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.

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.

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

Antithrombin (AT) is a glycoprotein produced by the liver and a principal antagonist of active clotting proteases. A deficit in AT function leads to AT qualitative deficiency, challenging to diagnose. Here we report that active AT may travel physiosorbed on the surface of plasma extracellular vesicles (EVs), contributing to form the \"EV-protein corona.\" The corona is enriched in specific AT glycoforms, thus suggesting glycosylation to play a key role in AT partitioning between EVs and plasma. Differences in AT glycoform composition of the corona of EVs separated from plasma of healthy and AT qualitative deficiency-affected subjects were also noticed. This suggests deconstructing the plasma into its nanostructured components, as EVs, could suggest novel directions to unravel pathophysiological mechanisms.

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.

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.
CONCLUSIONS: 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.

Background: Antithrombin (AT) replacement is occasionally utilized in the setting of extracorporeal membrane oxygenation (ECMO)-associated heparin resistance. Although past studies emphasized the high costs and limited clinical benefit of AT supplementation,  guidance on strategies to prevent unnecessary use remain lacking.Methods: In this retrospective study, we evaluated the cost, efficacy, and safety outcomes three years pre- and post-implementation of an AT restriction protocol in adult ECMO patients. The primary endpoint was the cost spent on anticoagulation and AT normalized to ECMO duration. Secondary endpoints included thromboembolic and bleeding outcomes.Results: 175 patients were included for analysis (pre-restriction protocol n = 87; post-restriction protocol n = 88). Implementation of the restriction resulted in complete elimination of AT use and significantly reduced the primary cost endpoint from $1009.20 to $42.99 per ECMO day (p < .001). There was no significant change in occurrence of new Venous Thromboembolism (VTE) (p = .099). Those in the pre-implementation group had significantly higher rates of transfusions (p < .001) and ISTH major bleeding (p < .001). Outcomes remained significant after exclusion of patients with coronavirus infections.Conclusion: Results of this study exemplify how AT restriction can be successfully implemented to decrease anticoagulation-associated costs without jeopardizing the risk of bleeding and thrombosis in ECMO patients.

BACKGROUND: Antithrombin (AT) is a natural anticoagulant essential to enhancing the unfractionated heparin (UFH) anticoagulant effect. Its supplementation in the management of UFH-based anticoagulation during veno-venous extracorporeal membrane oxygenation (VV ECMO) has a strong pathophysiological rationale.
METHODS: This is a single-center, retrospective cohort study of adult VV ECMO patients with anticoagulation maintained by UFH targeting an activated partial thromboplastin time (aPTT) of 40-50 s and AT activity >80%. We compare anticoagulation management and survival outcomes between AT subpopulations, defined by a threshold AT activity ≥80%. Linear and logistic regression analyses were used to evaluate the variation in AT activity and its association with ICU survival.
RESULTS: In 244 patients enrolled from 2009 to 2022, anticoagulation was maintained by a median heparin dose of 11.4 IU/kg/h [IQR: 8.2-14.7] with a mean aPTT of 46.1 s (±7.3) and AT activity of 88.9% (±17.0). A lower mean aPTT, higher dose of UFH and shorter fraction of time without UFH were associated with higher AT activity (p < .01). Higher AT activity showed a consistent association with ICU survival (for 10% increase of AT, odds ratio for ICU mortality: 0.95; 95% CI 0.93-0.97; p value <.01).
CONCLUSIONS: There is a positive association between AT activity and UFH requirements but no significant difference in the rate of bleeding events. A higher mean AT during VV ECMO was associated with ICU survival. Future studies are needed to differentiate between exogenously supplemented versus endogenous AT effect.

Despite their low morbidity, thromboembolic events in hyperadrenocorticism are associated with high mortality. Identifying the main hemostatic abnormalities will improve the prophylactic approach of these canine patients. The aim of this study was to evaluate hemostatic alterations related with ACTH-dependent HAC and its association with hypercoagulable state. For this purpose, 25 dogs diagnosed with ACTH-dependent HAC were compared with 28 healthy dogs as a control group. The hemostatic variables included platelet count, antithrombin, fibrinogen, D-dimer, PT, aPTT, rotational thromboelastometry (ROTEM) and platelet aggregation. Results showed a hypercoagulable state in 32% (8/25) dogs by ROTEM, which had at least 2 of the next features: decreased coagulation time (CT) or clot formation time (CFT) on INTEM (5/25) or EXTEM (4/25); increased maximum clot firmness (MCF) on INTEM (9/25), EXTEM (6/25) and FIBTEM (9/25). These same variables had a significant difference (P≤ 0.05) compared with the control group, as well as the parameters of α-angle and CT. Median fibrinogen levels (310 vs.178 mg/dL), mean platelet aggregation (11.1 vs. 7.9 Ohms), median platelet count (360 vs. 225 ×103/µL) and mean antithrombin activity (140 vs. 119%) were increased in ACTH-dependent HAC dogs compared to control group. PT (7.1 vs. 8.0 seconds) and aPTT (11.6 vs. 15.2 seconds) were also shortened in ACTH-dependent HAC dogs. Our findings confirm the presence of a hypercoagulable tendency in dogs with HAC. Although multifactorial, fibrinogen concentration and MCF FIBTEM showed the relevance of this protein for hypercoagulability in HAC.

During inflammation and situations of cellular stress protein disulfide isomerase (PDI) is released in the blood plasma from the platelet and endothelial cells to influence thrombosis. The addition of exogenous PDI makes the environment pro-thrombotic by inducing disulfide bond formation in specific plasma protein targets like vitronectin, factor V, and factor XI. However, the mechanistic details of PDI interaction with its target remain largely unknown. A decrease in the coagulation time was detected in activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) on addition of the purified recombinant PDI (175 nM). The coagulation time can be controlled using an activator (quercetin penta sulfate, QPS) or an inhibitor (quercetin 3-rutinoside, Q3R) of PDI activity. Likewise, the PDI variants that increase the PDI activity (H399R) decrease, and the variant with low activity (C53A) increases the blood coagulation time. An SDS-PAGE and Western blot analysis showed that the PDI does not form a stable complex with either thrombin or antithrombin (ATIII) but it uses the ATIII-thrombin complex as a template to bind and maintain its activity. A complete inhibition of thrombin activity on the formation of ATIII-thrombin-PDI complex, and the complex-bound PDI-catalyzed disulfide bond formation of the target proteins may control the pro- and anti-thrombotic role of PDI.