Activation Of Blood Coagulation Research Articles

Biophysical characterization of blood coagulation factor VIII binding to lipid nanodiscs that mimic activated platelet surfaces.

Following proteolytic activation, activated blood coagulation factor VIII (FVIIIa) binds to activated platelet membranes, forming the intrinsic tenase complex with activated factor IX (FIXa). Previous studies have identified the C1 and C2 domains as the membrane binding domains of FVIII through conserved arginine residues. A membrane binding model for the FVIII C domains proposes that surface exposed hydrophobic and positively charged residues at each C domain interact with the membrane, yet a comprehensive thermodynamic and structural description of this interaction is lacking. To determine residues of interaction, thermodynamics, and membrane binding preference for FVIII membrane association. Binding of FVIII constructs to lipid nanodiscs were characterized with nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC), bio-layer interferometry (BLI), and X-ray crystallography. The thermodynamics of FVIII membrane binding indicate that the C1 domain associates through an enthalpically driven process while the C2 domain is entropically driven. Alanine mutations to surface-exposed hydrophobic residues in the C2 domain reveal differential effects on membrane binding, highlighting important determinants at the residue-level. The structure of a C2 double mutant, L2251A/L2252A, demonstrates that its decreased affinity is likely due to decreasing the surface area hydrophobicity. NMR studies with the C2 domain identified residues of interaction with soluble O-phospho-L-serine (OPLS) as well as lipid nanodiscs. Lastly, increasing phosphatidylethanolamine (PE) and decreasing PS content decreases overall FVIII affinity for membrane surfaces. This study provides further insight into the molecular basis for how FVIII interacts with platelets to form the intrinsic tenase complex.

Efficacy and Safety of Rivaroxaban, Apixaban, and Edoxaban for Nonvalvular Atrial Fibrillation Based on Blood Coagulation Activity and Drug Plasma Concentration: SETtsu and North Osaka Multicenter Direct Oral AntiCoagulant (SET DOAC) Registry

Background: The therapeutic effects of oral anticoagulant drugs for nonvalvular atrial fibrillation (NVAF) suggest that the three factor Xa (FXa) inhibitors may have distinct safety profiles, though this is not yet fully conclusive. This study investigated the current dosing of rivaroxaban, apixaban, and edoxaban by monitoring drug plasma concentration (PC) and coagulation activity from the viewpoint of the safety. Methods and results: This multicenter clinical study monitored the drug PC and two coagulation biomarkers (fibrinogen and fibrin monomer complex [FMC]) at peak and trough timing in 268 outpatients taking rivaroxaban (n = 72), apixaban (n = 71), and edoxaban (n = 125) for NVAF. Doses were adjusted based on the dose-adjustment criteria of each drug. Referencing our previous study, peak drug PC remained below the cut-off level for predicting bleeding events except in eight patients (rivaroxaban, n = 3; apixaban, n = 2; edoxaban, n = 3) in whom bleeding events occurred. Among them, two (one each on rivaroxaban and edoxaban) had a peak drug PC below the cut-off level. Drug PCs widely varied from peak to trough, whereas FMC levels, reflecting thrombin activity, remained within the normal range (<6.1 µg/mL) regardless of PC variations. These results indicated that the anticoagulant effects of these drugs persisted throughout the day regardless of the drug PC levels, dosage, and dosing frequency. Regarding the change over time in peak PC, the elevation over time developed more in rivaroxaban (29/57; 50.9%, p < 0.05) than in edoxaban (32/101; 31.7%), and rivaroxaban tended to accumulate more than edoxaban. Conclusions: Although drug PC levels of once-daily FXa inhibitors widely varied from peak to trough, FMC levels were maintained within the normal range without daily variations. Rivaroxaban also tended to accumulate over time. The results indicate the low risk of thrombotic events with once-daily FXa inhibitors and its correspondence to the twice-daily regimen.

Modulating Coagulation via Bioinspired Mesoporous Calcium-Decorated Silica Nanoparticles for Efficient Fibrin Clot Formation.

Blood clotting is vital for preventing bleeding after an injury. Hemostasis is a complex cascade involving numerous plasma proteins. Uncontrolled bleeding leads to mortality. The presence of Ca (calcium) activates and promotes the different phases in the coagulation cascade. Even nonbiological surfaces such as silicates may activate coagulation factor XII (FXII). This causes the clotting of the blood. The exceptional hemostatic ability of the mesoporous calcium-decorated silica nanoparticles (MCSNs) is achieved by stimulating the factors needed to form fibrin mesh, a durable clot, thereby establishing hemostasis. This may be used as a hemostatic agent during an accident surgical procedure and other bleeding-related trauma conditions. This study investigates the mechanistic activation of the coagulation cascade by MCSN through blood coagulation index, clotting time, and coagulation activation studies like PT and aPTT. Our finding demonstrates that MCSN induces platelet adhesion and RBC aggregation and activates thrombin generation through distinct pathways.

Blood Coagulation Activities and Influence on DNA Condition of Alginate-Calcium Composites Prepared by Freeze-Drying Technique.

The aim of this research was to synthesize and characterize alginate-calcium composites using a freeze-drying method, with a focus on their potential applications in biomedicine. This study specifically explored the biochemical properties of these composites, emphasizing their role in blood coagulation and their capacity to interact with DNA. Additionally, the research aimed to assess how the cross-linking process influences the structural and chemical characteristics of the composites. Detailed analyses, including microscopic examination, surface area assessment, and atomic absorption spectrometry, yielded significant results. The objective of this study was to examine the impact of calcium chloride concentration on the calcium content in alginate composites. Specifically, the study assessed how varying concentrations of the cross-linking solution (ranging from 0.5% to 2%) influence the calcium ion saturation within the composites. This investigation is essential for understanding the physicochemical properties of the materials, including calcium content, porosity, and specific surface area. The results are intended to identify the optimal cross-linking conditions that maximize calcium enrichment efficiency while preserving the material's structural integrity. The study found that higher calcium chloride concentrations in alginate cross-linking improve the formation of a porous structure, enhanced by two-stage freeze-drying. Increased calcium levels led to a larger surface area and pore volume, and significantly higher calcium content. Furthermore, assays of activated partial thromboplastin time (aPTT) showed a reduction in clotting time for alginate composites containing calcium ions, indicating their potential as hemostatic agents. The aPTT test showed shorter clotting times with higher calcium ion concentrations, without enhanced activation of the extrinsic clotting pathway. The developed alginate material with calcium effectively supports hemostasis and reduces the risk of infection. The study also explored the capacity of these composites to interact with and modify the structure of plasmid DNA, underscoring their potential for future biomedical applications.

Investigation of a Serine Protease Inhibitor Active in the Infectious Stage of the Human Liver Fluke Opisthorchis viverrini.

Serine protease inhibitors (serpins) participate in the regulation of inflammation, blood coagulation, and complement activation in humans. This research aimed to identify and characterize such inhibitors of the human liver fluke Opisthorchis viverrini. Parasite proteins that might contribute to the modulation of host physiology are of particular interest, especially as chronic opisthorchiasis increases the risk of developing biliary cancer. BLAST was used to find hypothetical serpins predicted from the parasite genome data. RNA extraction and reverse transcriptase PCR were used to isolate a serpin cDNA and to determine developmental transcript abundance. The evolutionary relation to other trematode serpins was revealed by phylogenetic analysis. Recombinant serpin was expressed in Escherichia coli and used to test the immunoreactivity of human opisthorchiasis sera and the inhibition of human serine proteases. A substantial serpin family with high sequence divergence among the members was found in the genus Opisthorchis. A serpin, different from previously analyzed trematode serpins, was cloned. The transcript was only detected in metacercariae and newly excysted juveniles. Human opisthorchiasis sera showed statistically significant reactivity to recombinant serpin. The serpin caused moderate inhibition of thrombin and low inhibition of kallikrein and chymotrypsin. This parasite serpin could be further evaluated as a diagnostic tool for early infection. Kallikrein and thrombin are involved in fibrinolysis; therefore, further research should explore the effects of the parasite serpin on this process.

15-lipoxygenase and serine protease inhibitory activities of Heritiera fomes (buch. -ham), a mangrove plant of the Sundarbans

15-Lipoxygenase (15-Lox) oxidizes fatty acid, thereby producing inflammatory mediators which cause diverse inflammatory diseases such as atherosclerosis, asthma, cancer etc. Serine proteases are enzymes that activate tissue factor VII by proteolytic cleavage, eventually activating blood coagulation pathway. Antioxidants play role in 15-Lox and serine protease inhibition to dampen elevation of cytokine levels, thereby function as anti-inflammation and anticoagulation agent. In this study, we examined 15-Lox and serine protease inhibitory activities of the ethanolic bark and leaf crude extract of Heritiera fomes. The crude bark extract (HFB) exhibited 15-Lox and serine protease inhibition with IC50 117.14 and 409.61 μg/ml, respectively. Chromatographically separated active fraction HFBPF1 demonstrated highest 15-Lox and serine protease inhibitory activity with IC50 62.68 and 72.62 μg/ml, respectively. The antioxidant activity and total flavonoid content were substantially increased in the chromatographic fractions compared to the crude extracts. The LC-MS/MS analysis of bark and leaf active fractions revealed the presence of 2,4,6-octatrienal, apigenin 7 (4˝-Z-p-coumerylglucoside), brosimacutin B, gambrin B2, kandelin A1 and tetraneurin A which we report for the first time in H. fomes. Therefore, we conclude the significant augmentation in the 15-Lox and serine protease inhibition activity of the fractions which could be exerted by the enriched flavonoid content.

Modeling cancer-associated hypercoagulability using glioblastoma spheroids in microfluidic chips

BackgroundCancer increases the risk of venous thromboembolism, and glioblastoma is one of the cancer types with the highest risk of venous thromboembolism (10%-30%). Tumor-intrinsic features are believed to affect vascular permeability and hypercoagulability, but novel models are required to study the pathophysiological dynamics underlying cancer-associated thrombosis at the molecular level. ObjectivesWe have developed a novel cancer-on-a-chip model to examine the effects of glioblastoma cells on the deregulation of blood coagulation. MethodsThis was accomplished by coculturing vessel-forming human umbilical vein endothelial cells with glioblastoma spheroids overexpressing tissue factor (TF), the initiator of coagulation (U251 lentivirus, LV-TF) or an LV-control (U251 LV-Ctrl) in an OrganoPlate Graft platform. ResultsUsing a modified thrombin generation assay inside the cancer-on-a-chip, we found that U251 LV-Ctrl and U251 LV-TF spheroids promoted an increased procoagulant state in plasma, as was shown by a 3.1- and 7.0-fold increase in endogenous thrombin potential, respectively. Furthermore, the anticoagulant drug rivaroxaban and TF coagulation-blocking antibody 5G9 inhibited the activation of blood coagulation in U251 LV-TF spheroid-containing graft plates, as was shown by a reduced endogenous thrombin potential (4.0- and 4.4-fold, respectively). ConclusionWith this study, we present a novel 3-dimensional cancer-on-a-chip model that has the potential to be used in the discovery of new anticoagulant drugs and identification of optimal anticoagulant strategies for glioblastoma and other cancer types.

Mesh-shaped absorbable hemostatic hydrogel patch fabricated with marine organism-derived protein biomaterials with contact-activated blood coagulation for application in visceral surgery

The utilization of efficacious hemostatic agents in surgical procedures has the potential to enhance operational efficacy, expedite patient recovery, and diminish the need for blood transfusions. However, in cases of excessive bleeding, few commercial hemostatic agents can maintain contact with the bleeding site and perform hemostatic functions effectively. In the present work, we propose a mesh-shaped absorbable lyophilized hemostatic hydrogel patch with excellent biodegradability, wet tissue adhesion ability, strong structural properties, and good blood coagulation activity by integrating the beneficial properties of two marine organism-derived bioengineered protein biomaterials, sea anemone silk-like protein (aneroin) and mussel adhesive protein (MAP). The hemostatic efficacy of the developed hydrogel patch was thoroughly evaluated using in vitro and in vivo experiments. The mesh-shaped hemostatic hydrogel patch exhibited the ability to absorb plasma upon contact with blood, adhere to surfaces, and facilitate aggregation of blood components, hence enhancing the process of blood coagulation. Using animal liver damage models, we confirmed that the hemostatic hydrogel patch could significantly enhance hemostatic capabilities by reducing clotting time and blood loss while exhibiting minimal inflammation and toxicity when remaining within the body. Thus, our proposed absorbable hemostatic hydrogel patch demonstrated great potential for use as a novel adhesive hemostatic agent for application in visceral surgery.

ГЕМОСТАТИЧЕСКИЕ НАРУШЕНИЯ ПРИ НЕОПУХОЛЕВОЙ МЕХАНИЧЕСКОЙ ЖЕЛТУХЕ И ХОЛАНГИТЕ В ЗАВИСИМОСТИ ОТ ТЯЖЕСТИ И ПОЛИМОРФИЗМА ГЕНА ФИБРИНОГЕНА

Background. Mechanical jaundice (MF) and cholangitis are considered to be of great significance in emergency surgery due to their complex pathogenesis, risk of complications and high mortality. Objective. To establish the severity of hemostatic disorders in association with fibrinogen gene polymorphism (FGB (G(-455)A)) in patients with non-cancerous MF and cholangitis of varying severity. Material and methods. The subjects were 25 patients with benign MF and cholangitis divided into 2 groups: group 1 (n=12) – those with a mild form of the disease; group 2 (n=13) – those with a severe form. The research methods included the assessment of endogenous intoxication, liver function as well as blood coagulation activity. Genetic analysis of the FGB (G(-455) geneA)) was performed using a polymerase chain reaction by Real–time PCR. The severity of the disease was determined using the V.D. Fedorov scale (2000). Results. The early stage of MF and cholangitis is accompanied by endotoxicosis, resulting in the damage of a number of organs and systems, in particular the liver. The latter plays an important role in changing the activity of blood coagulation system. These disorders had a strong correlation with the severity of the disease: in mild cases, the character was less pronounced and reversible, and in severe cases – persistent and severe. Hemostatic disorders recorded in the early period of the disease depend on the severity: in mild cases, hypercoagulation and normofibrinolysis are noted, and in severe cases – hypocoagulation and hypofibrinolysis. Genetic polymorphism of the fibrinogen gene FGB (G(-455)A) had a significant impact on the pathogenetic process of MF and cholangitis. Patients with the mutant genotype (A/A) of the FGB gene were found to show the greatest severity of homeostasis system violation when compared with patients with genotypes G/G and G/A – with minimal imbalance. Conclusions. Hemostatic disorders associated with polymorphism of the fibrinogen gene FGB (G(-455)A) play an important role in the pathogenesis of MF and cholangitis.

Site-Specific Analysis of Core and Antenna Fucosylation on Serum Glycoproteins.

Fucosylation is an important structural feature of glycans and plays an essential role in the regulation of glycoprotein functions. Fucosylation can be classified into core- (CF) and antenna-fucosylation (AF, also known as (sialyl-) Lewis) based on the location on N-glycans, and they perform distinct biological functions. In this study, core- and antenna-fucosylated N-glycans on human serum glycoproteins that hold great clinical application values were systematically characterized at the site-specific level using StrucGP combined with the recently developed fucosylation assignment method. The results showed that fucosylation was widely distributed on serum glycoproteins, with 50% of fucosylated glycopeptides modified by AF N-glycans, 37% by CF N-glycans, and 13% by dual-fucosylated N-glycans. Interestingly, CF and AF N-glycans preferred to modify different groups of serum glycoproteins with different tissue origins and were involved in distinctive biological processes. Specifically, AF N-glycoproteins are mainly from the liver and participated in complement activation, blood coagulation, and endopeptidase activities, while CF N-glycoproteins originate from diverse tissues and are mainly involved in cell adhesion and signaling transduction. These data further enhanced our understanding of fucosylation on circulation glycoproteins.

The Role of Myeloid Cells in Thromboinflammatory Disease.

Inflammation contributes to the development of thrombosis, but the mechanistic basis for this association remains poorly understood. Innate immune responses and coagulation pathways are activated in parallel following infection or injury, and represent an important host defense mechanism to limit pathogen spread in the bloodstream. However, dysregulated proinflammatory activity is implicated in the progression of venous thromboembolism and arterial thrombosis. In this review, we focus on the role of myeloid cells in propagating thromboinflammation in acute inflammatory conditions, such as sepsis and coronavirus disease 2019 (COVID-19), and chronic inflammatory conditions, such as obesity, atherosclerosis, and inflammatory bowel disease. Myeloid cells are considered key drivers of thromboinflammation via upregulated tissue factor activity, formation of neutrophil extracellular traps (NETs), contact pathway activation, and aberrant coagulation factor-mediated protease-activated receptor (PAR) signaling. We discuss how strategies to target the intersection between myeloid cell-mediated inflammation and activation of blood coagulation represent an exciting new approach to combat immunothrombosis. Specifically, repurposed anti-inflammatory drugs, immunometabolic regulators, and NETosis inhibitors present opportunities that have the potential to dampen immunothrombotic activity without interfering with hemostasis. Such therapies could have far-reaching benefits for patient care across many thromboinflammatory conditions.

The Inhibition of Serine Proteases by Serpins Is Augmented by Negatively Charged Heparin: A Concise Review of Some Clinically Relevant Interactions.

Serine proteases are members of a large family of hydrolytic enzymes in which a particular serine residue in the active site performs an essential role as a nucleophile, which is required for their proteolytic cleavage function. The array of functions performed by serine proteases is vast and includes, among others, the following: (i) the ability to fight infections; (ii) the activation of blood coagulation or blood clot lysis systems; (iii) the activation of digestive enzymes; and (iv) reproduction. Serine protease activity is highly regulated by multiple families of protease inhibitors, known collectively as the SERine Protease INhibitor (SERPIN). The serpins use a conformational change mechanism to inhibit proteases in an irreversible way. The unusual conformational change required for serpin function provides an elegant opportunity for allosteric regulation by the binding of cofactors, of which the most well-studied is heparin. The goal of this review is to discuss some of the clinically relevant serine protease-serpin interactions that may be enhanced by heparin or other negatively charged polysaccharides. The paired serine protease-serpin in the framework of heparin that we review includes the following: thrombin-antithrombin III, plasmin-anti-plasmin, C1 esterase/kallikrein-C1 esterase inhibitor, and furin/TMPRSS2 (serine protease Transmembrane Protease 2)-alpha-1-antitrypsin, with the latter in the context of COVID-19 and prostate cancer.

Transcriptome analysis of cultured human vascular endothelial cells after γ-ray irradiation and correlation analysis with ATP, ADP, and adenosine as secondary soluble factors.

Vascular endothelial cells serve as barriers between blood components and subendothelial tissue and regulate the blood coagulation-fibrinolytic system. Ionizing radiation is a common physical stimulant that induces a bystander effect whereby irradiated cells influence neighboring cells through signalings, including purinergic receptor signaling, activated by adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine as secondary soluble factors. Human vascular endothelial EA.hy926 cells were cultured and irradiated with γ-rays or treated with ATP, ADP, or adenosine under non-toxic conditions. RNA-seq, gene ontology, and hierarchical clustering analyses were performed. The transcriptome analysis of differentially expressed genes in vascular endothelial cells after γ-ray irradiations suggests that the change of gene expression by γ-irradiation is mediated by ATP and ADP. In addition, the expression and activity of the proteins related to blood coagulation and fibrinolysis systems appear to be secondarily regulated by ATP and ADP in vascular endothelial cells after exposure to γ-irradiation. Although it is unclear whether the changes of the gene expression related to blood coagulation and fibrinolysis systems by γ-irradiation affected the increased hemorrhagic tendency through the exposure to γ-irradiation or the negative feedback to the activated blood coagulation system, the present data indicate that toxicity associated with γ-irradiation involves the dysfunction of vascular endothelial cells related to the blood coagulation-fibrinolytic system, which is mediated by the signalings, including purinergic receptor signaling, activated by ATP and ADP.

Dependence of coagulation activity of chicken blood IN VITRO on temperature

The paper presents the results of examine the relationship between blood plasma incubation temperature and the hemostasis parameters in chickens. The purpose of this research was to study of the blood coagulation parameters in chickens during hypo-, normo- and hyperthermia in vitro. In addition, to assess the possibility of using a coagulometer to identify hemostasis activity and the use of hemostatic reactions in chickens as models for human medicine. The Vologda region was where the study was conducted. The coagulogram was examined for the following parameters: thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen activity, and antithrombin III activity (AT-III). It was found that at 46° C activation of blood clotting occurs, manifested as an acceleration of the TT by 29% (13 seconds), PT by 72% (111 seconds), and decrease in the antithrombin III activity by 6% (1.5 seconds) compared to normothermia. The parameters of secondary hemostasis also responded to hypothermia (18 °C) with hypercoagulation. It was an acceleration of PT of 81% (125 sec), an increase in fibrinogen activity by 77% (84 sec) and a decrease in AT-III activity by 55% (14 sec), with the exception of thrombin time, which was extended by 70% (104 seconds). There were no significant changes in the APTT at different temperatures. The correlation-regression and one-factor analysis of variance revealed a significant correlation between the temperature and the TT and the fibrinogen activity, where the coefficients of determination for which were 71% and 39%, respectively. Plasma hemostasis activity measured on a coagulometer (at 37 °C) showed no significant differences from those measured at 43 °C, except for prothrombin time, which was 71.51 seconds (47%) less than that measured at 43 °C.

Green Solvent-Based Antifouling Polymer Brushes Demonstrate Excellent Hemocompatibility.

Medical devices are crucial for patient care, yet even the best biomaterials lead to infections and unwanted activation of blood coagulation, potentially being life-threatening. While hydrophilic polymer brushes are the best coatings to mitigate these issues, their reliance on fossil raw materials underscores the urgency of bio-based alternatives. In this work, we introduce polymer brushes of a green solvent-based monomer, prohibiting protein adsorption, bacterial colonization, and blood clot formation at the same level as fossil-based polymer brushes. The polymer brushes are composed of N,N-dimethyl lactamide acrylate (DMLA), can be polymerized in a controlled manner, and show strong hydrophilicity as determined by thermodynamic analysis of the surface tension components. The contact of various challenging protein solutions results in repellency on the poly(DMLA) brushes. Furthermore, the poly(DMLA) brushes completely prevent the adhesion and colonization of Escherichia coli. Remarkably, upon blood contact, the poly(DMLA) brushes successfully prevent the formation of a fibrin network and leukocyte adhesion on the surface. While showcasing excellent antifouling properties similar to those of N-hydroxypropyl methacrylamide (HPMA) polymer brushes as one of the best antifouling coatings, the absence of hydroxyl groups prevents activation of the complement system in blood. We envision the polymer brushes to contribute to the future of hemocompatible coatings.

Blood Coagulation Activities of Cotton-Alginate-Copper Composites.

Alginate-based materials have gained significant attention in the medical industry due to their biochemical properties. In this article, we aimed to synthesize Cotton-Alginate-Copper Composite Materials (COT-Alg(-)Cu(2+)). The main purpose of this study was to assess the biochemical properties of new composites in the area of blood plasma coagulation processes, including activated partial thromboplastin time (aPTT), prothrombin time (PT), and thrombin time (TT). This study also involved in vitro antimicrobial activity evaluation of materials against representative colonies of Gram-positive and Gram-negative bacteria and antifungal susceptibility tests. The materials were prepared by immersing cotton fibers in an aqueous solution of sodium alginate, followed by ionic cross-linking of alginate chains within the fibers with Cu(II) ions to yield antimicrobial activity. The results showed that the obtained cotton-alginate-copper composites were promising materials to be used in biomedical applications, e.g., wound dressing.

A Syngeneic Murine Model of Antiphospholipid Syndrome (APS) Recapitulates Aspects of Human APS

Background Antiphospholipid syndrome (APS) is characterized by arterial or venous thrombosis, or recurrent pregnancy loss, in the presence of persistently positive tests for antiphospholipid antibodies (aPL). aPL include lupus anticoagulants (LAC), anti-β2-glycoprotein 1 (β2GPI) and anti-cardiolipin antibodies (aCL). Among them, antibodies directed toward β2GPI have been best characterized and shown in most studies to have a significant association with thrombosis. Most murine models of APS are generated by infusion of either polyclonal IgG isolated from APS patients, or monoclonal IgG directed against human or murine β2GPI. However, one or more of the following limitations may limit their clinical relevance: 1) Unlike chronically elevated autologous aPL in APS patients, cross-species IgG infused into mice circulate for only limited periods of time and may provoke allogeneic responses; 2) Monoclonal human or murine anti-β2GPI IgG, unlike polyclonal aPL from patients, tend to be high affinity, monospecific, and may recognize epitopes different than those recognized by clinical aPL. Hence, we sought to develop a relevant syngeneic murine model of APS that employed polyclonal murine anti-β2GPI IgG that would circulate in mice for extended periods, and not induce allogeneic inflammatory responses. Methods β2GPI-deficient ( apoh -/-) mice were previously developed in our lab on a C57/BL6 background using CRISPR/cas9. IgG isolated from apoh -/- mice immunized with human plasma β2GPI was used as a source of polyclonal murine aPL IgG, while IgG isolated from apoh -/- mice treated with adjuvant alone served as control IgG in all experiments. Wild-type (WT) C57/BL6 mice (two groups of 8 mice each) were injected intravenously with 20 µg of either murine polyclonal aPL or control IgG, and after 72 hours were evaluated for venous thrombosis using the IVC stasis model, with thrombi harvested and measured 48 h after IVC ligation. In parallel studies, we injected mice with aPL or control IgG and evaluated 1) activation of washed platelets, 2) aortic wall gene expression, and 3) levels of circulating neutrophil extracellular traps (NETs) using blood and tissue samples isolated at identical time points as IVC ligation studies (mice in these studies did not undergo IVC ligation). Platelet activation was assessed by measuring the binding of JON/A and expression of platelet P-selectin by flow-cytometry. Total RNA isolated from aortic walls of 3 mice each treated with murine aPL or control IgG was analyzed by RNA sequencing. Results WT C57/BL6 mice that received 20 µg of polyclonal murine aPL sustained plasma anti-β2GPI IgG levels >500 ng/mL for more than 120 h after IgG injection. In the IVC stasis model, six out of eight mice receiving aPL IgG and four out of eight mice receiving control IgG developed thrombi (lack of thrombi in all mice was likely due to bleeding of extensive IVC back branches in C57/BL6 mice). Thrombi weight in aPL-treated mice were significantly larger than those from mice that received control IgG (19.7 ± 1.7 mg vs 13.5 ± 1.0 mg, P = 0.015). Thrombin (0.1 U/ml)-induced platelet activation was significantly greater for platelets isolated from aPL-administered mice compared to those from control mice 72h after IgG injection as determined by binding of JON/A (1246.7 ± 55.3 MFI vs 879 ± 37.9 MFI, P = 0.005) and expression of P-selectin (5729.7 ± 553.9 MFI vs 3777.3 ± 402.5 MFI, P=0.046). Gene enrichment analysis of mRNA from aortas demonstrated significant upregulation of gene pathways related to complement and coagulation cascades, blood coagulation, and oxidoreductase activity in the aPL-treated mice. Individually upregulated genes included C8, C9 fga and fgb. Elevated levels of circulating nucleosomes were also observed in aPL treated mice but did not reach significance due to small numbers. Conclusion These results demonstrate that anti-β2GPI polyclonal aPL raised in apoh-/- mice circulate for extended periods when reinfused into WT mice, enhance thrombosis, induce platelet hyperreactivity, and cause changes in mRNA expression in the vascular wall. Further characterization of anti-β2GPI IgG for LAC activity, anti-PS/PT reactivity and β2GPI domain 1 reactivity is in progress. While all animal models of APS have weaknesses, this syngeneic model provides an additional approach that recapitulates many aspects of vascular inflammation seen in human APS.

Sterilized Polyhexanide-Releasing Chitosan Membranes with Potential for Use in Antimicrobial Wound Dressings.

Wound infection is a common complication of chronic wounds. It can impair healing, which may not occur without external help. Antimicrobial dressings (AMDs) are a type of external help to infected chronic wounds. In this study, highly porous membranes made of only chitosan and containing the antiseptic polyhexanide (poly(hexamethylene biguanide); PHMB) were prepared by cryogelation, aiming to be used in AMDs. These membranes exhibited a water swelling capacity of 748%, a water drop penetration time of 11 s in a dry membrane and a water vapor transmission rate of 34,400 g H2O/m2/24 h when in contact with water. The best drug loading method involved simultaneous loading by soaking in a PHMB solution and sterilization by autoclaving, resulting in sterilized, drug-loaded membranes. When these membranes and a commercial PHMB-releasing AMD were assayed under the same conditions, albeit far from the in vivo conditions, their drug release kinetics were comparable, releasing PHMB for ca. 6 and 4 h, respectively. These membranes exhibited high antibacterial activity against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, which are bacterial species commonly found in infected wounds and blood clotting activity. The obtained results suggest that these membranes may have potential for use in the development of AMDs.

GRSF1 antagonizes age-associated hypercoagulability via modulation of fibrinogen mRNA stability

Age-associated hypercoagulability is accompanied by the increase of plasma levels of some coagulation factors including fibrinogen which may contribute to the increased risk of cardiovascular, cerebrovascular, and thrombotic diseases in elderly people. However, the underlying mechanism of increased plasma fibrinogen concentration during aging is still elusive. GRSF1 belongs to the heterogeneous nuclear ribonucleoproteins F/H (hnRNP F/H) subfamily. Here, we report that GRSF1 attenuates hypercoagulability via negative modulation of fibrinogen expression. We demonstrated that GRSF1 negatively regulated fibrinogen expression at both mRNA and protein levels. GRSF1 directly interacted with the coding region (CDS) of FGA, FGB, and FGG mRNAs, and decreased their stability thus mitigating fibrinogen expression. We further identified that only a few G-tracts within the Fib C domain of FGA, FGB, and FGG CDS and the qRRM2 domain of GRSF1 were required for their interaction. Moreover, we confirmed hypercoagulability and the decrease of GRSF1 expression level during mice aging. Functionally, GRSF1 overexpression in old mice liver decreased fibrinogen plasma level, reduced hypercoagulability, and mitigated blood coagulation activity, whereas GRSF1 knockdown in young mice liver increased fibrinogen plasma level and promoted blood coagulation activity. Collectively, our findings unveil a novel posttranscriptional regulation of fibrinogen by GRSF1 and uncover a critical role of GRSF1 in regulating blood coagulation activity.

Leukemia Inhibitory Factor, Tissue Factor, Thrombomodulin, P-Selectine, D-Dimer, Thrombin Generation and Procoagulant Phospholipid Dependent Clotting Time Are Predictive for in Vitro Fertilization Outcome. the Prospective Roadmap-IVF Study

Background The link between blood hypercoagulability, endothelial cell activation and infertility or in vitro fertilization (IVF) failure is a puzzling issue. Activation of platelets, endothelial cells and blood coagulation, proinflammatory and angiogenetic potential play important role in implantation and early embryonic development. Biomarkers of cellular hypercoagulability are associated with high risk of IVF failure. Soluble Leukemia Inhibitory Factor (LIF), an interleukin 6 class cytokine expressed in the trophectoderm of the developing embryo is an important regulator in the establishment of pregnancy. Endoglin (Eng), a co-receptor for TGF-β1 and TGF-β3 is implicated in vascular complications of pregnancy and particularly in preeclampsia. Aim: The prospective longitudinal monocentric observational cohort study ROADMAP-IVF, evaluated the synergistic prognostic value of LIF and Eng with the biomarkers of cellular hypercoagulability in predicting the endometrial receptivity of fresh IVF cycles. Materials and Methods. The ROADMAP-IVF enrolled 40 women eligible for IVF with normal blood count, PT, aPTT, Fg, renal and liver function. The control group (CG) consisted of 30 healthy women with history of uncomplicated pregnancies. Exclusion criteria: Use of anticoagulant or antiplatelet agents during the last 30 days before inclusion. Known cardiovascular disease, active cancer. Active corticosteroid treatment. Primary end-point: Echographically documented pregnancy at 7 weeks from implantation. Blood was collected on day 2 (D2) from the natural cycle and 6 days after hormone treatment initiation corresponding to D8 of the cycle. Procoagulant phospholipid-dependent clotting time (PPL-ct), tissue factor (TF), thrombomodulin (TM), von Willebrand factor (vWF) and D-Dimers (DDi) were measured with assays from Diagnostica Stago (Asnieres, France). P- and E-Selectin, LIF and Eng were measured with ELISA. Thrombin generation (TG) with the TF 5 pM PPP-Reagent ® was assessed on Calibrated Automated Thrombogram (Stago, France). The cut-off values for studied biomarkers have been selected on the basis of ROC analysis. Univariate and multivariate logistic regression analysis examined the associations between the biomarkers and the study outcome. Results: 40 women were enrolled in the study. In 11 women pregnancy was confirmed with ultrasound and in 23 women no pregnancy was echographically documented. Age was not significantly different between the IVF (41 ys; range 22 - 48 ys) and the control group (40 ys; range 20 - 46 ys). At D2 and D8 the DDi and LIF were significantly higher in IVFG as compared to the CG. At D2 most of the patients had PPL-ct, DDi and MRI of TG higher than the upper normal limit (UNL). At D8 only TF was significantly increased as compared to the CG. Analytical data are presented in Table 1. Age did not correlate with the levels of the studied biomarkers. Significant correlations (p<0.05) were found between DDi and ETP (r=0.35); TM, TF and P-Selectin (r=0.4) at D2; TF, LIF and Eng (r=0.5) at D2 and D8; P-Selectin and PPL-ct (r=-0.4) at D2; P-Selectin and TM (r=0.4), E-Selectin (r=0.5), LIF (0.4) at D2; E-Selectin and P-Selectin (r=0.5), TF (r=0.3), and LIF (r=0.4) at D2. The ROC analysis showed that IVF failure was associate with DDi increase at D2 (AUC = 0.64); P-Selectin increase at D2(AUC = 0.57) and D8 (AUC = 0.61); TM increase at D8 (AUC = 0.61); TF increase at D8 (AUC = 0.57); LIF increase at D8 (AUC = 0.59); ETP increase at D2 (AUC = 0.67) and D8 (AUC = 0.67); Peak increase at D2 (AUC = 0.64) and D8 (AUC = 0.61). Prolongation of PPL-ct on D8 was predictive for positive pregnancy outcome(AUC 0.64). Conclusion The ROADMAP-IVF study showed that at least one out of four women eligible for IVF present biological evidence of activation of platelets, endothelial cells, blood coagulation or LIF upregulation. Hormone treatment administration does not significantly alter the proinflammatory or the hypercoagulable state. Among the studied biomarkers the levels of LIF, DDi, P-Selectine, TM and TF as well as the status of thrombin generation and PPL-ct showed a significant predictive value for the IVF outcome, particularly when measured at 6 days after hormone treatment administration. These findings will lead to the elaboration of an risk assessment model for IVF failure combining the selected biomarkers of hypercoagulability with LIF.