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Background: Clinical translation of the extracorporeal artificial placenta (AP) is impeded by the high risk for intracranial hemorrhage in extremely premature newborns. The Nitric Oxide Surface Anticoagulation (NOSA) system is a novel non-thrombogenic extracorporeal circuit. This study aims to test the NOSA system in the AP without systemic anticoagulation., Methods: Ten extremely premature lambs were delivered and connected to the AP. For the NOSA group, the circuit was coated with DBHD-N 2 O 2 /argatroban, 100 ppm nitric oxide was blended into the sweep gas, and no systemic anticoagulation was given. For the Heparin control group, a non-coated circuit was used and systemic anticoagulation was administered., Results: Animals survived 6.8 ± 0.6 days with normal hemodynamics and gas exchange. Neither group had any hemorrhagic or thrombotic complications. ACT (194 ± 53 vs. 261 ± 86 s; p < 0.001) and aPTT (39 ± 7 vs. 69 ± 23 s; p < 0.001) were significantly lower in the NOSA group than the Heparin group. Platelet and leukocyte activation did not differ significantly from baseline in the NOSA group. Methemoglobin was 3.2 ± 1.1% in the NOSA group compared to 1.6 ± 0.6% in the Heparin group (p < 0.001)., Conclusions: The AP with the NOSA system successfully supported extremely premature lambs for 7 days without significant bleeding or thrombosis., Impact: The Nitric Oxide Surface Anticoagulation (NOSA) system provides effective circuit-based anticoagulation in a fetal sheep model of the extracorporeal artificial placenta (AP) for 7 days. The NOSA system is the first non-thrombogenic circuit to consistently obviate the need for systemic anticoagulation in an extracorporeal circuit for up to 7 days. The NOSA system may allow the AP to be implemented clinically without systemic anticoagulation, thus greatly reducing the intracranial hemorrhage risk for extremely low gestational age newborns. The NOSA system could potentially be applied to any form of extracorporeal life support to reduce or avoid systemic anticoagulation., (© 2023. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Introduction: Trypsin inhibitors (TIs) have the ability to competitively or non-competitively bind to trypsin and inhibit its action. These inhibitors are commonly found in plants and are used in protease inhibition studies involved in biochemical pathways of pharmacological interest., Objectives: This work aimed to purify a trypsin inhibitor from Bauhinia pulchella seeds ( Bpu TI), describing its kinetic mechanism and anticoagulant effect., Methods: Affinity chromatography, protein assay, and SDS-PAGE were used to purify the inhibitor. Mass spectrometry, inhibition assays, and enzyme kinetics were used to characterize the inhibitor. In vitro assays were performed to verify its ability to prolong blood clotting time., Results: Affinity chromatography on a Trypsin-Sepharose 4B column gave a yield of 43.1. Bpu TI has an apparent molecular mass of 20 kDa with glycosylation (1.15%). Protein identification was determined by MS/MS, and Bpu TI showed similarity to several Kunitz-type trypsin inhibitors. Bpu TI inhibited bovine trypsin as an uncompetitive inhibitor with IC50 (3 x 10 -6 M) and Ki (1.05 x 10 -6 M). Additionally, Bpu TI showed high stability to temperature and pH variations, maintaining its activity up to 100ºC and in extreme pH ranges. However, the inhibitor was susceptible to reducing agents, such as DTT, which completely abolished its activity. Bpu TI showed an anticoagulant effect in vitro at a concentration of 33 μM, prolonging clotting time by 2.6 times., Conclusion: Our results suggest that Bpu TI can be a biological tool to be used in blood clotting studies., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Thrombosis of extracorporeal circuits causes significant morbidity and mortality worldwide. In this study, plasma treatment technology and chemical grafting method were used to construct heparinized surfaces on the PVC substrate, which could not only reduce thrombosis but also decrease the side effects of the direct injection of anticoagulants. The PVC substrate was modified by plasma treatment technology firstly to obtain the active surface with the hydroxyl groups used for grafting. Then, heparin was grafted onto the modified PVC surface using different grafting strategies to prepare different heparinized surfaces. The experimental results indicated that the sodium alginate (SA) and carboxymethyl chitosan (CCS) used as interlayers could significantly increase the graft density of heparin to improve the anticoagulant effects and hemocompatibility of heparinized surfaces. In addition, the modification of heparin can further improve the anticoagulant effects. The CCS/low-molecular-weight heparin (LWMH) surface has the best anticoagulant properties, which can prolong the activated partial thromboplastin time (APTT) values of human plasma for about 35 s, reduce the hemolysis rates to <0.3 %, and perform well in the in-vitro blood circulation test. The heparinized surfaces prepared in this work have great application potential in anticoagulant treatment for medical devices., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Cancer-associated thrombosis is a frequent complication in patients with malignancies. While factor XI (FXI)/FXIa inhibition is efficacious in preventing postoperative venous thromboembolism, its role in tumor cell-induced coagulation is less defined., Objectives: We thus aimed to provide mechanistic insights into FXI/FXIa inhibition in tumor cell-induced coagulation activation., Methods: Procoagulant activity (PCA) of 4 different tissue factor (TF) expressing tumor cell lines was analyzed by single-stage clotting and thrombin generation assay in the presence of a FXIa inhibitor, BMS-262084 (BMS), an inhibitory FXI antibody (anti-FXI), or peak and trough concentrations of rivaroxaban or tinzaparin. Further, tumor cell-induced platelet aggregation was recorded. Recombinant human TF served as positive control., Results: Although BMS and anti-FXI potently inhibited FXIa amidolytic activity, both inhibitors efficiently mitigated recombinant human TF- and tumor cell-induced fibrin clot formation and platelet aggregation only in the presence of low TF PCA. The anticoagulant effects showed an inverse correlation with the magnitude of cellular TF PCA expression. Similarly, BMS markedly interfered with tumor cell-induced thrombin generation, with the most prominent effects on peak and total thrombin. In addition, anticoagulant effects of FXIa inhibition by 10 μM BMS were in a similar range to those obtained by 600 nM rivaroxaban and 1.6 μM tinzaparin at low TF PCA levels. However, rivaroxaban and tinzaparin also exerted marked anticoagulant activity at high TF PCA levels., Conclusion: Our findings indicate that FXI/FXIa inhibition interferes with tumor cell-induced coagulation activation only at low TF PCA expression levels, a finding with potential implications for future in vivo studies., Competing Interests: Declaration of competing interests J.M., A.S., C.L., J.R., and L.B. declare no conflicts of interest relevant to the content of this article. C.C.R. has received travel support from Pfizer. M.V. declares personal fees for lectures from Bristol-Myers Squibb and travel support from Bayer, Bristol-Myers Squibb, and LEO Pharma. C.B. has received personal fees for consultancy, research support, and/or travel support from Bayer, Boehringer Ingelheim, Bristol-Myers Squibb, Daiichi Sankyo, Novartis, Pfizer, Roche, and Sanofi. F.L. has received personal fees for lectures or consultancy and/or research support from Bayer, Boehringer Ingelheim, Bristol-Myers Squibb, Daiichi Sankyo, LEO Pharma, Pfizer, Sanofi, and Viatris., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

The use of biomaterials in implanted medical devices remains hampered by platelet adhesion and blood coagulation. Thrombus formation is a prevalent cause of failure of these blood-contacting devices. Although systemic anticoagulant can be used to support materials and devices with poor blood compatibility, its negative effects such as an increased chance of bleeding, make materials with superior hemocompatibility extremely attractive, especially for long-term applications. This review examines blood-surface interactions, the pathogenesis of clotting on blood-contacting medical devices, popular surface modification techniques, mechanisms of action of anticoagulant coatings, and discusses future directions in biomaterial research for preventing thrombosis. In addition, this paper comprehensively reviews several novel methods that either entirely prevent interaction between material surfaces and blood components or regulate the reaction of the coagulation cascade, thrombocytes, and leukocytes., (© 2023 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

Warfarin, an oral anticoagulant, has been used for decades to prevent thromboembolic events. The complex interplay between CYP2C9 and VKORC1 genotypes on warfarin PK and PD properties is not fully understood in special sub-groups of patients. This study aimed to externally validate a population pharmacokinetic/pharmacodynamic (PK/PD) model for the effect of warfarin on international normalized ratio (INR) and to evaluate optimal dosing strategies based on the selected covariates in Caribbean Hispanic patients. INR, and CYP2C9 and VKORC1 genotypes from 138 patients were used to develop a population PK/PD model in NONMEM. The structural definition of a previously published PD model for INR was implemented. A numerical evaluation of the parameter-covariate relationship was performed. Simulations were conducted to determine optimal dosing strategies for each genotype combinations, focusing on achieving therapeutic INR levels. Findings revealed elevated IC 50 for G/G, G/A, and A/A VKORC1 haplotypes (11.76, 10.49, and 9.22 mg/L, respectively), in this population compared to previous reports. The model-guided dosing analysis recommended daily warfarin doses of 3-5 mg for most genotypes to maintain desired INR levels, although subjects with combination of CYP2C9 and VKORC1 genotypes * 2/* 2-, * 2/* 3- and * 2/* 5-A/A would require only 1 mg daily. This research underscores the potential of population PK/PD modeling to inform personalized warfarin dosing in populations typically underrepresented in clinical studies, potentially leading to improved treatment outcomes and patient safety. By integrating genetic factors and clinical data, this approach could pave the way for more effective and tailored anticoagulation therapy in diverse patient groups., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

In unfractionated heparin (UFH) monitoring during extracorporeal circulation, the traditional measures of activated clotting time (ACT) or activated partial thromboplastin time (APTT) may diverge, confounding anticoagulant adjustments. We aimed to explore the factors explaining this discrepancy in children and young adults. This retrospective observational study, conducted at an urban regional tertiary hospital, included consecutive pediatric patients who received UFH during extracorporeal circulation (continuous kidney replacement therapy or extracorporeal membrane oxygenation) between April 2017 and March 2021. After patients whose ACT and APTT were not measured simultaneously or who were also taking other anticoagulants were excluded, we analyzed 94 samples from 23 patients. To explain the discrepancy between ACT and APTT, regression equations were created using a generalized linear model (family  =  gamma, link  =  logarithmic) with ACT as the response variable. Other explanatory variables included age, platelet count, and antithrombin. Compared to APTT alone as an explanatory variable, the Akaike information criterion and pseudo-coefficient of determination improved from 855 to 625 and from 0.01 to 0.42, respectively, when these explanatory variables were used. In conclusion, we identified several factors that may explain some of the discrepancy between ACT and APTT in the routinely measured tests. Evaluation of these factors may aid in appropriate adjustments in anticoagulation therapy., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Sea cucumbers contain a wide range of biomolecules, including sulfated polysaccharides (SPs), with immense therapeutic and nutraceutical potential. SPs in sea cucumbers are mainly fucosylated chondroitin sulfate (FCS) and fucan sulfate (FS) which exhibit a series of pharmacological effects, including anticoagulant activity, in several biological systems. FCS is a structurally distinct glycosaminoglycan in the sea cucumber body wall, and its biological properties mainly depend on the degree of sulfation, position of sulfate group, molecular weight, and distribution of branches along the backbone. So far, FCS and FS have been recognized for their antithrombotic, anti-inflammatory, anticancer, antidiabetic, anti-hyperlipidemic, anti-obesity, and antioxidant potential. However, the functions of these SPs are mainly dependent on the species, origins, harvesting season, and extraction methods applied. This review focuses on the SPs of sea cucumbers and how their structural diversities affect various biological activities. In addition, the mechanism of actions of SPs, chemical structures, factors affecting their bioactivities, and their extraction methods are also discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Sulfated polysaccharides of brown algae, fucoidans, are known for their anticoagulant properties, similar to animal heparin. Their complex and irregular structure is the main bottleneck in standardization and in defining the relationship between their structure and bioactivity. Fucoidan-active enzymes can be effective tools to overcome these problems. In the present work, we identified the gene fwf5 encoding the fucoidan-active endo-fucanase of the GH168 family in the marine bacterium Wenyingzhuangia fucanilytica CZ1127 T . The biochemical characteristics of the recombinant fucanase FWf5 were investigated. Fucanase FWf5 was shown to catalyze the endo-type cleavage of the 1→4-O-glycosidic linkages between 2-O-sulfated α-L-fucose residues in fucoidans composed of the alternating 1→3- and 1→4-linked residues of sulfated α-L-fucose. This is the first report on the endo-1→4-α-L-fucanases (EC 3.2.1.212) of the GH168 family. The endo-fucanase FWf5 was used to selectively produce high- and low-molecular-weight fucoidan derivatives containing either regular alternating 2-O- and 2,4-di-O-sulfation or regular 2-O-sulfation. The polymeric 2,4-di-O-sulfated fucoidan derivative was shown to have significantly greater in vitro anticoagulant properties than 2-O-sulfated derivatives. The results have demonstrated a new type specificity among fucanases of the GH168 family and the prospects of using such enzymes to obtain standard fucoidan preparations with regular sulfation and high anticoagulant properties.

Glycosaminoglycans (GAGs) with unique structures from marine animals show intriguing pharmacological activities and negligible biological risks, providing more options for us to explore safer agents. The swim bladder is a tonic food and folk medicine, and its GAGs show good anticoagulant activity. In this study, two GAGs, CMG-1.0 and GMG-1.0, were extracted and isolated from the swim bladder of Cynoscion microlepidotus and Gadus morhua . The physicochemical properties, precise structural characteristics, and anticoagulant activities of these GAGs were determined for the first time. The analysis results of the CMG-1.0 and GMG-1.0 showed that they were chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chains with molecular weights of 109.3 kDa and 123.1 kDa, respectively. They were mainly composed of the repeating disaccharide unit of -{IdoA-α1,3-GalNAc 4S -β1,4-}- (DS-A). The DS-B disaccharide unit of -{IdoA 2S -α1,3-GalNAc 4S -β1,4-}- also existed in both CMG-1.0 and GMG-1.0. CMG-1.0 had a higher proportion of CS-O disaccharide unit -{-GlcA-β1,3-GalNAc-β1,4-}- but a lower proportion of CS-E disaccharide unit -{-GlcA-β1,3-GalNAc 4S6S -β1,4-}- than GMG-1.0. The disaccharide compositions of the GAGs varied in a species-specific manner. Anticoagulant activity assay revealed that both CMG-1.0 and GMG-1.0 had potent anticoagulant activity, which can significantly prolong activated partial thromboplastin time. GMG-1.0 also can prolong the thrombin time. CMG-1.0 showed no intrinsic tenase inhibition activity, while GMG-1.0 can obviously inhibit intrinsic tenase with EC 50 of 58 nM. Their significantly different anticoagulant activities may be due to their different disaccharide structural units and proportions. These findings suggested that swim bladder by-products of fish processing of these two marine organisms may be used as a source of anticoagulants.

Cardiovascular diseases (CVDs) caused by thrombotic events are a significant global health concern, affecting millions of people worldwide. The international normalized ratio (INR) is the most widely used measure of coagulation status, and frequent testing is required to adjust blood-thinning drug dosage, requiring hospital visits and experts to perform the test. Here we present a low-cost and portable smartphone-based device for screening INR levels from whole blood samples at the point of care. Our device uses a 3D printed platform and light-emitting diode backlight modules to create a uniform optical environment, and its foldable design allows for easy transport. Our device also features an algorithm that allows users to acquire and process video of sample flow in a microfluidic channel on their smartphone, providing a cost-effective and convenient option for blood coagulation monitoring at the point of care. We tested the performance of our smartphone-based INR device using both commercially available control samples and clinical human blood samples, demonstrating high accuracy and reliability. Our device has the potential to improve patient outcomes by enabling more frequent monitoring and, as appropriate, dosage adjustments of blood-thinning drugs, providing an affordable and portable option for screening INR levels at the point of care., Competing Interests: Declaration of competing interest W. X. and H. C. K. have a patent application for the invention reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Liquid infused surfaces (LIS) hold remarkable potential in anticoagulation. However, liquid loss of LIS in the bloodstream remains a challenge toward its clinical application. Here, micronano hierarchy structures are obtained on the titanium alloy substrate by regulating the microspheres' distribution. When the gap between the microspheres is smaller than the diameter of the red blood cell (RBC), the LIS is more stable under the blood wash and presents a better anticoagulation performance. The proper interval is found to prevent the RBCs from entering the gap and remove the liquid on the surface. The retained thickness of the liquid film is measured by the atomic force microscopy (AFM) technique. The LIS is applied on the front guide vane of an artificial heart pump and exhibits significant improvement on anticoagulation in the blood circulation in vitro for 25 h. The techniques and findings can be used to optimize the anticoagulation performance of LIS-related biomedical implant devices.

Fucosylated chondroitin sulfate (FCS) extracted from Phyllophorella kohkutiensis (PkFCS) is composed of d-GalNAc, d-GlcA, l-Fuc and -SO 4 2- . According to the defined structures revealed by NMR spectra of the branches released by mild acid hydrolysis and oligosaccharides generated by β-eliminative depolymerization, the backbone of PkFCS is CS-E, and the branch types attached to C-3 of d-GlcA include l-Fuc 2S4S , l-Fuc 3S4S , l-Fuc 4S, and the disaccharide α-d-GalNAc-1,2-α-l-Fuc 3S4S with the ratio of 43:13:22:22. Notably, novel heptasaccharide and hendecasaccharide were identified that are branched with continuous distribution of the disaccharide. The structural sequences of the oligosaccharides indicate that three unique structural motifs are present in the entire PkFCS polymer, including a motif branched with randomly distributed different sulfated l-Fuc units, a motif containing regular l-Fuc 2S4S branches and a motif enriched in α-d-GalNAc-1,2-α-l-Fuc 3S4S . This is the first report about the distribution pattern of diverse branches in natural FCS. Natural PkFCS exhibited potent anticoagulant activity on APTT prolonging and anti-iXase activity. Regarding the structurally defined oligosaccharides with sulfated fucosyl side chains, octasaccharide (Pk4b) is the minimum fragment responsible for its anticoagulant activity correlated with anti-iXase. However, further glycosyl modification with a non-sulfated d-GalNAc at the C-2 position of l-Fuc 3S4S could significantly decrease the anticoagulant and anti-iXase activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Zingiber mioga is a highly economic crop that is used to produce vegetables, spices and herbal pharmaceuticals. Its edible flower bud contributes most to the economic value, but the big leaves were discarded as agricultural waste, which urgently needs to be exploited. In this work, polysaccharides from waste Z. mioga leaves (PWZMLs) were extracted using ultrasonic-microwave-assisted extraction (UMAE). After purification and characterization, the antioxidation and anticoagulation of PWZMLs were evaluated to appraise the potential in cardiovascular protection. Under the liquid-solid ratio of 26: 1 mL/g, after ultrasonication at 495 W for 10 min, followed by microwaving at 490 W for 5 min, the yield of PWZMLs achieved to 6.22 ± 0.14 %, notably higher (P < 0.01) than other methods, and ultrasound contributed more to the yield than microwave. Various analyses confirmed that PWZMLs were negatively charged polysaccharides with galacturonic acid the dominant uronic acid. PWZMLs exerted excellent antioxidant capacity, especially for scavenging 1, 1-diphenyl-2-picrylhydrazyl radical. PWZMLs also elicited promising anticoagulant property, particularly for prolonging activated partial thromboplastin time and lowering fibrinogen, which were almost equivalent to heparin at the same concentration. PWZMLs contained two polysaccharide fractions (199.53 and 275.42 kDa) that could synergistically contribute to the pronounced antioxidant and anticoagulant activities. The PWZMLs extracted with optimized UMAE have great potential in cardiovascular protection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Elucidating epigenetic mechanisms could provide new biomarkers for disease diagnosis and prognosis. Technological advances allow genome-wide profiling of 5-hydroxymethylcytosines (5hmC) in liquid biopsies. 5hmC-Seal followed by NGS is a highly sensitive technique for 5hmC biomarker discovery in cfDNA. Currently, 5hmC Seal is optimized for EDTA blood collection. We asked whether heparin was compatible with 5hmC Seal as many clinical and biobanked samples are stored in heparin., Methods: We obtained 60 samples in EDTA matched to 60 samples in heparin from the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial. Samples were comprised of 30 controls and 30 individuals who were later diagnosed with colon cancer. We profiled genome-wide 5hmC in cfDNA using 5hmC-Seal assay followed by NGS. The 5hmC profiling data from samples collected in EDTA were systematically compared to those in heparin across various genomic features., Results: cfDNA isolation and library construction appeared comparable in heparin vs. EDTA. Typical genomic distribution patterns of 5hmC, including gene bodies and enhancer markers, were comparable in heparin vs. EDTA. 5hmC analysis of cases and controls yielded highly correlated differential features suggesting that both anticoagulants were compatible with 5hmC Seal assay., Conclusions: While not currently recommended for the 5hmC-Seal protocol, blood samples stored in heparin were successfully used to generate analysable and biologically relevant genome-wide 5hmC profiling. Our findings are the first to support opportunities to expand the biospecimen resource to heparin samples for 5hmC Seal and perhaps other PCR-based technologies in epigenetic research.

Background: Sample processing methods and storage time affect the outcome of biochemical analysis., Objectives: We aimed to assess the effects of dipotassium-ethylenediaminetetraacetic acid (K2-EDTA) and lithium-heparin treatments and storage times on selected analytes in equine synovial fluid (SF)., Methods: Approximately 2 mL of SF from each horse (n = 7) were collected via femoropatellar joint arthrocentesis into K2-EDTA-treated bottles (K2-EDTA group), lithium-heparin-treated bottles (heparin group), and plain bottles (control group). The pH was determined using an electronic bench pH meter. The total nucleated cell count (TNCC) of samples was determined by hemocytometer method, while total protein (TP) concentrations, and lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activities of the samples were determined spectrophotometrically at 2, 8, 24, 48, and 168 hours postcollection while being maintained at approximately 4°C., Results: TP concentrations in the anticoagulant-treated groups remained stable for 48 hours. TNCCs were stable for 8 hours. However, after 2 hours, ALP, LDH, and pH varied significantly (P < 0.05). At 2  hours, mean ALP and LDH activities were significantly elevated in the lithium-heparin treatment samples, while the activity of these analytes was similar in the K2-EDTA and control groups. At 8  hours, the TNCC and pH were significantly elevated in K2-EDTA treated groups, while values were similar in lithium-heparin and control groups. No significant variation was seen in TP values at 2  hours, irrespective of treatment., Conclusions: The analytes-except for TP-became unstable within a few hours postcollection. Lithium-heparin and K2-EDTA treatments significantly altered ALP, LDH, TNCCs, and pH but not the TP concentrations of equine SF. Studies establishing reference intervals for these analytes based on the anticoagulant used are warranted to limit misinterpretations in clinical or research settings., (© 2023 American Society for Veterinary Clinical Pathology.)

Background:  Cirrhotic patients display an increased risk for both bleeding and thrombosis. We investigated platelet activation across Child-Pugh stages (CPSs) and portal hypertension (PH) severity., Material and Methods:  A total of 110 cirrhotic patients were prospectively included. CPS and hepatic venous pressure gradient (HVPG) were determined. Platelet surface expression of P-selectin and activated glycoprotein (GP) IIb/IIIa were measured by flow cytometry before/after stimulation with protease-activated receptor (PAR)-1 (thrombin receptor activating peptide, TRAP) and PAR-4 (AYPGKF) agonists, epinephrine, and lipopolysaccharide (LPS)., Results:  Platelet count was similar across CPS but lower with increasing PH severity. Expression of P-selectin and activated GPIIb/IIIa in response to TRAP and AYPGKF was significantly reduced in platelets of CPS-B/C versus CPS-A patients (all p <  0.05). Platelet P-selectin expression upon epinephrine and LPS stimulation was reduced in CPS-C patients, while activated GPIIb/IIIa in response to these agonists was lower in CPS-B/C (all p <  0.05). Regarding PH severity, P-selectin and activated GPIIb/IIIa in response to AYPGKF were lower in HVPG ≥20 mmHg patients (both p <  0.001 vs. HVPG < 10 mmHg). Similarly, activated GPIIb/IIIa was lower in HVPG ≥20 mmHg patients after TRAP stimulation ( p <  0.01 vs. HVPG < 10 mmHg). The lower platelet surface expression of P-selectin and activated GPIIb/IIIa upon stimulation of thrombin receptors (PAR-1/PAR-4) in CPS-B/C and HVPG ≥20 mmHg patients was paralleled by reduced antithrombin-III levels in those patients (all p <  0.05). Overall, PAR-1- and PAR-4-mediated platelet activation correlated with antithrombin-III levels ( p <  0.001)., Conclusion:  Platelet responsiveness decreases with increasing severity of liver cirrhosis and PH but is potentially counterbalanced by lower antithrombin-III levels., Competing Interests: K.B., B.S.H., P.S., and T.R. were co-supported by the Austrian Federal Ministry for Digital and Economic Affairs, the National Foundation for Research, Technology and Development, Boehringer Ingelheim, and the Christian Doppler Research Association. B.S. was supported by the Medical Scientific Fund of the Mayor of the City of Vienna Philips, Gilead, and Siemens and has received speaker fees from AbbVie and Siemens and travel support from AbbVie and Gilead. D.B. has received travel support from Gilead and AbbVie and speaker fees from AbbVie. P.S. was supported by the Medical Scientific Fund of the Mayor of the City of Vienna (Project:18070), received consulting fees from PharmaIN, and travel support from Falk and Phenex Pharmaceuticals. M.M. served as a speaker and/or consultant and/or advisory board member for AbbVie, Bristol-Myers Squibb, Gilead, Collective Acumen, and W. L. Gore & Associates and received travel support from AbbVie, Bristol-Myers Squibb, and Gilead. T.R. received grant support from AbbVie, Boehringer-Ingelheim, Gilead, MSD, Philips Healthcare, and Gore; speaker fees from AbbVie, Gilead, Gore, Intercept, Roche, MSD; consulting/advisory board fees from AbbVie, Bayer, Boehringer-Ingelheim, Gilead, Intercept, MSD, Siemens; travel support from Boehringer-Ingelheim, Gilead, and Roche. T.G. received speaker fees from Amgen, Bayer, Boehringer-Ingelheim, Bristol Myers Squibb, Daiichi-Sankyo, Novartis, and Pfizer, and grant support from Boehringer-Ingelheim, Bristol Myers Squibb, Medtronic, and Abbott., (Thieme. All rights reserved.)

Background and Objectives: Due to the high individual variability of anticoagulant warfarin, this study aimed to investigate the effects of vitamin K concentration and gut microbiota on individual variability of warfarin in 246 cardiac surgery patients., Methods: The pharmacokinetics and pharmacodynamics (PKPD) model predicted international normalized ratio (INR) and warfarin concentration. Serum and fecal samples were collected to detect warfarin and vitamin K [VK1 and menaquinone-4 (MK4)] concentrations and gut microbiota diversity, respectively. In addition, the patient's medical records were reviewed for demographic characteristics, drug history, and CYP2C9, VKORC1, and CYP4F2 genotypes., Results: The PKPD model predicted ideal values of 62.7% for S-warfarin, 70.4% for R-warfarin, and 76.4% for INR. The normal VK1 level was 1.34±1.12 nmol/ml (95% CI: 0.33-4.08 nmol/ml), and the normal MK4 level was 0.22±0.18 nmol/ml (95% CI: 0.07-0.63 nmol/ml). The MK4 to total vitamin K ratio was 16.5±9.8% (95% CI: 4.3-41.5%). The S-warfarin concentration of producing 50% of maximum anticoagulation and the half-life of prothrombin complex activity tended to increase with vitamin K. Further, Prevotella and Eubacterium of gut microbiota identified as the main bacteria associated with individual variability of warfarin. The results suggest that an increase in vitamin K concentration can decrease anticoagulation, and gut microbiota may influence warfarin anticoagulation through vitamin K2 synthesis., Conclusion: This study highlights the importance of considering vitamin K concentration and gut microbiota when prescribing warfarin. The findings may have significant implications for the personalized use of warfarin. Further research is needed to understand better the role of vitamin K and gut microbiota in warfarin anticoagulation., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Purpose of Review: Rebalanced hemostasis describes the precarious balance of procoagulant and antithrombotic proteins in patients with severe liver failure. This review is aimed to discuss currently available coagulation monitoring tests and pertinent decision-making process for plasma coagulation factor replacements during liver transplantation (LT)., Recent Findings: Contemporary viscoelastic coagulation monitoring systems have demonstrated advantages over conventional coagulation tests in assessing the patient's coagulation status and tailoring hemostatic interventions. There is increasing interest in the use of prothrombin complex and fibrinogen concentrates, but it remains to be proven if purified factor concentrates are more efficacious and safer than allogeneic hemostatic components. Furthermore, the decision to use antifibrinolytic therapy necessitates careful considerations given the risks of venous thromboembolism in severe liver failure., Summary: Perioperative hemostatic management and thromboprophylaxis for LT patients is likely to be more precise and patient-specific through a better understanding and monitoring of rebalanced coagulation. Further research is needed to refine the application of these tools and develop more standardized protocols for coagulation management in LT., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Citrate is widely used as an anticoagulant for platelet function tests (PFTs). Due to an intrinsic inhibitory effect of citrate on platelet function, hirudin is used as an alternative. However, studies comparing the effect of these anticoagulants on rotational thromboelastometry (ROTEM) platelet whole blood impedance aggregometry in thrombocytopenic patients are scant. Cross-sectional study was done in 105 patients who entered the critical phase of Dengue hemorrhagic fever with plasma leakage and severe thrombocytopenia (<100 × 10 9 /L). Samples were collected on two consecutive days and considered as a combined data set for analysis, out of which 200 have been included in the data analysis. Platelet count was used from routine full blood count. ROTEM platelet used TRAPTEM assay, which was performed with 3.2% sodium citrate and 525 ATU/ml hirudin anticoagulated blood. Means of all the TRAPTEM parameters were significantly higher in hirudin, compared to citrate samples ( p  < .05). Significantly higher overall platelet aggregation was observed in hirudinized samples with a significant mean difference ( p  < .05) compared to citrate in each quartile of platelet count. Higher platelet aggregation was observed with hirudin compared to citrate in ROTEM platelet whole blood impedance aggregometry in thrombocytopenic patients elaborating the importance of using hirudin anticoagulation in PFTs, particularly in patients with severe thrombocytopenia.

Statins are powerful lipid-lowering drugs that inhibit cholesterol biosynthesis via downregulation of hydroxymethylglutaryl coenzyme-A reductase, which are largely used in patients with or at risk of cardiovascular disease. Available data on thromboembolic disease include primary and secondary prevention as well as bleeding and mortality rates in statin users during anticoagulation for VTE. Experimental studies indicate that statins alter blood clotting at various levels. Statins produce anticoagulant effects via downregulation of tissue factor expression and enhanced endothelial thrombomodulin expression resulting in reduced thrombin generation. Statins impair fibrinogen cleavage and reduce thrombin generation. A reduction of factor V and factor XIII activation has been observed in patients treated with statins. It is postulated that the mechanisms involved are downregulation of factor V and activated factor V, modulation of the protein C pathway and alteration of the tissue factor pathway inhibitor. Clinical and experimental studies have shown that statins exert antiplatelet effects through early and delayed inhibition of platelet activation, adhesion and aggregation. It has been postulated that statin-induced anticoagulant effects can explain, at least partially, a reduction in primary and secondary VTE and death. Evidence supporting the use of statins for prevention of arterial thrombosis-related cardiovascular events is robust, but their role in VTE remains to be further elucidated. In this review, we present biological evidence and experimental data supporting the ability of statins to directly interfere with the clotting system.

We previously enabled a direct insight into the quality of citrate anticoagulant tubes before their intended use for specimen collection by introducing an easy-to-perform UV spectrometric method for citrate determination on a purified water model. The results revealed differences between the tubes of three producers, Greiner BIO-ONE (A), LT Burnik (B), and BD (C). It became apparent that tubes C contain an additive, which absorbs light in the ultraviolet range and prevents reliable evaluation of citrate anticoagulant concentration with the suggested method. In this research, we re-evaluate the quality of citrate-evacuated blood collection tubes by complementing UV spectrometry with ion chromatography. (1) Comparable results were obtained for tubes B at 220 nm. (2) Citrate concentrations determined with ion chromatography were lower for tubes A and C. Chromatograms reveal additional peaks for both. (3) Influences of heparin on absorption spectra and chromatograms of citrate were studied. Some similarities with the shape of the anticoagulant spectra of tubes A and C were observed, and the lithium heparin peak in chromatograms is close to them, but a confident judgment was not possible. (4) Contamination of anticoagulant solution with potassium, magnesium, and calcium was confirmed for all the brands, and contamination with lithium for B and C.

Leeches are well-known annelids due to their obligate blood-feeding habits. Some leech species secrete various biologically active substances which have important medical and pharmaceutical value in antithrombotic treatments. In this study, we provided a high-quality genome of the Asian buffalo leech ( Hirudinaria manillensis ), based on which we performed a systematic identification of potential antithrombotic genes and their corresponding proteins. Combining automatic and manual prediction, we identified 21 antithrombotic gene families including fourteen coagulation inhibitors, three platelet aggregation inhibitors, three fibrinolysis enhancers, and one tissue penetration enhancer. A total of 72 antithrombotic genes, including two pseudogenes, were identified, including most of their corresponding proteins forming three or more disulfide bonds. Three protein families (LDTI, antistasin, and granulin) had internal tandem repeats containing 6, 10, and 12 conserved cysteines, respectively. We also measured the anticoagulant activities of the five identified hirudins (hirudin&#95;Hman1 ~ hirudin&#95;Hman5). The results showed that three (hirudin&#95;Hman1, hirudin&#95;Hman2, and hirudin&#95;Hman5), but not the remaining two, exhibited anticoagulant activities. Our study provides the most comprehensive collection of antithrombotic biomacromolecules from a leech to date. These results will greatly facilitate the research and application of leech derivatives for medical and pharmaceutical purposes in the treatment of thrombotic diseases.

Introduction: Emicizumab is used as hemostatic prophylaxis for patients with hemophilia A (PwHA), irrespective of the presence of inhibitors. Although bacterial infection can lead to a procoagulant state, there is limited information on coagulation and fibrinolysis potentials in emicizumab-treated PwHA and on the use of anticoagulants in such cases., Aim: We examined whether anticoagulants affect the coagulation and fibrinolysis potentials in plasma from PwHA spiked with emicizumab., Methods: Plasma from PwHA was in vitro supplemented with emicizumab (50 μg/mL; emi-plasma) and anticoagulants (recombinant thrombomodulin (rTM), nafamostat mesylate (NM), unfractionated heparin (UFH), or low-molecular-weight heparin (LMH)). PwHA plasma spiked with rFVIII (1 IU/mL) was used as a reference (ref-plasma). The coagulation and fibrinolysis potentials in plasma was measured by thrombin and plasmin generation assay (T/P-GA) and clot-fibrinolysis waveform analysis (CFWA)., Results: In T/P-GA and CFWA, coagulation potentials (maximum coagulation velocity; |min1|, and peak thrombin; Th-Peak) in plasma rose with increasing concentrations of emicizumab and rFVIII, but fibrinolytic potentials (peak plasmin; Plm-Peak, and maximum fibrinolytic velocity; |FL-min1|) remained unchanged. Adding rTM, NM, and UFH to emi-plasma suppressed coagulation and fibrinolysis potentials, similar to ref-plasma. Regarding the heparin, UFH and LMH inhibited the improved coagulation in emi-plasma. UFH inhibited fibrinolysis as well, but LMH did not., Conclusions: Anticoagulants could exhibit the inhibitory effects on the coagulation and fibrinolysis potentials in plasma from PwHA spiked with emicizumab, similar to those in normal plasma., (© 2023 John Wiley & Sons Ltd.)

In this study, anti-melanogenic, anti-inflammatory and anti-coagulant potentials of eighteen selected constituents of Ammi visnaga L. were investigated by Induced Fit Docking (IFD) and molecular dynamic simulation with Schrödinger software. The binding free energies of the selected natural compounds were computed by means of ΔG MM-GBSA studies. Anti-melanogetic activity of the constituent against agaricus bisporus tyrosinase, Priestia megaterium tyrosinase and Homo sapiens tyrosinase were evaluated. The result showed that apiumetin had more negative binding free energy against three tyrosinase enzymes than cognate ligands, tropolone and kojic acid. Docking analysis was also performed to predict the constituents with anti-inflammatory activity against human Tumor necrosis factor, Cyclooxygenase-2, Prostaglandin D 2 11-ketoreductase AKR1C3 and Prostaglandin reductase PTGR2. The results showed that pyranocoumarins (visnadin, dihydrosamidin, samidin) have more negative binding free energy against Cyclooxygenase-2 and Prostaglandin D 2 11-ketoreductase receptors than cognate drugs, rofecoxib and indomethacin. In addition, docking analysis shows that pyranocoumarins, apiumetin and cimifugin have more negative binding free energy against Vitamin K epoxide reductase than S-warfarin drug, predicting that they have anticoagulant activity. Furthermore, the constituents and their cognate drugs were subjected to 100 ns MD Simulation to predict their stability at the active sites of the enzymes., (© 2023 Wiley-VHCA AG, Zurich, Switzerland.)

Objectives: The optimal method for monitoring of anticoagulation in patients on extracorporeal life support (ECLS) is unknown. The objective of this study was to assess the relationship between anti-factor Xa level (anti-Xa; IU/mL) and activated partial thromboplastin time (aPTT; seconds) for monitoring intravenous unfractionated heparin anticoagulation in adult ECLS patients., Methods: Charts of all adult patients cannulated for ECLS from 2015 through 2017 were reviewed and laboratory and heparin infusion data were extracted for analysis. Time matched pairs of anti-Xa and aPTT were considered concordant if both laboratory values were within the same clinically utilized range. A hierarchical logistic regression model was used to determine factors associated with discordance while accounting for patient level effects., Results: A total of 1016 paired anti-Xa and aPTT values from 65 patients were evaluated. 500 (49.2%) paired samples were discordant with a degree of variability on linear regression ( r 2 = 0.315). The aPTT fell into a higher therapeutic range compared to the anti-Xa in 31.6% and lower in 17.3%. Logistic regression demonstrated that discordance was independently associated with time from initiation of ECLS (OR 1.17 per day, p < 0.001), average heparin infusion rate (OR 1.25 per U/kg/hr, p < 0.001), and INR (OR 3.22, p < 0.001)., Conclusions: Nearly half of all aPTT and anti-Xa values were in discordant ranges and discordance is more likely as the time on ECLS and the INR level increase. The use of either assay in isolation to guide heparin anticoagulation may lead to misestimation of the degree of anticoagulation in complex ECLS patients., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Anticoagulant drugs that are currently used to prevent and/or treat thrombosis have some limitations that hinder their ability to meet specific clinical requirements. While these drugs effectively reduce the rates of thrombotic events, they simultaneously increase the risk of bleeding. Moreover, their risk-to-benefit balance is problematic in some patients, such as those with severe chronic kidney disease or those at high bleeding risk. A novel anticoagulation method, FXI inhibition has emerged as a promising alternative. It demonstrates a strong rationale for the prevention and treatment of venous thromboembolism and the potential fulfillment of unmet clinical needs in the cardiovascular field. A number of FXI inhibitors are currently undergoing clinical investigation. The objective of this review is to provide an overview of early results of research on FXI inhibitors in the cardiovascular setting, offering valuable insights into their potential role in shaping the future of anticoagulation., Competing Interests: Declaration of Competing Interest Santagata D. MD: no conflict to disclose. Professor Donadini M.P. MD, PhD: no conflict to disclose. Professor Ageno W. MD: Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Bayer, BMS/Pfizer, Viatris, Leo Pharma, Sanofi. Participation on a Data Safety Monitoring Board or Advisory Board for Bayer, Norgine, Leo Pharma, Sanofi, Techdow., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

In the former report, the casein peptide TKLTEEEKNR (PfCN) exhibits strong thrombin inhibitory activity in vitro. Its absorption capabilities, however, are unclear. Therefore, we studied its absorption characteristics both in vivo and in vitro. PfCN was carried by cells from the apical chamber to the basolateral chamber via active translocation in Caco-2 cells. Meanwhile, it can also be transported by HUVECs. We found that PfCN can be taken up by HUVECs using confocal laser imaging. PfCN has been proven to have good absorption properties in in vivo experiments. After five minutes of oral treatment, PfCN was identified in the blood, peaking at 82.75 ± 36.52 ng/mL in 30 min. And PfCN vanished from the blood circulation after 120 min. According to in vivo experiments, excessive concentrations of PfCN will alter the permeability of HUVECs. As a result, there is a foundation for PfCN application in the food sector. Meanwhile, we also hope this article can give an idea to the researchers who studying the absorption of functional peptides., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Fucosylated glycosaminoglycans (FGs) derived from sea cucumbers exhibit potent intrinsic Xase (iXase) inhibition, anticoagulation, and antithrombosis. Plasma activated partial thromboplastin time (APTT), a widely used screening test worldwide, is crucial for evaluating anticoagulant efficacy. However, the applicability of these commercially available APTT reagents for assessing anticoagulation of FGs remains unreported. In this study, we investigated the disparity between ellagic acid and colloidal silica APTT reagents in evaluating anticoagulation of dHG-5 and dHLFG-4, two depolymerized FGs, and elucidated the underlying rationale. The results demonstrated that dHG-5 and dHLFG-4 exhibited heightened sensitivity to the ellagic acid APTT reagent both in vitro and in vivo, and did not significantly affect the activation of APTT reagents for plasma. In addition, both ellagic acid and colloidal silica APTT reagents inhibited the anti-iXase of dHG-5 and dHLFG-4, and the inhibition of the ellagic acid APTT reagent was less pronounced compared to the colloidal silica APTT reagent. These findings suggest that the reduced impact of the ellagic acid APTT reagent on the anti-iXase activity of dHG-5 and dHLFG-4 is responsible for the increased sensitivity in plasma APTT analysis. This study offers valuable insights into the characteristics of two APTT reagents applied for assessing the anticoagulant activity of FG-related compounds.

Ethnopharmacological Relevance: Traditional medicine recommends the use of Rheum rhaponticum L. and R. rhabarbarum L. to treat over thirty complaints, including disorders related to the cardiovascular system such as heartache, pains in the pericardium, epistaxis and other types of haemorrhage, blood purification as well as disorders of venous circulation., Aim of the Study: This work was dedicated to examining for the first time the effects of extracts from petioles and roots of R. rhaponticum and R. rhabarbarum, as well as two stilbene compounds (rhapontigenin and rhaponticin) on the haemostatic activity of endothelial cells and functionality of blood plasma components of the haemostatic system., Materials and Methods: The study was based on three main experimental modules, including the activity of proteins of the human blood plasma coagulation cascade and the fibrinolytic system as well as analyses of the haemostatic activity of human vascular endothelial cells. Additionally, interactions of the main components of the rhubarb extracts with crucial serine proteases of the coagulation cascade and fibrinolysis (i.e. thrombin, the coagulation factor Xa and plasmin) were analyzed in silico., Results: The examined extracts displayed anticoagulant properties and significantly reduced the tissue factor-induced clotting of human blood plasma (by about 40%). Inhibitory effects of the tested extracts on thrombin and the coagulation factor Xa (FXa) were found as well. For the extracts, the IC 50 was ranging from 20.26 to 48.11 μg/ml. Modulatory effects on the haemostatic response of endothelial cells, including the release of von Willebrand factor, tissue-type plasminogen activator and the plasminogen activator inhibitor-1, have been also found., Conclusions: Our results indicated for the first time that the examined Rheum extracts influenced the haemostatic properties of blood plasma proteins and endothelial cells, with the prevalence of the anticoagulant action. The anticoagulant effect of the investigated extracts may be partly attributed to the inhibition of the FXa and thrombin activities, the key serine proteases of the blood coagulation cascade., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Thrombin-binding aptamer (TBA) is one of the best-known G-quadruplex (G4)-forming aptamers. By adopting its peculiar chair-like G4 structure, TBA can efficiently bind to thrombin, thus producing an anticoagulant effect. The major limit to its therapeutic application is represented by its poor thermal and biological resistance. Therefore, numerous research studies have been focused on the design of TBA analogues with chemical modifications to improve its pharmacokinetic and pharmacodynamic properties. To maintain the functional recognition to protein surface on which TBA anticoagulant activity depends, it is essential to preserve the canonical antiparallel topology of the TBA quadruplex core. In this paper, we have designed three TBA variants with modified G-tetrads to evaluate the effects of nucleobase and sugar moiety chemical modifications on biological properties of TBA, preserving its chair-like G-quadruplex structure. All derivatives contain 8-bromo-2'-deoxyguanosine (G Br ) in syn positions, while in the anti-positions, locked nucleic acid guanosine (G LNA ) in the analogue TBABL, 2'-O-methylguanosine (G OMe ) in TBABM, and 2'-F-riboguanosine (G F ) in TBABF is present. CD (Circular Dichroism), CD melting, 1H-NMR (Nuclear Magnetic Resonance), and non-denaturing PAGE (Polyacrylamide Gel Electrophoresis), nuclease stability, prothrombin time (PT) and fibrinogen-clotting assays have been performed to investigate the structural and biological properties of these TBA analogues. The most interesting results have been obtained with TBABF, which revealed extraordinary thermal stability (T m approximately 40 °C higher than that of TBA), anticoagulant activity almost doubled compared to the original aptamer, and, above all, a never-observed resistance to nucleases, as 50% of its G4 species was still present in 50% FBS at 24 h. These data indicate TBABF as one of the best TBA analogue ever designed and investigated, to the best of our knowledge, overcoming the main limitations to therapeutic applications of this aptamer.

Xuebijing injection (XBJ) has a good therapeutic effect on the patients with severe coronavirus disease, but the material basis of XBJ with the anticoagulant effect to improve the coagulopathy and thromboembolism is still unclear. Herein, we developed a new strategy based on aggregation-induced emission (AIE) for monitoring thrombin activity and screening thrombin inhibitors from XBJ. The molecule AIE 603 and the thrombin substrate peptide S-2238 were formed into AIE nanoparticle (AIENP) which emitted notable fluorescence due to the restriction of intramolecular motions. In the presence of thrombin, AIENP was specifically hydrolyzed and AIE 603 was released from AIENP, leading to the decrease of fluorescence intensity. Furthermore, AIENP was combined with ultra-high performance liquid chromatography-fraction collector (UHPLC-FC) and ultra-high performance liquid chromatography quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) for separation, preparation, screening and identification of the thrombin inhibitors from XBJ, a total of 58 chemical constituents were identified, among which 6 compounds possessed higher anticoagulant activity. Notably, the overall inhibition rate of the 6 mixed standards was equivalent to about 60% of the inhibition rate of XBJ. Therefore, this work provides a novel, cheap and simple method for monitoring thrombin activity and is promising to screen active substances from traditional Chinese medicines., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Antithrombin III is an important anticoagulant factor with anti-inflammatory properties. However, few studies have explored its anti-inflammatory actions in ATIII overexpressed transgenic animals. In this study, the dairy goats with mammary overexpression of ATIII were used to investigate their general health, milk quality and particularly their response to inflammatory challenge. The results showed that transgenic goats have a normal phenotype regarding their physiological and biochemical parameters, including whole blood cells, serum protein levels, total cholesterol, urea nitrogen, uric acid, and total bilirubin, compared to the WT. In addition, the quality of milk also improved in transgenic animals compared to the WT, as indicated by the increased milk fat and dry matter content and the reduced somatic cell numbers. Under the stimulation of an LPS injection, the transgenic goats had elevated contents of IGA, IGM and superoxide dismutase SOD, and had reduced proinflammatory cytokine release, including IL-6, TNF-α and IFN-β. A 16S rDNA sequencing analysis also showed that the transgenic animals had a similar compositions of gut microbiota to the WT goats under the stimulation of LPS injections. Mammary gland ATIII overexpression in dairy goats is a safe process, and it did not jeopardize the general health of the transgenic animals; moreover, the compositions of their gut microbiota also improved with the milk quality. The LPS stimulation study suggests that the increased ATIII expression may directly or indirectly suppress the inflammatory response to increase the resistance of transgenic animals to pathogen invasion. This will be explored in future studies.

Background: Carboxymethylated Lasiodiplodan (LaEPS-C), Lasiodiplodia theobromae β-glucan exopolysaccharide derivative, has a well-known range of biological activities. Compared to LaEPS-C, its fractions, Linear (LLaEPS-C) and Branched (BLaEPS-C), have biological potentialities scarcely described in the literature. So, in this study, we investigate the immunomodulatory, antiviral, antiproliferative, and anticoagulant activities of LLaEPS-C and BLaEPS-C and compare them to the LaEPS-C., Methods: LaEPS was obtained from L. theobromae MMBJ. After carboxymethylation, LaEPS-C structural characteristics were confirmed by Elementary Composition Analysis by Energy Dispersive X-Ray Detector (EDS), Fourier Transform Infrared (FTIR), and Nuclear Magnetic Resonance (NMR). The immunomodulatory activity on cytokine secretion was evaluated in human monocyte-derived macrophage cultures. The antiviral activity was evaluated by Hep-2 cell viability in the presence or absence of hRSV (human respiratory syncytial virus). In vitro antiproliferative activity was tested by sulforhodamine B assay. The anticoagulant activity was determined by APTT (Activated Partial Thromboplastin Time) and PT (Prothrombin Time)., Results: LaEPS-C showed low macrophage cell viability only at 100 µg/mL (52.84 ± 24.06, 48 h), and LLaEPS-C presented no effect. Conversely, BLaEPS-C showed cytotoxicity from 25 to 100 µg/mL (44.36 ± 20.16, 40.64 ± 25.55, 33.87 ± 25.16; 48 h). LaEPS-C and LLaEPS-C showed anti-inflammatory activity. LaEPS-C presented this at 100 µg/mL (36.75 ± 5.53, 48 h) for IL-10, and LLaEPS-C reduces TNF-α cytokine productions at 100 µg/mL (18.27 ± 5.80, 48 h). LLaEPS-C showed an anti-hRSV activity (0.7 µg/ml) plus a low cytotoxic activity for Hep-2 cells (1.4 µg/ml). LaEPS-C presented an antiproliferative activity for NCI-ADR/RES (GI 50 65.3 µg/mL). A better PT was achieved for LLaEPS-C at 5.0 µg/mL (11.85 ± 0.87s)., Conclusions: These findings demonstrated that carboxymethylation effectively improves the biological potential of the LaEPS-C and their fractions. From those polysaccharides tested, LLaEPS provided the best results with low toxicity for anti-inflammatory, antiviral, and anticoagulant activities., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Almost all commercial bioprosthetic heart valves (BHVs) are crosslinked with glutaraldehyde (GLUT); however, issues such as immune responses, calcification, delayed endothelialization, and especially severe thrombosis threaten the service lifespan of BHVs. Surface modification is expected to impart GLUT-crosslinked BHVs with versatility to optimize service performance. Here, a postfunctionalization strategy was established for GLUT-crosslinked BHVs, which were firstly modified with metal-phenolic networks (MPNs) to shield the exposed calcification site, and then anticoagulant recombinant humanized type III collagen (rhCOLIII) was immobilized to endow them with long-term antithrombogenicity and enhanced endothelialization properties. The postfunctionalization coating exhibited promising mechanical properties and resistance to enzymatic degradation capability resembling that of GLUT-crosslinked porcine pericardium (GLUT-PP). With the introduction of meticulously tailored rhCOLIII, the anti-coagulation and re-endothelialization properties of TA/Fe-rhCOLIII were significantly improved. Furthermore, the mild inflammatory response and reduced calcification were evidenced in TA/Fe-rhCOLIII by subcutaneous implantation. In conclusion, the efficacy of the proposed strategy combining anti-inflammatory MPNs and multifunctional rhCOLIII to improve anticoagulation, reduce the inflammatory response, and ultimately achieve rapid reendothelialization was supported by both ex vivo and in vivo experiments. Altogether, the current findings may provide a simple strategy for enhancing the service function of BHVs after implantation and show great potential in clinical applications.

Tyrosine sulfation is a post-translational modification (PTM) that modulates function by mediating key protein-protein interactions. One of the early proteins shown to possess this PTM was hirudin, produced in the salivary glands of the medicinal leech Hirudo medicinalis , whereby tyrosine sulfation led to a ∼10-fold improvement in α-thrombin inhibitory activity. Outside of this pioneering discovery, the involvement of tyrosine sulfation in modulating the activity of salivary proteins from other hematophagous organisms was unknown. We hypothesized that the intrinsic instability of the tyrosine sulfate functionality, particularly under the acidic conditions used to isolate and analyze peptides and proteins, has led to poor detection during the isolation and/or expression of these molecules.Herein, we summarize our efforts to interrogate the functional role of tyrosine sulfation in the thrombin inhibitory and anticoagulant activity of salivary peptides and proteins from a range of different blood feeding organisms, including leeches, ticks, mosquitoes, and flies. Specifically, we have harnessed synthetic chemistry to efficiently generate homogeneously sulfated peptides and proteins for detailed structure-function studies both in vitro and in vivo .Our studies began with the leech protein hirudin P6 (from Hirudinaria manillensis ), which is both sulfated on tyrosine and O -glycosylated at a nearby threonine residue. Synthetically, this was achieved through solid-phase peptide synthesis (SPPS) with a late-stage on-resin sulfation, followed by native chemical ligation and a folding step to generate six differentially modified variants of hirudin P6 to assess the functional interplay between O -glycosylation and tyrosine sulfation. A one-pot, kinetically controlled ligation of three peptide fragments was used to assemble homogeneously sulfoforms of madanin-1 and chimadanin from the tick Haemaphysalis longicornis . Dual tyrosine sulfation at two distinct sites was shown to increase the thrombin inhibitory activity by up to 3 orders of magnitude through a novel interaction with exosite II of thrombin. The diselenide-selenoester ligation developed by our lab provided us with a means to rapidly assemble a library of different sulfated tick anticoagulant proteins: the andersonins, hyalomins, madanin-like proteins, and hemeathrins, thus enabling the generation of key structure-activity data on this family of proteins. We have also confirmed the presence of tyrosine sulfation in the anticoagulant proteins of Anopheles mosquitoes (anophelins) and the Tsetse fly (TTI) via insect expression and mass spectrometric analysis. These molecules were subsequently synthesized and assessed for thrombin inhibitory and anticoagulant activity. Activity was significantly improved by the addition of tyrosine sulfate modifications and led to molecules with potent antithrombotic activity in an in vivo murine thrombosis model.The Account concludes with our most recent work on the design of trivalent hybrids that tandemly occupy the active site and both exosites (I and II) of α-thrombin, with a TTI-anophelin hybrid ( K i = 20 fM against α-thrombin) being one of the most potent protease inhibitors and anticoagulants ever generated. Taken together, this Account highlights the importance of the tyrosine sulfate post-translational modification within salivary proteins from blood feeding organisms for enhancing anticoagulant activity. This work lays the foundation for exploiting native or engineered variants as therapeutic leads for thrombotic disorders in the future.

In clinical practice, high-effective antithrombosis remains a challenge for blood-contacting medical devices. Inspired by the enhanced antithrombogenicity of anticoagulant and antiplatelet combination therapy, a strategy is proposed to synthesize dual-pathway antithrombotic polymers by incorporating anticoagulant and antiplatelet dual functional groups into a single thermosetting polymer chain. The synthesized polymer shows increased antithrombogenicity in vitro, with prolonged activated partial thromboplastin time (APTT) and decreased platelet adhesion. Additionally, it downregulates the expression of coagulation- and inflammation-related factors in rabbit plasma after ex vivo arteriovenous shunt assay and maintains patency of small vascular grafts for at least 6 months without thrombosis on the luminal surface after in vivo replacement of rabbit carotid artery. This work provides a new approach to producing novel antithrombotic polymers for blood-contacting medical devices., (© 2023 Wiley-VCH GmbH.)

Preservation of blood through use of anticoagulants allows delayed assessment of hematologic health and is commonly employed in veterinary health assessments. The two most common anticoagulants are lithium heparin (LH) and dipotassium ethylenediaminetetraacetic acid (EDTA), and their effects can vary widely between species. The hematologic effects of these anticoagulants on blood from European starlings ( Sturnus vulgaris ) have not been established, and these birds could serve as models for passerine species both in managed collections and in the wild. Blood was drawn from 45 European starlings and immediately divided into either LH or EDTA microtainers. For each sample, packed cell volume (PCV), total solids (TS), and complete blood counts were performed. There were no significant differences between EDTA and LH anticoagulated blood for PCV, white blood cell count (WBC) slide estimates, WBC determined by Leukopet, absolute heterophils, absolute lymphocytes, absolute monocytes, absolute eosinophils, or absolute basophils. Blood anticoagulated with EDTA had higher total solids than blood mixed with LH. For both anticoagulants, Leukopet-measured total WBC were consistently higher than blood film estimates. There were no subjective morphologic differences for WBC and no hemolysis observed in the samples. Thrombocyte clumping was prominent for LH blood samples and minimal for EDTA samples. These results reveal that LH and EDTA are both suitable anticoagulants for use in European starlings, and EDTA may be superior for diagnostic purposes or for qualitative evaluation of thrombocyte quantity.

Background: Extracorporeal life support (ECLS) has extensive applications in managing patients with acute cardiac and pulmonary failure. Two primary modalities of ECLS, cardiopulmonary bypass (CPB) and extracorporeal membrane oxygenation (ECMO), include several similarities in their composition, complications, and patient outcomes. Both CPB and ECMO pose a high risk of thrombus formation and platelet activation due to the large surface area of the devices and bleeding due to system anticoagulation. Therefore, novel methods of anticoagulation are needed to reduce the morbidity and mortality associated with extracorporeal support. Nitric oxide (NO) has potent antiplatelet properties and presents a promising alternative or addition to anticoagulation with heparin during extracorporeal support., Methods: We developed two ex vivo models of CPB and ECMO to investigate NO effects on anticoagulation and inflammation in these systems., Results: Sole addition of NO as an anticoagulant was not successful in preventing thrombus formation in the ex vivo setups, therefore a combination of low-level heparin with NO was used. Antiplatelet effects were observed in the ex vivo ECMO model when NO was delivered at 80 ppm. Platelet count was preserved after 480 min when NO was delivered at 30 ppm., Conclusion: Combined delivery of NO and heparin did not improve haemocompatibility in either ex vivo model of CPB and ECMO. Anti-inflammatory effects of NO in ECMO systems have to be evaluated further., (© 2023 The Authors. Artificial Organs published by International Center for Artificial Organ and Transplantation (ICAOT) and Wiley Periodicals LLC.)

Heparinase is the only mammalian endoglycosidase that breaks down the commonly used blood-anticoagulant heparin into therapeutically relevant low-molecular-weight-heparin. Importantly, heparinase has been considered a malignant disease diagnostic marker. Thus, it is essential to develop detection scheme for heparinase. However, optical methods for heparinase determination are limited. In the present work, we report a turn-on fluorescence sensor for detection of heparinase that utilizes heparin-templated aggregation of a tetra-cationic porphyrin derivative, TMPyP 4+ , as a sensing framework. Heparinase cleaves the glycosidic linkage between hexosamine and uronic acid in the structure of heparin to destroy its polyelectrolytic nature that originally causes the aggregation of TMPyP 4+ . Thus, heparinase leads to dissociation of TMPyP 4+ aggregates and generates an optical signal. This system leads to a sensitive and selective response towards heparinase with a Limit of Detection (LOD) of 0.3 pmol/L. Further, the same system is demonstrated to sense a trace amount of Oversulfated Chondrootin Sulphate (OSCS) in heparin, which is a heparin adulterant, by utilizing the fact that OSCS serves as an inhibitor for heparinase activity, which leads to reverse modulation in the photo-physical features of the monomer/aggregate equilibrium of the TMPyP 4+ -heparin-heparinase system. The sensing mechanism has been thoroughly demonstrated by ground-state absorption, steady-state emission, and time-resolved emission measurements. The selectivity of the sensor was tested using lysozyme, α-amylase, pepsin, trypsin, lipase, and glucose oxidase in the heparinase selectivity study and the method is also validated using another method reported in the literature. The study provides a new approach for the development of optical methods for the detection of heparinase and oversulfated chondroitin sulfate, which is currently limited., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Heparin is the most common anticoagulant used in clinical practice but shows some downsides such as short half-life (for the high molecular weight heparin) and secondary effects. On the other hand, its low molecular weight analogue cannot be neutralized with protamine, and therefore cannot be used in some treatments. To address these issues, we conjugated polyethylene glycol (PEG) to heparin reducing end (end-on) via oxime ligation and studied the interactions of the conjugate (Hep-b-PEG) with antithrombin III (AT) and protamine. Isothermal titration calorimetry showed that Hep-b-PEG maintains the affinity to AT. Dynamic light scattering demonstrated that the Hep-b-PEG formed colloidal stable nanocomplexes with protamine instead of large multi-molecular aggregates, associated with heparin side effects. The in vitro (human plasma) and in vivo experiments (Sprague Dawley rats) evidenced an extended half-life and higher anticoagulant activity of the conjugate when compared to unmodified heparin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sandra Amaral, Tamara Lozano-Fernández, Juan Sabin, Amanda Gallego, Alain da Silva Morais, Rui L. Reis, África González-Fernández., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Acute pancreatitis (AP) is a serious inflammatory disorder and still lacks effective therapy globally. In this study, a novel Ranacyclin peptide, Ranacin, was identified from the skin of Pelophylax nigromaculatus frog. Ranacin adopted a compact β-hairpin conformation with a disulfide bond (Cys5-Cys15). Ranacin was also demonstrated effectively to inhibit trypsin and have anticoagulant and antioxidant activities in vitro. Furthermore, the severity of pancreatitis was significantly alleviated in l-Arg-induced AP mice after treatment with Ranacin. In addition, structure-activity studies of Ranacin analogues confirmed that the sequences outside the trypsin inhibitory loop (TIL), especially at the C-terminal side, might be closely associated with the efficacy of its trypsin inhibitory activity. In conclusion, our data suggest that Ranacin can improve pancreatic injury in mice with severe AP through its multi-activity. Therefore, Ranacin is considered a potential drug candidate in AP therapy.

Objective: Effective dosing of anticoagulants aims to prevent blood clot formation while avoiding hemorrhages. This complex task is challenged by several disturbing factors and drug-effect uncertainties, requesting frequent monitoring and adjustment. Biovariability in drug absorption and action further complicates titration and calls for individualized strategies. In this paper, we propose an adaptive closed-loop control algorithm to assist in warfarin therapy management., Methods: The controller was designed and tested in silico using an established pharmacometrics model of warfarin, which accounts for inter-subject variability. The control algorithm is an adaptive Model Predictive Control (a-MPC) that leverages a simplified patient model, whose parameters are updated with a Bayesian strategy. Performance was quantitatively evaluated in simulations performed on a population of virtual subjects against an algorithm reproducing medical guidelines (MG) and an MPC controller available in the literature (l-MPC)., Results: The proposed a-MPC significantly (p 0.05) lowers rising time (2.8 vs. 4.4 and 11.2 days) and time out of range (3.3 vs. 7.2 and 12.9 days) with respect to both MG and l-MPC, respectively. Adaptivity grants a significantly (p 0.05) lower number of subjects reaching unsafe INR values compared to when this feature is not present (8.9% vs.15% of subjects presenting an overshoot outside the target range and 0.08% vs. 0.28% of subjects reaching dangerous INR values)., Conclusion: The a-MPC algorithm improve warfarin therapy compared to the benchmark therapies., Significance: This in-silico validation proves effectiveness of the a-MPC algorithm for anticoagulant administration, paving the way for clinical testing.

Background: Whether anticoagulant therapy should be used after spinal-cord injury (SCI) surgery was controversial. The anticoagulation characteristics of a newly developed anticoagulant, recombinant neorudin (EPR-hirudin (EH)), were explored using a rat model of SCI to provide a basis for clinical anticoagulation therapy of SCI., Methods: A rat model of SCI was developed by Allen's method. Then, thrombosis in the inferior vena cava was induced by ligation. The low-bleeding characteristics of EH were explored by investigating dose-response and time-effect relationships, as well as multiple administration of EH, on thrombus formation complicated with SCI., Results: EH inhibited thrombosis in a dose-dependent manner by reducing the wet weight and dry weight of the thrombus. An inhibiting action of EH on thrombosis was most evident in the group given EH 2 h after SCI. After multiple intravenous doses of EH, thrombosis inhibition was improved to that observed with low molecular weight heparin (LMWH) (87% vs 90%). EH administration after SCI neither increased bleeding in the injured spine nor damaged to nerve function. Bleeding duration and activated partial thromboplastin time were increased in the high-dose EH group compared with that in the normal-saline group, but were lower than those in the LMWH group., Conclusion: EH can reduce thrombus formation in a rat model of SCI, and bleeding is decreased significantly compared with that using LMWH. EH may prevent thrombosis after SCI or spinal surgery., Competing Interests: The authors report no conflicts of interest in this work., (© 2023 Liu et al.)

Chlorella is one of the most widely cultivated species of microalgae and has been consumed as a "green healthy food". In this study, a novel polysaccharide (CPP-1) was isolated from Chlorella pyrenoidosa, structurally analyzed, and sulfated as a promising anticoagulant. Structural analyses by chemical and instrumental methods such as monosaccharide composition, methylation-GC-MS and 1D/2D NMR spectroscopy analysis revealed that CPP-1 had a molecular weight of ~13.6 kDa, and mainly consisted of d-mannopyranose (d-Manp), 3-O-methylated d-Manp (3-O-Me-d-Manp), and d-galactopyranose (d-Galp). The molar ratio of d-Manp and d-Galp was 1.0:2.3. CPP-1 consisted of a (1→6)-linked β-d-Galp backbone substituted at C-3 by the d-Manp and 3-O-Me-d-Manp residues in a molar ratio of 1:1, which was a regular mannogalactan. The sulfated Chlorella mannogalactan (SCM) with sulfated group content of 40.2 % equivalent to that of unfractionated heparin was prepared and analyzed. NMR analysis confirmed its structure, indicating that most free hydroxyl groups in the side chains and partial hydroxyl groups in the backbone were sulfated. Anticoagulant activity assays indicated that SCM exhibited strong anticoagulant activity by inhibiting intrinsic tenase (FXase) with IC 50 of 13.65 ng/mL, which may be a safer anticoagulant as an alternative to heparin-like drugs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Heparin is a glycosaminoglycans (GAGs) member and well-known FDA-approved anticoagulant that has been widely used in the clinic for 100 years. It has also been evaluated in various fields for further clinical applications, such as in anti-cancer or anti-inflammatory therapy beyond its anticoagulant effect. Here, we sought to utilize heparin molecules as drug carriers by directly conjugating the anticancer drug doxorubicin to the carboxyl group of unfractionated heparin. Given the molecular action of doxorubicin in intercalating DNA, it is expected to be less effective when structurally combined with other molecules. However, by utilizing doxorubicin molecules to produce reactive oxygen species (ROS), we found that the heparin-doxorubicin conjugates have significant cytotoxic ability to kill CT26 tumor cells with low anticoagulant activity. Several doxorubicin molecules were bound to heparin to provide sufficient cytotoxic capability and self-assembly ability due to their amphiphilic properties. The self-assembled formation of these nanoparticles was demonstrated through DLS, SEM and TEM. The cytotoxic ROS-generating doxorubicin-conjugated heparins could inhibit tumor growth and metastasis in CT26-bearing Balb/c animal models. Our results demonstrate that this cytotoxic doxorubicin-based heparin conjugate can significantly inhibit tumor growth and metastasis, thus showing promise as a potential new anti-cancer therapeutic., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jooho Park reports financial support was provided by Research Foundation of Korea. Jooho Park reports a relationship with National Research Foundation of Korea that includes: funding grants., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Clinical use of intraoperative auto-transfusion requires the removal of platelets and plasma proteins due to pump-based suction and water-soluble anticoagulant administration, which causes dilutional coagulopathy. Herein, we develop a carboxylated and sulfonated heparin-mimetic polymer-modified sponge with spontaneous blood adsorption and instantaneous anticoagulation. We find that intrinsic coagulation factors, especially XI, are inactivated by adsorption to the sponge surface, while inactivation of thrombin in the sponge-treated plasma effectively inhibits the common coagulation pathway. We show whole blood auto-transfusion in trauma-induced hemorrhage, benefiting from the multiple inhibitory effects of the sponge on coagulation enzymes and calcium depletion. We demonstrate that the transfusion of collected blood favors faster recovery of hemostasis compared to traditional heparinized blood in a rabbit model. Our work not only develops a safe and convenient approach for whole blood auto-transfusion, but also provides the mechanism of action of self-anticoagulant heparin-mimetic polymer-modified surfaces., (© 2023. Springer Nature Limited.)