plasma. Our design employs thrombin-binding aptamers and a fluorescence "turn on" technology such that a signal is produced upon the interaction of AT with the otherwise "turned off" aptamer-thrombin complex. The prominent signal-background interference originating from plasma is remarkably diminished by using a heparin-bead solid-phase extraction of AT. We achieved highly sensitive and rapid detection of AT in 5 to 20 min with a limit of detection of 15.11 nM. This approach offers a promising alternative to traditional AT tests in clinical settings, potentially facilitating personalized anticoagulant therapy.

Reduced Plasma Selenoprotein P Is Associated With Type I Antithrombin Deficiency and a Prothrombotic State.

A positive association between antithrombin activity and selenium level was reported. Selenoprotein P, the most important selenium carrier, was identified within human plasma fibrin clots. To investigate the relationship between selenoprotein P and antithrombin and its role in modulation of fibrin clot properties in antithrombin-deficient patients. Proteomic analysis of plasma fibrin clots was performed with mass spectrometry. In 108 patients with genetically confirmed type I (57%) or type II (43%) antithrombin deficiency and in healthy controls (n = 50), we assessed plasma selenoprotein P levels and thiobarbituric acid-reactive substances by enzyme-linked immunosorbent assay, along with fibrin clot permeability, clot lysis time, and thrombin generation. Clot-bound antithrombin concentration was 0.46 ± 0.32 mg/g protein, while selenoprotein P level was 30-fold lower (0.015 ± 0.012 mg/g). Type I compared to type II antithrombin-deficient patients had higher clot-bound antithrombin and selenoprotein P levels (both P < .001), associated together (ρ = 0.93, P < .001). Individuals with type I compared to type II antithrombin deficiency or controls had about 40% lower plasma selenoprotein P levels (P < .001). In antithrombin-deficient patients, plasma selenoprotein P was associated with antithrombin antigen (ρ = 0.35, P < .001) and thiobarbituric acid-reactive substances (ρ = 0.42, P < .001). Plasma selenoprotein P correlated also with endogenous thrombin potential (r = -0.33, P < .001), fibrin clot permeability (r = 0.43, P < .001), and clot lysis time (r = -0.40, P < .001) in antithrombin-deficient patients but not in controls. Patients with type I antithrombin deficiency had higher clot-bound selenoprotein P and reduced plasma selenoprotein P levels. Plasma selenoprotein P was associated with prothrombotic fibrin clot phenotype and enhanced thrombin generation.

A retrospective study of the management and outcomes of pregnancies with inherited antithrombin deficiency.

Antithrombin (AT) deficiency is a rare but highly thrombogenic inherited thrombophilia. Its association with adverse pregnancy outcomes (APO) is undefined. There is limited guidance on managing AT deficiency in pregnancy. Some significant issues remain controversial, including risk assessment for prophylactic anticoagulation, anticoagulant therapy, and monitoring. Our goal was to examine if the antepartum management of patients with AT deficiency affected their pregnancy outcomes. This retrospective, single-center observational study included pregnant women with inherited AT deficiency in Peking Union Medical College Hospital between 2013 and 2024. Seventeen pregnancies in 6 women with AT deficiency were identified. A total of 7 pregnancies received adjusted-dose low-molecular-weight heparin (LMWH) and were monitored by anti-Xa level, AT activity, and D-dimer. There were 5 live births (all received LMWH), 7 second-trimester abortions (1 received LMWH), and 5 early pregnancy losses (1 received LMWH). There were 5 abruptio placentae events (3 received LMWH) and 7 thrombotic events (2 received LMWH). AT deficiency is at least an important partial factor contributing to APO. It is suggested to make a full assessment of AT patients both for venous thrombus embolism and APO risk. We observed a high prevalence of heparin resistance and a positive correlation between adequate anticoagulation and pregnancy outcome based on tight monitoring with anti-Xa level and timely adjustment of the LMWH dosage.

Acquired antithrombin deficiency in adult patients with postcardiotomy extracorporeal membrane oxygenation.

This study aimed to investigate the relationship between acquired antithrombin deficiency in patients undergoing postcardiotomy extracorporeal membrane oxygenation (PC-ECMO) and thromboembolic or haemorrhagic events such as bleeding, peripheral arterial thromboembolism, and ischemic cerebrovascular events. The study was designed as a single-center, prospective study and conducted at our hospital between November 2019 and June 2021. 50 patients who underwent ECMO due to postcardiotomy cardiogenic shock were included in the study. Antithrombin (AT) activity testing was performed immediately after ECMO placement and continued for 5 days. The total of haemorrhagic or thromboembolic events was defined as morbidity. The entire patient population was assessed daily for AT measurements according to morbidity status, and ROC analysis was applied to determine the cut-off point. The correlation between clinical outcomes and morbidities with antithrombin levels was analysed. In our study, we identified a cut-off for AT levels on the first postoperative day. The risk of both bleeding (p = .006) and thromboembolism (p = .012) was significantly higher in patients below the 48.9% cut-off value. AT levels were compared with data on separation from PC-ECMO. The rate of separation from ECMO was 7.969 times higher in cases with AT levels above 51.8 on the third postoperative day and 5.6 times higher in cases with AT levels above 47.5 on the fourth postoperative day. Acquired antithrombin deficiency may develop in adults undergoing PC-ECMO. In our study, we demonstrated that in patients with low antithrombin levels, the risk of bleeding and thromboembolism increased. Additionally, since AT levels were higher in survivors, this can be considered an indicator of severity. This study is the first prospective study related to determining target antithrombin levels in adult patients undergoing PC-ECMO.

The Efficacy and Safety of DOACs in Inherited Antithrombin Deficiency: A Cohort Study from a Tertiary Referral Center.

Individuals with inherited antithrombin deficiency (IATD) have a high risk of venous thromboembolism (VTE). Most VTEs are managed with direct oral anticoagulants (DOACs), but the utility of DOACs in antithrombin deficiency (ATD) is unreported. Patients with IATD treated with DOAC were identified from our institutions' IATD registry. We assessed patients' characteristics, ATD type, and initial VTE characteristics, thrombosis recurrence and bleeding rates. Thirty-three patients received DOACs for 73 (38.5-111.5) months (median (interquartile range)). Prior to taking DOACs, 12 (36%) patients had VTE recurrence: these occurred after anticoagulation was ceased (4), nonadherence to VKA prior to DOAC use (3), or during heparin use in pregnancy (5). There were no VTE recurrences on standard-dose DOAC, except in a noncompliant patient receiving dabigatran. There was one recurrence with compliant DOAC use-a patient receiving rivaroxaban 10 mg. Six (18%) patients experienced clinically relevant bleeding, which was predominantly menorrhagia (5/6). One major bleeding event, intracranial hemorrhage, occurred in a patient receiving full-dose rivaroxaban who had refractory hypertension (0.5 events/100 patient-years). In this cohort, compliant DOAC users had an overall VTE recurrence rate of 0.5/100 patient-years, whereas with low-dose DOACs the event rate was 3.5/100 patient-years. Standard-dose DOACs appear efficacious and relatively safe in IATD.

Venous thromboembolism risk in adults with hereditary thrombophilia: a systematic review and meta-analysis.

This systematic review and meta-analysis assesses venous thromboembolism (VTE) risk in adults with hereditary thrombophilia, including Factor V Leiden (FVL) mutation, prothrombin G20210A (FII) mutation, compound heterozygosity, protein C (PC), protein S (PS), and antithrombin (AT) deficiency. Eligibility criteria included studies suitable for quantitative synthesis with extractable information on VTE risk in adults (> 15 years). There were no restrictions on VTE type, location, or occurrence. Two authors reviewed all studies and extracted data from 107 publications, encompassing 107,130 individuals (21,560 experiencing VTE). We used a random effects model and calculated odds ratios (ORs) with 95% confidence intervals (CIs). The highest risk was associated with homozygous FVL (OR 5.58, 95% CI 4.61-6.74), homozygous FII (OR 5.16, 95% CI 3.12-8.52), and compound heterozygosity (OR 4.64, 95% CI 2.25-9.58). In contrast, VTE risk was lowest for FVL heterozygosity (OR 2.97, 95% CI 2.41-3.67) and FII heterozygosity (OR 2.21, 95% CI 1.70-2.87), whereas PC (OR 3.23, 95% CI 2.05-5.08), PS (OR 3.01, 95% CI 2.26-4.02), and AT deficiency (OR 4.01, 95% CI 2.50-6.44) demonstrated an intermediate VTE risk. These results highlight an increased risk of venous thromboembolism in adults with hereditary thrombophilia. However, the risk for patients with PC, PS, and AT deficiency appears to be lower than previously stated, likely due to varying thrombogeneity of the underlying genetic mutations. Further research addressing this aspect of VTE risk in hereditary thrombophilia is imperative to improve patient management. TRIAL REGISTRATION: PROSPERO registration number CRD42022376757.

Heterozygosity for the Budapest 3 mutation in SERPINC1 in a family with thrombophilia and structural anomalies of the inferior vena cava

BackgroundAtresia of the infrarenal inferior vena cava (IVC) is associated with thrombophilia and antithrombin (AT) deficiency (ATD) due to homozygosity for the so-called Budapest 3 variant, c.391C > T, in the gene, SERPINC1.Case presentationWe report on a father and his two sons that had severe thrombosis at a young age. One son had absence of, and the other had very gracile infrarenal IVC. The father had gracile vena iliaca. All had significant collateral building. AT activity was determined with four different methods and varied between moderately reduced and borderline normal values, depending on the method. While all were heterozygous for c.391C > T, the father was also heterozygous for a variant of uncertain significance in SERPINC1.ConclusionsThe findings support the association between c.391C > T in SERPINC1, thrombophilia, and atresia of the IVC system and indicate that even heterozygosity for c.391C > T may contribute to such anomalies. ATD detection was hampered by the varying sensitivity of methods used for AT activity measurement.

Long-range and real-time PCR identification of a large SERPINC1 deletion in a patient with antithrombin deficiency.

Congenital antithrombin (AT) or serpin C1 deficiency, caused by a SERPINC1 abnormality, is a high-risk factor for venous thrombosis. SERPINC1 is prone to genetic rearrangement, because it contains numerous Alu elements. In this study, a Japanese patient who developed deep vein thrombosis during pregnancy and exhibited low AT activity underwent SERPINC1 gene analysis using routine methods: long-range polymerase chain reaction (PCR) and real-time PCR. Sequencing using long-range PCR products revealed no pathological variants in SERPINC1 exons or exon-intron junctions, and all the identified variants were homozygous, suggesting a deletion in one SERPINC1 allele. Copy number quantification for each SERPINC1 exon using real-time PCR revealed half the number of exon 1 and 2 copies compared with controls. Moreover, a deletion region was deduced by quantifying the 5'-upstream region copy number of SERPINC1 for each constant region. Direct long-range PCR sequencing with primers for the 5'-end of each presumed deletion region revealed a large Alu-mediated deletion (∼13kb) involving SERPINC1 exons 1 and 2. Thus, a large deletion was identified in SERPINC1 using conventional PCR methods.

Hereditary antithrombin deficiency pilot project registry from the American Thrombosis and Hemostasis Network

BackgroundPatients with hereditary antithrombin deficiency (HAD) have an increased risk of venous thromboembolism (VTE). The American Thrombosis and Hemostasis Network (ATHN) 12: HAD Pilot Project established a registry to collect data on patients with HAD. ObjectivesTo inform current practice and serve as a platform to design a multicenter global registry for patients with HAD. MethodsThe HAD registry was designed in 2020 to identify 100 patients with HAD receiving care at ATHN-affiliated centers. Demographics, type of HAD, thrombotic events, risk factors, anticoagulants, and antithrombin (AT) concentrate administration were recorded. ResultsNinety-four patients were included; 65% were females; 51% had type 1 HAD. Mean age at diagnosis was 26 years (SD, 18 years); 61% had VTE: 55% deep vein thrombosis and 27% pulmonary embolisms. Eight patients had arterial thrombosis. Recurrent thrombosis occurred in 58.6% of patients (44.8%) despite anticoagulation. The main risk factor for thrombosis in females was estrogen. Direct oral anticoagulants were prescribed in 30%, heparin in 34%, and warfarin in 32%. There were 139 pregnancies. Low-molecular-weight heparin was administered in 33% and AT concentrate in 19% and 11% prior to and after delivery, respectively. Twelve patients developed thrombosis in pregnancy. Seventy-nine patients underwent 239 surgeries or procedures, mainly gastrointestinal and vascular. Overall, 35% of participants received AT concentrate without adverse events. ConclusionIn ATHN 12, VTE was the predominant manifestation, frequently recurrent. There was a trend toward using direct oral anticoagulants. Low-molecular-weight heparin was administered in one-third of pregnancies and AT concentrate in one-fifth without adverse events. These data should encourage prospective studies to optimize the management of these patients.

Pharmacokinetics of Human Plasma-Derived Antithrombin in Pediatric Patients Supported on Extracorporeal Membrane Oxygenation.

Extracorporeal membrane oxygenation (ECMO) support of critically ill pediatric patients is associated with increased risk of thromboembolic events, and unfractionated heparin is used commonly for anticoagulation. Given reports of acquired antithrombin (AT) deficiency in this patient population and associated concern for heparin resistance, AT activity measurement and off-label AT replacement have become common in pediatric ECMO centers despite limited optimal dosing regimens. We conducted a retrospective cohort study of pediatric ECMO patients (0 to <18 years) at a single academic center to characterize the pharmacokinetics (PK) of human plasma-derived AT. We demonstrated that a two-compartment turnover model appropriately described the PK of AT, and the parameter estimates for clearance, central volume, intercompartmental clearance, peripheral volume, and basal AT input under non-ECMO conditions were 0.338 dL/h/70 kg, 38.5 dL/70 kg, 1.16 dL/h/70 kg, 40.0 dL/70 kg, and 30.4 units/h/70 kg, respectively. Also, ECMO could reduce bioavailable AT by 50% resulting in 2-fold increase of clearance and volume of distribution. To prevent AT activity from falling below predetermined thresholds of 50% activity in neonates and 80% activity in older infants and children, we proposed potential replacement regimens for each age group, accompanied by therapeutic drug monitoring.

Antithrombin Deficiency during Venoarterial Extracorporeal Membrane Oxygenation: Extremely Common, but Does It Matter?

Antithrombin Deficiency during Venoarterial Extracorporeal Membrane Oxygenation: Extremely Common, but Does It Matter?

Thrombophilia Screening: Not So Straightforward.

Although inherited thrombophilias are lifelong risk factors for a first thrombotic episode, progression to thrombosis is multifactorial and not all individuals with inherited thrombophilia develop thrombosis in their lifetimes. Consequently, indiscriminate screening in patients with idiopathic thrombosis is not recommended, since presence of a thrombophilia does not necessarily predict recurrence or influence management, and testing should be selective. It follows that a decision to undertake laboratory detection of thrombophilia should be aligned with a concerted effort to identify any significant abnormalities, because it will inform patient management. Deficiencies of antithrombin and protein C are rare and usually determined using phenotypic assays assessing biological activities, whereas protein S deficiency (also rare) is commonly detected with antigenic assays for the free form of protein S since available activity assays are considered to lack specificity. In each case, no single phenotypic assay is capable of detecting every deficiency, because the various mutations express different molecular characteristics, rendering thrombophilia screening repertoires employing one assay per potential deficiency, of limited effectiveness. Activated protein C resistance (APCR) is more common than discrete deficiencies of antithrombin, protein C, and protein S and also often detected initially with phenotypic assays; however, some centres perform only genetic analysis for factor V Leiden, as this is responsible for most cases of hereditary APCR, accepting that acquired APCR and rare F5 mutations conferring APCR will go undetected if only factor V Leiden is evaluated. All phenotypic assays have interferences and limitations, which must be factored into decisions about if, and when, to test, and be given consideration in the laboratory during assay performance and interpretation. This review looks in detail at performance and limitations of routine phenotypic thrombophilia assays.

Outcomes of venous thromboembolism in patients with inherited thrombophilia treated with direct oral anticoagulants: insights from the RIETE registry.

While direct oral anticoagulants (DOACs) are frequently used to treat venous thromboembolism (VTE), the outcomes of patients with inherited thrombophilia (IT) receiving DOACs for VTE remain understudied. We used data from the international RIETE registry to compare the rates of VTE recurrences, major bleeding, and mortality during anticoagulant treatment in VTE patients with and without IT, grouped by the use of DOACs or standard anticoagulant therapy. Among 103,818 enrolled patients, 21,089 (20.3%) were tested for IT, of whom 8422 (39.9%) tested positive: Protein C deficiency 294, Protein S deficiency 726, Antithrombin deficiency 240, Factor V Leiden 2248, Prothrombin gene mutation 1434, combined IT 3480. Overall, 14,189 RIETE patients (6.2% with IT) received DOACs, and 89,629 standard anticoagulation (8.4% with IT), mostly with heparins followed by vitamin K antagonists. Proportions of patients receiving DOACs did not differ between IT-positive and IT-negative patients. Rates of VTE recurrence on anticoagulant treatment were highest in patients with AT deficiency (P < 0.01). Rates of on-treatment major bleeding and all-cause mortality were lowest among patients with Factor V Leiden (FVL) or PT G20210A mutations, compared with patients who tested negative. Patients with IT who received DOACs had lower rates of major bleeding than those receiving standard anticoagulation. Excluding FVL and Protein S deficiency, patients with IT had lower rates of VTE recurrence with DOACs than with standard anticoagulation. DOACs are equally safe and effective in VTE patients with IT, with lower bleeding rates than those on standard anticoagulation.

Phenotypic and genetic analysis of a Chinese pedigree affected with Hereditary antithrombin deficiency due to a novel variant of SERPINC1 gene

To analyze the clinical phenotype and genetic characteristics of a Chinese pedigree affected with Hereditary antithrombin deficiency. A pedigree diagnosed at the the Second Affiliated Hospital of Wenzhou Medical University, Yuying Children's Hospital in June, 2020 was selected as the study subject. Plasma prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), and thrombin time (TT) of the probands and their pedigree members were determined using a STA-R automatic coagulation analyzer. Antithrombin activity (AT: A) and antithrombin antigen (AT: Ag) in plasma were determined with chromogenic substrate and immunonephelometry assays. All exons and flanking sequences of the anticoagulant protein gene SERPINC1 were amplified by PCR and subjected to Sanger sequencing. Candidate variants were verified with bioinformatic tools (PolyPhen-2, SIFT, Mutation Taster and PYMOL) to explore their effect on the function and structural conformation of the protein. The probands (II-2, II-10), their brother (II-5) and sons (III-1, III-8) had shown normal PT, APTT, FIB, and TT, but significantly decreased AT: A and AT: Ag, with their levels being 34%, 57%, 56%, 48%, 53% and 13.51 mg/dL, 13.44 mg/dL, 18.39 mg/dL, 17.36 mg/dL, 17.71 mg/dL, respectively. The remaining pedigree members had normal values. Sanger sequencing revealed that the probands and all affected pedigree members had harbored a heterozygous c.851T>C (p.Met284Thr) missense variant in exon 5 of the SERPINC1 gene. Bioinformatic analysis and simulation suggested that the variant has resulted in alteration of hydrogen bonds at the c.851 position, which may affect the structure of the protein. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was classified as pathogenic (PS1+PM1+PM5+PP1+PP4). The probands and other affected members were all diagnosed with type I hereditary AT deficiency, for which the c.851T>C (p.Met284Thr) variant of the SERPINC1 gene may be accountable.

Clinical and Molecular Characterization of Nine Novel Antithrombin Mutations.

Antithrombin (AT) is the major plasma inhibitor of thrombin (FIIa) and activated factor X (FXa), and antithrombin deficiency (ATD) is one of the most severe thrombophilic disorders. In this study, we identified nine novel AT mutations and investigated their genotype-phenotype correlations. Clinical and laboratory data from patients were collected, and the nine mutant AT proteins (p.Arg14Lys, p.Cys32Tyr, p.Arg78Gly, p.Met121Arg, p.Leu245Pro, p.Leu270Argfs*14, p.Asn450Ile, p.Gly456delins_Ala_Thr and p.Pro461Thr) were expressed in HEK293 cells; then, Western blotting, N-Glycosidase F digestion, and ELISA were used to detect wild-type and mutant AT. RT-qPCR was performed to determine the expression of AT mRNA from the transfected cells. Functional studies (AT activity in the presence and in the absence of heparin and heparin-binding studies with the surface plasmon resonance method) were carried out. Mutations were also investigated by in silico methods. Type I ATD caused by altered protein synthesis (p.Cys32Tyr, p.Leu270Argfs*14, p.Asn450Ile) or secretion disorder (p.Met121Arg, p.Leu245Pro, p.Gly456delins_Ala_Thr) was proved in six mutants, while type II heparin-binding-site ATD (p.Arg78Gly) and pleiotropic-effect ATD (p.Pro461Thr) were suggested in two mutants. Finally, the pathogenic role of p.Arg14Lys was equivocal. We provided evidence to understand the pathogenic nature of novel SERPINC1 mutations through in vitro expression studies.