Search

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cyclic peptides present a robust platform for drug design, offering high specificity and stability due to their conformationally constrained structures. In this study, we introduce an updated version of the Cyclic Peptide Matching program (cPEPmatch) tailored for the identification of cyclic peptides capable of mimicking protein-glycosaminoglycan (GAG) binding sites. We focused on engineering cyclic peptides to replicate the GAG-binding affinity of antithrombin III (ATIII), a protein that plays a crucial role in modulating anticoagulation through interaction with the GAG heparin. By integrating computational and experimental methods, we successfully identified a cyclic peptide binder with promising potential for future optimization. MD simulations and MM-GBSA calculations were used to assess binding efficacy, supplemented by umbrella sampling to approximate free energy landscapes. The binding specificity was further validated through NMR and ITC experiments. Our findings demonstrate that the computationally designed cyclic peptides effectively target GAGs, suggesting their potential as novel therapeutic agents. This study advances our understanding of peptide-GAG interactions and lays the groundwork for future development of cyclic peptide-based therapeutics.

Abstract Background Inherited antithrombin, protein C, and protein S deficiency increase the risk of venous thromboembolism. The presence of defects can be identified by clinical laboratory assays. In most Chinese clinical laboratories, the screening tests for antithrombin, protein C, and protein S deficiency are their activity assays. Ensuring appropriate pre-analytical storage conditions for activity tests is essential. This study aimed to assess the effects of storage conditions on antithrombin, protein C, and protein S activity in frozen plasma. Methods We collected the remaining plasma of 29 patients. The baseline of antithrombin, protein C, and protein S activity values were tested within 4 h. Then, each sample was sub-packaged into 4 EP tubes, and was stored at -20 °C for 3 days, -20 °C for 7 days, -80 °C for 3 days, and − 80 °C for 7 days, respectively. After thawing, samples were tested by two systems. Results The percentage deviation of antithrombin and protein C activity assay was 10%. Some samples, initially within the normal range, were classified as abnormal after freezing storage. Conclusions Our study indicated that antithrombin and protein C remain stable when stored at -20 °C or -80 °C in a week. We argued that Protein S activity is not stable in frozen plasma. The use of frozen-thawed plasma for PS activity assay may result in overdiagnosis of protein S deficiency.

Objective: Hypercoagulopathy related to hyperinflammation may be responsible for mortality and organ failure in coronavirus disease-2019 (COVID-19) patients. Abnormal coagulation profiles were associated with poor outcomes. In this study, we aimed to evaluate the prognostic value of antithrombin (AT) levels in critically ill COVID-19 patients on the intensive care unit (ICU) admission. Materials and Methods: Four hundred ten critically ill COVID-19 patients were retrospectively analyzed. Inflammatory and conventional coagulation parameters as well as AT activity levels were recorded on the ICU admission. The clinical outcomes of patients were analyzed. Results: AT levels on the ICU admission were significantly lower in non-survivors than survivors (77.9% vs. 82.5%; p=0.027). Besides AT, D-dimer values of non-survivors were significantly higher than survivors (2775 vs. 1495 µg/L; p

Abstract Background There is no reliable indicator that can assess the treatment effect of anticoagulant therapy for sepsis-associated disseminated intravascular coagulation (DIC) in the short term. The aim of this study is to develop and validate a prognostic index identifying 28-day mortality in septic DIC patients treated with antithrombin concentrate after a 3-day treatment. Methods The cohort for derivation was established utilizing the dataset from post-marketing surveys, while the cohort for validation was acquired from Japan’s nationwide sepsis registry data. Through univariate and multivariate analyses, variables that were independently associated with 28-day mortality were identified within the derivation cohort. Risk variables were then assigned a weighted score based on the risk prediction function, leading to the development of a composite index. Subsequently, the area under the receiver operating characteristic curve (AUROC). 28-day survival was compared by Kaplan–Meier analysis. Results In the derivation cohort, 252 (16.9%) of the 1492 patients deceased within 28 days. Multivariable analysis identified DIC resolution (hazard ratio [HR]: 0.31, 95% confidence interval [CI]: 0.22–0.45, P

Background: Inherited antithrombin, protein C, and protein S deficiency increase the risk of venous thromboembolism. The presence of defects can be identified by clinical laboratory assays. In most Chinese clinical laboratories, the screening tests for antithrombin, protein C, and protein S deficiency are their activity assays. Ensuring appropriate pre-analytical storage conditions for activity tests is essential. This study aimed to assess the effects of storage conditions on antithrombin, protein C, and protein S activity in frozen plasma. Methods: We collected the remaining plasma of 29 patients. The baseline of antithrombin, protein C, and protein S activity values were tested within 4 h. Then, each sample was sub-packaged into 4 EP tubes, and was stored at -20 °C for 3 days, -20 °C for 7 days, -80 °C for 3 days, and − 80 °C for 7 days, respectively. After thawing, samples were tested by two systems. Results: The percentage deviation of antithrombin and protein C activity assay was<10% compared with the initial values. Protein S activity showed a significant reduction in frozen plasma, with a deviation > 10%. Some samples, initially within the normal range, were classified as abnormal after freezing storage. Conclusions: Our study indicated that antithrombin and protein C remain stable when stored at -20 °C or -80 °C in a week. We argued that Protein S activity is not stable in frozen plasma. The use of frozen-thawed plasma for PS activity assay may result in overdiagnosis of protein S deficiency. [ABSTRACT FROM AUTHOR]

Background: There is no reliable indicator that can assess the treatment effect of anticoagulant therapy for sepsis-associated disseminated intravascular coagulation (DIC) in the short term. The aim of this study is to develop and validate a prognostic index identifying 28-day mortality in septic DIC patients treated with antithrombin concentrate after a 3-day treatment. Methods: The cohort for derivation was established utilizing the dataset from post-marketing surveys, while the cohort for validation was acquired from Japan's nationwide sepsis registry data. Through univariate and multivariate analyses, variables that were independently associated with 28-day mortality were identified within the derivation cohort. Risk variables were then assigned a weighted score based on the risk prediction function, leading to the development of a composite index. Subsequently, the area under the receiver operating characteristic curve (AUROC). 28-day survival was compared by Kaplan–Meier analysis. Results: In the derivation cohort, 252 (16.9%) of the 1492 patients deceased within 28 days. Multivariable analysis identified DIC resolution (hazard ratio [HR]: 0.31, 95% confidence interval [CI]: 0.22–0.45, P < 0.0001) and rate of Sequential Organ Failure Assessment (SOFA) score change (HR: 0.42, 95% CI: 0.36–0.50, P < 0.0001) were identified as independent predictors of death. The composite prognostic index (CPI) was constructed as DIC resolution (yes: 1, no: 0) + rate of SOFA score change (Day 0 SOFA score–Day 3 SOFA score/Day 0 SOFA score). When the CPI is higher than 0.19, the patients are judged to survive. Concerning the derivation cohort, AUROC for survival was 0.76. As for the validation cohort, AUROC was 0.71. Conclusion: CPI can predict the 28-day survival of septic patients with DIC who have undergone antithrombin treatment. It is simple and easy to calculate and will be useful in practice. [ABSTRACT FROM AUTHOR]

Antithrombin (AT) is a critical regulator of the coagulation cascade by inhibiting multiple coagulation factors including thrombin and FXa. Binding of heparinoids to this serpin enhances the inhibition considerably. Mutations located in the heparin binding site of AT result in thrombophilia in affected individuals. Our aim was to study 10 antithrombin mutations known to affect their heparin binding in a heparin pentasaccharide bound state using two molecular dynamics (MD) based methods providing enhanced sampling, GaMD and LiGaMD2. The latter provides an additional boost to the ligand and the most important binding site residues. From our GaMD simulations we were able to identify four variants (three affecting amino acid Arg47 and one affecting Lys114) that have a particularly large effect on binding. The additional acceleration provided by LiGaMD2 allowed us to study the consequences of several other mutants including those affecting Arg13 and Arg129. We were able to identify several conformational types by cluster analysis. Analysis of the simulation trajectories revealed the causes of the impaired pentasaccharide binding including pentasaccharide subunit conformational changes and altered allosteric pathways in the AT protein. Our results provide insights into the effects of AT mutations interfering with heparin binding at an atomic level and can facilitate the design or interpretation of in vitro experiments. [ABSTRACT FROM AUTHOR]

Objective: Hypercoagulopathy related to hyperinflammation may be responsible for mortality and organ failure in coronavirus disease-2019 (COVID-19) patients. Abnormal coagulation profiles were associated with poor outcomes. In this study, we aimed to evaluate the prognostic value of antithrombin (AT) levels in critically ill COVID-19 patients on the intensive care unit (ICU) admission. Materials and Methods: Four hundred ten critically ill COVID-19 patients were retrospectively analyzed. Inflammatory and conventional coagulation parameters as well as AT activity levels were recorded on the ICU admission. The clinical outcomes of patients were analyzed. Results: AT levels on the ICU admission were significantly lower in non-survivors than survivors (77.9% vs. 82.5%; p=0.027). Besides AT, D-dimer values of non-survivors were significantly higher than survivors (2775 vs. 1495 µg/L; p<0.001). The cut-off levels for AT and D-dimer were 70.5% and 1585 µg/L, respectively. When AT and D-dimer were analyzed together, mortality estimation was better than only D-dimer or AT. Conclusion: Low AT levels may be indicative of severe disease and mortality together with high D-dimer levels in COVID-19 patients. Diagnosing and managing AT deficiency in COVID-19 patients could be beneficial for survival. So AT level measurements should be included in the routine panel of laboratory investigation. [ABSTRACT FROM AUTHOR]

Andexanet alfa is a Gla-domainless mutant (S195A) factor Xa (GDXa) approved for acute reversal of oral factor Xa inhibitors. Cardiac surgery patients exposed to andexanet before cardiopulmonary bypass often exhibit severe heparin resistance. There is a paucity of data on the effectiveness and optimal dosage of antithrombin use in this setting. The objective of this study was to evaluate the in vitro effect of increased heparin with antithrombin levels on attenuating heparin resistance induced by GDXa. Heparinised normal pooled plasma and cardiopulmonary bypass plasma were spiked with GDXa 4 μM. Tissue factor-activated thrombin generation was used to assess heparin reversal effects of GDXa and restoration of anticoagulation with additional heparin with and without antithrombin. Serum thrombin–antithrombin complex, antithrombin activity, and tissue factor pathway inhibitor were also measured in tissue factor-activated, recalcified cardiopulmonary bypass plasma spiked with GDXa. In normal pooled plasma, GDXa-induced heparin reversal was mitigated by maintaining a high heparin concentration (12 U ml−1) and supplementing antithrombin (1.5–4.5 μM) based on peak and velocity of thrombin generation. Heparin reversal by GDXa was also demonstrated in cardiopulmonary bypass plasma, but supplementing both heparin (8 U ml−1) and antithrombin (3 μM) attenuated GDXa-induced changes in peak and velocity of thrombin generation by 72.5% and 72.2%, respectively. High heparin and antithrombin levels attenuated thrombin–antithrombin complex formation in tissue factor-activated, GDXa-spiked cardiopulmonary bypass plasma by 85.7%, but tissue factor pathway inhibitor remained depleted compared with control cardiopulmonary bypass plasma. Simultaneous supplementation of heparin and antithrombin mitigate GDXa-induced heparin resistance by compensating for the loss of tissue factor pathway inhibitor. [ABSTRACT FROM AUTHOR]

Aim: to assess adaptive hemostasis changes in multiple dichorionic pregnancy after in vitro fertilization (IVF). Materials and Methods. A prospective observational randomized controlled trial was conducted by examining 58 and 46 pregnant women with multiple dichorionic diamniotic twins resulting from applying assisted reproductive technologies (ART) and spontaneous delivery (comparison group), respectively. Hemostasis parameters were studied as follows: activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen, antithrombin, protein C, protein S, functions of protein С (РrоС Global test), D-dimer, platelet aggregation with adenosine-5-diphosphate (ADP), ristocetin, and collagen. Results. A high coagulation potential was revealed, more prominent after using ART (p < 0.05). Fibrinogen level gradually increased while gestation age increased, whereas APTT, PT and TT level decreased. In the group with natural conception, fibrinogen increased by 22 % in the second trimester, reaching 4.5 g/L (95 % CI = 4,2–4,8) and by 6 % in the third trimester, reaching 4.8 g/L (95 % CI = 4,3–5,4), whereas in the IVF group – by 26 %, reaching 5.3 g/L (95 % CI = 4,7–5,6) and by 21 %, reaching (6.5 g/L; 95 % CI = 5,2–6,8) in relevant trimester of pregnancy, respectively. Antithrombin level was lower in IVF patients – 76.8 % (95 % CI = 72.6 – 81.0) in the second trimester, reaching 70.6 % (95 % CI = 64.8–76.4) in the third trimester (p < 0.001). Protein C level did not differ significantly between groups and was low within the reference range. The aggregatogram demonstrated a high platelet hemostatic potential in IVF patients (p < 0.05) as early as in the first trimester: ADP-induced aggregation – 68.3 % (95 % CI = 62.9–73.7), ristocetin-induced aggregation – 53.1 % (95 % = CI 48.7–58.5), collagen-induced aggregation – 58.4 % (95 % CI = 52.1–64.7). In the third trimester, both platelet aggregation and functional activity (ADP-induced aggregation – 64.5 % [95 % CI = 59.3–69.7], ristocetin-induced aggregation – 68.4 % [95 % CI = 63.2–73.6], collagen-induced aggregation – 50.7 % [95 % CI = 44.3–57.1]; p < 0.05) and D-dimer level persistently increased, also more prominently in the IVF group (1.60 ± 0.46 ng/ml; p < 0.05). Conclusion. Gestational adaptation in induced multiple pregnancies is at high risk of breach in compensatory mechanisms and requires monitoring for timely detection of decompensation signs and their correction to prolong pregnancy till optimal delivery time frame.

• NXT007 is a bispecific IgG engineered from emicizumab to potentiate the activity. • The natural anticoagulant function of AT is unaffected by the presence of emicizumab. • NXT007 was here found not to affect the AT-function despite its enhanced FX-binding. • The results suggest that NXT007 would not increase the AT-related thrombotic risk. [ABSTRACT FROM AUTHOR]

Introduction: Extracorporeal membrane oxygenation (ECMO) may act as a driver or propagator of systemic inflammation. In turn, cytokine release can modify thromboelastographic (TEG) tests which are commonly used for anticoagulation monitoring. In this context, antithrombin (AT) supplementation might further modify TEG. Methods: This is a pre-specified sub-study of the “Randomized Controlled Trial of Antithrombin Supplementation During Extracorporeal Membrane Oxygenation” study (investigator-initiated, randomized, single-blind, two-arm trial) conducted in two Italian ECMO referral ICUs. Adult patients requiring vv-ECMO for respiratory failure and undergoing unfractioned heparin (UFH) administration were enrolled and randomized whether to receive AT supplementation. Plasma samples for cytokine assay (IL-8, IL-10, IL-6, IL-1β, TNF-α and Pro-ADM) and heparinase TEG were collected from every patient before ECMO start, 24 h and 72 h after ECMO start, before ECMO removal, and 7 days after ECMO removal or upon ICU discharge whichever happened first. AT concentration, coagulation and clinical data were collected before ECMO start and at pre-fixed time points. Results: Thirty-nine patients were enrolled (21 treatments, 18 controls). TEG-R had a weak-to-moderate positive correlation with IL-8, IL-6, IL-10 and TNF-α and a moderate positive correlation with Pro-ADM. TEG-ANG showed a weak negative correlation with IL-8, IL-6 and TNF-α, while TEG-MA negatively correlated with IL-8, TNF-α and Pro-ADM. AT supplementation seemed to modify the association between TEG-MA and IL-8, IL-10 and Pro-ADM; conversely, AT did not affect the relationship among TEG-R or TEG-ANG and the studied cytokines. Conclusions: High concentrations of systemic cytokines correlated with longer reaction times and decreased angle and amplitude at TEG, suggesting that an increase in inflammation is related with hypocoagulability as revealed by thromboelastography. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Background Andexanet alfa, an anti-Xa inhibitor antagonist, induces heparin resistance. Here, we report a case of successful management of cardiopulmonary bypass with andexanet alfa-induced heparin resistance using nafamostat mesylate. Case presentation An 84-year-old female, with Stanford type A acute aortic dissection, underwent an emergency surgery for total aortic arch replacement. Andexanet alfa 400 mg was administered preoperatively to antagonize edoxaban, an oral Xa inhibitor. Heparin 300 IU/kg was administered before cardiopulmonary bypass, and the activated clotting time (ACT) was 291 s. The ACT was 361 s after another administration of heparin 200 IU/kg. According to our routine therapy for heparin resistance, an initial dose of nafamostat mesylate 10 mg was administered intravenously, followed by a continuous infusion of 20–30 mg/h. The ACT was prolonged to 500 s, and cardiopulmonary bypass was successfully established thereafter. Conclusions This case report presents the successful management of cardiopulmonary bypass with andexanet alfa-induced heparin resistance using nafamostat mesilate. This report presents the successful management of cardiopulmonary bypass with andexanet alfa-induced heparin resistance using nafamostat mesilate.

Abstract Background Disseminated intravascular coagulation (DIC) syndrome is a highly lethal condition characterized by the complication of multiple organ damage. Although the effects of combined antithrombin (AT) and recombinant thrombomodulin (rTM) on DIC syndrome have previously been examined, the results are inconsistent and inconclusive. Therefore, we conducted a systematic review on the combined administration of AT and rTM for the treatment of septic DIC to investigate the superiority of the combination therapy over either AT or rTM monotherapy using a random-effects analysis model. Method We searched electronic databases, including Medline, Cochrane Central Register of Controlled Trials, Scopus, and Igaku-Chuo Zasshi (ICHU-SHI) Japanese Central Review of Medicine Web from inception to January 2022. Studies assessing the efficacy of combined AT and rTM were included. The primary outcome was all-cause mortality, and the secondary outcome was occurrence of serious bleeding complications compared to monotherapy. We presented the pooled odds ratio (OR) or hazard ratio (HR) with 95% confidence intervals (CI) depending on reporting results in each primary study. Results We analyzed seven enrolled clinical trials, all of which were observational studies. Combination therapy had a non-significant favorable association with lower 28-day mortality compared to monotherapy (HR 0.67 [0.43–1.05], OR 0.73 [0.45–1.18]). The I 2 values were 60% and 72%, respectively, suggesting high heterogeneity. As a secondary outcome, bleeding complications were similar between the two groups (pooled OR 1.11 [0.55–2.23], I 2 value 55%). Conclusions Although the findings in this analysis could not confirm a statistically significant effect of AT and rTM combination therapy for septic DIC, it showed a promising effect in terms of improving mortality. The incidence of bleeding was low and clinically feasible. Further research is warranted to draw more conclusive results. Trial registration This study was registered in the University Hospital Medical Information Network (UMIN) Clinical Trials Registry (UMIN ID: 000049820).

Rationale: Definitive guidelines for anticoagulation management during veno-venous extracorporeal membrane oxygenation (VV ECMO) are lacking, whereas bleeding complications continue to pose major challenges. Objectives: To describe anticoagulation modalities and bleeding events in adults receiving VV ECMO. Methods: This was an international prospective observational study in 41 centers, from December 2018 to February 2021. Anticoagulation was recorded daily in terms of type, dosage, and monitoring strategy. Bleeding events were reported according to site, severity, and impact on mortality. Measurements and Main Results: The study cohort included 652 patients, and 8,471 days on ECMO were analyzed. Unfractionated heparin was the initial anticoagulant in 77% of patients, and the most frequently used anticoagulant during the ECMO course (6,221 d; 73%). Activated partial thromboplastin time (aPTT) was the most common test for monitoring coagulation (86% of days): the median value was 52 seconds (interquartile range, 39 to 61 s) but dropped by 5.3 seconds after the first bleeding event (95% confidence interval, −7.4 to −3.2; P < 0.01). Bleeding occurred on 1,202 days (16.5%). Overall, 342 patients (52.5%) experienced at least one bleeding event (one episode every 215 h on ECMO), of which 10 (1.6%) were fatal. In a multiple penalized Cox proportional hazard model, higher aPTT was a potentially modifiable risk factor for the first episode of bleeding (for 20-s increase; hazard ratio, 1.07). Conclusions: Anticoagulation during VV ECMO was a dynamic process, with frequent stopping in cases of bleeding and restart according to the clinical picture. Future studies might explore lower aPTT targets to reduce the risk of bleeding. [ABSTRACT FROM AUTHOR]

The protein C (PC) pathway involves physiological anticoagulant factors (PC, protein S [PS], and factor V) and performs major anticoagulant functions in adults. Variations in overall PC pathway function due to dynamic changes in PC and PS in early childhood are poorly understood. We aimed to evaluate the contributions of PC pathway function during early childhood by measuring changes in plasma thrombin generation (TG) after administration of the PC activator protac. We evaluated correlations between anticoagulant factors and percentage of protac-induced coagulation inhibition (PiCi%). Before protac addition, TG in newborns (n = 35), infants (n = 42), young children (n = 35), and adults (n = 20) were 525 ± 74, 720 ± 96, 785 ± 53, and 802 ± 64 mOD/min, and PiCi% were 42.1 ± 9.9, 69.8 ± 11.0, 82.9 ± 4.4, and 86.9 ± 3.4%, respectively. The distribution of PiCi% on the two axes of TG (with or without protac) changed continuously with age and differed from that of warfarin-treated plasma and adult PC- or PS-deficient plasma. PiCi% increased dynamically during infancy and correlated with PS levels in newborns and PC levels in young children. Addition of PC or fresh frozen plasma equivalent to approximately 25% PC to PC-deficient plasma improved PiCi%. This automatic measurement requires only a small sample volume and is useful for analysis of developmental hemostasis. [ABSTRACT FROM AUTHOR]

Patients with hemophilia A (PwHA) may have concurrent deficiency of representative anticoagulant proteins, protein (P)C, PS, and antithrombin (AT), which reduces bleeding frequency. However, emicizumab-driven hemostasis in PwHA with such thrombophilic potential remains unclarified. This study investigated the influence of natural anticoagulants on emicizumab-driven coagulation in HA model plasma. Various concentrations of PS and AT were added to PS-deficient plasma and AT-deficient plasma in the presence of anti-FVIII antibody (FVIIIAb; 10BU/mL). PC-deficient plasma was mixed with normal plasma at various concentrations in the presence of FVIIIAb. Emicizumab (50 µg/mL) was added to these thrombophilic HA model plasmas, prior to tissue factor/ellagic acid-triggered thrombin generation assays. Co-presence of emicizumab increased peak thrombin values (PeakTh) dependent on PS, AT, and PC concentrations. Maximum coagulation potentials in the PS-reduced HA model plasmas remained normal in the presence of emicizumab. PeakTh were close to normal in the presence of 50%AT irrespective of emicizumab, but were higher than normal in the presence of 25%AT. Addition of recombinant FVIIa (corresponding to an administered dose of 90 μg/kg) enhanced coagulation potential to normal levels. Our findings provide novel information on hemostatic regulation in emicizumab-treated PwHA with a possible thrombophilic disposition. [ABSTRACT FROM AUTHOR]

Background: Andexanet alfa, an anti-Xa inhibitor antagonist, induces heparin resistance. Here, we report a case of successful management of cardiopulmonary bypass with andexanet alfa-induced heparin resistance using nafamostat mesylate. Case presentation: An 84-year-old female, with Stanford type A acute aortic dissection, underwent an emergency surgery for total aortic arch replacement. Andexanet alfa 400 mg was administered preoperatively to antagonize edoxaban, an oral Xa inhibitor. Heparin 300 IU/kg was administered before cardiopulmonary bypass, and the activated clotting time (ACT) was 291 s. The ACT was 361 s after another administration of heparin 200 IU/kg. According to our routine therapy for heparin resistance, an initial dose of nafamostat mesylate 10 mg was administered intravenously, followed by a continuous infusion of 20–30 mg/h. The ACT was prolonged to 500 s, and cardiopulmonary bypass was successfully established thereafter. Conclusions: This case report presents the successful management of cardiopulmonary bypass with andexanet alfa-induced heparin resistance using nafamostat mesilate. This report presents the successful management of cardiopulmonary bypass with andexanet alfa-induced heparin resistance using nafamostat mesilate. [ABSTRACT FROM AUTHOR]

Background: Disseminated intravascular coagulation (DIC) syndrome is a highly lethal condition characterized by the complication of multiple organ damage. Although the effects of combined antithrombin (AT) and recombinant thrombomodulin (rTM) on DIC syndrome have previously been examined, the results are inconsistent and inconclusive. Therefore, we conducted a systematic review on the combined administration of AT and rTM for the treatment of septic DIC to investigate the superiority of the combination therapy over either AT or rTM monotherapy using a random-effects analysis model. Method: We searched electronic databases, including Medline, Cochrane Central Register of Controlled Trials, Scopus, and Igaku-Chuo Zasshi (ICHU-SHI) Japanese Central Review of Medicine Web from inception to January 2022. Studies assessing the efficacy of combined AT and rTM were included. The primary outcome was all-cause mortality, and the secondary outcome was occurrence of serious bleeding complications compared to monotherapy. We presented the pooled odds ratio (OR) or hazard ratio (HR) with 95% confidence intervals (CI) depending on reporting results in each primary study. Results: We analyzed seven enrolled clinical trials, all of which were observational studies. Combination therapy had a non-significant favorable association with lower 28-day mortality compared to monotherapy (HR 0.67 [0.43–1.05], OR 0.73 [0.45–1.18]). The I2 values were 60% and 72%, respectively, suggesting high heterogeneity. As a secondary outcome, bleeding complications were similar between the two groups (pooled OR 1.11 [0.55–2.23], I2 value 55%). Conclusions: Although the findings in this analysis could not confirm a statistically significant effect of AT and rTM combination therapy for septic DIC, it showed a promising effect in terms of improving mortality. The incidence of bleeding was low and clinically feasible. Further research is warranted to draw more conclusive results. Trial registration: This study was registered in the University Hospital Medical Information Network (UMIN) Clinical Trials Registry (UMIN ID: 000049820). [ABSTRACT FROM AUTHOR]

• We identified seven variants in antithrombin-deficient patients. • Two de novo variants were detected by droplet digital PCR and pyrosequencing. • Our findings provide new insights on the diverse variants in antithrombin deficiency. [ABSTRACT FROM AUTHOR]

Abstract Background Disseminated intravascular coagulation (DIC) is a common and critical complication in sepsis. Antithrombin activity, which is considered a biomarker for disease severity, was measured in septic DIC treated with antithrombin concentrates in this study. Methods We conducted a retrospective analysis of post-marketing survey data that included 1,800 patients with sepsis-associated DIC and antithrombin activity of 70% or less who were treated with antithrombin concentrates. The changes in sequential organ failure assessment (SOFA) score, DIC score, and antithrombin activity were sequentially assessed. Logistic regression analysis and receiver operating characteristic (ROC) curve analysis were performed to evaluate the performance of antithrombin activity to assess 28-day survival. Furthermore, the relationship between post-treatment antithrombin activity and survival was examined by Logistic regression analysis. Results Sex, baseline SOFA score, baseline antithrombin activities, and the presence of pneumonia and soft tissue infection were significantly associated with 28-day mortality. The area under the curve for mortality was 0.639 for post-treatment antithrombin activity, and higher than those of baseline- and delta antithrombin activities. Logistic regression analysis revealed that higher post-treatment antithrombin activity was associated with better 28-day survival. When post-treatment antithrombin activity was more than 80%, the estimated survival was 88.2%. Whereas, the survival was 74.4% when the antithrombin activity was 80% or less (P

Abstract Mixed phenotype acute leukaemia (MPAL) is associated with worse overall survival, compared with other acute leukaemias in adults. Lack of clear treatment guidelines makes the therapy challenging. ALL-like induction and consolidation treatment followed by allo-HSCT is the preferred first-line treatment. We present a case of a 36-year-old woman diagnosed with MPAL (EGIL Myelo/B) with KMT2A rearrangement, treated with the PALG-ALL-7 (including PEG-asparaginase) protocol. On day 25 after the induction therapy initiation, numbness of limbs and dizziness were observed. Therefore, the imaging studies (CT and MRI) were performed and a diagnosis of thrombosis of superior sagittal sinus of the brain was established. Routinely performed blood coagulation tests showed prolonged APTT and PT, decreased antithrombin III activity and decreased free protein S concentration. LMWH treatment and substitutional therapy with antithrombin III were started, which resulted in a significant reduction in the thrombosis associated symptoms and improvement of the neurological status after 3 days. After induction and consolidation therapy, the patient obtained complete haematological remission and negative measurable residual disease. Six months after the diagnosis, allo-HSCT was successfully performed. During the 4 months follow-up, the patient remained MRD negative and thrombotic symptoms free. To the best of our knowledge, our communication has been the first report of such complication in an MPAL patient treated with PEG-asparaginase containing protocol in adults. We recommend increased vigilance in patients manifesting any mild neurological symptoms and early decision about the MRI study performance.

Makoto Kobayashi,1 Shun Takai,2 Kyohei Sakurai,3 Yoshimatsu Ehama3 1Director of Surgery and Intensive Care Center, Hakodate Goryokaku Hospital, Hakodate City, Hokkaido, Japan; 2Division of Gastroenterology, Hakodate Goryokaku Hospital, Hakodate City, Hokkaido, Japan; 3Division of Emergency Medicine, Hakodate Goryokaku Hospital, Hakodate City, Hokkaido, JapanCorrespondence: Makoto Kobayashi, Director of Surgery and Intensive Care Center, Hakodate Goryokaku Hospital, 38-3 Goryokaku-cho, Hakodate City, Hokkaido, 040-8611, Japan, Tel +81-138-51-2295, Fax +81-56-2695, Email mobilecoba@me.comObjective: Sepsis caused by severe acute cholangitis requires biliary drainage to decrease the intra-biliary pressure. Furthermore, several studies showed that anticoagulant treatment can improve the outcomes of patients with sepsis-associated disseminated intravascular coagulation (DIC). There were reports examining the efficacy of anti-DIC drugs in patients undergoing biliary drainage with sepsis-associated DIC, and no reports compared the efficacy of DIC treatments when no drainage is performed. In this study, the influence of antithrombin (AT) replacement therapy and recombinant thrombomodulin (rTM) preparations on the overall survival (OS) of patients with and without biliary drainage was analyzed.Patients and Methods: This retrospective cohort study in a single institution involved patients with sepsis-associated DIC caused by severe biliary tract infection. In total, 71 patients treated by either AT replacement therapy or rTM preparation were assessed for inclusion. The two groups were patients with biliary drainage (n = 45) and without drainage (n = 26). To assess the clinical efficacy of anti-DIC drugs in each group, the 60-day OS was determined through estimated survival analysis.Results: Focusing on the effects of different therapeutic agents for DIC, in the group of patients with biliary drainage, OS showed no difference between patients treated by rTM and AT. However, in patients without biliary drainage, the survival curves of patients treated with AT replacement were lower than those of patients with rTM preparation.Conclusion: This study revealed that the OS of patients without biliary drainage differed depending on the DIC therapeutic agent for sepsis-associated DIC caused by acute cholangitis. We would recommend the use of rTM preparation over AT replacement therapy for patients who cannot undergo biliary drainage.Keywords: sepsis, antithrombin, recombinant thrombomodulin, DIC treatment, acute cholangitis

Antithrombin (AT) is a critical regulator of the coagulation cascade by inhibiting multiple coagulation factors including thrombin and FXa. Binding of heparinoids to this serpin enhances the inhibition considerably. Mutations located in the heparin binding site of AT result in thrombophilia in affected individuals. Our aim was to study 10 antithrombin mutations known to affect their heparin binding in a heparin pentasaccharide bound state using two molecular dynamics (MD) based methods providing enhanced sampling, GaMD and LiGaMD2. The latter provides an additional boost to the ligand and the most important binding site residues. From our GaMD simulations we were able to identify four variants (three affecting amino acid Arg47 and one affecting Lys114) that have a particularly large effect on binding. The additional acceleration provided by LiGaMD2 allowed us to study the consequences of several other mutants including those affecting Arg13 and Arg129. We were able to identify several conformational types by cluster analysis. Analysis of the simulation trajectories revealed the causes of the impaired pentasaccharide binding including pentasaccharide subunit conformational changes and altered allosteric pathways in the AT protein. Our results provide insights into the effects of AT mutations interfering with heparin binding at an atomic level and can facilitate the design or interpretation of in vitro experiments.

Recent therapeutic strategies for hemophilia include long-term therapeutic gene expression using adeno-associated virus (AAV) and rebalancing therapy via the downregulation of anticoagulant pathways. However, these approaches have limitations in immune responses or insufficiency to control acute bleeding. Thus, we developed a therapeutic strategy for hemophilia B by a combined rebalancing and human factor 9 (hF9) gene knockin (KI) using a lipid nanoparticle (LNP) and AAV. Antithrombin (AT; Serpin Family C Member 1 [Serpinc1]) was selected as the target anticoagulation pathway for the gene KI. First, the combined use of LNP-clustered regularly interspaced short palindromic repeats (CRISPR) and AAV donor resulted in 20% insertions or deletions (indels) in Serpinc1 and 67% reduction of blood mouse AT concentration. Second, hF9 coding sequences were integrated into approximately 3% of the target locus. hF9 KI yielded approximately 1,000 ng/mL human factor IX (hFIX) and restored coagulation activity to a normal level. LNP-CRISPR injection caused sustained AT downregulation and hFIX production up to 63 weeks. AT inhibition and hFIX protein-production ability could be maintained by the proliferation of genetically edited hepatocytes in the case of partial hepatectomy. The co-administration of AAV and LNP showed no severe side effects except random integrations. Our results demonstrate hemophilia B therapy by a combination of rebalancing and hF9 KI using LNP and AAV.

Mixed phenotype acute leukaemia (MPAL) is associated with worse overall survival, compared with other acute leukaemias in adults. Lack of clear treatment guidelines makes the therapy challenging. ALL-like induction and consolidation treatment followed by allo-HSCT is the preferred first-line treatment. We present a case of a 36-year-old woman diagnosed with MPAL (EGIL Myelo/B) with KMT2A rearrangement, treated with the PALG-ALL-7 (including PEG-asparaginase) protocol. On day 25 after the induction therapy initiation, numbness of limbs and dizziness were observed. Therefore, the imaging studies (CT and MRI) were performed and a diagnosis of thrombosis of superior sagittal sinus of the brain was established. Routinely performed blood coagulation tests showed prolonged APTT and PT, decreased antithrombin III activity and decreased free protein S concentration. LMWH treatment and substitutional therapy with antithrombin III were started, which resulted in a significant reduction in the thrombosis associated symptoms and improvement of the neurological status after 3 days. After induction and consolidation therapy, the patient obtained complete haematological remission and negative measurable residual disease. Six months after the diagnosis, allo-HSCT was successfully performed. During the 4 months follow-up, the patient remained MRD negative and thrombotic symptoms free. To the best of our knowledge, our communication has been the first report of such complication in an MPAL patient treated with PEG-asparaginase containing protocol in adults. We recommend increased vigilance in patients manifesting any mild neurological symptoms and early decision about the MRI study performance. [ABSTRACT FROM AUTHOR]

Background: Disseminated intravascular coagulation (DIC) is a common and critical complication in sepsis. Antithrombin activity, which is considered a biomarker for disease severity, was measured in septic DIC treated with antithrombin concentrates in this study. Methods: We conducted a retrospective analysis of post-marketing survey data that included 1,800 patients with sepsis-associated DIC and antithrombin activity of 70% or less who were treated with antithrombin concentrates. The changes in sequential organ failure assessment (SOFA) score, DIC score, and antithrombin activity were sequentially assessed. Logistic regression analysis and receiver operating characteristic (ROC) curve analysis were performed to evaluate the performance of antithrombin activity to assess 28-day survival. Furthermore, the relationship between post-treatment antithrombin activity and survival was examined by Logistic regression analysis. Results: Sex, baseline SOFA score, baseline antithrombin activities, and the presence of pneumonia and soft tissue infection were significantly associated with 28-day mortality. The area under the curve for mortality was 0.639 for post-treatment antithrombin activity, and higher than those of baseline- and delta antithrombin activities. Logistic regression analysis revealed that higher post-treatment antithrombin activity was associated with better 28-day survival. When post-treatment antithrombin activity was more than 80%, the estimated survival was 88.2%. Whereas, the survival was 74.4% when the antithrombin activity was 80% or less (P < 0.0001). However, the relationship between post-treatment antithrombin activity and 28-day survival was considerably different between patients who recovered from DIC by Day 6 compared to those who did not. Similarly, the estimated 28-day survival, based on antithrombin activity, varied among patients with high and low SOFA scores, and the calculation needs to be adjusted based on the severity of the condition. Conclusions: Post-treatment antithrombin activity measurement was helpful in estimating the 28-day survival in patients with sepsis-associated DIC. However, patient outcomes vary considerably depending on factors that include baseline SOFA score, age, and baseline antithrombin activity. These variables play a substantial role in determining patient prognosis and should be considered when evaluating and interpreting the results. [ABSTRACT FROM AUTHOR]

• Anti-Xa and aPTT are more suitable for patients supported on ECMO than ACT. • Anti-Xa was a better test for adjusting heparin dose. • Combination strategies showed some advantages over a single test. • Thromboelastography and combination strategies are less frequently applied. Anticoagulation is critical in patients supported on extracorporeal membrane oxygenation (ECMO). The appropriate monitoring strategies for heparin remain unclear. This systematic review aimed to compare the accuracy and safety of various monitoring strategies for patients supported on ECMO. The PubMed and Web of Science databases were searched for articles in March 2023 without restrictions on publication date. Anticoagulation monitoring strategies for adults supported on ECMO were compared across all included studies. The incidence of bleeding, thrombosis, mortality, blood transfusion, correlation between tests and heparin dose, and the discordance between different tests were discussed in the included studies. The risk of bias was assessed using the Newcastle–Ottawa Scale and Cochrane Collaboration's tool. Twenty-six studies, including a total of 1,684 patients, met the inclusion criteria. The monitoring of anticoagulation by activated partial thromboplastin time (aPTT) resulted in less blood product transfusion than that by activated clotting time (ACT). Moreover, the monitoring of anticoagulation by anti-factor Xa (Anti-Xa) resulted in a more stable anticoagulation than that by aPTT. Anti-Xa and aPTT correlated with heparin dose better than ACT, and the discordance between different monitoring tests was common. Finally, combined monitoring showed some advantages in reducing mortality and blood product transfusion. Anti-Xa and aPTT are more suitable for anticoagulation monitoring for patients supported on ECMO than ACT. Thromboelastography and combination strategies are less applied. Most of the studies were retrospective, and their sample sizes were relatively small; thus, more appropriate monitoring strategies and higher quality research are needed. [ABSTRACT FROM AUTHOR]

Abstract Background The International Society on Thrombosis and Haemostasis (ISTH) released overt disseminated intravascular coagulation (DIC) diagnostic criteria in 2001. Since then, DIC has been understood as the end-stage consumptive coagulopathy and not the therapeutic target. However, DIC is not merely a decompensated coagulation disorder, but also includes early stages with systemic activation in coagulation. Thus, the ISTH has recently released sepsis-induced coagulopathy (SIC) criteria that can diagnose compensated-phase of coagulopathy with readily available biomarkers. Main body DIC is a laboratory-based diagnosis due to various critical conditions, although sepsis is the most common underlying disease. The pathophysiology of sepsis-associated DIC is multifactorial, and in addition to coagulation activation with suppressed fibrinolysis, multiple inflammatory responses are initiated by activated leukocytes, platelets, and vascular endothelial cells as part of thromboinflammation. Although overt DIC diagnostic criteria were established by ISTH to diagnose the advanced stage of DIC, additional criteria that can detect an earlier stage of DIC were needed for potential therapeutic considerations. Accordingly, the ISTH introduced SIC criteria in 2019 that are easy to use and require only platelet count, prothrombin time-international normalized ratio, and Sequential Organ Failure Assessment Score. SIC score can be used to evaluate disease severity and determine the timing of potential therapeutic interventions. One of the major disadvantages in treating sepsis-associated DIC is the lack of availability of specific therapeutic approaches beyond treating the underlying infection. Clinical trials to date have failed because included patients who were not coagulopathic. Nevertheless, in addition to infection control, anticoagulant therapy will be the choice for sepsis-associated DIC. Therefore, the efficacy of heparin, antithrombin, and recombinant thrombomodulin has to be proven in future clinical studies. Conclusion It is necessary to develop a novel therapeutic strategy against sepsis-associated DIC and improve the outcomes. Consequently, we recommend screening and monitoring DIC using SIC scoring system.

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.

Study objective: Antithrombin (AT) activity is reduced during cardiopulmonary bypass (CPB) surgery. Guidelines has demonstrated that perioperative AT supplementation contributed to blood conservation and prevent perioperative thrombotic complications and target organ injury owing to its role in reducing thrombin generation. But these recommends is lack of support of meta-analysis in the guidelines. This meta-analysis aims to include all the relevant randomized controlled trails (RCT) on patients who experienced cardiac surgeries with CPB and investigate the effect of perioperative AT on blood conservation and complications after cardiac surgery. Methods: Standard published RCTs were searched from bibliographic databases to identify all evidence reporting perioperative AT supplementation for patients undergoing cardiovascular surgeries. The primary outcome was postoperative blood loss, the secondary outcomes were blood component transfusion (red blood cell (RBC), fresh frozen plasma (FFP), platelet and autologous blood), postoperative morbidity and in hospital mortality. The relative risk (RR) for dichotomous outcomes and the standardized mean difference (SMD) for continuous outcomes were estimated using a random-effects model. Trial sequential analysis (TSA) was performed using TSA software 0.9.5.10. Results: 13 RCTs with 996 participants undergoing different cardiovascular surgeries were included. Meta-analysis showed AT did not decrease postoperative blood loss (SMD -0.01, 95%CI -0.2 to 0.19). Subgroup analysis showed the effect of AT on postoperative blood loss was not associated with age, RCT type, surgery type, injection time of AT and AT deficiency. TSA further suggested that no additional studies were required for the stable result. Perioperative AT also did not reduce RBC ((SMD 0.10, 95%CI -0.66 to 0.85), (RR 0.99, 95%CI 0.83 to 1.19)), FFP ((SMD 0.11, 95%CI -0.19 to 0.41), (RR 1.30, 95%CI 0.90 to 1.87)), platelet (RR 1.10, 95%CI 0.83 to 1.46) and autologous blood (SMD 0.46, 95%CI -0.12 to 1.8504) transfusions. Perioperative AT significantly increased in hospital mortality (RR 2.53, 95%CI 1.02 to 6.28) and acute kidney injury (AKI) (RR 3.72, 95%CI 1.73 to 8.04) incidence. There was no significant difference in postoperative reexploration, thromboembolism, ECMO/IABP support, and stroke incidence between AT and non-AT group. Conclusions: With the improvement of AT level and heparin sensitivity, perioperative AT has no significant effect on blood conservation. And it is noteworthy that the treatment increased in hospital mortality and the incidence of AKI after cardiac surgery.

Background: Heparin resistance is a common complication of surgical patients requiring anticoagulation, such as those undergoing cardiopulmonary bypass (CPB). Treatments to address heparin resistance include supplementation of antithrombin (AT) or fresh frozen plasma (FFP). This retrospective database analysis compared key outcomes in suspected heparin-resistant patients undergoing CPB treated with AT or FFP. Methods: De-identified United States electronic health records (Cerner Health Facts®) were queried. International Classification of Diseases (ICD-9/10) codes were used to determine CPB procedures and FFP administration. AT administration was identified using medication data, while a combination of lab and medication data examining activated clotting times detected heparin resistance in FFP patients. Adult inpatients (≥18 years old) seen between 2001 and 2018 were included. Differences in mortality, intensive care unit (ICU) length of stay (LOS), and hospital-free days (using a 30-day post-discharge period) were assessed with univariate models as well as adjusted logistic regression models controlling for patient characteristics and Charlson Comorbidity Index (CCI) scores. Results: Of the 502 patients identified, 247 received AT and 255 received FFP. The FFP cohort was associated with a higher CCI compared to the AT cohort (3.3 ± 2.4 vs. 2.3 ± 2.0, P

Aims: There remains a paucity of literature regarding best practice for antithrombin (AT) monitoring, dosing and dose–response in paediatric extracorporeal membrane oxygenation (ECMO) patients. Methods: We conducted a retrospective cohort study at a quaternary care paediatric intensive care unit in all patients <18 years of age supported on ECMO from 1 June 2011 to 30 April 2020. Adverse events and outcomes were characterized for all ECMO runs. AT activity and replacement were characterized and compared between two clinical protocols. AT activities measured post‐ vs. pre‐AT replacement were compared in order to characterize a dose–response relationship. Results: The final cohort included 191 patients with 201 ECMO runs and 2028 AT activity measurements. The median AT activity was 65% (interquartile range [IQR], 51–82) and 879 (43.3%) measurements met the criteria of deficient. The overall median AT dose and increase in AT activity were 50.6 units/kg/dose (IQR, 39.5–67.2) and 23.5% (IQR, 9.8–36.0), respectively. In the protocol that restricted AT activity measurements to clinical scenarios concerning for heparin resistance, there was significantly higher dosing in conjunction with significantly fewer overall administrations. Approximately one third of AT activity remained deficient after repletion. There was no difference in mechanical complications, reasons for discontinuation of ECMO support, time on ECMO or survival between protocols. Conclusions: There was a high prevalence of AT deficiency in paediatric ECMO patients. An AT replacement protocol based on evaluating heparin resistance is associated with fewer AT administrations, with similar circuit and patient outcomes. Further data are needed to identify optimal dosing strategies. [ABSTRACT FROM AUTHOR]

Infectious pneumonia common among neonates has morbidity rate as high as 25%. Proper clinical diagnosis with treatment for respiratory and circulatory failure may prevent mortality. Neonates with infectious pneumonia are mostly treated with mechanical ventilation, antibiotics and pulmonary surfactant. Here, we assessed the effects of exogenous pulmonary surfactant on neonatal infectious pneumonia (NIP). A total of 120 neonates treated from October 2019 to December 2020 were randomly divided into control and observation groups (n=60). Control group received conventional treatment for 7 d, based on which observation group was given 100 mg/kg PS. Their blood gas indices, oxygenation status, lung consolidation and levels of serum procalcitonin (PCT), high-sensitivity C-reactive protein (hs-CRP) and interleukin-6 (IL-6) were compared. Partial pressure of oxygen (PaO2) increased, while that of carbon dioxide (PaCO2) decreased 24 and 48 h after treatment compared with those before treatment, and the observation group had higher PaO2 and lower PaCO2 (P <0.05). Oxygenation index was lower, but arterial/alveolar partial pressure was higher in the observation group than those in the control group 24 and 48 h after treatment (P <0.05). The clinical pulmonary infectious score was lower in the observation group 7 d after treatment (P <0.05). The levels of serum antithrombin III and lung surfactant protein B rose, whereas D-dimer level declined in both groups after treatment, especially in the observation group (P <0.05). PCT, hs-CRP and IL-6 levels 7 d after treatment were lower than those before treatment, and lower in the observation group (P <0.05). For the treatment of severe infectious pneumonia in neonates, exogenous PS can effectively ameliorate blood gas indices, oxygenation status and expression levels of AT-III, SP-B and D-D, relieve pulmonary infection and reduce the levels of inflammatory factors. [ABSTRACT FROM AUTHOR]

Background: Serpin is a superfamily of serine proteinase inhibitors. They have anticoagulative activities and immunoregulatory effects. The family has been widely studied in stroke patients and animal stroke models. However, results from clinical and preclinical studies are controversial. The systematic review and meta‐analysis aimed to determine whether serpin activities are affected by stroke and whether members of the serpin family could be used in stroke treatment. Methods: Literature was systematically searched in six databases until September 5, 2022. In the included studies, 47 clinical studies (8276 subjects) reported concentrations of serpin proteins in stroke patients and healthy controls. In total, 41 preclinical studies (742 animals) reported neurological outcomes in animal models with serpin treatment and vehicle. Results: Meta‐analysis of clinical studies showed that both ischemic (IS) and hemorrhagic stroke patients had higher thrombin‐antithrombin complex (TAT) levels and lower antithrombin (AT) levels which were persistent in the acute and subacute phase of IS. Meta‐analysis of preclinical studies reported the efficacy of serpins in treating stroke. C1‐INH and FUT175 reduced brain infarct size and improved sensorimotor and motor behavior in a dose‐ and time‐dependent manner in the MCAO models. Conclusions: Our study confirmed the important roles serpin family proteins played in the onset, progression, and treatment of stroke. Among serpins, AT and TAT may be used as blood biomarkers in the early diagnosis of stroke. C1‐INH and FUT175 could be potential medications for IS. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate whether early antifactor Xa (anti‐Xa) achieved the target range when venous thromboembolism (VTE) was treated with low‐molecular‐weight heparin (LMWH), based on body weight and renal function in patients treated in intensive care units (ICUs). Anti‐Xa levels in patients treated with LMWH for VTE in ICU and medical wards between January 1, 2021, and June 30, 2022, were retrospectively assessed. The demographics, laboratory parameters, and early anti‐Xa peak levels of patients were collected. All patients were followed up for 3 months to collect VTE recurrence/bleeding events. Univariate and multivariate linear regression analyses were used to identify the factors affecting anti‐Xa levels. A total of 108 patients were enrolled in this study, including 70 patients in ICU and 38 patients on medical wards. The early anti‐Xa level (0.36 vs 0.61 IU/mL, P <.001) and target achievement rate (21.4% vs 39.5%, P =.015) of patients in ICU were lower than those in medical wards. Multivariate linear regression showed that only antithrombin (AT) significantly affected anti‐Xa levels in patients in ICU (β = 0.008, 95%CI 0.005 to 0.011, P <.001). There was no significant difference in VTE recurrence events (11.8% vs 7.5%, P =.628) and bleeding events (29.4% vs 17.0%, P =.304) between the early anti‐Xa within‐target group and the early anti‐Xa below‐target group. Low anti‐Xa peak levels are common in patients in ICU. AT activity should be monitored when using LMWH in critically ill patients. [ABSTRACT FROM AUTHOR]

Persistent inflammation, immunosuppression, and catabolism syndrome (PICS) is a serious condition after critical care. We examined the efficacy of antithrombin, which may attenuate coagulopathy with the control of inflammation, for PICS among patients with sepsis-induced disseminated intravascular coagulation (DIC). The present study used the inpatient claims database with laboratory findings to identify patients admitted to intensive care units and diagnosed with sepsis and DIC. A composite of the incidence of PICS on day 14 or 14-day mortality as the primary outcome was compared between the antithrombin and control groups using a propensity-score-matched analysis. Secondary outcomes were the incidence of PICS on day 28, 28-day mortality, and in-hospital mortality. A total of 324 well-balanced matched pairs were generated from 1622 patients. The primary outcome did not differ between the antithrombin and control groups (63.9% vs. 68.2%, respectively, p = 0.245). However, the incidences of 28-day and in-hospital mortality were significantly lower in the antithrombin group (16.0% vs. 23.5% and 24.4% vs. 35.8%, respectively). Similar results were obtained in a sensitivity analysis using overlap weighting. Antithrombin did not reduce the occurrence of PICS on day 14 in patients with sepsis-induced DIC; however, it was associated with a better mid-term (day 28) prognosis. [ABSTRACT FROM AUTHOR]

Introduction: The sample matrix composition, which is greatly affected by the type of blood collection tube used during phlebotomy, is of major importance in laboratory testing as it can influence test results. We developed an LC-MRM-MS test to molecularly characterize antithrombin in citrate plasma. The test principle differs greatly from traditional laboratory tests and the influence of varying plasma sample matrices is largely unknown. Objectives: To identify whether variations in sample matrix affect the LC-MRM-MS test for antithrombin and assess whether sample pre-processing by immunocapture reduces matrix-specific effects. Methods: Samples (n = 45) originating from four different blood collection tubes (sodium citrate, lithium heparin, K2-EDTA and K2-EDTA with protease inhibitors) were processed directly or after immunocapture. Antithrombin was digested into proteotypic peptides, which were monitored by LC-MRM-MS. Results from lithium heparin and the K2-EDTA matrices were compared to the standard sample matrix, sodium citrate, using Deming regression analysis and repeated measures one-way ANOVA. Results: Deming regression analysis of directly processed samples revealed slopes deviating >5% from the line of identity for at least six out of 22 peptides in all matrices. Significant differences between all matrices were found upon analysis by ANOVA for at least 10 peptides. Pre-processing by immunocapture led to slopes within 5% of the line of identity for nearly all peptides of the matrices. Furthermore, significant differences between matrices after immunocapture were only observed for four peptides. Conclusion: Variations in the sample matrix affect the measurement of antithrombin by LC-MRM-MS, but observed effects are greatly reduced upon pre-processing by immunocapture.

Abstract Background Antithrombin (AT) is the main physiological anticoagulant involved in hemostasis. Hereditary AT deficiency is a rare autosomal dominant thrombotic disease mainly caused by mutations in SERPINC1, which was usually manifested as venous thrombosis and pulmonary embolism. In this study, we analyzed the clinical characteristics and screened for mutant genes in two pedigrees with hereditary AT deficiency, and the functional effects of the pathogenic mutations were evaluated. Methods Candidate gene variants were analyzed by next-generation sequencing to screen pathogenic mutations in probands, followed by segregation analysis in families by Sanger sequencing. Mutant and wild-type plasmids were constructed and transfected into HEK293T cells to observe protein expression and cellular localization of SERPINC1. The structure and function of the mutations were analyzed by bioinformatic analyses. Results The proband of pedigree A with AT deficiency carried a heterozygous frameshift mutation c.1377delC (p.Asn460Thrfs*20) in SERPINC1 (NM000488.3), a 1377C base deletion in exon 7 resulting in a backward shift of the open reading frame, with termination after translation of 20 residues, and a different residue sequence translated after the frameshift. Bioinformatics analysis suggests that the missing amino acid sequence caused by the frameshift mutation might disrupt the disulfide bond between Cys279 and Cys462 and affect the structural function of the protein. This newly discovered variant is not currently included in the ClinVar and HGMD databases. p.Arg229* resulted in a premature stop codon in exon 4, and bioinformatics analysis suggests that the truncated protein structure lost its domain of interaction with factor IX (Ala414 site) after the deletion of nonsense mutations. However, considering the AT truncation protein resulting from the p.Arg229* variant loss a great proportion of the molecule, we speculate the variant may affect two functional domains HBS and RCL and lack of the corresponding function. The thrombophilia and decreased-AT-activity phenotypes of the two pedigrees were separated from their genetic variants. After lentiviral plasmid transfection into HEK293T cells, the expression level of AT protein decreased in the constructed c.1377delC mutant cells compared to that in the wild-type, which was not only reduced in c.685C > T mutant cells but also showed a significant band at 35 kDa, suggesting a truncated protein. Immunofluorescence localization showed no significant differences in protein localization before and after the mutation. Conclusions The p.Asn460Thrfs*20 and p.Arg229* variants of SERPINC1 were responsible for the two hereditary AT deficiency pedigrees, which led to AT deficiency by different mechanisms. The p.Asn460Thrfs*20 variant is reported for the first time.

Background: The International Society on Thrombosis and Haemostasis (ISTH) released overt disseminated intravascular coagulation (DIC) diagnostic criteria in 2001. Since then, DIC has been understood as the end-stage consumptive coagulopathy and not the therapeutic target. However, DIC is not merely a decompensated coagulation disorder, but also includes early stages with systemic activation in coagulation. Thus, the ISTH has recently released sepsis-induced coagulopathy (SIC) criteria that can diagnose compensated-phase of coagulopathy with readily available biomarkers. Main body: DIC is a laboratory-based diagnosis due to various critical conditions, although sepsis is the most common underlying disease. The pathophysiology of sepsis-associated DIC is multifactorial, and in addition to coagulation activation with suppressed fibrinolysis, multiple inflammatory responses are initiated by activated leukocytes, platelets, and vascular endothelial cells as part of thromboinflammation. Although overt DIC diagnostic criteria were established by ISTH to diagnose the advanced stage of DIC, additional criteria that can detect an earlier stage of DIC were needed for potential therapeutic considerations. Accordingly, the ISTH introduced SIC criteria in 2019 that are easy to use and require only platelet count, prothrombin time-international normalized ratio, and Sequential Organ Failure Assessment Score. SIC score can be used to evaluate disease severity and determine the timing of potential therapeutic interventions. One of the major disadvantages in treating sepsis-associated DIC is the lack of availability of specific therapeutic approaches beyond treating the underlying infection. Clinical trials to date have failed because included patients who were not coagulopathic. Nevertheless, in addition to infection control, anticoagulant therapy will be the choice for sepsis-associated DIC. Therefore, the efficacy of heparin, antithrombin, and recombinant thrombomodulin has to be proven in future clinical studies. Conclusion: It is necessary to develop a novel therapeutic strategy against sepsis-associated DIC and improve the outcomes. Consequently, we recommend screening and monitoring DIC using SIC scoring system. [ABSTRACT FROM AUTHOR]

The interaction between coagulation factors Xa and IXa and the activated state of their inhibitor, antithrombin (AT),have been investigated using X-ray diffraction studies. However, only mutagenesis data are available for non-activated AT. Our aim was to propose a model based on docking and advanced-sampling molecular dynamics simulations that can reveal the conformational behavior of the systems when AT is not binding a pentasaccharide. We built the initial structure for non-activated AT-FXa and AT-FIXa complexes using HADDOCK 2.4. The conformational behavior was studied using Gaussian accelerated molecular dynamics simulations. In addition to the docked complexes, two systems based on the X-ray structures were also simulated, with and without the ligand. The simulations revealed large variability in conformation for both factors. In the docking-based complex of AT-FIXa, conformations with stable Arg150–AT interactions can exist for longer time periods but the system also has a higher tendency for reaching states with very limited interaction with the "exosite" of AT. By comparing simulations with or without the pentasaccharide, we were able to gain insights into the effects of conformational activation on the Michaelis complexes. RMSF analysis and correlation calculations for the alpha-carbon atoms revealed important details of the allosteric mechanisms. Our simulations provide atomistic models for better understanding the conformational activation mechanism of AT against its target factors. [ABSTRACT FROM AUTHOR]

OBJECTIVES: To assess the wholistic costs of systemic anticoagulation delivery in heparin versus bivalirudin-based maintenance of adult patients supported on extracorporeal membrane oxygenation (ECMO). DESIGN: Single-center retrospective cohort study. SETTING: Large academic ECMO center. PATIENTS: Adults on ECMO receiving heparin or bivalirudin for primary maintenance systemic anticoagulation. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Electronic data were abstracted from a database maintained by our ECMO center, which transitioned to a preferred bivalirudin-based anticoagulation management in 2017. The pretransition group consisted of 126 patients (123 heparin and three bivalirudin), whereas the posttransition group included 275 patients (82 heparin and 193 bivalirudin). Drug costs were estimated using the wholesale acquisition cost, and laboratory assays costs were estimated using reimbursement fee schedules. Cost data were normalized to the duration of the ECMO run and reported in U.S. Dollar per ECMO day. Following the practice change, bivalirudin patients were less likely to receive AT supplementation (31.0 vs 12.4%; p < 0.0001) and had fewer coagulation assays ordered (6.1 vs 5.4 per ECMO day; p = 0.0004). After the transition, there was a dramatic decrease in costs related to AT assay assessments ($11.78 [interquartile range {IQR}, $9.48–$13.09] vs $1.03 [IQR, $0–$5.75]; p < 0.0001) and AT supplementation ($0 [IQR, $0–$312.82] vs $0 [IQR, $0–$0]; p < 0.0001) per ECMO day. Unadjusted survival at 28 days was higher posttransition (64.3 vs 74.9%; p = 0.0286). CONCLUSIONS: Antithrombin assays and supplementation compromise a significant proportion of heparin-based anticoagulation costs in ECMO patients and is substantially reduced when a bivalirudin-based anticoagulation strategy is deployed. A favorable association exists between the aggregate cost of administration of bivalirudin compared with heparin-based systemic anticoagulation in adults supported on ECMO driven by reductions in antithrombin activity assessments and the cost of antithrombin replacement. [ABSTRACT FROM AUTHOR]

Introduction: We performed in vitro experiments using whole human blood without anticoagulants to clarify the activity of anticoagulant proteins on membranes coated with acrylate-copolymer (ACP) with a hydrophilic blood-contacting layer compared to those coated by immobilizing heparin (IHP) in extracorporeal circulation. Methods: Whole human blood from healthy volunteers was recirculated in two types of experimental circuits with an ACP-coated reservoir and tubes and an ACP-coated or IHP-coated membrane. To compare the fluctuation of anticoagulant proteins, the circuit pressure at the inlet and outlet of the membrane was measured every 5 min; antithrombin antigen (ATQ), antithrombin activity, protein-C quantitation (PCQ), protein-C activity, protein-S free antigen (PSQ), and protein-S activity were measured at 0, 30, 60, 120, and 180 min in each experiment (n = 5). Results: The time taken to achieve high circuit pressure (> 300 mmHg) at the inlet of the membrane was significantly shorter in the ACP-coated membrane circuit (28 ± 2.7 min) than in the IHP-coated membrane circuit (54 ± 24 min); however, the ATQ, PCQ, and PSQ at 180 min of recirculation were significantly higher in the former than in the latter (all p <.05). Conclusions: ACP-coated membranes can prevent the consumption of anticoagulant proteins but cannot delay circuit thrombogenicity compared to IHP-coated membranes. Considering patient care during the post-extracorporeal circulation period, the use of ACP coating, which can preserve anticoagulant protein, is better in extracorporeal circulation circuits. [ABSTRACT FROM AUTHOR]