Antithrombin (AT) is a serine protease inhibitor which exerts its anticoagulant effect through binding to serine residues in the active centers of procoagulant serine proteases. Its deficiency is associated with increased risk of venous thrombosis. We aim to investigate the pathogenic mechanism of two natural mutants (W221C and M284R) in inherited AT deficiency.

We analyzed 9 unrelated patients with inherited AT deficiency by extracting peripheral blood DNA and sequencing the SERPINC1 gene after amplification by polymerase chain reaction. Enzyme-linked immunosorbent assay and heparin affinity chromatography were used to assess AT secretion and purification efficiency. The mutant AT models were evaluated via computational simulations.

Among the 9 patients with inherited AT deficiency, 8 patients had type I AT deficiency, and one patient had type II AT deficiency with subtype of reactive site mutation. Seven of them experienced venous thrombotic events and all patients were found genetic mutations including missense (n = 6), deletion (n = 2) and insertion (n = 1). Two point mutations, W221C and M284R, were identified and were hypothesized to affect AT by destabilizing the central β-sheet. Based on immunoassays and heparin purification, the W221C mutant may impair AT secretion, whereas M284R mutant decreased the total AT production (696.8 ± 151.6 ng/ml versus 3833.72 ± 315.4 ng/ml, p = 0.029). Both mutants delayed the peak of AT release in heparin affinity chromatography.

Our study demonstrates that two mutations in SERPINC1 gene altered the production and structure of AT by in vitro protein expression and functional studies, including protein secretion and production. These findings enhance our understanding of the genetic basis of AT deficiency and its possible clinical implications.

Venous thromboembolism (VTE) represents a worldwide health challenge, impacting millions of people each year. The genesis of venous thrombosis is influenced in part by genetic components. Hereditary thrombosis is described as a genetically determined susceptibility to VTE. In the present study, a male patient was referred to our department presenting with multiple venous thrombosis events in different locations. Given a lack of identifiable risk factors, we aimed to investigate the possible genetic factor underlying venous thrombosis. Whole-exome sequencing was employed to examine genes linked to inherited thrombophilia in the proband. Putative variants were subsequently confirmed through Sanger sequencing within the family. The proband was identified as carrying two genetic mutations. One is the novel c.400G > C (p.E134Q) mutation affecting the final nucleotide of exon 5 in the PROC gene, potentially impacting splicing. The other is a previously reported heterozygous nonsense variant c.1016G > A (p.W339X) in the SERPINC1 gene. The proband inherited the former from her mother and the latter from her father. The presence of digenic inheritance in the patient reflects the complex phenotype of venous thrombosis and demonstrates the significance of an unbiased approach to detect pathogenic variants, especially in patients with a high risk of hereditary thrombosis.

Inherited AT deficiency is an autosomal-dominant thrombophilic disorder usually caused by various SERPINC1 defects associated with a high risk of recurrent venous thromboembolism. In this article, the phenotype, gene mutation, and molecular pathogenic mechanisms were determined in three pedigrees with inherited AT deficiency.

Coagulation indices were examined on STAGO STA-R-MAX analyzer. The AT:Ag was analyzed by ELISA. All exons and flanking sequences of SERPINC1 were amplified by PCR. AT wild type and three mutant expression plasmids were constructed and then transfected into HEK293FT cells. The expression level of AT protein was analyzed by ELISA and Western blot.

The AT:A and AT:Ag of probands 1 and 3 were decreased to 49% and 52 mg/dL, 38% and 44 mg/dL, respectively. The AT:A of proband 2 was decreased to 32%. The SERPINC1 gene analysis indicated that there was a p.Ile421Thr in proband 1, a p.Leu417Gln in proband 2, and a p.Met252Thr in proband 3, respectively. The AT mRNA expression level of the three mutants was not significantly different from AT-WT by qRT-PCR. The results of ELISA and Western blot tests showed that the AT-M252T and AT-I421T mutants had a higher AT expression than the AT wild type (AT-WT), and the AT protein expression of AT-L417Q mutants had no significant difference compared with AT-WT in the cell lysate. The AT expression levels of AT-M252T and AT-I421T mutants were lower than that of AT-WT, and there was no significant difference between AT-L417Q mutant and AT-WT in the supernatant.

The p.I421T and p.M252T mutations affected the secretion of AT protein leading to type I AT deficiency of probands 1 and 3. The p.Leu417Gln mutation was responsible for the impaired or ineffective activity AT protein in proband 2 and caused type II AT deficiency.

A 46-year-old Han man first had sigmoid sinus and transverse sinus venous thrombosis at the age of 42. At the age of 44, he once again developed thrombosis. Genetic testing showed heterozygous SERPINC1 mutation, bone marrow biopsy showed fibrosis grade 1 (MF-1), and JAK2 V617F mutation was positive, accompanied by UGT1A1 mutation and β-thalassemia gene mutation.

A 46-year-old Han man was first found to have sigmoid sinus and transverse sinus venous thrombosis at the age of 42 but had no individual or family thrombosis history, and he had been regularly taking warfarin anticoagulant therapy for a long period of time. At the age of 44, venous thrombosis reappeared in parts of the intrahepatic vein, main portal vein, splenic vein, and superior mesenteric vein, and his spleen was obviously enlarged. He had a history of jaundice for many years, and genetic testing revealed that he carried a heterozygous SERPINC1 mutation. Bone marrow biopsy showed multifocal fibrous tissue hyperplasia among trabeculae and focal fibrosis. He was positive for the JAK2 V617F mutation. At the same time, UGT1A1 and β-thalassemia gene mutations existed, and a SERPINC1 mutation and UGT1A1 mutation were both found in his parents.

The patient in this case had thrombophilia as the primary symptom, JAK2V617-positive myeloproliferative neoplasm (MPN) was the main potential cause, and hereditary AT-III deficiency may have been one of multiple secondary causes. It remains to be determined whether UGT1A1 and β-thalassemia gene mutations are related to thrombophilia. However, the clinical features of MPN in this patient were hidden, and the relevant clinical features of coexisting thalassemia and hereditary Gilbert syndrome, reported here for the first time domestically and abroad, were complicating factors, causing great difficulties for a clear diagnosis. Thus, when thrombophilia has been determined, it is necessary to screen the relevant latent problems overall. When the clinical features cannot be perfectly explained by one etiology, a relevant comprehensive examination should also be initiated from the perspective of multiple etiologies.

o explore the phenotype and genotype of a hereditary antithrombin deficient Chinese family. Functional and molecular analysis of the proband and his family members was performed. Online bioinformatics software was used to predict the pathogenicity of the novel mutation. ClustalX-2.1-win and PyMol software were applied to conservative analysis and generate molecular graphic images, respectively. Functional analysis had shown that the antithrombin (AT):A of the proband was reduced to 32% whereas AT:Ag was normal. Molecular analysis revealed a heterozygous missense mutation p. Leu417Gln in exon 7 of SERPINC1 gene. Bioinformatics and model analysis indicated that this mutation could affect the integrity of local intermolecular structures, resulting in a mild type of antithrombin deficiency but when combined with other genetic or acquired thrombophilic factors, patients may develop venous thrombosis. The p.Leu417Gln mutation was responsible for the decrease of AT:A in this family and caused type II antithrombin deficiency.

Antithrombin (AT) is one of the physiological anticoagulants that are mainly synthesized in the liver. As a protease inhibitor belonging to the serpin superfamily, AT is able to inactivate thrombin and inhibit activated coagulation factors IX, X, XI, and XII (FIXa, FXa, FXIa, and FXIIa).1 Moreover, it has been found that AT can inhibit activated FVII (FVIIa) by accelerating dissociation of FVIIa-tissue factor complex and preventing it from recombining.2 The AT gene (SERPINC1), located on chromosome 1 at q23.1-23.9 and spreads 13.5 kb, is composed of seven extrons and six introns.3 Hereditary AT deficiency is a rare thrombotic disorder caused by defects in SERPINC1 gene.4 It is inherited in an autosomal-dominant manner with an incidence of roughly 0.02 to 0.25% in the general population and 2 to 5% in patients with a history of venous thromboembolism (VTE).1 5 The most common thrombotic manifestations of patients with AT deficiency are VTEs, and their risks of VTE are approximately 20 times higher than those of nondeficient individuals.6 And the consequences of thrombophilia caused by AT deficiency are more severe than those of protein C and S protein deficiency,2 so it should be given sufficient attention.

Different types of in silico approaches can be used to predict the phenotypic consequence of missense variants. Such algorithms are often categorized as sequence based or structure based, when they necessitate 3D structural information. In addition, many other in silico tools, not dedicated to the analysis of variants, can be used to gain additional insights about the possible mechanisms at play.

Here we applied different computational approaches to a set of 20 known missense variants present on different proteins (CYP, complement factor B, antithrombin and blood coagulation factor VIII). The tools that were used include fast computational approaches and web servers such as PolyPhen-2, PopMusic, DUET, MaestroWeb, SAAFEC, Missense3D, VarSite, FlexPred, PredyFlexy, Clustal Omega, meta-PPISP, FTMap, ClusPro, pyDock, PPM, RING, Cytoscape, and ChannelsDB.

We observe some conflicting results among the methods but, most of the time, the combination of several engines helped to clarify the potential impacts of the amino acid substitutions.

Combining different computational approaches including some that were not developed to investigate missense variants help to predict the possible impact of the amino acid substitutions. Yet, when the modified residues are involved in a salt-bridge, the tools tend to fail, even when the analysis is performed in 3D. Thus, interactive structural analysis with molecular graphics packages such as Chimera or PyMol or others are still needed to clarify automatic prediction.

Deep vein thrombosis (DVT) is the formation of a blood clot formed in the deep veins of the lower limbs. Known genetic factors of DVT include deficiencies of antithrombin (AT), protein C, protein S, factor V Leiden mutation, and prothrombin G20210A mutation. Here, a 5-generation Chinese family with inherited DVT was recruited for genetic analysis.

The patient came to see a doctor because of leg swelling. A color Doppler ultrasound examination showed extensive thrombosis within the deep veins of her left leg. Computed tomography angiography showed a pulmonary embolism in her right lower pulmonary artery.

Type II AT deficiency lead to inherited DVT.

Whole-exome sequencing and cosegregation analysis were carried for the DVT family.

An unreported heterozygous missense variation, c.281T>C, was identified within the SERPINC1 gene. This missense variation of SERPINC1 leads to type II AT deficiency.

This result further enriched the variation spectrum of the SERPINC1 gene.

Acute unprovoked idiopathic fatal pulmonary embolism (IFPE) causes sudden death without an identifiable thrombogenic risk. We aimed to investigate the underlying genomic risks of IFPE through whole exome sequencing (WES).

We reviewed 14years of consecutive out-of-hospital fatal pulmonary embolism records (n=1478) from the ethnically diverse population of New York City. We selected 68 qualifying IFPE cases for WES. We compared the WES data of IFPE cases to those of 9332 controls to determine if there is an excess of rare damaging variants in the genome using ethnicity-matched controls in collapsing analyses.

We found nine of the 68 decedents (13·2%) who died of IFPE had at least one pathogenic or likely pathogenic variant in one of the three anti-coagulant genes: SERPINC1 (Antithrombin III), PROC, and PROS1. The odds ratio of developing IFPE as a variant carrier for SERPINC1 is 144·2 (95% CI, 26·3-779·4; P=1·7×10-7), for PROC is 85·6 (95% CI, 13·0-448·9; P=2.0×10-5), and for PROS1 is 56·4 (95% CI, 5·3-351·1; P=0·001). The average age-at-death of anti-coagulant gene variant carriers is significantly younger than that of non-carriers (28·56years versus 38·02years; P=0·01).

This study showed the important role of severe thrombophilia due to natural anti-coagulant deficiency in IFPE. Evaluating severe thrombophilia in out-of-hospital fatal PE beyond IFPE is warranted.

Antithrombin (AT) deficiency is an autosomal dominant disorder, and identification of mutation AT variants would improve our understanding of the anticoagulant function of this serine protease inhibitor (SERPIN) and the molecular pathways underlying this disorder. In the present study, we performed whole-exome sequencing of a Chinese family with deep vein thrombosis, and identified a new small deletion that eliminates four amino acids (INEL) from exon 4 of SERPINC1 gene. This causes type I AT deficiency by enhancing the intracellular retention of this protein. AT retention leads to endoplasmic reticulum (ER) stress, which further inhibits AT release. In addition, ER stress activates ER-associated degradation, which promotes AT degradation. Suppression of ER stress enhanced the secretion of AT, while inhibition of ER-associated degradation suppressed AT release. Thus, our study identified a new mutation (INEL deletion) causing type I AT deficiency, and uncovered a novel mechanism for AT retention through enhanced ER stress, which may provide an innovative approach for treating AT deficiency.

Inherited antithrombin (AT) deficiency is a rare autosomal disease that could increase the risk of venous thromboembolism (VTE) and usually caused by mutations of SERPINC1. Although a number of mutations of SERPINC1 have been reported in Chinese Han population, the impact of different subtypes of these mutations on the thrombotic phenotype is still unknown. Here, we performed a retrospective cohort study including 169 AT patients from 63 families to compare the clinical features between null mutation carriers and missense mutation carriers. We found that patients carrying null mutations have a higher risk of VTE (HR 2.29, 95% CI 1.16-4.69, P=0.02 adjusted for sex and VTE family history) and earlier median onset age of VTE (27 vs. 32years, P=0.045) as well as lower AT activities (47.6±1.0% vs. 59.1±2.3%, P<0.001) than those with missense mutations. We also observed that thrombus location sites showed no difference between null mutation carriers and missense mutation carriers, gene locations of the mutations did not relate with the incidence rate of VTE. This study demonstrated that different types of SERPINC1 mutations may play different roles in the development of VTE and should be considered in the prevention of VTE.

Congenital (hypo)dysfibrinogenemia patients may have obstetric complications during their pregnancies. This study aimed to evaluate thromboelastography (TEG) as a potential tool for assessing the tendency for obstetric complications in those patients in a non-pregnant state. A total of 22 female subjects with congenital (hypo)dysfibrinogenemia were recruited. Nine subjects had histories of obstetric complications and the other 13 subjects had at least one uneventful pregnancy without obstetric complications as yet. Detailed clinical investigation and phenotype/genotype detection were carried out, and both kaolin-activated TEG and functional fibrinogen TEG (FF-TEG) were applied in all subjects. Significant differences were identified in all TEG parameters except for R and angle between these two groups (P < 0.05) by covariance analysis. Receiver operating characteristic (ROC) analysis of discrimination between these two groups of patients was performed for TEG parameters. Significantly high odds ratio (OR) of obstetric complications occurrence were demonstrated in K ≥ 3.8 min, maximum amplitude (MA) ≤ 54.2 mm, comprehensive index (CI) ≤ -3 (11.67, 95% CI 1.527-89.121, P < 0.05 in all), and MA-CFF ≤ 12.1 mm (20.00, 95% confidence interval (95% CI) 1.967-203.322, P = 0.002). Moreover, MA-CFF had better prognostic performance, with a corresponding area under the receiver operating curve of 0.923 (range 0.815-1.031, P = 0.001). This study suggests that (hypo)dysfibrinogenemia patients with values outside of the cut-off values of TEG assays under non-pregnant state may have a higher risk of obstetric complications occurring when they are pregnant. No parameters under non-pregnant state in clinical laboratory have ever been reported to be risk factors for obstetric complication occurrence in (hypo)dysfibrinogenemia patients. This study explored such parameters in TEG assays and found that parameters of TEG assays under non-pregnant status might predict the occurrence of obstetric complications, which could provide physicians with important information about whether fibrinogen replacement therapy is required, so as to prevent the occurrence of obstetric complications, especially for patients who are asymptomatic in daily life.

Having encountered several subjects with venous thromboembolism (VTE) in 1 family in which 1 proband has recurrent VTE (rVTE), we aimed to assess the risk of VTE in first-degree relatives, especially the children of individuals with rVTE, and to investigate the association of endothelial nitric oxide synthase (eNOS) G894T polymorphism between Chinese persons with rVTE and their offspring.We collected information about family histories and blood samples from 126 individuals with rVTE who had presented to our institute from 2003 to 2014, and 126 population-based controls and the first-degree relatives of subjects in these 2 groups. We tested blood samples for heritable thrombophilia and calculated odds ratios (ORs) and kappa coefficients.First-degree relatives of individuals with rVTE patients had a statistically significant risk of developing VTE (OR 2.62, 95% confidence interval [CI] 1.61-4.26, P < 0.001). For siblings, the OR was 2.72 (95% CI 1.56-4.73, P < .001). Moreover, for each year that the patient was older, the OR was 0.98 (95% CI 0.97-0.99, P = 0.03). One (11.2%) of the 9 individuals who had the same eNOS G894T polymorphism as their probands had a history of VTE, whereas none of the 17 relatives without the same polymorphism had developed VTE. The associations between patients and their children were statistically significant for VTE (kappa = 0.23, P < 0.001) and for eNOS G894T (kappa = 0.03, P = 0.04).In this case-controlled study, we demonstrated a higher risk of VTE among first-degree relatives of individuals with rVTE, especially in siblings of younger subjects with rVTE. We also found that eNOS G894T polymorphism may be a predictor of VTE in offspring of individuals with rVTE.

A novel heterozygous variant, FGA c.169_180+2 del (designated fibrinogen Shanghai), was identified in a patient with dysfibrinogenemia with antiphospholipid antibody syndrome (APS) and recurrent venous thrombosis, and in his asymptomatic father. We aimed to reveal the functional implication of structural change caused by this variant.

Transcription analysis was performed with FGA minigene transfection assay to evaluate the impact of nucleosides deletion on mRNA editing. The fibrinogen isolated from propositus' plasma was used to characterise its functional defects. Fibrin polymerization and clot lysis experiments were performed by optical measurement of turbidity. Thrombin-catalysed fibrinopeptide release was analysed by the reversed-phase, high-performance liquid chromatography. The ultrastructures of fibrin clots were visualised by scanning electron microscopy.

FGA c.169_180+2 del led to an aberrant mRNA with exon 2 skipping and encoded an shortened Aα chain with 42 amino acids truncation at its N-terminal. The propositus' fibrinogen had an impaired release of fibrinopeptide A and abnormal polymerization with a significantly prolonged lag time, a slower maximum slope and reduced final turbidity. The fibrin clot formed with propositus' fibrinogen showed thicker fibres with looser network structure. Clot lysis was normal using the purified fibrinogen but was significantly impaired using the plasma sample from propositus, compared with that from his father.

Fibrinogen Shanghai results in N-terminal truncation of Aα chain, which does not interfere with synthesis, assembly or secretion of fibrinogen, but compromises fibrin polymerization and clot formation. APS at least partially contributes to the development of thrombosis in the propositus.

Congenital dysfibrinogenemia (CD) is a rare qualitative disorder of fibrinogen (Fg) with heterogeneous clinical manifestations. We aimed to analyze clinical phenotype and molecular basis of 102 Chinese CD patients and to evaluate the application of thromboelastography (TEG).

Clinical manifestations were recorded and quantified using the consensus ISTH bleeding assessment tool. Kaolin activated TEG and functional Fg TEG were applied in 30 patients. Genetic analysis of Fg genes were performed by direct sequencing.

27.5% patients experienced bleeding, 3.9% had thrombosis and 68.6% were asymptomatic. Females were more prone to experience bleeding (P=0.01). Significant difference (P<0.05) in TEG results were found between patients with hot-spot mutations at AαArg35(16) and γArg301(275), but were not identified between patients with and without bleeding. Normal TEG results were found in patients with mutations at AαArg35(16), AαPro37(18) or AαArg38(19). Six novel mutations were identified, including AαGly33(14)del, AαAsp57(38)_Trp60(41)delIVS2+1_+2GTdel, AαPhe742(723)Tyr, γAsn334(308)Thr, γGly335(309)Cys and γTrp395(369)Leu.

CD patients have similar clinical manifestations and hot-spot mutations worldwide with no ethnic difference. TEG results could not indicate the bleeding risk in patients, but priority of mutation screening at thrombin cleavage site or polymerization site on Aа chain may be given if TEG results are normal.