OBJECTIVE: To derive systematic review informed, modified Delphi consensus regarding monitoring and replacement of specific coagulation factors during pediatric extracorporeal membrane oxygenation (ECMO) support for the Pediatric ECMO Anticoagulation CollaborativE.
METHODS: A structured literature search was performed using PubMed, Embase, and Cochrane Library (CENTRAL) databases from January 1988 to May 2020, with an update in May 2021.
METHODS: Included studies assessed monitoring and replacement of antithrombin, fibrinogen, and von Willebrand factor in pediatric ECMO support.
METHODS: Two authors reviewed all citations independently, with conflicts resolved by a third reviewer if required. Twenty-nine references were used for data extraction and informed recommendations. Evidence tables were constructed using a standardized data extraction form.
RESULTS: Risk of bias was assessed using the Quality in Prognosis Studies tool. The evidence was evaluated using the Grading of Recommendations Assessment, Development, and Evaluation system. A panel of 48 experts met over 2 years to develop evidence-based recommendations and, when evidence was lacking, expert-based consensus statements. A web-based modified Delphi process was used to build consensus via the Research And Development/University of California Appropriateness Method. Consensus was defined as greater than 80% agreement. We developed one weak recommendation and four expert consensus statements.
CONCLUSIONS: There is insufficient evidence to formulate recommendations on monitoring and replacement of antithrombin, fibrinogen, and von Willebrand factor in pediatric patients on ECMO. Optimal monitoring and parameters for replacement of key hemostasis parameters is largely unknown.

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.

The use of medicinal leeches in clinical therapy has been employed for a long time, as it was originally recognized for exerting antithrombin effects. These effects were due to the ability of the leech to continuously suck blood while attached to human skin. According to Chinese Pharmacopoei, leeches used in traditional Chinese medicine mainly consist of Whitmania pigra Whitman, Hirudo nipponia Whitman, and Whitmania acranulata, but the latter two species are relatively scarce. The main constituents of leeches are protein and peptide macromolecules. They can be categorized into two categories based on their pharmacological effects. One group consists of active ingredients that directly target the coagulation system, such as hirudin, heparin, and histamine, which are widely known. The other group comprises protease inhibitor components like Decorsin and Hementin. Among these, hirudin secreted by the salivary glands of the leech is the most potent thrombin inhibitor and served as the sole remedy for preventing blood clotting until the discovery of heparin. Additionally, leeches play a significant role in various traditional Chinese medicine formulations. In recent decades, medicinal leeches have been applied in fields including anti-inflammatory treatment, cardiovascular disease management, antitumor treatment, and many other medical conditions. In this review, we present a comprehensive overview of the historical journey and medicinal applications of leeches in various medical conditions, emphasizing their pharmaceutical significance within traditional Chinese medicine. This review offers valuable insights for exploring additional therapeutic opportunities involving the use of leeches in various diseases and elucidating their underlying mechanisms for future research.

Data on the pathophysiological mechanisms of hemostatic alterations in the thrombotic events that occur during Ramadan intermittent fasting (RIF), particularly in the natural coagulation inhibitors, are very limited. Thus, our objective was to evaluate the effect of RIF on the natural anticoagulants level, antithrombin, protein C, and total and free protein S (PS) in healthy participants. Participants were divided into two groups. Group I consisted of 29 healthy fasting participants whose blood samples were taken after 20 days of fasting. Group II included 40 healthy non-fasting participants whose blood samples were taken 2-4 weeks before the month of Ramadan. Coagulation screening tests including prothrombin time (PT), activated partial thromboplastin time (APTT) and plasma fibrinogen level, natural anticoagulants; antithrombin, protein C, free and total PS and C4 binding protein (C4BP) levels were evaluated in the two groups. High levels of total and free PS without change in antithrombin, protein C, and C4BP levels were noted in the fasting group as compared with non-fasting ones (p < 0.05). PT and APTT showed no difference between the two groups. However, the fibrinogen level was higher in the fasting group. In conclusion, RIF was found to be associated with improved anticoagulant activity in healthy participants, which may provide temporal physiological protection against the development of thrombosis in healthy fasting people.

Low molecular weight heparin and synthetic mimetics such as fondaparinux show different binding kinetics, protease specificity, and clinical effects. A combination of allosteric and template-mediated bridging mechanisms have been proposed to explain the differences in rate acceleration and specificity. The difficulty in working with heterogeneous heparin species has rendered a crystallographic interpretation of the differences in antithrombin activation between mimetics and natural heparin inaccessible. In this study, we examine the allosteric changes in antithrombin caused by binding fondaparinux, enoxaparin and depolymerized natural heparins using millisecond hydrogen deuterium exchange mass spectrometry (TRESI-HDX MS) and relate these conformational changes to complex stability in the gas phase using collision induced unfolding (CIU). This exploration reveals that in addition to the dynamic changes caused by fondaparinux, long chain heparins reduce structural flexibility proximal to Arg393, the cleavable residue in the reactive centre loop of the protein. These local changes in protein dynamics are associated with an increase in overall complex stability that increases with heparin chain length. Ultimately, these results shed light on the molecular mechanisms underlying differences in activity and specificity between heparin mimetics and natural heparins.

Disseminated intravascular coagulation (DIC) is a pathologic state that follows systemic injury and other diseases. Often a complication of sepsis or trauma, DIC causes coagulopathy associated with paradoxical thrombosis and hemorrhage. DIC upregulates the thrombotic pathways while simultaneously downregulating the fibrinolytic pathways that cause excessive fibrin deposition, microcirculatory thrombosis, multiorgan dysfunction, and consumptive coagulopathy with excessive bleeding. Given these opposing disease phenotypes, DIC management is challenging and includes treating the underlying disease and managing the coagulopathy. Currently, no therapies are approved for DIC. We have developed clot-targeted therapeutics that inhibit clot polymerization and activate clot fibrinolysis to manage DIC. We hypothesize that delivering both an anticoagulant and a fibrinolytic agent directly to clots will inhibit active clot polymerization while also breaking up pre-existing clots; therefore, reversing consumptive coagulopathy and restoring hemostatic balance. To test this hypothesis, we single- and dual-loaded fibrin-specific nanogels (FSNs) with antithrombinIII (ATIII) and/or tissue plasminogen activator (tPA) and evaluated their clot preventing and clot lysing abilities in vitro and in a rodent model of DIC. In vivo, single-loaded ATIII-FSNs decreased fibrin deposits in DIC organs and reduced blood loss when DIC rodents were injured. We also observed that the addition of tPA in dual-loaded ATIII-tPA-FSNs intensified the antithrombotic and fibrinolytic mechanisms, which proved advantageous for clot lysis and restoring platelet counts. However, the addition of tPA may have hindered wound healing capabilities when an injury was introduced. Our data supports the benefits of delivering both anticoagulants and fibrinolytic agents directly to clots to reduce the fibrin load and restore hemostatic balance in DIC.

BACKGROUND: Antithrombin is a small plasma glycoprotein synthesized in the liver that belongs to the serpin family of serine protease inhibitors and inactivates several enzymes in the coagulation pathway. It plays a leading major factor on coagulation pathway, therefore administration of antithrombin is essential to treat serious clinical conditions such as disseminated intravascular coagulation (DIC). Despite the theoretical benefits of antithrombin supplementation, the optimal antithrombin activity for heparin efficacy and the benefits of antithrombin supplementation in various disease entities are not yet fully understood.
METHODS: The strict administration guidelines on antithrombin III in cases of DIC by the National Health Insurance Service and the Ministry of Food and Drug Safety complied as follows: antithrombin levels below 20 mg/dL in adults; antithrombin activity below 70% of normal in adults; total administration period of antithrombin must be carefully limited to within maximum 3 days, and the total administration dose must be below 7,000 international unit (IU), (loading dose, 1,000 IU in 1 hour: maintenance dose, 500 IU every 6 hours for 3 days).
RESULTS: We identified 76 eligible for analysis according to the above-mentioned criteria in our institution (male/female, 59/17). Forty-four were identified to the non-survivor group and 32 patients were recognized as the survivor group. The baseline parameters in the non-survivor and survivor groups were comparable with no significant differences in age (66.5±18.1 vs. 66.0±16.2 years, P=0.90), sex (32/12 vs. 27/5, P=0.35), hospital length of stay (31.1±34.5 vs. 31.2±26.1 days, P=0.99), sequential organ failure assessment (SOFA) (7.3±2.5 vs. 6.6±2.0, P=0.22), simplified acute physiology score II (SAPS II) (46.0±8.8 vs. 43.5±9.2, P=0.23), cause for DIC (P=0.95), and underlying disease (P=0.38). The levels of antithrombin III on the day just before the administration significantly lower in the non-survivor groups than in the survivor groups (50.1%±13.6% vs. 57.6%±12.5%, P=0.01). The hemoglobin level in the 2nd day and 7th day after antithrombin III administration was significantly different between the non-survivor and survivor groups (9.9±1.9 vs. 11.0±2.0 g/dL, P=0.01, and 9.4±1.8 vs. 10.5±1.6 g/dL, P=0.006). The antithrombin III levels on the day of administration [area under the curve (AUC) =0.672] demonstrated significantly better prediction of mortality than the A antithrombin III levels on 1st day (AUC =0.552), the 2nd day (AUC =0.624), and 7th day (AUC =0.593).
CONCLUSIONS: Our study suggests that the antithrombin administration may be effective tools for DIC treatment, and may be more positively considered, especially in the cases of DIC, which is a frequent complication of septic shock, sepsis, and other critical disease entities and which is associated with a high level of mortality. Furthermore, our study also suggests that the total doses and periods of antithrombin administration, which recommended by national guidelines, may be insufficient, therefore prolongation of period and increase of total dose of antithrombin supplement might be necessary.

The interaction of heparin with antithrombin (AT) involves a specific sequence corresponding to the pentasaccharide GlcNAc/NS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S (AGA*IA). Recent studies have revealed that two AGA*IA-containing hexasaccharides, which differ in the sulfation degree of the iduronic acid unit, exhibit similar binding to AT, albeit with different affinities. However, the lack of experimental data concerning the molecular contacts between these ligands and the amino acids within the protein-binding site prevents a detailed description of the complexes. Differential epitope mapping (DEEP)-STD NMR, in combination with MD simulations, enables the experimental observation and comparison of two heparin pentasaccharides interacting with AT, revealing slightly different bound orientations and distinct affinities of both glycans for AT. We demonstrate the effectiveness of the differential solvent DEEP-STD NMR approach in determining the presence of polar residues in the recognition sites of glycosaminoglycan-binding proteins.

The direct-acting oral anticoagulant dabigatran etexilate (DE) targets thrombin and is used widely to prevent thromboembolism. A 79-year-old man was admitted to the Emergency Department due to anuria for 2 days. An urgent laboratory examination revealed a serum creatinine concentration of 888 µmol/L. He was diagnosed with acute exacerbation of chronic renal insufficiency. During continuous renal replacement therapy (CRRT), the coagulation test showed a severe reduction in the fibrinogen level as well as a significantly prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT). The patient had been taking DE (110 mg twice daily) for a long time and had not suspended the medication or reduced the dose during the worsening of anuria. Therefore, it should be evaluated before considering plasma replacement therapy for the patient, whether the abnormal coagulation parameters were induced by interference of excessive DE. Tentatively, we used activated charcoal to treat the plasma and then retested the fibrinogen, PT, and APTT. Results showed that the coagulation indices nearly returned to normal. The present case indicated that activated charcoal could adsorb DE in plasma effectively and eliminate its interference with coagulation test results, thereby providing support for clinical diagnosis and treatment.

Injectable anticoagulants are widely used in medical procedures to prevent unwanted blood clotting. However, many lack safe, effective reversal agents. Here, we present new data on a previously described RNA origami-based, direct thrombin inhibitor (HEX01). We describe a new, fast-acting, specific, single-molecule reversal agent (antidote) and present in vivo data for the first time, including efficacy, reversibility, preliminary safety, and initial biodistribution studies. HEX01 contains multiple thrombin-binding aptamers appended on an RNA origami. It exhibits excellent anticoagulation activity in vitro and in vivo. The new single-molecule, DNA antidote (HEX02) reverses anticoagulation activity of HEX01 in human plasma within 30 s in vitro and functions effectively in a murine liver laceration model. Biodistribution studies of HEX01 in whole mice using ex vivo imaging show accumulation mainly in the liver over 24 h and with 10-fold lower concentrations in the kidneys. Additionally, we show that the HEX01/HEX02 system is non-cytotoxic to epithelial cell lines and non-hemolytic in vitro. Furthermore, we found no serum cytokine response to HEX01/HEX02 in a murine model. HEX01 and HEX02 represent a safe and effective coagulation control system with a fast-acting, specific reversal agent showing promise for potential drug development.