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Purpose of Review: In humans, tissue factor pathway inhibitor (TFPI) exists in two alternatively spliced isoforms, TFPIα and TFPIβ. TFPIα consists of three Kunitz domains (K1, K2 and K3) and a highly basic C-terminal tail. K1 inhibits the tissue factor-activated factor VII complex, K2 specifically inhibits activated factor X, K3 is essential for interaction with its cofactor, protein S, and the basic C-terminus is binds factor V-short (FV-short) with high affinity. TFPIβ consists of K1 and K2 that is glycosylphosphatidylinositol anchored directly to cell surfaces. This review explores the structure/function of TFPI and its cofactors (protein S and FV-short), and the relative contributions that different TFPI isoforms may play in haemostatic control., Recent Findings: Recent data have underscored the importance of TFPIα function and its reliance on its cofactors, protein S and FV-short, in influencing haemostatic control as well as bleeding and thrombotic risk., Summary: TFPIα is likely the most important pool of TFPI in modifying the risk of thrombosis and bleeding. TFPIα forms a trimolecular complex with FV-short and protein S in plasma. FV-short expression levels control the circulating levels of TFPIα, whereas protein S exerts essential cofactor mediated augmentation of it anticoagulant function., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Platelets are small cell fragments that play a crucial role in hemostasis, requiring fast response times and fine signaling pathway regulation. For this regulation, platelets require a balance between two pathway types: the activatory and negative signaling pathways. Activatory signaling mediators are positive responses that enhance stimuli initiated by a receptor in the platelet membrane. Negative signaling regulates and controls the responses downstream of the same receptors to roll back or even avoid spontaneous thrombotic events. Several blood-related pathologies can be observed when these processes are unregulated, such as massive bleeding in activatory signaling inhibition or thrombotic events for negative signaling inhibition. The study of each protein and metabolite in isolation does not help to understand the role of the protein or how it can be contrasted; however, understanding the balance between active and negative signaling could help develop effective therapies to prevent thrombotic events and bleeding disorders.

Patients with arterial embolic disease have benefited greatly from antiplatelet therapy. However, hemorrhage risk of antiplatelet agents cannot be ignored. Herein, we describe the discovery of 2,3-dihydro[1,4]dioxino[2,3- g ]benzofuran compounds as novel PAR4 antagonists. Notably, the isomers 36 and 37 with the chemotype of phenoxyl methylene substituted on the 2,3-dihydro-1,4-dioxine ring exhibited potent in vitro antiplatelet activity (IC 50 = 26.13 nM for 36 and 14.26 nM for 37 ) and significantly improved metabolic stability in human liver microsomes ( T 1/2 = 97.6 min for 36 and 11.1 min for BMS-986120). 36 also displayed good oral PK profiles (mice: T 1/2 = 7.32 h and F = 45.11%). Both of them showed overall potent ex vivo antiplatelet activity at concentrations of 6 and 12 mg/kg, with no impact on the coagulation system and low bleeding liability. Our work will facilitate development of novel PAR4 antagonists as a safer therapeutic option for arterial embolism.

Background: A safe and efficacious hemostatic product with a long shelf-life is needed to reduce mortality from hemorrhage due to trauma and improve surgical outcomes for persons with platelet deficiency or dysfunction. Thrombosomes, a trehalose-stabilized, leukoreduced, pooled blood group-O freeze-dried platelet-derived hemostatic (FPH) with a 3-year shelf-life, may satisfy this need., Objectives: To characterize the mechanism of action of FPH., Methods: FPH's ability to adhere to collagen, aggregate with and without platelets, and form clots was evaluated in vitro. Nonobese diabetic-severe combined immunodeficiency mouse models were used to assess circulation persistence and hemostatic efficacy., Results: FPH displays the morphology and surface proteins of activated platelets. FPH adheres to collagen, aggregates, and promotes clots, producing an insoluble fibrin mesh. FPH is rapidly cleared from circulation, has hemostatic efficacy comparable to apheresis platelets in a murine tail-cut, and acts in a dose-dependent manner., Conclusion: FPH is a first-in-class investigational treatment and shows strong potential as a hemostatic agent that is capable of binding exposed collagen, coaggregating with endogenous platelets, and promoting the coagulation cascade. These properties may be exploited to treat active platelet-related or diffuse vascular bleeding. FPH has the potential to fulfill a large unmet patient need as an acute hemostatic treatment in severe bleeding, such as surgery and trauma., Competing Interests: Declaration of competing interests All authors are employees and option holders at Cellphire Therapeutics, Inc. W.A.A. has a patent application for the use of FPH (Thrombosomes) for the treatment of rare platelet disorders. G.M.F., B.K., and K.M. are named on several patent applications for the use of FPH (Thrombosomes) and CPP., (Copyright © 2023 Cellphire Therapeutics, Inc. Published by Elsevier Inc. All rights reserved.)

Background: Platelet-rich thrombi occlude arteries causing fatal infarcts like heart attacks and strokes. Prevention of thrombi by current antiplatelet agents can cause major bleeding. Instead, we propose using N-acetyl cysteine (NAC) to act against the protein VWF (von Willebrand factor), and not platelets, to prevent arterial thrombi from forming., Methods: NAC was assessed for its ability to prevent arterial thrombosis by measuring platelet accumulation rate and occlusion time using a microfluidic model of arterial thrombosis with human blood. Acute clot formation, clot stability, and tail bleeding were measured in vivo with the murine modified Folts model. The effect of NAC in the murine model after 6 hours was also measured to determine any persistent effects of NAC after it has been cleared from the blood., Results: We demonstrate reduction of thrombi formation following treatment with NAC in vitro and in vivo. Human whole blood treated with 3 or 5 mmol/L NAC showed delayed thrombus formation 2.0× and 3.7× longer than control, respectively ( P <0.001). Blood treated with 10 mmol/L NAC did not form an occlusive clot, and no macroscopic platelet aggregation was visible ( P <0.001). In vivo, a 400-mg/kg dose of NAC prevented occlusive clots from forming in mice without significantly affecting tail bleeding times. A lower dose of NAC significantly reduced clot stability. Mice given multiple injections showed that NAC has a lasting and cumulative effect on clot stability, even after being cleared from the blood ( P <0.001)., Conclusions: Both preclinical models demonstrate that NAC prevents thrombus formation in a dose-dependent manner without significantly affecting bleeding time. This work highlights a new pathway for preventing arterial thrombosis, different from antiplatelet agents, using an amino acid derivative as an antithrombotic therapeutic., Competing Interests: Disclosures C.A. Bresette and D.N. Ku have filed a patent on the use of N-acetyl cysteine for the prevention of arterial thrombosis. The other authors report no conflicts.

Background: Single-nucleotide polymorphisms linked with the rs1474868 T allele ( MFN2 [mitofusin-2] T/T) in the human mitochondrial fusion protein MFN2 gene are associated with reduced platelet MFN2 RNA expression and platelet counts. This study investigates the impact of MFN2 on megakaryocyte and platelet biology., Methods: Mice with megakaryocyte/platelet deletion of Mfn2 ( Mfn2 -/- [ Mfn2 conditional knockout]) were generated using Pf4-Cre crossed with floxed Mfn2 mice. Human megakaryocytes were generated from cord blood and platelets isolated from healthy subjects genotyped for rs1474868. Ex vivo approaches assessed mitochondrial morphology, function, and platelet activation responses. In vivo measurements included endogenous/transfused platelet life span, tail bleed time, transient middle cerebral artery occlusion, and pulmonary vascular permeability/hemorrhage following lipopolysaccharide-induced acute lung injury., Results: Mitochondria was more fragmented in megakaryocytes derived from Mfn2 -/- mice and from human cord blood with MFN2 T/T genotype compared with control megakaryocytes. Human resting platelets of MFN2 T/T genotype had reduced MFN2 protein, diminished mitochondrial membrane potential, and an increased rate of phosphatidylserine exposure during ex vivo culture. Platelet counts and platelet life span were reduced in Mfn2 -/- mice accompanied by an increased rate of phosphatidylserine exposure in resting platelets, especially aged platelets, during ex vivo culture. Mfn2 -/- also decreased platelet mitochondrial membrane potential (basal) and activated mitochondrial oxygen consumption rate, reactive oxygen species generation, calcium flux, platelet-neutrophil aggregate formation, and phosphatidylserine exposure following dual agonist activation. Ultimately, Mfn2 -/- mice showed prolonged tail bleed times, decreased ischemic stroke infarct size after cerebral ischemia-reperfusion, and exacerbated pulmonary inflammatory hemorrhage following lipopolysaccharide-induced acute lung injury. Analysis of MFN2 SNPs in the iSPAAR study (Identification of SNPs Predisposing to Altered ALI Risk) identified a significant association between MFN2 and 28-day mortality in patients with acute respiratory distress syndrome., Conclusions: Mfn2 preserves mitochondrial phenotypes in megakaryocytes and platelets and influences platelet life span, function, and outcomes of stroke and lung injury., Competing Interests: Disclosures None.

Introduction: Major traumatic injury is associated with early hemorrhage-related and late-stage deaths due to multiple organ failure (MOF). While improvements to hemostatic resuscitation have significantly reduced hemorrhage-related deaths, the incidence of MOF among trauma patients remains high. Dysregulation of vascular endothelial cell (EC) barrier function is a central mechanism in the development of MOF; however, the mechanistic triggers remain unknown. Accelerated fibrinolysis occurs in a majority of trauma patients, resulting in high circulating levels of fibrin(ogen) degradation products, such as fragment X. To date, the relationship between fragment X and EC dysregulation and barrier disruption is unknown. The goal of this study was to determine the effects of fragment X on EC barrier integrity and expression of paracellular junctional proteins that regulate barrier function., Methods: Human lung microvascular endothelial cells (HLMVECs) were treated with increasing concentrations of fragment X (1, 10, and 100 μg/mL), and barrier function was monitored using the xCELLigence live-cell monitoring system. Quantitative PCR (qPCR) was performed to measure changes in EC expression of 84 genes. Immunofluorescent (IF) cytostaining was performed to validate qPCR findings., Results: Fragment X treatment significantly increased endothelial permeability over time (P < 0.05). There was also a significant reduction in VE-cadherin mRNA expression in fragment X-treated HLMVECs compared to control (P = 0.01), which was confirmed by IF staining., Conclusions: Fragment X may induce EC hyperpermeability by reducing VE-cadherin expression. This suggests that a targeted approach to disrupting EC-fragment X interactions could mitigate EC barrier disruption, organ edema, and MOF associated with major trauma., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The formation of vascular networks consisting of arteries, capillaries, and veins is vital in embryogenesis. It is also crucial in adulthood for the formation of a functional vasculature. Cerebral arteriovenous malformations (CAVMs) are linked with a remarkable risk of intracerebral hemorrhage because arterial blood is directly shunted into the veins before the arterial blood pressure is dissipated. The underlying mechanisms responsible for arteriovenous malformation (AVM) growth, progression, and rupture are not fully known, yet the critical role of inflammation in AVM pathogenesis has been noted. The proinflammatory cytokines are upregulated in CAVM, which stimulates overexpression of cell adhesion molecules in endothelial cells (ECs), leading to improved leukocyte recruitment. It is well-known that metalloproteinase-9 secretion by leukocytes disrupts CAVM walls resulting in rupture. Moreover, inflammation alters the angioarchitecture of CAVMs by upregulating angiogenic factors impacting the apoptosis, migration, and proliferation of ECs. A better understanding of the molecular signature of CAVM might allow us to identify biomarkers predicting this complication, acting as a goal for further investigations that may be potentially targeted in gene therapy. The present review is focused on the numerous studies conducted on the molecular signature of CAVM and the associated hemorrhage. The association of numerous molecular signatures with a higher risk of CAVM rupture is shown through inducing proinflammatory mediators, as well as growth factors signaling, Ras-mitogen-activated protein kinase-extracellular signal-regulated kinase, and NOTCH pathways, which are accompanied by cellular level inflammation and endothelial alterations resulting in vascular wall instability. According to the studies, it is assumed that matrix metalloproteinase, interleukin-6, and vascular endothelial growth factor are the biomarkers most associated with CAVM and the rate of hemorrhage, as well as diagnostic methods, with respect to enhancing the patient-specific risk estimation and improving treatment choices., (© 2023 John Wiley & Sons Ltd.)

When platelet concentrates (PCs) were first introduced in the 1960s as a blood component therapy, they were stored in the cold. As platelet transfusion became more important for the treatment of chemotherapy-induced thrombocytopenia, research into ways to increase supply intensified. During the late 1960s/early 1970s, it was demonstrated through radioactive labeling of platelets that room temperature platelets (RTP) had superior post-transfusion recovery and survival compared with cold-stored platelets (CSP). This led to a universal switch to room temperature storage, despite CSP demonstrating superior hemostatic effectiveness upon being transfused. There has been a global resurgence in studies into CSP over the last two decades, with an increase in the use of PC to treat acute bleeding within hospital and pre-hospital care. CSP demonstrate many benefits over RTP, including longer shelf life, decreased bacterial risk and easier logistics for transport, making PC accessible in areas where they have not previously been, such as the battlefield. In addition, CSP are reported to have greater hemostatic function than RTP and are thus potentially better for the treatment of bleeding. This review describes the history of CSP, the functional and metabolic assays used to assess the platelet storage lesion in PC and the current research, benefits and limitations of CSP. We also discuss whether the application of new technology for studying mitochondrial and glycolytic function in PC could provide enhanced understanding of platelet metabolism during storage and thus contribute to the continued improvements in the manufacturing and storage of PC.

Achieving rapid and effective hemostasis remains a multidisciplinary challenge. Here, distinctive functional carbon dots derived from carbonized Platycladus orientalis (CPO-CDs) are developed using one-step hydrothermal method. The negatively charged surface of CPO-CDs retains partial functional groups from CPO precursor, exhibiting excellent water solubility and high biocompatibility. Both rat liver injury model and tail amputation model have confirmed the rapid and effective hemostatic performance of CPO-CDs on exogenous hemorrhage. Further, on endogenous blood-heat hemorrhage syndrome rat model, CPO-CDs could inhibit hemorrhage and alleviate inflammation response. Interestingly, the excellent hemostasis performance of CPO-CDs is ascribed to activate exogenous coagulation pathway and common coagulation pathway. More importantly, metabolomics of rat plasma suggests that the hemostasis effect of CPO-CDs is closely related to platelet functions. Therefore, the designed in vitro experiments are performed and it is discovered that CPO-CDs significantly promote platelets adhesion, activation, and aggregation. Further, the underlying mechanism investigation suggests that Src/Syk signal pathway plays a key role in platelets activation triggered by CPO-CDs. Overall, CPO-CDs with rapid and excellent hemostatic performance are discovered for the first time, which could be an excellent candidate for the treatment of hemorrhagic diseases., (© 2023 Wiley-VCH GmbH.)

BMP signaling is crucial to blood vessel formation and function, but how pathway components regulate vascular development is not well-understood. Here, we find that inhibitory SMAD6 functions in endothelial cells to negatively regulate ALK1-mediated responses, and it is required to prevent vessel dysmorphogenesis and hemorrhage in the embryonic liver vasculature. Reduced Alk1 gene dosage rescued embryonic hepatic hemorrhage and microvascular capillarization induced by Smad6 deletion in endothelial cells in vivo. At the cellular level, co-depletion of Smad6 and Alk1 rescued the destabilized junctions and impaired barrier function of endothelial cells depleted for SMAD6 alone. Mechanistically, blockade of actomyosin contractility or increased PI3K signaling rescued endothelial junction defects induced by SMAD6 loss. Thus, SMAD6 normally modulates ALK1 function in endothelial cells to regulate PI3K signaling and contractility, and SMAD6 loss increases signaling through ALK1 that disrupts endothelial cell junctions. ALK1 loss-of-function also disrupts vascular development and function, indicating that balanced ALK1 signaling is crucial for proper vascular development and identifying ALK1 as a 'Goldilocks' pathway in vascular biology that requires a certain signaling amplitude, regulated by SMAD6, to function properly., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Platelets are key vascular effectors in hemostasis, with activation signals leading to fast recruitment, aggregation, and clot formation. The canonical process of hemostasis is well-characterized and shares many similarities with pathological thrombus formation. However, platelets are also crucially involved in the maintenance of vascular integrity under both steady-state and inflammatory conditions by ensuring blood vessel homeostasis and preventing microbleeds. In these settings, platelets use distinct receptors, signaling pathways, and ensuing effector functions to carry out their deeds. Instead of simply forming clots, they mainly act as individual sentinels that swiftly adapt their behavior to the local microenvironment. In this review, we summarize previously recognized and more recent studies that have elucidated how anucleate, small platelets manage to maintain vascular integrity when faced with challenges of infection, sterile inflammation, and even malignancy. We dissect how platelets are recruited to the vascular wall, how they identify sites of injury, and how they prevent hemorrhage as single cells. Furthermore, we discuss mechanisms and consequences of platelets' interaction with leukocytes and endothelial cells, the relevance of adhesion as well as signaling receptors, in particular immunoreceptor tyrosine-based activation motif receptors, and cross talk with the coagulation system. Finally, we outline how recent insights into inflammatory hemostasis and vascular integrity may aid in the development of novel therapeutic strategies to prevent hemorrhagic events and vascular dysfunction in patients who are critically ill., (© 2023 by The American Society of Hematology.)

Intraventricular hemorrhage is a potentially life-threatening condition. Approximately 20% of patients develop posthemorrhagic hydrocephalus with increased ventricular volume and intracranial pressure. Hydrocephalus develops partially due to increased secretion of cerebrospinal fluid by the choroid plexus. During hemorrhage a multitude of factors are released into the cerebrospinal fluid. Many of these have been implicated in the hypersecretion. In this study, we have investigated the isolated effect of inflammatory components, on the abundance of two membrane transporters involved in cerebrospinal fluid secretion by the choroid plexus: the Na + -dependent Cl - /HCO 3 - exchanger, Ncbe, and the Na + , K + , 2Cl - cotransporter, NKCC1. We have established a primary choroid plexus epithelial cell culture from 1 to 7 days old mouse pups. Seven days after seeding, the cells formed a monolayer. The cells were treated with either tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL-1β), or interleukin 6 (IL-6) to mimic inflammation. The data show that treatment with TNFα, and IL-1β only transiently increased NKCC1 abundance whereas the effect on Ncbe abundance was a transient decrease. IL-6 however significantly increased NKCC1 (242%), the phosphorylated NKCC1 (147%), as well as pSPAK (406%) abundance, but had no effect on Ncbe. This study suggests that the inflammatory pathway involved in hypersecretion primarily is mediated by activation of basolateral receptors in the choroid plexus, mainly facilitated by IL-6. This study highlights the complexity of the pathophysiological circumstances occurring during intraventricular hemorrhage., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Introduction: Obesity is associated with higher mortality following trauma, although the pathogenesis is unclear. Both obesity and trauma are associated with syndecan-1 shedding and metalloproteinase-9 (MMP-9) activation, which can adversely affect endothelial cell function. We recently demonstrated that fibrinogen stabilizes endothelial cell surface syndecan-1 to reduce shedding and maintain endothelial barrier integrity. We thus hypothesized that MMP-9 activation and syndecan-1 shedding would be exacerbated by obesity after trauma but attenuated by fibrinogen-based resuscitation., Materials and Methods: ApoE null ( -/- ) mice were fed a Western diet to induce obesity. Mice were subjected to hemorrhage shock and laparotomy then resuscitated with Lactated Ranger's (LR) or LR containing fibrinogen and compared to null and lean sham wild type mice. Mean arterial pressure (MAP) was monitored. Bronchial alveolar lavage protein as an indicator of permeability and lung histopathologic injury were assessed. Syndecan-1 protein and active MMP-9 protein were measured., Results: MAP was similar between lean sham and ApoE -/- sham mice. However, following hemorrhage, ApoE -/- mice resuscitated with fibrinogen had significantly higher MAP than LR mice. Lung histopathologic injury and permeability were increased in LR compared to fibrinogen resuscitated animals. Compared with lean sham mice, both active MMP-9 and cleaved syndecan-1 level were significantly higher in ApoE -/- sham mice. Resuscitation with fibrinogen but not lactated Ringers largely reduced these changes., Conclusions: Fibrinogen as a resuscitative adjunct in ApoE -/- mice after hemorrhage shock augmented MAP and reduced histopathologic injury and lung permeability, suggesting fibrinogen protects the endothelium by inhibiting MMP-9-mediated syndecan-1 cleavage in obese mice., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Down syndrome is the most common chromosomal abnormality in humans. Patients with Down syndrome have hematologic disorders, including mild to moderate thrombocytopenia. In case of Down syndrome, thrombocytopenia is not associated with bleeding, and it remains poorly characterized regarding molecular mechanisms. We investigated the effects of overexpression of Dyrk1A, an important factor contributing to some major Down syndrome phenotypes, on platelet number and bleeding in mice. Mice overexpressing Dyrk1A have a decrease in platelet number by 20%. However, bleeding time was found to be reduced by 50%. The thrombocytopenia and the decreased bleeding time observed were not associated to an abnormal platelet receptors expression, to a defect of platelet activation by ADP, thrombin or convulxin, to the presence of activated platelets in the circulation or to an abnormal half-life of the platelets. To propose molecular mechanisms explaining this discrepancy, we performed a network analysis of Dyrk1A interactome and demonstrated that Dyrk1A, fibronectin and fibrinogen interact indirectly through two distinct clusters of proteins. Moreover, in mice overexpressing Dyrk1A, increased plasma fibronectin and fibrinogen levels were found, linked to an increase of the hepatic fibrinogen production. Our results indicate that overexpression of Dyrk1A in mice induces decreased bleeding consistent with increased plasma fibronectin and fibrinogen levels, revealing a new role of Dyrk1A depending on its indirect interaction with these two proteins., (© 2023 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

It is found that a hot environment aggravates hemorrhagic shock-induced internal environment and organ dysfunction. Meanwhile mitochondria show over-fission. Whether inhibition of mitochondrial fission benefits from the early treatment of hemorrhagic shock under a hot environment is unclear. An uncontrolled hemorrhagic shock model in rats is used, and the effects of mitochondrial fission inhibitor mdivi-1 on mitochondrial function, organ function, and survival rate of rats are measured. The results show that 0.1-3 mg/kg mdivi-1 antagonizes hemorrhagic shock-induced mitochondrial fragment. In addition, mdivi-1 improves mitochondrial function, and alleviates hemorrhagic shock-induced oxidative stress and inflammation under a hot environment. Further studies show that 0.1-3 mg/kg Mdivi-1 reduces blood loss, and maintains a mean artery pressure (MAP) of 50-60 mmHg before bleeding-stops after hemorrhagic shock, compared with single Lactate Ringer's (LR) resuscitation. Notably, 1 mg/kg of Mdivi-1 extends the time of hypotensive resuscitation to 2-3 h. During 1 or 2 h of ligation, Mdivi-1 prolongs survival time and protects vital organ function by rescuing mitochondrial morphology and improving mitochondrial function. These results suggest Mdivi-1 is suitable for the early treatment of hemorrhagic shock under a hot environment and can extend the golden treatment time to 2-3 hour for hemorrhagic shock under a hot environment., (© 2023 The Authors. Advanced Biology published by Wiley-VCH GmbH.)

Platelets play a central role in the arrest of bleeding. The ability of platelets to engage with extracellular matrix proteins of the subendothelium has long been recognized as a pivotal platelet attribute, underpinning adequate hemostasis. The propensity of platelets to rapidly bind and functionally respond to collagen was one of the earliest documented events in platelet biology. The receptor primarily responsible for mediating platelet/collagen responses was identified as glycoprotein (GP) VI and successfully cloned in 1999. Since that time, this receptor has held the attention of many research groups, and through these efforts, we now have an excellent understanding of the roles of GPVI as a platelet- and megakaryocyte-specific adheso-signaling receptor in platelet biology. GPVI is considered a viable antithrombotic target, as data obtained from groups across the world is consistent with GPVI being less involved in physiological hemostatic processes but participating in arterial thrombosis. This review will highlight the key aspects of GPVI contributions to platelet biology and concentrate on the interaction with recently identified ligands, with a focus on fibrin and fibrinogen, discussing the role of these interactions in the growth and stability of thrombi. We will also discuss important therapeutic developments that target GPVI to modulate platelet function while minimizing bleeding outcomes., Competing Interests: Declaration of competing interests Martine Jandrot-Perrus is a co-founder of Acticor Biotech. P.H.M, E.E.G, and R.A.S.A. have no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Sialic acid (SA) is present at the terminal ends of carbohydrate chains in glycoproteins and glycolipids and is involved in various biological phenomena. The biological function of the disialyl-T (SAα2-3Galβ1-3(SAα2-6)GalNAcα1-O-Ser/Thr) structure is largely unknown. To elucidate the role of disialyl-T structure and determine the key enzyme from the N-acetylgalactosaminide α2,6-sialyltransferase (St6galnac) family involved in its in vivo synthesis, we generated St6galnac3- and St6galnac4-deficient mice. Both single-knockout mice developed normally without any prominent phenotypic abnormalities. However, the St6galnac3::St6galnact4 double knockout (DKO) mice showed spontaneous hemorrhage of the lymph nodes (LN). To identify the cause of bleeding in the LN, we examined podoplanin, which modifies the disialyl-T structures. The protein expression of podoplanin in the LN of DKO mice was similar to that in wild-type mice. However, the reactivity of MALII lectin, which recognizes disialyl-T, in podoplanin immunoprecipitated from DKO LN was completely abolished. Moreover, the expression of vascular endothelial cadherin was reduced on the cell surface of high endothelial venule (HEV) in the LN, suggesting that hemorrhage was caused by the structural disruption of HEV. These results suggest that podoplanin possesses disialyl-T structure in mice LN and that both St6galnac3 and St6galnac4 are required for disialyl-T synthesis., (© 2023. The Author(s).)

The lack of innovation in von Willebrand disease (VWD) originates from many factors including the complexity and heterogeneity of the disease but also from a lack of recognition of the impact of the bleeding symptoms experienced by patients with VWD. Recently, a few research initiatives aiming to move past replacement therapies using plasma-derived or recombinant von Willebrand factor (VWF) concentrates have started to emerge. Here, we report an original approach using synthetic platelet (SP) nanoparticles for the treatment of VWD type 2B (VWD-2B) and severe VWD (type 3 VWD). SP are liposomal nanoparticles decorated with peptides enabling them to concomitantly bind to collagen, VWF, and activated platelets. In vitro, using various microfluidic assays, we show the efficacy of SPs to improve thrombus formation in VWF-deficient condition (with human platelets) or using blood from mice with VWD-2B and deficient VWF (VWF-KO, ie, type 3 VWD). In vivo, using a tail-clip assay, SP treatment reduced blood loss by 35% in mice with VWD-2B and 68% in mice with VWF-KO. Additional studies using nanoparticles decorated with various combinations of peptides demonstrated that the collagen-binding peptide, although not sufficient by itself, was crucial for SP efficacy in VWD-2B; whereas all 3 peptides appeared necessary for mice with VWF-KO. Clot imaging by immunofluorescence and scanning electron microscopy revealed that SP treatment of mice with VWF-KO led to a strong clot, similar to those obtained in wild-type mice. Altogether, our results show that SP could represent an attractive therapeutic alternative for VWD, especially considering their long half-life and stability., (© 2023 by The American Society of Hematology.)

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with enhanced NETosis and impaired degradation of neutrophil extracellular traps (NETs). Galectin-3 is a β-galactoside binding protein and is associated with neutrophil functions as well as involved in mediating autoimmune disorders. In this study, we plan to examine the associations of galectin-3 with the pathogenesis of SLE and NETosis. Galectin-3 expression levels were determined in peripheral blood mononuclear cells (PBMCs) of SLE patients for the association with lupus nephritis (LN) or correlation of SLE disease activity index 2000 (SLEDAI-2K). NETosis was observed in human normal and SLE and murine galectin-3 knockout (Gal-3 KO) neutrophils. Gal-3 KO and wild-type (WT) mice induced by pristane were used to evaluate disease signs, including diffuse alveolar haemorrhage (DAH), LN, proteinuria, anti-ribonucleoprotein (RNP) antibody, citrullinated histone 3 (CitH3) levels, and NETosis. Galectin-3 levels are higher in PBMCs of SLE patients compared with normal donors and positively correlated with LN or SLEDAI-2K. Gal-3 KO mice have higher percent survival and lower DAH, LN proteinuria, and anti-RNP antibody levels than WT mice induced by pristane. NETosis and citH3 levels are reduced in Gal-3 KO neutrophils. Furthermore, galectin-3 resides in NETs while human neutrophils undergo NETosis. Galectin-3-associated immune complex deposition can be observed in NETs from spontaneously NETotic cells of SLE patients. In this study, we provide clinical relevance of galectin-3 to the lupus phenotypes and the underlying mechanisms of galectin-3-mediated NETosis for developing novel therapeutic strategies targeting galectin-3 for SLE.

The feeding of high-energy and low-protein diets often induces fatty liver hemorrhagic syndrome (FLHS) in laying hens. However, the mechanism of hepatic fat accumulation in hens with FLHS remains uncertain. In this research, a comprehensive hepatic proteome and acetyl-proteome analysis was performed in both normal and FLHS-affected hens. The results indicated that the upregulated proteins were primarily associated with fat digestion and absorption, the biosynthesis of unsaturated fatty acids, and glycerophospholipid metabolism, while the downregulated proteins were mainly related to bile secretion and amino acid metabolism. Furthermore, the significant acetylated proteins were largely involved in ribosome and fatty acid degradation, and the PPAR signaling pathway, while the significant deacetylated proteins were related to valine, leucine, and isoleucine degradation in laying hens with FLHS. Overall, these results demonstrate that acetylation inhibits hepatic fatty acid oxidation and transport in hens with FLHS, and mainly exerts its effects by affecting protein activity rather than expression. This study provides new nutritional regulation options to alleviate FLHS in laying hens.

Background: The risk of military and civilian radiation exposure is increasing, and determining the effects of exposure is a high priority. Irradiation of the nearby blood supply after a nuclear event may impede mobilization of blood products for resuscitation at a time of great need. RBCs are administered to patients with trauma and hemorrhage to transport and deliver oxygen and avoid tissue hypoxia. Here we determine the effects of ionizing radiation on the energy metabolome of RBCs isolated from cold stored whole blood to determine if their stability is compromised by radiation exposure., Study Design and Methods: Whole blood from healthy volunteers was subjected to 0, 25, or 75 Gy of X-irradiation, and stored at 4°C. RBCs were isolated from stored WB at 0, 1, 7, 14, and 21 days of storage. The levels of extracted Krebs cycle intermediates, nicotinamide adenine dinucleotides, and phosphorylated derivatives of adenosine and guanosine were determined by tandem mass spectroscopy., Results: Irradiation at either 25Gy or 75Gy had no significant effect on any parameter measured compared to control (0Gy). However, there was a significant change over time in storage for ATP, GDP, and guanosine., Discussion: Irradiation at doses up to 75Gy had no effect on the energy metabolome of RBCs prepared from blood stored at 4°C for up to 21 days, suggesting that the RBC energy metabolome is not affected by radiation exposure and the blood can still be used for resuscitation in trauma patients., (Published 2023. This article is a U.S. Government work and is in the public domain in the USA.)

Purpose: The objective of this study is to develop a bleeding risk model for assessing device-induced bleeding risk in patients supported with blood contact medical devices (BCMDs)., Methods: The mathematical model for evaluating bleeding risk considers the effects of shear stress on von Willebrand factor (vWF) unfolding, high molecular weight multimers-vWF (HMWM-vWF) degradation, platelet activation and receptor shedding and platelet-vWF binding ability. Functions of the effect of shear stress on the above factors are fitted/employed and solved by the Eulerian transport equation. An axial flow-through Couette device and two clinical VADs which are HeartWare Ventricular Assist Device (HVAD) and HeartMate II (HM II) blood pump were employed to perform the simulation to evaluate platelet receptor shedding (GPIbα and GPIIb/IIIa), loss of HWMW-vWF, platelet-vWF binding ability and bleeding risk for validating the accuracy of our model., Results: The platelet-vWF binding ability after being subjected to high shear region in the axial flow-through Couette device predicted by our bleeding model was highly consistent with reported experimental data. As indicated by our CFD simulation results in the axial flow-through Couette device, it can find that an increase in shear stress led to a decrease in the adhesion ability of platelets on vWF, while the binding ability of vWF with platelets first increase and then decrease as shear stress elevates gradually beyond a threshold. The factor of exposure time can enhance the effect of shear stress. Additionally, the shear-induced bleeding risk predicted by our model increases with increasing shear stress and exposure time in an axial flow-through Couette device. As indicated by our numerical model, the bleeding risk in HVAD was higher than HMII, which is highly consistent with the meta-analysis based on clinical statistics. Our simulation investigations in these two clinical VADs also found that HVAD caused a higher rate of platelet receptor shedding and lower damage to HWMW-vWF than HeartMate II. The high shear stress generated in the narrow and turbulent regions of both VADs was the underlying cause of device-induced bleeding., Conclusion: In this study, the shear-induced bleeding risk predicted by our bleeding model in axial flow-through Couette device and two clinical VADs is consistent or highly correlated with experimental and clinical findings, which proves the accuracy of our bleeding model. Our bleeding model can be used to aid the development of new BCMDs with improved functional characteristics and biocompatibility, and help to reduce risk of device-induced adverse events in patients., Competing Interests: Declaration of Competing Interest There is no conflict of interest in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Diffuse alveolar haemorrhage (DAH) is a rapidly developing condition owing to a lack of effective treatment and resulting in a high mortality rate in systemic lupus erythematosus (SLE). Neutrophil extracellular traps (NETs) contain numerous antigens and proinflammatory substances that directly damage the vascular endothelium and aggravate vascular inflammation, which is considered an important pathogenic factor of DAH in SLE. Therefore, blocking the release of NETs from neutrophils is an important target for the treatment of DAH in SLE. In this study, we investigated whether the inhibition of neutrophils releasing NETs could relieve DAH in SLE. Necrostatin-1 (Nec-1), a small molecule, has been reported to inhibit the release of NETs by neutrophils. In vitro experiments revealed that Nec-1 inhibited alveolar epithelial cell damage by preventing the release of NETs. Furthermore, vivo studies showed that Nec-1 alleviated lupus pulmonary haemorrhage in mice by reducing lung pathology severity, body weight, and serum inflammatory cytokine levels. Mechanistically, Nec-1 prevented NET release by inhibiting neutrophil elastase (NE) activation and N-Gasdermin D (N-GSDMD) expression. Additionally, immunohistochemistry and immunofluorescence findings showed that Nec-1 decreased NE expression in the lung tissues of mice with lupus pulmonary haemorrhage. Thus, NETs released by neutrophils contributed to the pathogenesis of DAH in SLE, and Nec-1 showed protective effects by the inhibition of NET production via the reduction of NE activation and N-GSDMD expression., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2023 Xinai Han et al.)

The purpose of this study was to explore whether conjugated linoleic acid (CLA) could alleviate fatty liver hemorrhagic syndrome (FLHS) induced by estradiol benzoate intramuscular injection in laying hens. One hundred male Hy-Line white chickens were randomly divided into two groups, namely, the control (CON) and estradiol benzoate (E) groups, and both groups were fed the same basal diet. After injections of estradiol benzoate at 2 mg/kg every two days for a total of 7 times, chickens in the E group showed FLHS symptoms, including liver enlargement, hemorrhage, and steatosis. Then half of the chickens in the E group received an additional diet containing 5000 mg/kg CLA for 8 weeks. The results of morphological observations, hematoxylin and eosin staining, and Oil Red O staining showed that CLA alleviated liver enlargement, hemorrhage, and lipid accumulation in FLHS chickens. In addition, we measured liver function and lipid metabolism indicators, including ALT, AST, TG, TCH, HDL-C, and LDL-C, which further suggested that CLA mitigated the disturbance of serum and liver metabolism in FLHS chickens. Mechanistically, CLA inhibited hepatic de novo lipogenesis, cholesterol synthesis, and TG accumulation and increased TG hydrolysis in FLHS chickens by regulating the gene expression of CD36, ACC, FAS, SCD 1, DGAT2, LIPE, ATGL, CPT1A, SREBP-1c, SREBP-2, PPARγ, and PPARα. Furthermore, CLA ameliorated hepatic oxidative stress and inhibited NF-κB signaling pathway-mediated inflammation in FLHS chickens. In conclusion, CLA regulated lipid metabolism, thus further alleviating oxidative stress and inflammation to alleviate FLHS induced by estrogen in chickens., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The aim of this study was to evaluate the beneficial effects and potential mechanisms of genistein (GEN) on production performance impairments and lipid metabolism disorders in laying hens fed a high-energy and low-protein (HELP) diet. A total of 120 Hy-line Brown laying hens were fed with the standard diet and HELP diet supplemented with 0, 50, 100, and 200 mg/kg GEN for 80 d. The results showed that the declines in laying rate (P < 0.01), average egg weight (P < 0.01), and egg yield (P < 0.01), and the increase of the ratio of feed to egg (P < 0.01) induced by HELP diet were markedly improved by 100 and 200 mg/kg of GEN treatment in laying hens (P < 0.05). Moreover, the hepatic steatosis and increases of lipid contents (P < 0.01) in serum and liver caused by HELP diet were significantly alleviated by treatment with 100 and 200 mg/kg of GEN in laying hens (P < 0.05). The liver index and abdominal fat index of laying hens in the HELP group were higher than subjects in the control group (P < 0.01), which were evidently attenuated by dietary 50 to 200 mg/kg of GEN supplementation (P < 0.05). Dietary 100 and 200 mg/kg of GEN supplementation significantly reduced the upregulations of genes related to fatty acid transport and synthesis (P < 0.01) but enhanced the downregulations of genes associated with fatty acid oxidation (P < 0.01) caused by HELP in the liver of laying hens (P < 0.05). Importantly, 100 and 200 mg/kg of GEN supplementation markedly increased G protein-coupled estrogen receptor (GPER) mRNA and protein expression levels and activated the AMP-activated protein kinase (AMPK) signaling pathway in the liver of laying hens fed a HELP diet (P < 0.05). These data indicated that the protective effects of GEN against the decline of production performance and lipid metabolism disorders caused by HELP diet in laying hens may be related to the activation of the GPER-AMPK signaling pathways. These data not only provide compelling evidence for the protective effect of GEN against fatty liver hemorrhagic syndrome in laying hens but also provide the theoretical basis for GEN as an additive to alleviate metabolic disorders in poultry., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Severe COVID-19 has been associated with a high rate of thrombotic events but also of bleeding events, particularly when the level of prophylactic anticoagulation was increased. Data on the contribution of platelets to these thrombotic events are discordant between reports, while the involvement of platelets in bleeding events has never been investigated. The objective of the present study was to assess platelet function during the first week of ICU hospitalization in patients with severe COVID-19 pneumonia. A total of 35 patients were prospectively included and blood samples were drawn on day (D) 0, D2 and D7. COVID-19 pneumonia was severe with a median PaO 2 /FiO 2 ratio of 91 [68-119] on D0. Platelets from these patients showed evidence of pre-activation and exhaustion with a significant reduction in the surface expression of GPVI, GPIb and GPIIbIIIa, together with a decrease in serotonin content. Platelets from patients with severe COVID-19 were hyporesponsive with a reduced maximal aggregation response to several platelet agonists and decreased adhesion to immobilized fibrinogen. Aggregation of washed platelets and plasma substitution experiments indicated that a plasma factor was at least partially responsible for this hyporeactivity of platelets. Blood flow experiments showed that severe COVID-19 platelets formed smaller, less stable aggregates on a collagen-coated surface, which could explain why some patients develop bleeding events. These findings should prompt us to carefully evaluate the risks and benefits of high-dose prophylactic anticoagulation, and to decrease the level of anticoagulation once the initial phase of the disease has resolved. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT04359992., Competing Interests: Declaration of competing interest CT reports personal fees paid by Sanofi for conference attendance., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

One of the principles of prevention and non-medicamentous treatment of liver diseases, including hepatitis of different etiology, is the normalization of the diet through the consumption of food with physiologically active ingredients, in particular betulin, which helps to eliminate the causes of metabolic and oxidative disorders within liver cells. The aim of the research was to assess in vivo the influence of triterpene alcohol betulin extracted from Betula pendula Roth. birch bark in fat-containing products (for example mayonnaise) on the blood biochemical parameters and liver morphological structure of rats with initiated acute toxic hepatitis. Material and methods . Hepatoprotective and antioxidant activities of betulin as part of mayonnaise samples has been investigated in vivo on the model of toxic hepatitis initiated by carbon tetrachloride in male Wistar rats weighing 210-265 g. The animals were divided into 4 groups of 10 animals each: CG-1 - intact, CG-2 and MG - with carbon tetrachloride initiated toxic hepatitis. rats of the main groups were orally administered mayonnaise once a day at a dosage of 1 ml for 21 days after the formation of the model pathology: OG-1 with the added betulin (1 mg per 1 kg of body weight), OG-2 without betulin. Disorders of metabolic and oxidative processes in liver cells of animals were evaluated by biochemical indicators of blood plasma: the level of glucose, albumin, total cholesterol, triglycerides and urea and the activity of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and γ-glutamyltransferase. Oxidative stress in rats was estimated by the activity of catalase and superoxide dismutase in blood hemolysate (at a dilution of 1:200 and 1:10, respectively); the total prooxidant (in blood plasma) and total antioxidant (in blood hemolysate at a dilution of 1:10) activity were determined spectrophotometrically (colored complexes of TWIN-80 oxidation products with thiobarbituric acid). The morphological structure of rats' liver was estimated by microscopy of prepared cuts of hepatic tissue. Results . Based on biochemical parameters of rat blood plasma, it has been established that the administration of mayonnaise with betulin prevents the development of cytolic syndrome and suppresses the process of peroxidation by directly neutralizing free radicals. Aspartate aminotransferase and alkaline phosphatase activity in blood plasma of the experimental animals of the main group MG-1 reduced by 20.7 and 35.2% compared with indicators of the rats of the main group MG-2. Glucose concentration normalized to the level of the control group CG-1. The concentration of bilirubin and triglycerides decreased by 22.9 and by 48.1%, which indicates a significant reduction in the indicators of cholestatic syndrome in the group of animals OG-1 compared to OG-2. The total prooxidant activity and the concentration of thiobarbiturate-reactive products decreased compared to the CG-2 and MG-2 groups, which indicates the suppression of oxidative stress and, as a result, an improvement in liver conditions of animals with toxic hepatitis even when taking a fat-containing product. In liver histopeparates of animals receiving mayonnaise with betulin, necrobotic changes were less pronounced in comparison with the group MG-2. They were estimated at 1 point: small-drip dystrophy spots were found, haemorrhages in the interregional septum with inflammatory infiltration in the course of hemorrhages against the presence of necrosis hepatocytes with pronounced adipose dystrophy in the centres of the lobules, step necrosis with signs of replacing the damaged hepatocytes of the connective tissue, accompanied by centrolobular hemorrhages in MG-2 rats. Conclusion . Introduced into the composition of mayonnaise betulin, reduces the development of cytolic syndrome in toxic hepatitis and suppresses the process of peroxidation, on the basis of which fat-containing foods with betulin can be recommended for clinical examination as specialized products in acute and chronic liver diseases, including complicated cholestasis., Competing Interests: The author declares no conflicts of interest., (Copyright© GEOTAR-Media Publishing Group.)

Objectives: To determine the association between both visceral fat quantity and adipose tissue dysfunction, and major bleeding in patients with established cardiovascular disease., Methods: Patients from the Second Manifestations of ARTerial disease study with established cardiovascular disease were included. Visceral fat was measured using ultrasound and adipose tissue dysfunction was depicted using metabolic syndrome criteria (revised National Cholesterol Education Program). Cox regression models were fitted to study the relation with major bleeding defined as Bleeding Academic Research Consortium (BARC) type 3 or 5, or International Society on Thrombosis and Haemostasis (ISTH) major bleeding. Sensitivity analyses were performed using C-reactive protein levels to reflect adipose tissue dysfunction., Results: In 6927 patients during a median follow up of 9.2 years, a total of 237 BARC type 3 or 5 bleedings and 224 ISTH major bleedings were observed. Visceral fat quantity was not related to major bleeding (HR 1.01, 95%CI 0.88-1.16 for BARC type 3 or 5 bleeding and HR 1.00, 95%CI 0.87-1.15 for ISTH major bleeding), nor was metabolic syndrome (HR 0.97, 95%CI 0.75-1.26 for BARC type 3 or 5 bleeding and HR 0.98, 95%CI 0.75-1.28 for ISTH major bleeding). Sensitivity analyses using C-reactive protein levels showed similar results. No effect modification was observed by sex, antithrombotic therapy, presence of metabolic syndrome or diabetes., Conclusion: In patients with cardiovascular disease, no association was found between visceral fat quantity measured with ultrasound or measures of adipose tissue dysfunction and the risk of major bleeding, irrespective of antithrombotic agent use., Competing Interests: Declaration of Competing Interest All authors declare no conflict of interest. The UCC-SMART study was financially supported by a grant of the University Medical Center Utrecht, the Netherlands. The supporting sources had no involvement in study design, analysis, interpretation, writing of the results, or the decision to submit for publication., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Bleeding tendency, a prominent feature of patients with Gaucher disease (GD), is associated with abnormal platelet function. Brain-derived neurotrophic factor (BDNF) is a protein with neuroprotective potential stored in alpha granules of circulating platelets. Here we studied BDNF levels in 50 patients with type I GD (GD1) and their correlation with platelet activity and bleeding tendency. Flow cytometry was used to test unstimulated and stimulated measurement of platelet surface-activated expression of αIIbβ3 integrin, P-selectin and lysosomal-associated membrane protein (LAMP3/CD63). Serum and plasma BDNF levels were quantified using ELISA. The bleeding history was recorded by a bleeding questionnaire. Serum BDNF levels were positively correlated with platelet count and moderately correlated with unstimulated and stimulated platelet P-selectin expression. Patients with more than one bleeding manifestation were shown to have lower serum BDNF levels, albeit similar platelet count. Plasma BDNF levels were significantly elevated in splenectomized patients and showed a moderate positive correlation with stimulated platelet CD63 expression. These observations demonstrate the first association between BDNF levels in the peripheral blood with platelet dysfunction and increased bleeding manifestation. The role of measuring serum BDNF for assessing platelet alpha degranulation defects and bleeding risk in patients with GD and the general population needs further study.

Red blood cell (RBC) transfusion is a life-saving intervention for millions of trauma patients every year worldwide. While hemoglobin thresholds are clinically driving the need for RBC transfusion, limited information is available with respect to transfusion efficacy at the molecular level in clinically relevant cohorts. Here, we combined plasma metabolomic and proteomic measurements in longitudinal samples (n = 118; up to 13 time points; total samples: 690) from trauma patients enrolled in the control of major bleeding after trauma (COMBAT) study. Samples were collected in the emergency department and at continuous intervals up to 168 h (seven days) post-hospitalization. Statistical analyses were performed to determine omics correlate to transfusions of one, two, three, five, or more packed RBC units. While confounded by the concomitant transfusion of other blood components and other iatrogenic interventions (e.g., surgery), here we report that transfusion of one or more packed RBCs—mostly occurring within the first 4 h from hospitalization in this cohort—results in the increase in circulating levels of additive solution components (e.g., mannitol, phosphate) and decreases in the levels of circulating markers of hypoxia, such as lactate, carboxylic acids (e.g., succinate), sphingosine 1-phosphate, polyamines (especially spermidine), and hypoxanthine metabolites with potential roles in thromboinflammatory modulation after trauma. These correlations were the strongest in patients with the highest new injury severity scores (NISS > 25) and lowest base excess (BE < −10), and the effect observed was proportional to the number of units transfused. We thus show that transfusion of packed RBCs transiently increases the circulating levels of plasticizers—likely leaching from the blood units during refrigerated storage in the blood bank. Changes in the levels of arginine metabolites (especially citrulline to ornithine ratios) are indicative of an effect of transfusion on nitric oxide metabolism, which could potentially contribute to endothelial regulation. RBC transfusion was associated with changes in the circulating levels of coagulation factors, fibrinogen chains, and RBC-proteins. Changes in lysophospholipids and acyl-carnitines were observed upon transfusion, suggestive of an effect on the circulating lipidome—though cell-extrinsic/intrinsic effects and/or the contribution of other blood components cannot be disentangled. By showing a significant decrease in circulating markers of hypoxia, this study provides the first multi-omics characterization of RBC transfusion efficacy in a clinically relevant cohort of trauma patients.

Phosphorylcholine (PC) is one of the main oxLDL epitopes playing a central role in atherosclerosis, due to its atherogenic and proinflammatory effects. PC can be cleared by natural IgM antibodies and low levels of these antibodies have been associated with human vein graft (VG) failure. Although PC antibodies are recognized for their anti-inflammatory properties, their effect on intraplaque angiogenesis (IPA) and intraplaque hemorrhage (IPH)-interdependent processes contributing to plaque rupture-are unknown. We hypothesized that new IgG phosphorylcholine antibodies (PC-mAb) could decrease vulnerable lesions in murine VGs.Therefore, hypercholesterolemic male ApoE3*Leiden mice received a (donor) caval vein interposition in the carotid artery and weekly IP injections of (5 mg/kg) PCmAb (n = 11) or vehicle (n = 12) until sacrifice at day 28. We found that PCmAb significantly decreased vein graft media (13%), intima lesion (25%), and increased lumen with 32% compared to controls. PCmAb increased collagen content (18%) and decreased macrophages presence (31%). PCmAb resulted in 23% decreased CD163+ macrophages content in vein grafts whereas CD163 expression was decreased in Hb:Hp macrophages. PCmAb significantly lowered neovessel density (34%), EC proliferation and migration with/out oxLDL stimulation. Moreover, PCmAb enhanced intraplaque angiogenic vessels maturation by increasing neovessel pericyte coverage in vivo (31%). Together, this resulted in a 62% decrease in IPH. PCmAb effectively inhibits murine atherosclerotic lesion formation in vein grafts by reducing IPA and IPH via decreased neovessel density and macrophages influx and increased neovessel maturation. PC-mAb therefore holds promise as a new therapeutic approach to prevent vein graft disease.

Systemic lupus erythematosus (SLE)-associated diffuse alveolar hemorrhage (DAH) is a rare but extremely harmful condition. The current study sought to dissect the mechanisms underlying the effects of human umbilical cord mesenchymal stem cell (HUCMSC)-derived exosomes on M2 macrophage polarization in SLE-associated DAH via the microRNA (miR)-146a-5p/NOTCH1 axis. A DAH mouse model was established using pristane. Exosomes were isolated from HUCMSCs transfected or untransfected with the miR-146a-5p antagonist or agonist and their NCs and then injected into DAH mice. Additionally, miR-146a-5p was overexpressed in macrophages. Expression of miR-146a-5p, NOTCH1, M1 macrophage markers, and M2 macrophage markers was measured in mice and macrophages, and inflammatory factor levels were detected. Mouse lung injuries were evaluated, so was the binding of miR-146a-5p to NOTCH1. Rescue experiments were conducted in mice and macrophages using NOTCH1 shRNA and pcDNA3.1-NOTCH1, respectively. NOTCH1 expression was enhanced in DAH mice. HUCMSC-derived exosomes reduced NOTCH1 expression, bleeding, inflammation, and M1 macrophage polarization but elevated M2 macrophage polarization in lung tissues of DAH mice. Mechanistically, NOTCH1 is negatively targeted by miR-146a-5p. miR-146a-5p overexpression diminished M1 marker and inflammatory factor levels but enhanced M2 marker levels in macrophages, which was nullified by NOTCH1 overexpression. HUCMSC-derived exosomes with miR-146a-5p inhibition increased NOTCH1 expression, worsened bleeding and inflammation, and augmented M1 macrophage polarization while decreasing M2 macrophage polarization in lung tissues of DAH mice, which was abrogated by silencing NOTCH1. HUCMSC-derived exosomes diminished NOTCH1 expression to accelerate M2 macrophage polarization via delivery of miR-146a-5p, thus alleviating SLE-associated DAH in mice.

Intraplaque hemorrhage (IPH) accelerates atherosclerosis progression. To scavenge excessive red blood cells (RBCs), vascular smooth muscle cells (VSMCs) with great plasticity may function as phagocytes. Here, we investigated the erythrophagocytosis function of VSMCs and possible regulations involved. Based on transcriptional microarray analysis, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that genes up-regulated in human carotid atheroma with IPH were enriched in functions of phagocytic activities, while those down-regulated were enriched in VSMCs contraction function. Transcriptional expression of Milk fat globule-epidermal growth factor 8 (MFG-E8) was also down-regulated in atheroma with IPH. In high-fat diet-fed apolipoprotein E-deficient mice, erythrocytes were present in cells expressing VSMC markers αSMA in the brachiocephalic artery, suggesting VSMCs play a role in erythrophagocytosis. Using immunofluorescence and flow cytometry, we also found that eryptotic RBCs were bound to and internalized by VSMCs in a phosphatidylserine/MFG-E8/integrin α V β 3 dependent manner in vitro. Inhibiting S1PR2 signaling with specific inhibitor JTE-013 or siRNA decreased Mfge8 expression and impaired the erythrophagocytosis of VSMCs in vitro. Partial ligation was performed in the left common carotid artery (LCA) followed by intra-intimal injection of isolated erythrocytes to observe their clearance in vivo. Interfering S1PR2 expression in VSMCs with Adeno-associated virus 9 inhibited MFG-E8 expression inside LCA plaques receiving RBCs injection and attenuated erythrocytes clearance. Erythrophagocytosis by VSMCs increased vascular endothelial growth factor-a secretion and promoted angiogenesis. The present study revealed that VSMCs act as phagocytes for RBC clearance through S1PR2 activation induced MFG-E8 release., (Copyright © 2022. Published by Elsevier Inc.)

Platelets are anucleate cells that mediate hemostasis. This occurs via a primary signal that is reinforced by secreted products such as ADP that bind purinergic receptors (P2Y1 and P2Y12) on the platelet surface. We recently identified a human subject, whom we termed platelet defect subject 25 (PDS25) with a platelet functional disorder associated with the P2Y12 receptor. PDS25 has normal blood cell counts and no history of bleeding diathesis. However, platelets from PDS25 have virtually no response to 2-MeSADP (a stable analogue of ADP). Genetic analysis of P2Y12 from PDS25 revealed a heterozygous mutation of D121N within the DRY motif. Rap1b activity was reduced in platelets from PDS25, while VASP phosphorylation was enhanced, suggesting that signaling from the P2Y12 receptor was interrupted by the heterozygous mutation. To explore this further, we produced knock-in mice that mimic our subject. Bleeding failed to cease in homozygous KI mice during tail bleeding assays, while tail bleeding times did not differ between WT and heterozygous KI mice. Furthermore, occlusions failed to form in most homozygous KI mice following carotid artery injury via FeCl 3 . These data indicate that the aspartic acid residue found in the DRY motif of P2Y12 is essential for P2Y12 function.

Background: Complement lectin pathway components, in particular mannan-binding lectin (MBL) and MBL-associated serine proteases (MASPs) have been shown to interact with coagulation factors and contribute to clot formation. Here we investigated the role of MBL and MASP-1 in the haemostatic response following mechanical vessel injury in a human microfluidic bleeding model., Methods: We studied haemostasis in a microvascular bleeding model in the presence of human endothelial cells and human whole blood under flow conditions. We monitored incorporation of proteins into the clot with fluorescently labelled antibodies and studied their effects on clot formation, platelet activation, and bleeding time with specific inhibitors. Platelet activation was also studied by flow cytometry., Results: Upon vessel injury, MBL accumulated at the injury site in a well-defined wall-like structure. MBL showed partial colocalisation with fibrin, and strong colocalisation with von Willebrand factor and (activated) platelets. Flow cytometry ruled out direct binding of MBL to platelets, but confirmed a PAR4- and thrombin-dependent platelet-activating function of MASP-1. Inhibiting MBL during haemostasis reduced platelet activation, while inhibiting MASP-1 reduced platelet activation, fibrin deposition and prolonged bleeding time., Conclusion: We show in a microvascular human bleeding model that MBL and MASP-1 have important roles in the haemostatic response triggered by mechanical vessel injury: MBL recognises the injury site, while MASP-1 increases fibrin formation, platelet activation and shortens bleeding time. While the complement lectin pathway may be harmful in the context of pathological thrombosis, it appears to be beneficial during the physiological coagulation response by supporting the crucial haemostatic system., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Golomingi, Kohler, Jenny, Hardy, Dobó, Gál, Pál, Kiss, Lam and Schroeder.)

Cyclophosphamide (CYP) damages all mucosal defence lines and induces hemorrhagic cystitis (HC) leading to detrusor overactivity. Patients who undergo combined chemio-radiotherapy are at higher risk of HC. Potentilla chinensis extract (PCE) prevent oxidative stress-dependent diseases. Thus, the aim of the study was to investigate the effect of PCE on urinary bladder function in CYP-induced HC in preclinical study. 60 rats were divided into 4 groups, as follows: I-control, II-rats with CYP-induced HC, III-rats received PCE in dose of 500 mg/kg, and IV-rats with CYP-induced HC which received PCE in dose of 500 mg/kg. PCE or vehicle were administered orally for 14 days. The cystometry was performed 3 days after the last dose of the PCE. Next, urothelium thickness and oedema measurement and biochemical analyses were performed. Cyclophosphamide induced hemorrhagic cystitis. PCE had no influence on the urinary bladder function and micturition cycles in normal rats. PCE diminished the severity of CYP-induced hemorrhagic cystitis. In the urothelium the cyclophosphamide induced the elevation of CGRP, TNF-α, IL-6, IL-1β, OTC 3, NIT, and MAL. Also, the level of T-H protein, HB-EGF, and ZO1 was decreased. Moreover, the level of ROCK1 and VAChT in detrusor muscle increased. cyclophosphamide caused an increased concentration of BDNF and NGF in the urine. In turn, PCE in cyclophosphamide-induced hemorrhagic cystitis caused a reversal of the described biochemical changes within urothelium, detrusor muscle and urine. PCE attenuates detrusor overactivity. In conclusion, our results revealed that PCE attenuates detrusor overactivity in case of cyclophosphamide-induced hemorrhagic cystitis. The potential properties of PCE appear to be important in terms of preventing of oxidative stress-dependent dysfunction of urinary bladder. PCE may become a potential supportive treatment in patient to whom cyclophosphamide-based chemotherapy is used., (© 2022. The Author(s).)

Impairment of vascular integrity is a hallmark of inflammatory diseases. We recently reported that single immune-responsive platelets migrate and reposition themselves to sites of vascular injury to prevent bleeding. However, it remains unclear how single platelets preserve vascular integrity once encountering endothelial breaches. Here we demonstrate by intravital microscopy combined with genetic mouse models that procoagulant activation (PA) of single platelets and subsequent recruitment of the coagulation cascade are crucial for the prevention of inflammatory bleeding. Using a novel lactadherin-based compound, we detect phosphatidylserine (PS)-positive procoagulant platelets in the inflamed vasculature. We identify exposed collagen as the central trigger arresting platelets and initiating subsequent PA in a CypD- and TMEM16F-dependent manner both in vivo and in vitro. Platelet PA promotes binding of the prothrombinase complex to the platelet membrane, greatly enhancing thrombin activity and resulting in fibrin formation. PA of migrating platelets is initiated by costimulation via integrin αIIbβ3 (GPIIBIIIA)/Gα13-mediated outside-in signaling and glycoprotein VI signaling, leading to an above-threshold intracellular calcium release. This effectively targets the coagulation cascade to breaches of vascular integrity identified by patrolling platelets. Platelet-specific genetic loss of either CypD or TMEM16F as well as combined blockade of platelet GPIIBIIIA and glycoprotein VI reduce platelet PA in vivo and aggravate pulmonary inflammatory hemorrhage. Our findings illustrate a novel role of procoagulant platelets in the prevention of inflammatory bleeding and provide evidence that PA of patrolling platelet sentinels effectively targets and confines activation of coagulation to breaches of vascular integrity., (© 2022 by The American Society of Hematology.)

The aim of this study is to model classical Bernard Soulier Syndrome in the zebrafish by targeting Gp1ba. We obtained gp1ba mutant embryos from Zebrafish International Resource Center and grew them to adulthood. The tail clips from these fish were used to prepare DNA and sequenced to identify heterozygotes. They were then bred to obtain homozygotes. The mutation was confirmed by DNA sequencing as a termination codon UAA in place of AAA codon at position 886 in the gp1ba transcript. Thus, at the Pro-295, the Gp1ba protein could be terminated. The blood from gp1ba homozygous and heterozygous mutants showed decreased ristocetin-mediated agglutination in the whole blood agglutination assay. The gp1ba heterozygous and homozygous larvae were subjected to a laser-assisted arterial thrombosis assay, and the results showed the prolonged occlusion in the caudal artery. These results suggested that the gp1ba mutant had a bleeding phenotype. The blood smears from the adult gp1ba, heterozygous and homozygous mutants, showed macrothrombocytes, similar to the human GP1BA deficiency that showed giant platelets. The bleeding assay on these heterozygous and homozygous mutants showed greater bleeding than wildtype, confirming the above findings. Taken together, the characterization of gp1ba zebrafish mutant suggested an autosomal dominant mode of inheritance. The zebrafish gp1ba mutant models classical Bernard Soulier Syndrome and could be used for reversing this phenotype to identify novel factors by the genome-wide piggyback knockdown method., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

The expression levels of intracellular heat shock proteins (Hsps), specialized chaperone proteins, increase in cases of cellular stress with protein misfolding and aggregation. In a previous study, we demonstrated that there is an extensive increase in intracellular Hsp27 and 70 expression levels in renal tissues in fire fatality cases. Hsp expression can be induced by not only heat, but also by tissue hypoxia. In cases of fatal hemorrhage, the individual suffers hypoxemia and consequently tissue hypoxia. Here, we examined 43 cases of fatal hemorrhage and a control group of 85 deaths not related to blood loss or temperature exposure. We evaluated Hsp27 and 70 protein expression levels in renal tissue using immunohistochemistry. The results revealed that no extensive Hsp27 or 70 expression is induced in the fatal hemorrhage cases. The renal Hsp levels were similar to those of the control group. Fatal blood loss does not cause relevant cell stress., Competing Interests: Declarations of interest None., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Tumor necrosis factor-α-induced protein eight like 1 (TIPE1) plays important role in autophagy, immunity, and lipid metabolism. The potential role of TIPE1 in fatty liver hemorrhage syndrome (FLHS) is elusory. In the present study, the full-length coding sequence of TIPE1 was cloned, and the polyclonal antibody of TIPE1 was produced by the recombinant TIPE1 protein. The bioinformatic analysis showed that the chicken TIPE1 protein, which was predicted to be a hydrophobic and non-transmembrane protein without signal peptide was highly different from that of mammals. Furthermore, proceeded by using TIPE1 polyclonal antibody, the tissue distribution analysis showed that TIPE1 protein is ubiquitously expressed in various tissues in adult hens and chicks, with its level being higher in the liver and, spleen, moderate in intestinal, brain, and heart. Besides, immunohistochemistry and immunofluorescence observation demonstrated that TIPE1 mainly existed in the cytoplasm in liver, duodenum, and cecum cell. Notably, the TIPE1 expressions were significantly decreased in laying hens suffering from FLHS. Collectively, these results showed that the chicken TIPE1 polyclonal antibody was successfully prepared and further used to analyze the expression profiles of chicken. And the expression of TIPE1 was reduced in FLHS which provided the foundation for further investigation in FLHS., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Calcific aortic valve disease (CAVD) is the most frequent pathogeny of aortic valve replacement in developed countries. Iron deposits are found in the intraleaflet hemorrhage (IH) areas of calcific aortic valves. Ferroptosis is a form of regulated cell death that involves metabolic dysfunction resulting from iron overload-dependent excessive lipid peroxidation. In this study, histological analysis showed that ferroptosis occurs in the IH areas of calcific aortic valves. We also demonstrated that Slc7a11 is expressed at low levels in OM-treated valvular interstitial cells (VICs) and IH areas and that low Slc7a11 expression is associated with calcification in CAVD. However, iron overload treatment did not promote VIC calcification under osteogenic conditions in vitro. Using lentiviral transfection to knockdown Slc7a11 in VICs, we found that the degree of iron overload-induced ferroptosis was positively increased in vitro. Finally, we also found that Slc7a11 knockdown promoted the osteogenic differentiation of VICs in vitro. In summary, this study reports a novel mechanism linking ferroptosis and CAVD development in which iron may promote Slc7a11-deficient VIC osteogenic differentiation by aggravating ferroptosis in vitro, thereby accelerating the progression of aortic valve calcification., (Copyright © 2022. Published by Elsevier Inc.)

Oligodendrocyte progenitor cells (OPCs) differentiate to myelin-producing mature oligodendrocytes and enwrap growing or demyelinated axons during development and post central nervous diseases. Failure of remyelination owing to cell death or undifferentiation of OPCs contributes to severe neurologic deficits and motor dysfunction. However, how to prevent the cell death of OPCs is still poorly understood, especially in hemorrhagic diseases. In the current study, we injected autologous blood into the mouse lateral ventricular to study the hemorrhage-induced OPC cell death in vivo. The integrity of the myelin sheath of the corpus callosum was disrupted post intraventricular hemorrhage (IVH) assessed by using magnetic resonance imaging, immunostaining, and transmission electron microscopy. Consistent with the severe demethylation, we observed massive cell death of oligodendrocyte lineages in the periventricular area. In addition, we found that ferroptosis is the major cell death form in Hemin-induced OPC death by using RNA-seq analysis, and the mechanism was glutathione peroxidase 4 activity reduction-resulted lipid peroxide accumulation. Furthermore, inhibition of ferroptosis rescued OPC cell death in vitro, and in vivo attenuated IVH-induced white matter injury and promoted recovery of neurological function. These data demonstrate that ferroptosis is an essential form of OPC cell death in hemorrhagic stroke, and rescuing ferroptotic OPCs could serve as a therapeutic target for stroke and related diseases., (© 2022. The Author(s).)

Bleeding correlates with disease severity in viral hemorrhagic fevers. We found that the increase in type I interferon (IFN-I) in mice caused by infection with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV; an arenavirus) reduced the megakaryocytic expression of genes encoding enzymes involved in lipid biosynthesis ( cyclooxygenase 1 and thromboxane A synthase 1 ) and a thrombopoietic transcription factor ( Nf-e2 ). The decreased expression of these genes was associated with reduced numbers of circulating platelets and defects in the arachidonic acid synthetic pathway, thereby suppressing serotonin release from δ-granules in platelets. Bleeding resulted when severe thrombocytopenia and altered platelet function reduced the amount of platelet-derived serotonin below a critical threshold. Bleeding was facilitated by the absence of the activity of the kinase Lyn or the administration of aspirin, an inhibitor of arachidonic acid synthesis. Mouse platelets were not directly affected by IFN-I because they lack the receptor for the cytokine (IFNAR1), suggesting that transfusion of normal platelets into LCMV-infected mice could increase the amount of platelet-released serotonin and help to control hemorrhage.

Trauma hemorrhage (TH) and subsequent sepsis are well known to frequently result in severe organ damage. Although macrophage-activating lipopeptide-2 (MALP-2) has been described to exert beneficial effects on organ damage, and further clinical course after both isolated trauma and sepsis, little is known about the impact of MALP-2 in a clinically realistic two-hit scenario of TH and subsequent sepsis. As the liver represents a key organ for the posttraumatic immune response and development of complications, the effects of MALP-2 on the posttraumatic hepatic immunologic response and tissue damage were investigated in a murine "two-hit" model. In C57BL/6 mice, blood pressure-controlled (35 ± 5 mm Hg) TH was induced. Cecal ligation and puncture (CLP) was performed 48 h after TH. Mice were divided into two control groups (control 1, TH and laparotomy without CLP; control 2, TH and CLP) and three experimental groups (TH + CLP) treated with MALP-2 at different timepoints (ETH, end of TH; ECLP, end of CLP; 6CLP, 6 h after CLP). The observation time lasted for 168 h after induction of TH. Kupffer cells (KC) were isolated and cultured, and MPO activity was analyzed. Cell culture supernatants were taken for cytokine analysis (TNF-α, IL-6, MCP-1, GM-CSF, IL-10). Histological analysis was performed using the Hepatic Injury Severity Scoring (HISS). Statistical evaluation was carried out using SPSS (version 24.0.0; IBM, Armonk, NY, USA). MPO activity of control 1 group was lowest compared with all the other groups (p < 0.01). MPO activity of control 2 group was significantly higher than that in all experimental groups (ETH (p < 0.01), ECLP (p < 0.01), and 6CLP (p = 0.03)). Within the experimental groups, MPO activity was significantly reduced in the ETH (p = 0.04) and the ECLP (p < 0.01) groups compared with the 6CLP group. Moreover, ETH was also associated with the most pronounced reduction of cytokine expression by KC (p < 0.05). HISS revealed the largest damage in the group control 2. TH and subsequent sepsis lead to a distinct immunologic reaction in the liver with an increase of cytokine expression of KC and pronounced infiltration of granulocytes with associated severe tissue damage. MALP application decreases the hepatic immune response and liver damage, with the most pronounced effects if applied at the end of TH., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Cardiovascular disease is strongly influenced by platelet activation. Platelet activation and thrombus formation at atherosclerotic plaque rupture sites is a dynamic process regulated by different signaling networks. Therefore, there are now focused efforts to search for novel bioactive compounds which target receptors and pathways in the platelet activation process while preserving normal hemostatic function. The antiplatelet activity of numerous fruits and vegetables and their multiple mechanisms of action have recently been highlighted. In this review, we review the antiplatelet actions of bioactive compounds via key pathways (protein disulfide isomerase, mitogen-activated protein kinases, mitochondrial function, cyclic adenosine monophosphate, Akt, and shear stress-induced platelet aggregation) with no effects on bleeding time. Therefore, targeting these pathways might lead to the development of effective antiplatelet strategies that do not increase the risk of bleeding.

Background: Vascular injury induces the exposure of subendothelial extracellular matrix (ECM) important to serve as substrate for platelets to adhere to the injured vessel wall to avoid massive blood loss. Different ECM proteins are known to initiate platelet adhesion and activation. In atherosclerotic mice, the small, leucine-rich proteoglycan biglycan is important for the regulation of thrombin activity via heparin cofactor II. However, nothing is known about the role of biglycan for hemostasis and thrombosis under nonatherosclerotic conditions., Methods: The role of biglycan for platelet adhesion and thrombus formation was investigated using a recombinant protein and biglycan knockout mice., Results: The present study identified biglycan as important ECM protein for the adhesion and activation of platelets, and the formation of three-dimensional thrombi under flow conditions. Platelet adhesion to immobilized biglycan induces the reorganization of the platelet cytoskeleton. Mechanistically, biglycan binds and activates the major collagen receptor glycoprotein (GP)VI, because reduced platelet adhesion to recombinant biglycan was observed when GPVI was blocked and enhanced tyrosine phosphorylation in a GPVI-dependent manner was observed when platelets were stimulated with biglycan. In vivo, the deficiency of biglycan resulted in reduced platelet adhesion to the injured carotid artery and prolonged bleeding times., Conclusions: Loss of biglycan in the vessel wall of mice but not in platelets led to reduced platelet adhesion at the injured carotid artery and prolonged bleeding times, suggesting a crucial role for biglycan as ECM protein that binds and activates platelets via GPVI upon vessel injury.

Uncontrolled bleeding is thought to be the most deadly cause of pre-hospital, traffic, and military accidents death. However, the popular commercial hemostats can only realize the hemostasis of mild bleeding. Therefore, we developed polydopamine (PDA) composite materials (PMs), which applied hydroxyapatite as the parent body. The PMs were produced via lyophilization and functionalized with amino, phenol hydroxyls groups, which endowed hydrophobicity to materials. This ensured a high aggregation ability of blood cells to the PMs and they were tested to be as high as 300% compared with the negative control group. The clotting time was shortened to 79.7% compared with the usually used commercial hemostat (Celox) in the test of in vitro hemostasis. Through the results of PT and APTT tests, blood coagulation index test, and the analysis of intracellular Ca 2+ activation, we further understood the mechanism of the hemostasis of the materials, which explained the low blood loss and quick coagulation time of the PM hemostats in detail. Besides, the low hemolysis and cytotoxicity of the PMs suggested the good biocompatibility of the hemostats, which was further proved by the regular morphology maintained by erythrocytes in the hemolysis tests. The study of nanoscale composites led the research for the methods of hemostasis., (© 2021 Wiley Periodicals LLC.)

Rapid and effective hemorrhage control is essential to reduce mortality following traumatic injuries. Herein we developed an organic solvent-free process to prepare carboxymethyl chitin microsphere (CMCHm) in an aqueous two-phase system through heating and freeze-drying. To further enhance the hemostatic performance of CMCHm, we loaded calcium ions and in-situ polymerized dopamine to get modified hemostatic microspheres CMCHm-Ca 2+ and CMCHm-PDA, respectively. The size of these microspheres was mainly distributed between 50 μm and 150 μm, and the porous microstructure was observed by SEM. The data of in vitro degradation, cell cytotoxicity, and hemolysis test indicated good biocompatibility of these microspheres. Importantly, CMCHm-Ca 2+ and CMCHm-PDA displayed better hemostatic performance compared with CMCHm and the positive controls Yunnan baiyao® and Quickclean®. Especially, the bleeding time was reduced to 59 s (CMCHm-Ca 2+ ) and 45 s (CMCHm-PDA) in the femoral artery/vein cut model, respectively. All these demonstrate CMCHm-Ca 2+ and CMCHm-PDA hold great potential for rapid hemostasis., (Copyright © 2021. Published by Elsevier Ltd.)