Thr90Ser Mutation in Antithrombin is Associated with Recurrent Thrombosis in a Heterozygous Carrier.

Antithrombin (AT) is a serine protease inhibitor that regulates the activity of coagulation proteases of both intrinsic and extrinsic pathways. We identified an AT-deficient patient with a heterozygous Thr90Ser (T90S) mutation who experiences recurrent venous thrombosis. To understand the molecular basis of the clotting defect, we expressed AT-T90S in mammalian cells, purified it to homogeneity, and characterized its properties in established kinetics, binding, and coagulation assays. The possible effect of mutation on the AT structure was also evaluated by molecular modeling. Results demonstrate the inhibitory activity of AT-T90S toward thrombin and factor Xa has been impaired three- to fivefold in both the absence and presence of heparin. The affinity of heparin for AT-T90S has been decreased by four- to fivefold. Kinetic analysis revealed the stoichiometry of AT-T90S inhibition of both thrombin and factor Xa has been elevated by three- to fourfold in both the absence and presence of heparin, suggesting that the reactivity of coagulation proteases with AT-T90S has been elevated in the substrate pathway. The anticoagulant activity of AT-T90S has been significantly impaired as analyzed in the AT-deficient plasma supplemented with AT-T90S. The anti-inflammatory effect of AT-T90S was also decreased. Structural analysis predicts the shorter side-chain of Ser in AT-T90S has a destabilizing effect on the structure of AT and/or the AT-protease complex, possibly increasing the size of an internal cavity and altering a hydrogen-bonding network that modulates conformations of the allosterically linked heparin-binding site and reactive center loop of the serpin. This mutational effect increases the reactivity of AT-T90S with coagulation proteases in the substrate pathway.

Double-Blind, Randomized, Placebo-Controlled Trial Comparing the Effects of Antithrombin Versus Placebo on the Coagulation System in Infants with Low Antithrombin Undergoing Congenital Cardiac Surgery.

OBJECTIVES: To determine whether precardiopulmonary bypass (CPB) normalization of antithrombin levels in infants to 100% improves heparin sensitivity and anticoagulation during CPB and has beneficial effects into the postoperative period. DESIGN: Randomized, double-blinded, placebo-controlled prospective study. SETTING: Multicenter study performed in 2 academic hospitals. PARTICIPANTS: The study comprised 40 infants younger than 7 months with preoperative antithrombin levels <70% undergoing CPB surgery. INTERVENTIONS: Antithrombin levels were increased with exogenous antithrombin to 100% functional level intraoperatively before surgical incision. MEASUREMENTS AND MAIN RESULTS: Demographics, clinical variables, and blood samples were collected up to postoperative day 4. Higher first post-heparin activated clotting times (sec) were observed in the antithrombin group despite similar initial heparin dosing. There was an increase in heparin sensitivity in the antithrombin group. There was significantly lower 24-hour chest tube output (mL/kg) in the antithrombin group and lower overall blood product unit exposures in the antithrombin group as a whole. Functional antithrombin levels (%) were significantly higher in the treatment group versus placebo group until postoperative day 2. D-dimer was significantly lower in the antithrombin group than in the placebo group on postoperative day 4. CONCLUSION: Supplementation of antithrombin in infants with low antithrombin levels improves heparin sensitivity and anticoagulation during CPB without increased rates of bleeding or adverse events. Beneficial effects may be seen into the postoperative period, reflected by significantly less postoperative bleeding and exposure to blood products and reduced generation of D-dimers.

Thrombophilia Associated with Early Post-angioplasty Thrombosis of Dialysis Vascular Access.

AIM: Percutaneous transluminal angioplasty (PTA) is widely used as the primary treatment for dialysis vascular access dysfunction. Nonetheless, many patients develop early occlusion after angioplasty. Thus, we investigated the role of thrombophilia in access occlusion within 30 days of angioplasty. MATERIALS AND METHODS: This case-control study included patients who underwent PTA for dialysis vascular access dysfunction. Patients who experienced occlusion within 30 days of angioplasty were included in the case group and those without occlusion for at least 30 days after angioplasty were included in the control group. All patients were tested for protein C, protein S, antithrombin III, lupus anticoagulant, and anticardiolipin antibodies. RESULTS: From February to October 2015, 462 patients underwent PTA for dialysis vascular access dysfunction. Forty-one patients (8.9%) had early occlusion within 30 days of angioplasty. The case group had more graft accesses (73 vs. 31%, P < 0.001) and thrombotic occlusions (67 vs. 15%, P < 0.001). A higher incidence of protein C (10 vs. 2%), protein S (15 vs. 5%), and antithrombin III (10 vs. 2%) deficiency and elevated anticardiolipin antibody (22 vs. 10%) levels were observed in the case group. Overall, 26 patients (63%) in the case group had at least one thrombophilic factor, compared with 15 patients (37%) in the control group (unadjusted odds ratio [OR], 3.004; 95% confidence interval [CI], 1.223-7.380; P = 0.027). After adjustment for confounding factors, the association between thrombophilic factors and early occlusion remained (adjusted OR, 3.806; 95% CI, 1.018-14.220; P = 0.047). CONCLUSION: Thrombophilia is associated with early occlusion after angioplasty for hemodialysis vascular access.

ß-Antithrombin, subtype of antithrombin deficiency and the risk of venous thromboembolism in hereditary antithrombin deficiency: A family cohort study.

Hereditary antithrombin deficiency is associated with a high incidence of venous thromboembolism (VTE), but VTE risk differs between families. Beta-antithrombin is reported to be the most active isoform of antithrombin in vivo. Whether ß-antithrombin activity and subtypes in antithrombin deficiency have impact on VTE risk has not been investigated outside the proband setting. We performed a retrospective family cohort study to investigate whether subtypes of antithrombin deficiency or ß-antithrombin levels are associated with the risk of first or recurrent VTE. Eighty-one subjects from 21 families were included, of which 52 were antithrombin deficient. Βeta-antithrombin levels were decreased in most type I and type IIPE subjects, but normal levels were found in all subtypes of antithrombin deficiency. The annual incidence of VTE in antithrombin-deficient family members was 1.24%, 95%CI: 0.72-1.99%, in low ß-antithrombin 1.36% (95%CI: 0.76-2.25%) and in normal ß-antithrombin 0.79% (95%CI: 0.10-2.77). The annual incidence of recurrence in family members was 3.1% (95%CI: 0.9-7.1%). Duration of anticoagulation had an impact on recurrence risk: In family members annual recurrence with fixed duration was 10% (95%CI: 2.1-29.2%), with indefinite duration 1.5% (95%CI: 0.2-5.4%), p < 0.05. Beta-antithrombin levels were not associated with the risk for first or recurrent VTE in antithrombin deficient subjects. CONCLUSIONS: In this high-risk antithrombin-deficient population, both subjects with low and normal plasma ß-antithrombin activity had high risks of first and recurrent VTE. This puts the importance of ß-antithrombin into question. Long-term anticoagulation is warranted in antithrombin-deficient VTE patients.

Strategy for Cardiovascular Surgery in Patients with Antithrombin III Deficiency.

Antithrombin III (ATIII) deficiency is a rare disorder in which thrombosis can be induced by stimuli that do not usually lead to thrombus formation, including minor injuries and surgery. Therefore, patients with ATIII deficiency undergoing cardiovascular surgery that involves heparinization require careful perioperative management. We experienced five patients with ATIII deficiency who underwent cardiovascular surgery and were managed with ATIII replacement. By administration of ATIII concentrate, preoperative ATIII activity was maintained at ≥120% and postoperative ATIII activity at ≥80%. All five patients were treated successfully without postoperative complications such as hemorrhage or thrombosis. In patients with ATIII deficiency undergoing cardiac surgery, it is important to perform ATIII replacement to achieve preoperative ATIII activity ≥120% and postoperative ATIII activity ≥80%, while the activated clotting time (ACT) is maintained at >400 seconds during cardiopulmonary bypass. In addition, long-term postoperative anticoagulant therapy is necessary in hereditary ATIII deficiency patients with a history of thrombosis.

Mild antithrombin deficiency and risk of recurrent venous thromboembolism: results from the MEGA follow-up study.

Essentials Mild antithrombin deficiency may increase the risk of recurrent venous thromboembolism (VTE). In a cohort study, we stratified patients with VTE to various cut-off antithrombin levels. A 1.6-3.7-fold increased risk of recurrent VTE was observed in the lowest antithrombin categories. Mild antithrombin deficiency (activity < 5th percentile of normal) increases recurrent VTE risk. SUMMARY: Background Mild antithrombin deficiency (previously defined as antithrombin activity below 70% or 80%) has been associated with a 2.4-3.5-fold increased risk of recurrent venous thromboembolism (VTE). This finding may have implications for duration of antithrombotic therapy in VTE patients with mild antithrombin deficiency. Objectives To externally validate whether mild antithrombin deficiency is a risk factor for recurrent VTE. Methods In a population-based cohort study, patients with a first VTE (n = 2357) were stratified according to percentile cut-off antithrombin levels (< 5th [< 87%], 5-10th [87-92%], > 10th percentile [> 92%]) and functional antithrombin levels (< 70%, 70-80%, > 80%). Results During a median follow-up of 7.4 years, 361 recurrent events occurred (incidence rate, 2.5/100 patient-years). We observed an increased risk of recurrent VTE in the lowest antithrombin activity category (< 5th percentile; < 87%) as compared with antithrombin activity that was > 10th percentile (> 92%), with an adjusted hazard ratio (HR) of 1.5 (95%CI, 1.0-2.3). When analyses were stratified to antithrombin cut-off criteria of< 70% vs. patients with antithrombin activity > 80%, the adjusted HR for venous recurrence was 3.7 (95% CI, 1.4-9.9). Mild antithrombin deficiency was able to predict recurrent VTE over at least 8 years of follow-up and the association remained present when the population was stratified to the presence or absence of thrombosis risk factors. Restriction analyses, where patients who used anticoagulation at time of blood draw and those who reported drinking ≥ 5 glasses alcohol daily were excluded, did not materially affect these outcomes. Conclusion This study confirms that mild antithrombin deficiency is a risk factor for recurrent VTE.

Antithrombin Debrecen (p.Leu205Pro) - Clinical and molecular characterization of a novel mutation associated with severe thrombotic tendency.

INTRODUCTION: Hereditary antithrombin (AT) deficiency is a rare thrombophilic disorder with heterogeneous genetic background and various clinical presentations. In this study we identified a novel AT mutation. Genotype-phenotype correlations, molecular characteristics and thrombotic manifestations of the mutation were investigated. MATERIALS AND METHODS: Thirty-one members of a single family were included. Clinical data was collected regarding thrombotic history. The mutation was identified by direct sequencing of the SERPINC1 gene. HEK293 cells were transfected with wild type and mutant SERPINC1 plasmids. Western blotting, ELISA and functional amidolytic assay were used to detect wild type and mutant AT. After double immunostaining, confocal laser scanning microscopy was used to localize mutant AT in the cells. Molecular modeling was carried out to study the structural-functional consequences of the mutation. RESULTS: Unprovoked venous thrombotic events at early age, fatal first episodes and recurrences were observed in the affected individuals. The median AT activity was 59%. Genetic analysis revealed heterozygous form of the novel mutation p.Leu205Pro (AT Debrecen). The mutant AT was expressed and synthesized in HEK293 cells but only a small amount was secreted. The majority was trapped intracellularly in the trans­Golgi and 26S proteasome. The mutation is suspected to cause considerable structural distortion of the protein. The low specific activity of the mutant AT suggested functional abnormality. CONCLUSIONS: AT Debrecen was associated with very severe thrombotic tendency. The mutation led to misfolded AT, impaired secretion and altered function. Detailed clinical and molecular characterization of a pathogenic mutation might provide valuable information for individualized management.

Stroke or left atrial thrombus prediction using antithrombin III and mean platelet volume in patients with nonvalvular atrial fibrillation.

BACKGROUND: CHADS2 (congestive heart failure, hypertension, age ≥ 75 years, diabetes mellitus, stroke) and CHA2 DS2 -VASc (congestive heart failure, hypertension, age ≥ 75 years, diabetes mellitus, stroke, vascular disease, age 65 to 74 years, sex category) scores showed just moderate discrimination ability in predicting thromboembolic complications in patients with nonvalvular atrial fibrillation (AF). HYPOTHESIS: To determine the association of antithrombin III (AT-III) deficiency and mean platelet volume (MPV) with the development of stroke or left atrial (LA) thrombus in patients with AF. METHODS: AT-III and MPV were analyzed in 352 patients with AF. The primary endpoint was a composite of ischemic stroke event and incidental LA thrombus. RESULTS: There were 50 events (14.2%) during a mean 35.4 months of follow-up. A significantly higher stroke or LA thrombus rate was observed in the low-AT-III group (<70%) than that in the high-AT-III group (≥70%). A significantly higher stroke or LA thrombus rate was observed in the high-MPV group (≥7.0 fL) than that in the low-MPV group (<7.0 fL). AF patients with an MPV ≥7.0 fL and AT-III deficiency had higher stroke or LA thrombus risk than those without an MPV ≥7.0 fL and AT-III deficiency. In the Cox proportional hazard analysis, high MPV was found to be an independent predictor of stroke or LA thrombus risk (hazard ratio: 6.408; 95% confidence interval: 2.874-14.286). Although AT-III deficiency was not an independent predictor of stroke or LA thrombus risk, a trend was observed. CONCLUSIONS: High MPV and AT-III deficiency were predictive markers for stroke or LA thrombus. Their predictive power for stroke was independent of antiplatelet treatment, anticoagulation therapy, and a high CHA2 DS2 -VASc score in patients with AF.

Characterization of thrombosis in patients with Proteus syndrome.

Patients with overgrowth and complex vascular malformation syndromes, including Proteus syndrome have an increased risk of thromboembolism. Proteus syndrome is a mosaic, progressive overgrowth disorder involving vasculature, skin, and skeleton, and caused by a somatic activating mutation in AKT1. We conducted a comprehensive review of the medical histories and hematologic evaluations of 57 patients with Proteus syndrome to identify potential risk factors for thrombosis. We found that six of ten patients, who were deceased, died secondary to deep venous thrombosis and/or pulmonary embolism. Of the remaining 47 living patients, six had thromboembolic events that all occurred postoperatively and in an affected limb. Eleven of 21 patients had an abnormal hypercoagulable panel including Factor V Leiden heterozygotes, antithrombin III deficiency, positive lupus anticoagulant, or Protein C or S deficiencies. We observed that eight of 17 patients had an abnormal D-dimer level >0.5 mcg/dl, but deep venous thromboses occurred in only four of those with D-dimer >1.0 mcg/dl. We conclude that the predisposition to thrombosis is likely to be multifaceted with risk factors including vascular malformations, immobility, surgery, additional prothrombotic factors, and possible pathophysiologic effects of the somatic AKT1 mutation on platelet function or the vascular endothelium. The D-dimer test is useful as a screen for thromboembolism, although the screening threshold may need to be adjusted for patients with this disorder. We propose developing a registry to collect D-dimer and outcome data to facilitate adjustment of the D-dimer threshold for Proteus syndrome and related disorders, including PIK3CA-Related Overgrowth Spectrum.

High levels of latent antithrombin in plasma from patients with antithrombin deficiency.

Antithrombin is an anticoagulant serpin that efficiently inhibits multiple procoagulant proteases. The cost for the structural flexibility required for this function is the vulnerability to mutations that impact its folding pathway. Most conformational mutations identified in serpins cause polymerisation. Only three mutations in SERPINC1 affecting two residues have been found to favour transformation to the latent conformation of antithrombin, another hyperstable non-anticoagulant form with strong antiangiogenic activity that constitutes 3 % of plasma antithrombin in healthy subjects. The analysis of latent antithrombin in 141 unrelated patients with antithrombin deficiency carrying 89 different SERPINC1 mutations identified four cases with higher levels than that of controls: p.Pro439Thr, p.Pro461Ser, p.Met283Val, and p.His401Tyr, the last also with circulating polymers. Heating of plasma at 42ºC exacerbated the transformation to the latent conformation in p.Pro439Thr and p.Pro461Ser. The conformational effect of p.Met283Val, the mutation associated with the highest levels of latent antithrombin detected in four members of a family, was verified in a recombinant model. Antithrombin deficiency in these cases should be classified as pleiotropic based on the impaired reactivity and low heparin affinity of the variant. Despite high levels of latent antithrombin (up to 80 µg/ml in p.Met283Val carriers), no vascular defects were described in carriers of these mutations. In conclusion, our study identifies new residues involved in the structural stability of antithrombin (and potentially of all serpins). High levels of endogenous latent antithrombin seem to play a minor antiangiogenic effect. Finally, pleiotropic deficiencies may be caused by mutations inducing transformation to the latent conformation.

A novel frameshift mutation leading to inherited type I antithrombin deficiency.

Inherited antithrombin (AT) deficiency is an autosomal dominant thrombotic disorder. We encountered a case of inherited type I AT deficiency and identified the mutation responsible; a novel 5406delA mutation in the SERPINC1 gene appeared to have caused a frameshift with premature termination at amino acid +283. The recombinant AT protein including 5406delA was not detected in cell lysates or culture supernatants. These results will contribute to the creation of an accurate database and define the molecular basis for AT deficiency.

Venous Thromboembolism After Knee Arthroscopy in Undiagnosed Familial Thrombophilia.

Venous thromboembolism is uncommon after knee arthroscopy, and there are no guidelines for thromboprophylaxis in elective routine knee arthroscopy. Preoperative evaluation of common thrombophilias should provide guidance for postarthroscopy thromboprophylaxis in otherwise healthy patients who are at high risk for venous thromboembolism. This study assessed 10 patients with venous thromboembolism after total hip or knee arthroplasty. Patients were assessed if venous thromboembolism occurred within 6 months after knee arthroscopy (n=10) or total hip or knee arthroplasty (n=21). This study assessed gene mutations (factor V Leiden, prothrombin G20210A, plasminogen activator inhibitor, methylenetetrahydrofolate reductase) and serologic thrombophilias (high levels of factors VIII and XI, homocysteine, anticardiolipin immunoglobulin G and immunoglobulin M antibodies, and lupus anticoagulant; low antigenic protein C, S, and free S; and antithrombin III deficiency). The same coagulation data were obtained for normal subjects (n=110). The major thrombophilias in the arthroscopy group were factor V Leiden heterozygosity (40%), high factor VIII level (50%), and high homocysteine (30%). The respective values in control subjects were 2% (P=.0004), 7% (P=.0011), and 5% (P=.02). When the arthroscopy group was compared with the 21 patients who had venous thromboembolism after total hip or knee arthroplasty, the sole difference was factor V Leiden heterozygosity, which was 40% vs 0%, respectively (P=.007). Although venous thromboembolism after knee arthroscopy is uncommon, to identify high-risk patients and guide postoperative thromboprophylaxis, the authors suggest routine preoperative measurement of 3 common familial thrombophilias: factor V Leiden, factor VIII, and homocysteine. [Orthopedics. 2016; 39(6):e1052-e1057.].

Issues in the Diagnosis and Management of Hereditary Antithrombin Deficiency.

OBJECTIVE: To review insights gained in the past several years about hereditary antithrombin (AT) deficiency and to outline approaches to the management of patients with AT deficiency in the acute and chronic settings. DATA SOURCES: An extensive literature search of Scopus (January 2008-April 2016) was performed for the terms congenital antithrombin deficiency, inherited antithrombin deficiency, or hereditary antithrombin deficiency Additional references were identified by reviewing literature citations. STUDY SELECTION: All relevant English-language case reports, reviews, clinical studies, meeting abstracts, and book chapters assessing hereditary AT deficiency were included. DATA SYNTHESIS: AT deficiency significantly increases the risk of venous thromboembolism (VTE). The risk of VTE is particularly high during pregnancy, the postpartum period, and following major surgery. Effective clinical management includes determination of the appropriate type and duration of antithrombotic therapy (ie, AT replacement for acute situations) while minimizing the risk of bleeding. For persons newly diagnosed with AT deficiency, age, lifestyle, concurrent medical conditions, family history, and personal treatment preferences can be used to individualize patient management. Patients should be informed of the risks associated with hormonal therapy, pregnancy, surgical procedures, and immobility, which further increase the risk of VTE in patients with AT deficiency. CONCLUSION: AT deficiency poses the highest risk for VTE among the hereditary thrombophilias, often requiring long-term anticoagulation. Undertaking an evaluation for hereditary thrombophilia is controversial; however, a diagnosis of VTE in association with AT deficiency can have management implications. An important treatment option for patients with this disorder in high-risk situations is AT concentrate.

Antithrombin Dublin (p.Val30Glu): a relatively common variant with moderate thrombosis risk of causing transient antithrombin deficiency.

The key haemostatic role of antithrombin and the risk of thrombosis associated with its deficiency support that the low incidence of antithrombin deficiency among patients with thrombosis might be explained by underestimation of this disorder. It was our aim to identify mutations in SERPINC1 causing transient antithrombin deficiency. SERPINC1 was sequenced in 214 cases with a positive test for antithrombin deficiency, including 67 with no deficiency in the sample delivered to our laboratory. The p.Val30Glu mutation (Antithrombin Dublin) was identified in five out of these 67 cases, as well as in three out of 127 cases with other SERPINC1 mutations. Genotyping in 1593 patients with venous thrombosis and 2592 controls from two populations, revealed a low prevalent polymorphism (0.3 %) that moderately increased the risk of venous thrombosis (OR: 2.9; 95 % CI: 1.07-8.09; p= 0.03) and identified one homozygous patient with an early thrombotic event. Carriers had normal anti-FXa activity, and plasma antithrombin was not sensitive to heat stress or proteolytic cleavage. Analysis of one sample with transient deficit revealed a type I deficiency, without aberrant or increased latent forms. The recombinant variant, which lacked the two amino-terminal residues, had reduced secretion from HEK-EBNA cells, formed hyperstable disulphide-linked polymers, and had negligible activity. In conclusion, p.Val30Glu by affecting the cleavage of antithrombin's signal peptide, results in a mature protein lacking the N-terminal dipeptide with no functional consequences in normal conditions, but that increases the sensitivity to be folded intracellularly into polymers, facilitating transient antithrombin deficiency and the subsequent risk of thrombosis.