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Abstract: Coagulation factor IX plays a central role in hemostasis through interaction with factor VIIIa to form a factor X-activating complex at the site of injury. The absence of factor IX activity results in the bleeding disorder hemophilia B. This absence of activity can arise either from a lack of circulating factor IX protein or mutations that decrease the activity of factor IX. This review focuses on analyzing the structure of factor IX with respect to molecular mechanisms that are at the basis of factor IX function. The proteolytic activation of factor IX to form activated factor IX(a) and subsequent structural rearrangements are insufficient to generate the fully active factor IXa. Multiple specific interactions between factor IXa, the cofactor VIIIa, and the physiological substrate factor X further alter the factor IXa structure to achieve the full enzymatic activity of factor IXa. Factor IXa also interacts with inhibitors, extravascular proteins, and cellular receptors that clear factor IX(a) from the circulation. Hemophilia B is treated by replacement of the missing factor IX by plasma-derived protein, a recombinant bioequivalent, or via gene therapy. An understanding of how the function of factor IX is tied to structure leads to modified forms of factor IX that have increased residence time in circulation, higher functional activity, protection from inhibition, and even activity in the absence of factor VIIIa. These modified forms of factor IX have the potential to significantly improve therapy for patients with hemophilia B., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Deformability and sickling of red blood cells (RBCs) from individuals with sickle cell trait (SCT) was evaluated under harsh biophysical conditions that mimic certain vascular beds in vivo. RBC deformability in osmotic-gradient ektacytometry was decreased in HbAS (SCT) compared to HbAA (wild-type) RBCs at supraphysiological osmolalities. RBC deformability was also measured by oxygen-gradient ektacytometry. Whereas RBC sickling was not observed under isotonic and neutral pH conditions, hypertonicity and acidosis alone or in combination induced reversible sickling of SCT RBC. These data suggest that hyperosmolality and/or acidosis enhance hypoxia-induced sickling of SCT RBC., (© 2024 British Society for Haematology and John Wiley & Sons Ltd.)

Background: In healthy individuals, plasma levels of clotting proteins naturally vary within a range of 50% to 150% of their mean values. We do not know how these variations modify thrombin generation., Objectives: To assess the impact of protein level variations on simulated thrombin generation in normal and factor (F)VIII-, FIX-, or FXI-deficient blood., Methods: We used a mathematical model of flow-mediated coagulation to simulate thrombin generation with all possible combinations of clotting protein variations within the normal range and for various tissue factor levels. We selected, analyzed, and ranked combinations that enhanced thrombin generation compared with baseline., Results: Protein variations most strongly affected thrombin generation at intermediate tissue factor levels. Low tissue factor levels prevented coagulation initiation, while high tissue factor levels always triggered thrombin generation. At intermediate levels, we identified protein variations that substantially modified thrombin generation. Low-normal FV shortened lag times and increased thrombin generation, whereas high-normal FV lengthened lag times and reduced thrombin generation. With severe FVIII and FIX deficiencies, low-normal tissue factor pathway inhibitor α and antithrombin amplified the effect of low-normal FV. For moderate FVIII and FIX deficiencies, high-normal tissue factor pathway inhibitor α and antithrombin enhanced the impact of high-normal FV in reducing thrombin production. In normal and FXI-deficient blood, high-normal FVIII and FIX significantly boosted thrombin generation., Conclusion: Our mathematical model predicted how variations in clotting protein levels, within the normal range, could contribute to the variability of bleeding phenotypes observed with clotting factor deficiencies. Our study generated experimentally testable hypotheses that could aid in developing new therapies toward normal hemostasis., (© 2024 The Author(s).)

Blood coagulation is a network of biochemical reactions wherein dozens of proteins act collectively to initiate a rapid clotting response. Coagulation reactions are lipid-surface dependent, and this dependence is thought to help localize coagulation to the site of injury and enhance the association between reactants. Current mathematical models of coagulation either do not consider lipid as a variable or do not agree with experiments where lipid concentrations were varied. Since there is no analytic rate law that depends on lipid, only apparent rate constants can be derived from enzyme kinetic experiments. We developed a new mathematical framework for modeling enzymes reactions in the presence of lipid vesicles. Here the concentrations are such that only a fraction of the vesicles harbor bound enzymes and the rest remain empty. We call the lipid vesicles with and without enzyme TF:VIIa + and TF:VIIa - lipid, respectively. Since substrate binds to both TF:VIIa + and TF:VIIa - lipid, our model shows that excess empty lipid acts as a strong sink for substrate. We used our framework to derive an analytic rate equation and performed constrained optimization to estimate a single, global set of intrinsic rates for the enzyme-substrate pair. Results agree with experiments and reveal a critical lipid concentration where the conversion rate of the substrate is maximized, a phenomenon known as the template effect. Next, we included product inhibition of the enzyme and derived the corresponding rate equations, which enables kinetic studies of more complex reactions. Our combined experimental and mathematical study provides a general framework for uncovering the mechanisms by which lipid mediated reactions impact coagulation processes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Until recently, the treatment of hemophilia A relied on factor (F)VIII replacement. However, up to one-third of patients with severe hemophilia A develop neutralizing alloantibodies that render replacement therapies ineffective. The development of emicizumab, a bispecific antibody that partially mimics FVIIIa, has revolutionized the treatment of these patients. However, the use of an activated prothrombin complex concentrate [FEIBA (Takeda)] to treat breakthrough bleeding in patients on emicizumab has been associated with thrombotic complications including a unique microangiopathy., Objectives: We hypothesized that the thrombotic complications observed with the combination of emicizumab and FEIBA might be due to excessive expression of procoagulant activity on the surface of endothelial cells., Methods: We examined the ability of emicizumab to promote FX activation on endothelial cells using 2 cell culture models., Results: We found that endothelial cells readily support emicizumab-mediated activation of FX by FIXa. The level of FXa generation depends on the concentration of available FIXa. The addition of FEIBA to emicizumab increased FXa generation in a dose-dependent manner on endothelial cells in both models. The rate of FXa generation was further enhanced by endothelial cell activation. However, unlike emicizumab, we found limited FXa generation in the presence of FVIII(a), which followed a significant lag time and was not dependent on FIXa concentration under these conditions., Conclusion: Emicizumab promotes FXa generation on the surface of endothelial cells, which is markedly enhanced in the presence of FEIBA. These findings demonstrate a potential mechanism for the thrombotic complications seen with the combined use of emicizumab and FEIBA., Competing Interests: Declaration of competing interests M.H. received investigator-initiated funding from Takeda. There are no other competing interests to disclose., (Published by Elsevier Inc.)

Background: Factor (F) XI deficiency is associated with increased bleeding risk in some individuals. Neither FXI levels nor clinical clotting assays predict the bleeding risk. Compared with controls, FXI-deficient bleeders have reduced clot formation, decreased fibrin network density, and increased susceptibility to fibrinolysis. Tissue factor pathway inhibitor (TFPI) was recently implicated as a modifying factor in individuals with bleeding of unknown cause., Objectives: To determine the potential of TFPI in modifying the bleeding risk in FXI-deficient individuals., Methods: The effects of TFPI on thrombin generation and clot formation, structure, and fibrinolysis in FXI-deficient plasma were measured in vitro in the absence or presence of inhibitory anti-TFPI antibody or exogenous recombinant TFPIα. Total plasma TFPI concentration was measured in 2 independent cohorts of controls and FXI-deficient individuals classified as bleeders or nonbleeders (cohort 1: 10 controls and 16 FXI-deficient individuals; cohort 2: 48 controls and 57 FXI-deficient individuals) and correlated with ex vivo plasma clot formation and fibrinolysis parameters associated with bleeding risk., Results: In an in vitro FXI deficiency model, inhibition of TFPI enhanced thrombin generation and clot formation, increased the network density, and decreased fibrinolysis, whereas an increase in TFPI had the opposite effects. Compared with controls, plasma from FXI-deficient bleeders had higher TFPI concentration. Total plasma TFPI concentrations correlated with parameters from ex vivo clotting and fibrinolysis assays that differentiate FXI-deficient bleeders and nonbleeders., Conclusion: Coagulation and fibrinolysis parameters that differentiate FXI-deficient nonbleeders and bleeders were altered by plasma TFPIα. Total plasma TFPI was increased in FXI-deficient bleeders. TFPI may modify the bleeding risk in FXI-deficient individuals., Competing Interests: Declaration of competing interest A.E.M. receives research funding and honoraria from Novo Nordisk. No other authors have relevant competing interests to disclose., (Copyright © 2022 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

This review aims to consolidate the relevant published data exploring the amino acid (AA) requirements of Nile tilapia, Oreochromis niloticus , and to reach a new set of recommendations based on those data. There are still inconsistencies in lysine, sulfur-containing AA, threonine, tryptophan, branched-chain AA, and total aromatic AA recommendations in data that have appeared since 1988. This review finds that strain, size, basal diet composition, and assessment method may have contributed to the inconsistencies in AA recommendations. Currently, the expansion of precision AA nutrition diets for Nile tilapia is receiving more attention because of the demand for flexibility in widespread ingredient substitutions which will allow compliance with environmentally sustainable principles. Such approaches involve changes in diet ingredient composition with possible inclusions of non-bound essential and non-essential AAs. Increasing the inclusion of non-bound AAs into Nile tilapia diets may modify protein dynamics and influence AA requirements. Emerging evidence indicates that not only essential but also some non-essential amino acids regulate growth performance, fillet yield, and flesh quality, as well as reproductive performance, gut morphology, intestinal microbiota, and immune responses. Thus, this review considers current AA recommendations for Nile tilapia and proposes refinements that may better serve the needs of the tilapia industry.

Despite their limitations, unfractionated heparin (UFH) and bivalirudin remain standard-of-care parenteral anticoagulants for percutaneous coronary intervention (PCI). We discovered novel direct thrombin inhibitors (DTIs) from tick salivary transcriptomes and optimised their pharmacologic activity. The most potent, ultravariegin, inhibits thrombin with a K i of 4.0 pM, 445-fold better than bivalirudin. Unexpectedly, despite their greater antithrombotic effect, variegin/ultravariegin demonstrated less bleeding, achieving a 3-to-7-fold wider therapeutic index in rodent thrombosis and bleeding models. When used in combination with aspirin and ticagrelor in a porcine model, variegin/ultravariegin reduced stent thrombosis compared with antiplatelet therapy alone but achieved a 5-to-7-fold lower bleeding time than UFH/bivalirudin. Moreover, two antibodies screened from a naïve human antibody library effectively reversed the anticoagulant activity of ultravariegin, demonstrating proof-of-principle for antidote reversal. Variegin and ultravariegin are promising translational candidates for next-generation DTIs that may reduce peri-PCI bleeding in the presence of antiplatelet therapy., (© 2021. The Author(s).)

Computational models of various facets of hemostasis and thrombosis have increased substantially in the last decade. These models have the potential to make predictions that can uncover new mechanisms within the complex dynamics of thrombus formation. However, these predictions are only as good as the data and assumptions they are built upon, and therefore model building requires intimate coupling with experiments. The objective of this article is to guide the reader through how a computational model is built and how it can inform and be refined by experiments. This is accomplished by answering six questions facing the model builder: (1) Why make a model? (2) What kind of model should be built? (3) How is the model built? (4) Is the model a "good" model? (5) Do we believe the model? (6) Is the model useful? These questions are answered in the context of a model of thrombus formation that has been successfully applied to understanding the interplay between blood flow, platelet deposition, and coagulation and in identifying potential modifiers of thrombin generation in hemophilia A., Competing Interests: K.B.N. reports grants from National Institutes of Health, during the conduct of the study. D.M.M. reports grants from Army Research Office, during the conduct of the study., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Bleeding frequency and severity within clinical categories of hemophilia A are highly variable and the origin of this variation is unknown. Solving this mystery in coagulation requires the generation and analysis of large data sets comprised of experimental outputs or patient samples, both of which are subject to limited availability. In this review, we describe how a computationally driven approach bypasses such limitations by generating large synthetic patient data sets. These data sets were created with a mechanistic mathematical model, by varying the model inputs, clotting factor, and inhibitor concentrations, within normal physiological ranges. Specific mathematical metrics were chosen from the model output, used as a surrogate measure for bleeding severity, and statistically analyzed for further exploration and hypothesis generation. We highlight results from our recent study that employed this computationally driven approach to identify FV (factor V) as a key modifier of thrombin generation in mild to moderate hemophilia A, which was confirmed with complementary experimental assays. The mathematical model was used further to propose a potential mechanism for these observations whereby thrombin generation is rescued in FVIII-deficient plasma due to reduced substrate competition between FV and FVIII for FXa (activated factor X).

Mounting evidence suggests that a variety of disease states are pathophysiologically related to activation of the contact system in vivo. The plasma contact system is composed of a cascade of serine proteases initiated by surface activation of factor XII, which can then proceed through a procoagulant pathway by activating the intrinsic coagulation factor XI, or a proinflammatory pathway by activating prekallikrein. Serpins are the primary endogenous inhibitors of the contact system, which irreversibly inhibit their respective protease(s), forming a stable complex. We modified an existing assay strategy for detecting these complexes in plasma using ELISAs and determined the effect of preanalytical variation caused by anticoagulant selection and processing time. The assays were sensitive and specific to inherited deficiency of individual contact factors. We conclude that these assays are robust and represent a relatively simple approach to the assessment of contact factor activation in plasma samples., (© 2020 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Hemostasis is the normal process that produces a blood clot at a site of vascular injury. Mice are widely used to study hemostasis and abnormalities of blood coagulation because their hemostatic system is similar in most respects to that of humans, and their genomes can be easily manipulated to create models of inherited human coagulation disorders. Two of the most widely used techniques for assessing hemostasis in mice are the tail bleeding time (TBT) and saphenous vein bleeding (SVB) models. Here we discuss the use of these methods in the evaluation of hemostasis, and the advantages and limits of using mice as surrogates for studying hemostasis in humans.

Storage lesion-induced, red cell-derived microvesicles (RBC-MVs) propagate coagulation by supporting the assembly of the prothrombinase complex. It has also been reported that RBC-MVs initiate coagulation via the intrinsic pathway. To elucidate the mechanism(s) of RBC-MV-induced coagulation activation, the ability of storage lesion-induced RBC-MVs to activate each zymogen of the intrinsic pathway was assessed in a buffer system. Simultaneously, the thrombin generation (TG) assay was used to assess their ability to initiate coagulation in plasma. RBC-MVs directly activated factor XII (FXII) or prekallikrein, but not FXI or FIX. RBC-MVs initiated TG in normal pooled plasma and in FXII- or FXI-deficient plasma, but not in FIX-deficient plasma, suggesting an alternate pathway that bypasses both FXII and FXI. Interestingly, RBC-MVs generated FIXa in a prekallikrein-dependent manner. Similarly, purified kallikrein activated FIX in buffer and initiated TG in normal pooled plasma, as well as FXII- or FXI-deficient plasma, but not FIX-deficient plasma. Dual inhibition of FXIIa by corn trypsin inhibitor and kallikrein by soybean trypsin inhibitor was necessary for abolishing RBC-MV-induced TG in normal pooled plasma, whereas kallikrein inhibition alone was sufficient to abolish TG in FXII- or FXI-deficient plasma. Heating RBC-MVs at 60°C for 15 minutes or pretreatment with trypsin abolished TG, suggesting the presence of MV-associated proteins that are essential for contact activation. In summary, RBC-MVs activate both FXII and prekallikrein, leading to FIX activation by 2 independent pathways: the classic FXIIa-FXI-FIX pathway and direct kallikrein activation of FIX. These data suggest novel mechanisms by which RBC transfusion mediates inflammatory and/or thrombotic outcomes.

Study Design: Cross-sectional study OBJECTIVES: To determine clinical factors associated with telomere length in persons with chronic spinal cord injury (SCI)., Setting: Veterans Affairs Medical Center, Boston, MA., Methods: Two hundred seventy-eight participants with chronic SCI provided blood samples for measurement of C-reactive protein (CRP), interleukin-6 (IL-6), and telomere length, completed respiratory health questionnaires, underwent dual X-ray absorptiometry (DXA) to assess body fat, and completed spirometry. High-throughput real-time PCR assays were used to assess telomere length in leukocyte genomic DNA. Linear regression models were used to assess cross-sectional associations with telomere length., Results: Telomere length was inversely related to age (p < 0.0001). In age-adjusted models, gender, race, injury duration, %-total and %-trunk fat, body mass index (BMI), %-predicted forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV 1 ), chronic cough or phlegm, CRP, IL-6, wheeze, smoking, diabetes, heart disease, chronic obstructive pulmonary disease (COPD), skin ulcer, urinary tract infection (UTI), or chest illness history were not significantly associated with telomere length. There was a suggestive age-adjusted association between persons with the most severe SCI (cervical motor complete and AIS C) and shorter telomere length (p = 0.055), an effect equivalent to ~8.4 years of premature aging. There were similar age-adjusted associations with telomere length between persons using a wheelchair (p = 0.059) and persons with chronic urinary catheter use (p = 0.082) compared to persons without these characteristics., Conclusions: Our results suggest that clinical characteristics such as decreased mobility and bladder dysfunction that are common in individuals with more severe SCI are associated with shorter telomere length.

Chromogenic substrates (CS) are synthetic substrates used to monitor the activity of a target enzyme. It has been reported that some CSs display competitive product inhibition with their target enzyme. Thus, in assays where enzyme activity is continuously monitored over long periods of time, the product inhibition may significantly interfere with the reactions being monitored. Despite this knowledge, it is rare for CSs to be directly incorporated into mathematical models that simulate these assays. This devalues the predictive power of the models. In this study, we examined the interactions between a single enzyme, coagulation factor Xa, and its chromogenic substrate. We developed, and experimentally validated, a mathematical model of a chromogenic assay for factor Xa that explicitly included product inhibition from the CS. We employed Bayesian inference, in the form of Markov-Chain Monte Carlo, to estimate the strength of the product inhibition and other sources of uncertainty such as pipetting error and kinetic rate constants. Our model, together with carefully calibrated biochemistry experiments, allowed for full characterization of the strength and impact of product inhibition in the assay. The effect of CS product inhibition in more complex reaction mixtures was further explored using mathematical models., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Essentials Many mediators increase tissue factor (TF) expression in a wide variety of cell types. The only known example of TF downregulation is by pericytes during wound healing angiogenesis. Downregulation of TF mRNA and protein in cultured pericytes is Protein Kinase C (PKC) dependent. Pericyte TF regulation is unique, since PKC mediates increased TF in all other cell types tested. SUMMARY: Background Embryonic and tumor-associated angiogenesis are linked to elevated expression of the procoagulant transmembrane receptor tissue factor (TF). In contrast, we have reported that high baseline TF expression by perivascular cells (pericytes) is dramatically reduced during angiogenesis at sites of wound healing. This is the only setting in which active TF downregulation has been reported, thus revealing a novel mechanism of TF regulation. Objectives To define the mechanisms underlying the unique pattern of TF expression in pericytes. Methods TF expression in primary cultures of human pericytes is not altered by angiogenic cytokines or growth factors, but is actively downregulated by phorbol 12-myristate 13-acetate (PMA). We characterized TF transcription, protein stability and trafficking in response to PMA. Results Exposure to PMA reduced TF mRNA synthesis and shortened the half-life of TF protein from 11 h to 4.5 h. Addition of PMA rapidly triggered endocytosis of cell surface TF, followed by degradation in lysosomes. Cell surface TF coagulant activity was maintained until internal stores were depleted. Reduction of TF transcription, TF endocytosis and enhanced degradation of TF protein were all blocked by broad-spectrum inhibitors of protein kinase C (PKC). This was a surprising finding, because PKC activation increases TF expression in other cell types that have been tested. Conclusions The unique PKC-dependent pathway of TF downregulation in pericytes suggests that TF downregulation may play a functional role in angiogenesis. Distinct pathways regulating pathological and physiological TF expression could be utilized to modulate TF expression for therapeutic purposes., (© 2019 International Society on Thrombosis and Haemostasis.)

Essentials Mice lacking factor IX (FIX) or factor XI (FXI) were tested in a saphenous vein bleeding model. FIX-deficient mice displayed a hemostatic defect and FXI-deficient mice were similar to wild type mice. Infusion of FXI or over-expression of FXI in FIX-deficient mice improved hemostasis. FXI may affect the phenotype of FIX-deficiency (hemophilia B)., Summary: Background In humans, deficiency of coagulation factor XI may be associated with a bleeding disorder, but, until recently, FXI-deficient mice did not appear to have a hemostatic abnormality. A recent study, however, indicated that FXI-deficient mice show a moderate hemostatic defect in a saphenous vein bleeding (SVB) model. Objectives To study the effect of FXI on bleeding in mice with normal levels of the FXI substrate FIX and in mice lacking FIX (a murine model of hemophilia B). Methods Wild-type mice and mice lacking either FIX (F9 - ) or FXI (F11 -/- ) were tested in the SVB model. The plasma levels of FXI in F11 -/- mice were manipulated by infusion of FXI or its active form FXIa, or by overexpressing FXI by the use of hydrodynamic tail vein injection. Results F9 - mice showed a significant defect in the SVB model, whereas F11 -/- mice and wild-type mice were indistinguishable. Intravenous infusion of FXI or FXIa into, or overexpression of FXI in, F9 - mice improved hemostasis in the SVB model. Overexpression of a FXI variant lacking a FIX-binding site also improved hemostasis in F9 - mice. Conclusions Although we were unable to demonstrate a hemostatic defect in F11 -/- mice in the SVB model, our results support the premise that supraphysiological levels of FXI improve hemostasis in F9 - mice through FIX-independent pathways., (© 2018 International Society on Thrombosis and Haemostasis.)

The biochemical properties of the non-vitamin K antagonist oral anticoagulants (NOACs) and their differences from the mechanism of action of vitamin K antagonists contribute to their properties as anticoagulants. These properties include as follows: (1) Inhibiting a single protease is much less effective at inhibiting coagulation than is inhibiting at multiple steps. Thus, the dose-response relationship between NOAC level and intensity of anticoagulation is shallower and more linear than that of vitamin K antagonists. This partially accounts for the greater safety of NOACs than vitamin K antagonists reported in some studies. (2) Because they are small molecules, NOACs can reach their target proteases in locations that plasma protease inhibitors, such as antithrombin, cannot. (3) NOACs compete with substrates for binding at the active site of the target protease and that binding is reversible. When the drug level falls, the drug dissociates from its target, and protease activity is restored. Thus, there is the possibility of a rebound in procoagulant activity if the drug is abruptly terminated. (4) The effects of a NOAC can be overcome by increasing the amount of substrate available for the target protease or the amount of protease produced. This property may contribute to the safety of NOACs and their potential reversibility by coagulation factor concentrates. The biochemical properties of NOACs contribute to their suitability for use in conditions that require a predictable moderate degree of anticoagulation when administered orally at a consistent dose. Their effects can be overcome by a sufficiently strong procoagulant stimulus. This characteristic likely contributes to their generally reduced risk of serious bleeding. However, they are not well suited for use in settings that require a profound degree of anticoagulation., (© 2017 American Heart Association, Inc.)

Background: Haemophilia B is caused by genetic aberrations in the F9 gene. The majority of these are non-synonymous mutations that alter the primary structure of blood coagulation factor IX (FIX). However, a synonymous mutation c.459G>A (Val107Val) was clinically reported to result in mild haemophilia B (FIX coagulant activity 15%-20% of normal). The F9 mRNA of these patients showed no skipping or retention of introns and/or change in mRNA levels, suggesting that mRNA integrity does not contribute to the origin of the disease in affected individuals. The aim of this study is to elucidate the molecular mechanisms that can explain disease manifestations in patients with this synonymous mutation., Methods: We analyse the molecular mechanisms underlying the FIX deficiency through in silico analysis and reproducing the c.459G>A (Val107Val) mutation in stable cell lines. Conformation and non-conformation sensitive antibodies, limited trypsin digestion, activity assays for FIX, interaction with other proteins and post-translation modifications were used to evaluate the biophysical and biochemical consequences of the synonymous mutation., Results: The Val107Val synonymous mutation in F9 was found to significantly diminish FIX expression. Our results suggest that this mutation slows FIX translation and affects its conformation resulting in decreased extracellular protein level. The altered conformation did not change the specific activity of the mutated protein., Conclusions: The pathogenic basis for one synonymous mutation (Val107Val) in the F9 gene associated with haemophilia B was determined. A mechanistic understanding of this synonymous variant yields potential for guiding and developing future therapeutic treatments., Competing Interests: Competing interests: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Wound healing requires interactions between coagulation, inflammation, angiogenesis, cellular migration, and proliferation. Healing in dermal wounds of hemophilia B mice is delayed when compared with hemostatically normal wild-type (WT) mice, with abnormal persistence of iron deposition, inflammation, and neovascularity. We observed healing following induced joint hemorrhage in WT and factor IX (FIX) knockout (FIX -/- ) mice, examining also parameters previously studied in an excisional skin wound model. Hemostatically normal mice tolerated this joint bleeding challenge, cleared blood from the joint, and healed with minimal pathology, even if additional autologous blood was injected intra-articularly at the time of wounding. Following hemarthrosis, joint wound healing in hemophilia B mice was impaired and demonstrated similar abnormal histologic features as previously described in hemophilic dermal wounds. Therefore, studies of pathophysiology and therapy of hemophilic joint bleeding performed in hemostatically normal animals are not likely to accurately reflect the healing defect of hemophilia. We additionally explored the hypothesis that the use of a FIX replacement protein with extended circulating FIX activity could improve synovial and osteochondral wound healing in hemophilic mice, when compared with treatment with unmodified recombinant FIX (rFIX) in the established joint bleeding model. Significantly improved synovial wound healing and preservation of normal osteochondral architecture are achieved by extending FIX activity after hemarthrosis using glycoPEGylated FIX when compared with an equivalent dose of rFIX. These results suggest that treating joint bleeding only until hemostasis is achieved may not result in optimal joint healing, which is improved by extending factor activity.

NETosis is a physiologic process in which neutrophils release their nuclear material in the form of neutrophil extracellular traps (NETs). NETs have been reported to directly promote thrombosis in animal models. Although the effects of purified NET components including DNA, histone proteins, and neutrophil enzymes on coagulation have been characterized, the mechanism by which intact NETs promote thrombosis is largely unknown. In this study, human neutrophils were stimulated to produce NETs in platelet-free plasma (PFP) or in buffer using phorbol myristate actetate or calcium ionophore. DNA and histone proteins were also separately purified from normal human neutrophils and used to reconstitute chromatin using a salt-gradient dialysis method. Neutrophil stimulation resulted in robust NET release. In recalcified PFP, purified DNA triggered contact-dependent thrombin generation (TG) and amplified TG initiated by low concentrations of tissue factor. Similarly, in a buffer milieu, DNA initiated the contact pathway and amplified thrombin-dependent factor XI activation. Recombinant human histones H3 and H4 triggered TG in recalcified human plasma in a platelet-dependent manner. In contrast, neither intact NETs, reconstituted chromatin, individual nucleosome particles, nor octameric core histones reproduced any of these procoagulant effects. We conclude that unlike DNA or individual histone proteins, human intact NETs do not directly initiate or amplify coagulation in vitro. This difference is likely explained by the complex histone-histone and histone-DNA interactions within the nucleosome unit and higher-order supercoiled chromatin leading to neutralization of the negative charges on polyanionic DNA and modification of the binding properties of individual histone proteins., (© 2017 by The American Society of Hematology.)

Influenza A virus infection is a common respiratory tract infection. Alveolar hemorrhage has been reported in patients with influenza pneumonia and in mice infected with influenza A. In this study, we investigated the effect of two anticoagulants on alveolar hemorrhage after influenza A virus (IAV) infection of wild-type mice. Wild-type mice were anticoagulated with either warfarin or the direct thrombin inhibitor dabigatran etexilate and then infected with a mouse-adapted influenza virus (A/Puerto Rico/8/34 H1N1). Alveolar hemorrhage was assessed by measuring hemoglobin levels in the bronchoalveolar lavage fluid (BALF). We also measured vascular permeability and viral genomes in the lung, as well as white blood cells, inflammatory mediators, and protein in BALF Survival and body weight were monitored for 14 days after influenza A infection. In infected mice receiving either warfarin or dabigatran etexilate we observed decreased activation of coagulation in the BALF and increased alveolar hemorrhage. Warfarin but not dabigatran etexilate increased vascular permeability and mortality of influenza A-infected mice. Anticoagulation did not affect levels of influenza A genomes, white blood cells, inflammatory mediators, or protein in the BALF Our study indicates that systemic anticoagulation increases alveolar hemorrhage in influenza A-infected mice., (© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Competing Interests: Conflict-of-interest disclosure: The author declares no competing financial interests.

Essentials Sickle cell disease is increasingly being recognized as a chronic hypercoagulable state. Thrombin generation is elevated in the whole blood, but not the plasma of sickle cell patients. Whole blood thrombin generation inversely correlates to erythrocyte phosphatidylserine exposure. Acquired protein S deficiency is likely explained by binding of protein S to sickle red cells. Click to hear Dr Hillery discuss coagulation and vascular pathologies in mouse models of sickle cell disease., Summary: Introduction Sickle cell disease (SCD) is a hypercoagulable state with chronic activation of coagulation and an increased incidence of thromboembolic events. However, although plasma pre-thrombotic markers such as thrombin-anithrombin complexes and D-dimer are elevated, there is no consensus on whether global assays of thrombin generation in plasma are abnormal in patients with SCD. Based on our recent observation that normal red blood cells (RBCs) contribute to thrombin generation in whole blood, we hypothesized that the cellular components in blood (notably phosphatidylserine-expressing erythrocytes) contribute to enhanced thrombin generation in SCD. Methods Whole blood and plasma thrombin generation assays were performed on blood samples from 25 SCD patients in a non-crisis 'steady state' and 25 healthy race-matched controls. Results Whole blood thrombin generation was significantly elevated in SCD, whereas plasma thrombin generation was paradoxically reduced compared with controls. Surprisingly, whole blood and plasma thrombin generation were both negatively correlated with phosphatidylserine exposure on RBCs. Plasma thrombin generation in the presence of exogenous activated protein C or soluble thrombomodulin revealed deficiencies in the protein C/S anticoagulant pathway in SCD. These global changes were associated with significantly lower plasma protein S activity in SCD that correlated inversely with RBC phosphatidylserine exposure. Conclusion Increased RBC phosphatidylserine exposure in SCD is associated with acquired protein S deficiency. In addition, these data suggest a cellular contribution to thrombin generation in SCD (other than RBC phosphatidylserine exposure) that explains the elevated thrombin generation in whole blood., (© 2016 International Society on Thrombosis and Haemostasis.)

Unlabelled: Essentials Disorders of hemostasis can lead to delayed and defective wound healing. In hemophilia B (HB) mice, 7 days of Factor (F)IX or VIIa are needed to normalize wound healing. One dose of a highly active FVIIa variant (DVQ) restored normal wound closure time in HB mice. Coagulation factors with enhanced activity may acquire biological effects not due to hemostasis., Summary: Introduction We have previously reported that hemophilia B (HB) mice have delayed healing of cutaneous wounds and alterations in wound histology. Administration of a single dose of either factor IX or recombinant activated FVII (rFVIIa) (NovoSeven) prior to wounding did not improve wound closure time or histology. The FVIIa analog DVQ (V158D, E296V and M298Q mutations) was designed to have higher tissue factor-independent activity than rVIIa. We hypothesized that a single dose of DVQ would be more effective in restoring wound healing in HB mice. Methods Cutaneous punch wounds were made on the backs of HB and wild-type mice, and the time to wound closure was monitored. HB mice were treated with a dose of rFVIIa (10 mg kg(-1) ) or DVQ (1 mg kg(-1) ) that corrected the tail bleeding time. Skin samples were taken at various time points after wounding, fixed, and stained, and the histology was examined. Results As previously reported, wound closure times in HB mice given one dose of rFVIIa were not improved over those in untreated HB mice. Surprisingly, healing times in HB mice treated with an equally hemostatic dose of DVQ were normalized to that in wild-type mice. However, DVQ did not correct all histologic abnormalities in HB mice. Conclusions As the doses of DVQ and rFVIIa were chosen to support comparable levels of hemostasis, our data suggest that the improved healing seen with DVQ is not solely attributable to its hemostatic activity. It is possible that the improved wound healing arises through the effect of DVQ on cell signaling mechanisms., (© 2016 International Society on Thrombosis and Haemostasis.)

Introduction: Coated platelets are a subpopulation of platelets that possess highly prothrombotic properties. Previous observational data suggest that bleeding phenotype in severe haemophilia A is associated with coated platelet levels. Haemophilia A patients with higher coated platelet levels may have a mild bleeding phenotype; those with lower levels may have a more severe bleeding phenotype., Aim: The aim of the study was to test the hypothesis that coated platelet levels are correlated with clinical bleeding phenotype., Methods: This cross-sectional, observational study enrolled 20 severe haemophilia A patients, including 15 with severe and five with a mild bleeding phenotype, and a control group of 12 healthy volunteers. The haemophilia bleeding phenotype was determined by the patient's medical history and haemophilia treatment centre records. Blood was obtained from each patient by venipuncture and platelets were analysed by flow cytometry., Results: Patients categorized as having a severe bleeding phenotype experienced a median eight bleeds per year compared to one bleed annually in the mild bleeding phenotype group. Both groups had similar total platelet counts and fibrinogen levels. There was no difference in coated platelet percentage between severe and mild bleeding phenotype (17 and 16% respectively), however, both groups had significantly lower % coated platelets compared to controls (44%, P < 0.0001)., Conclusion: Coated platelet levels were not associated with bleeding phenotype in this study; however, these data may suggest coated platelet levels are lower in haemophilia patients relative to healthy volunteers., (© 2015 John Wiley & Sons Ltd.)

The goal of these studies was to extensively characterize the first recombinant FIX therapeutic corresponding to the threonine-148 (Thr-148) polymorph, IXINITY (trenonacog alfa [coagulation factor IX (recombinant)]). Gel electrophoresis, circular dichroism, and gel filtration were used to determine purity and confirm structure. Chromatographic and mass spectrometry techniques were used to identify and quantify posttranslational modifications. Activity was assessed as the ability to activate factor X (FX) both with and without factor VIIIa (FVIIIa) and in a standard clotting assay. All results were consistent across multiple lots. Trenonacog alfa migrated as a single band on Coomassie-stained gels; activity assays were normal and showed <0.002 IU of activated factor IX (FIXa) per IU of FIX. The molecule has >97%  γ-carboxylation and underwent the appropriate structural change upon binding calcium ions. Trenonacog alfa was activated normally with factor XIa (FXIa); once activated it bound to FVIIIa and FXa. When activated to FIXa, it was inhibited efficiently by antithrombin. Glycosylation patterns were similar to plasma-derived FIX with sialic acid content consistent with the literature reports of good pharmacokinetic performance. These studies have shown that trenonacog alfa is a highly pure product with a primary sequence and posttranslational modifications consistent with the common Thr-148 polymorphism of plasma-derived FIX.

All animals develop in association with complex microbial communities. It is now well established that commensal microbiota is essential for the correct functionality of each organ in the host. Particularly, the commensal gastro-intestinal microbiota (CGIM) is a key factor for development, immunity and nutrient conversion, rendering them bio-available for various uses. Thus, nutritional inputs generate a positive loop in maintaining host health and are essential in shaping the composition of the CGIM communities. Probiotics, which are live exogenous microorganisms, selectively provided to the host, are a promising concept for manipulating the microbiota and thus for increasing the host health status. Nevertheless, most mechanisms induced by probiotics to fortify the immune system are still a matter of debate. Alternatively, prebiotics, which are non-digestible food ingredients, can favor the growth of specific target groups of CGIM. Several metabolites are produced by the CGIM, one of the most important are the short-chain fatty acids (SCFAs), which emerge from the fermentation of complex carbohydrates. SCFAs have been recognized as key players in triggering beneficial effects elicited by simple diffusion and by specific receptors present, thus, far only in epithelial cells of higher vertebrates at different gastro-intestinal locations. However, both strategies have shown to provide resistance against pathogens during periods of high stress. In fish, knowledge about the action of pro- and prebiotics and SCFAs is still limited. Thus, in this review, we briefly summarize the mechanisms described on this topic for higher vertebrates and discuss why many of them may operate in the fish gut representing a model for different mucosal tissues.

In this issue of Blood, Zhu et al have established, in human blood, that factor XIa and polyphosphate make significant contributions to thrombus formation. This makes these molecules good targets for therapeutic intervention.

Transglutaminases are a superfamily of isoenzymes found in cells and plasma. These enzymes catalyze the formation of ε-N-(γ-glutamyl)-lysyl crosslinks between proteins. Cystamine blocks transglutaminase activity and is used in vitro in human samples and in vivo in mice and rats in studies of coagulation, immune dysfunction, and inflammatory disease. These studies have suggested cystamine blocks fibrin crosslinking and has anti-inflammatory effects, implicating transglutaminase activity in the pathogenesis of several diseases. We measured the effects of cystamine on fibrin crosslinking, tissue factor-triggered plasma clot formation and thrombin generation, and coagulation factor enzymatic activity. At concentrations that blocked fibrin crosslinking, cystamine also inhibited plasma clot formation and reduced thrombin generation. Cystamine inhibited the amidolytic activity of coagulation factor XI and thrombin towards chromogenic substrates. These findings demonstrate that cystamine exhibits anticoagulant activity during coagulation. Given the close relationship between coagulation and inflammation, these findings suggest prior studies that used cystamine to implicate transglutaminase activity in disease pathogenesis warrant re-examination.

Background: The oral thrombin inhibitor dabigatran has the drawbacks that it does not have a validated antidote. Data from animal studies and plasma coagulation assays suggest that prothrombin complex concentrate (PCC) or recombinant factor VIIa (FVIIa) might reverse dabigatran anticoagulation., Methods: Cellular elements make a significant contribution to hemostasis. Our goals were to (1) test the hypothesis that both FVIIa and a 4-factor PCC improve parameters of thrombin generation in the presence of dabigatran in a cell-based model; and (2) determine whether results in a cell-based model correlate with hemostasis in vivo., Results: PCC reversed dabigatran effects on the rate, peak, and total amount of thrombin but did not shorten the lag (n = 6 experiments in triplicate). By contrast, FVIIa shortened the lag, increased the rate and peak, but did not improve total thrombin (n = 6). Effects of PCC were seen at both therapeutic and markedly supratherapeutic dabigatran levels, whereas beneficial effects of FVIIa decreased as the dabigatran level increased. The PCC effect was reproduced by adding prothrombin, factor X, and factor IX. At therapeutic dabigatran levels, both PCC and FVIIa normalized hemostasis time in a mouse saphenous vein bleeding model., Conclusions: A cell-based model reflects the effects on thrombin generation of clinically relevant levels of FVIIa and PCC in the presence of dabigatran. Enhancing the rate of thrombin generation and peak thrombin level appear to correlate best with hemostasis in vivo. The ineffectiveness of FVIIa at supratherapeutic dabigatran levels may explain conflicting reports of its efficacy in dabigatran reversal.

Dabigatran, rivaroxaban, and apixaban are orally active anticoagulants that are approved in many countries. Dabigatran inhibits thrombin, whereas rivaroxaban and apixaban are factor Xa inhibitors. In clinical trials, these novel oral anticoagulants were at least as effective as warfarin for preventing stroke in patients with atrial fibrillation, but with a lower rate of serious bleeding. However, the lack of true antidotes for these agents has caused concern when patients suffer life-threatening bleeding or trauma or require emergent invasive procedures. True antidotes are under development for all of these agents. In the meantime, activated and nonactivated prothrombin complex concentrates have been used as reversal agents. Factor VIIa may also be effective for reversal of the factor Xa inhibitors. Reversal of novel oral anticoagulants by these hemostatic agents has not been studied in bleeding human patients, so their true efficacy and appropriate dosing are not known., (© 2014 by The American Society of Hematology. All rights reserved.)