Tissue Fibrin Deposition Research Articles

Extracellular Histones Trigger Disseminated Intravascular Coagulation by Lytic Cell Death.

Histones are cationic nuclear proteins that are essential for the structure and functions of eukaryotic chromatin. However, extracellular histones trigger inflammatory responses and contribute to death in sepsis by unknown mechanisms. We recently reported that inflammasome activation and pyroptosis trigger coagulation activation through a tissue-factor (TF)-dependent mechanism. We used a combination of various deficient mice to elucidate the molecular mechanism of histone-induced coagulation. We showed that histones trigger coagulation activation in vivo, as evidenced by coagulation parameters and fibrin deposition in tissues. However, histone-induced coagulopathy was neither dependent on intracellular inflammasome pathways involving caspase 1/11 and gasdermin D (GSDMD), nor on cell surface receptor TLR2- and TLR4-mediated host immune response, as the deficiency of these genes in mice did not protect against histone-induced coagulopathy. The incubation of histones with macrophages induced lytic cell death and phosphatidylserine (PS) exposure, which is required for TF activity, a key initiator of coagulation. The neutralization of TF diminished the histone-induced coagulation. Our findings revealed lytic cell death as a novel mechanism of histone-induced coagulation activation and thrombosis.

Abstract 487: Extracellularhistonestrigger Disseminated Intravascular Coagulation By Lytic Cell Death

Background: Histones are cationic nuclear proteins that are essential for the structure andfunctions of eukaryotic chromatin. However, extracellular histones trigger inflammatoryresponses and contribute to death in sepsis by unknown mechanisms. We recently reported thatinflammasome activation and pyroptosis trigger coagulation activation through a tissue factor(TF)-dependent mechanism. Objective and methods: We used a combination of various deficient mice to elucidate the molec-ular mechanism of histone-induced coagulation. Results: We show that histones trigger coagulation activation in vivo as evidenced bycoagulation parameters and fibrin deposition in tissues. However, histone-induced coagulopathywas neither dependent on intracelluar inflammasome pathwyas involving caspase 1/11 andgasdermin D (GSDMD), nor on cell surface TLR2 and TLR4 mediated host immune response, asdeficiency of these genes in mice did not protect against histone-induced coagulopathy.Incubation of histones with macrophages induced lytic cell death and phosphatidylserine (PS)exposure, which is required for TF activity, a key initiator of coagulation. Neutralization of TFdiminished histone-induced coagulation. Conclusion: Our findings reveal lytic cell death as a novel mechanism of histone-inducecoagulation activation and thrombosis.

Inhibition of contact-mediated activation of factor XI protects baboons against S aureus–induced organ damage and death

AbstractStaphylococcus aureus infections can produce systemic bacteremia and inflammation in humans, which may progress to severe sepsis or septic shock, even with appropriate antibiotic treatment. Sepsis may be associated with disseminated intravascular coagulation and consumptive coagulopathy. In some types of mouse infection models, the plasma coagulation protein factor XI (FXI) contributes to the pathogenesis of sepsis. We hypothesize that FXI also contributes to the pathogenesis of sepsis in primates, and that pharmacological interference with FXI will alter the outcome of Staphylococcus aureus–induced lethality in a baboon model. Pretreatment of baboons with the anti-FXI antibody 3G3, a humanized variant of the murine monoclonal 14E11 that blocks FXI activation by FXIIa, substantially reduced the activation of coagulation, as reflected by clotting times and plasma complexes of coagulation proteases (FXIIa, FXIa, FIXa, FXa, FVIIa, and thrombin) with serpins (antithrombin or C1 inhibitor) following infusion of heat-inactivated S aureus. 3G3 treatment reduced fibrinogen and platelet consumption, fibrin deposition in tissues, neutrophil activation and accumulation in tissues, cytokine production, kininogen cleavage, cell death, and complement activation. Overall, 3G3 infusion protected the structure and function of multiple vital organs, including lung, heart, liver, and kidney. All treated animals reached the end point survival (7 days), whereas all nontreated animals developed terminal organ failure within 28 hours. We conclude that FXI plays a role in the pathogenesis of S aureus–induced disseminated intravascular coagulation and lethality in baboons. The results provide proof of concept for future therapeutic interventions that may prevent sepsis-induced organ failure and save lives in certain forms of sepsis.

Targeting factor VIII expression to platelets for hemophilia A gene therapy does not induce an apparent thrombotic risk in mice

Background Targeting factor (F) VIII expression to platelets is a promising gene therapy approach for hemophilia A, and is successful even in the presence of inhibitors. It is well known that platelets play important roles not only in hemostasis, but also in thrombosis and inflammation. Objective To evaluate whether platelet-FVIII expression might increase thrombotic risk and thereby compromise the safety of this approach. Methods In this study, platelet-FVIII-expressing transgenic mice were examined either in steady-state conditions or under prothrombotic conditions induced by inflammation or the FV Leiden mutation. Native whole blood thrombin generation assay, rotational thromboelastometry analysis and ferric chloride-induced vessel injury were used to evaluate the hemostatic properties. Various parameters associated with thrombosis risk, including D-dimer, thrombin-antithrombin complexes, fibrinogen, tissue fibrin deposition, platelet activation status and activatability, and platelet-leukocyte aggregates, were assessed. Results We generated a new line of transgenic mice that expressed 30-fold higher levels of platelet-expressed FVIII than are therapeutically required to restore hemostasis in hemophilic mice. Under both steady-state conditions and prothrombotic conditions induced by lipopolysaccharide-mediated inflammation or the FV Leiden mutation, supratherapeutic levels of platelet-expressed FVIII did not appear to be thrombogenic. Furthermore, FVIII-expressing platelets were neither hyperactivated nor hyperactivatable upon agonist activation. Conclusion We conclude that, in mice, more than 30-fold higher levels of platelet-expressed FVIII than are required for therapeutic efficacy in hemophilia A are not associated with a thrombotic predilection.

Targeting FVIII Expression to Platelets for Hemophilia A Gene Therapy Does Not Bare an Apparent Thrombosis Risk

Our group has previously developed a gene therapy approach for hemophilia A in which FVIII expression is targeted to platelets. Platelet expressed FVIII successfully restores hemostasis in hemophilic mice even in the presence of high titer inhibitory anti-FVIII antibodies and induces immune tolerance to FVIII. Therapeutic efficacy was achieved in our original transgenic mouse line at a level of only 0.75mU FVIII per 108 platelets, which corresponded to an equivalent of 1.25% FVIII in plasma of wild-type (WT) mice. FVIII is not normally expressed in platelets but with platelet-FVIII gene therapy FVIII levels at the site of injury might dramatically increase due to platelet aggregation and activation, and consequent local FVIII release from platelets together with VWF. Furthermore, a higher embolism rate has been suggested in transgenic mice expressing FVIII in platelets on an otherwise FVIII deficient background. Thus, although this approach is very successful in restoring hemostasis, evaluating potential pathological consequences in conjunction with platelet-FVIII is of great importance. Here we explored whether a pro-thrombotic state was induced by platelet expressed FVIII in an attempt to define the breadth of the therapeutic window. To examine this, we analyzed high platelet-FVIII expressing mice using five techniques including 1) a native whole blood thrombin generation assay, 2) ex vivo clot formation using thomboelastometry (ROTEM), 3) assessment of plasma parameters linked with an increased thrombosis risk (D-Dimer, thrombin anti-thrombin complexes (TAT), fibrinogen), 4) in vivo clot formation using a ferric chloride carotid artery injury model, and 5) tissue fibrin deposition. In addition to steady state, mice were subjected to an inflammatory challenge to induce pro-thrombotic conditions.We generated transgenic mice, LV17/18tg, that expressed 18mU FVIII per 108 platelets on a FVIII deficient background. This platelet-FVIII level was 24-fold higher than in our originally described mouse line. While FVIII deficient mice had negligible thrombin generation, comparing WT control to LV17/18tg mice, neither thrombin generation nor ex vivo clot formation was increased above WT levels in the transgenic mice. In WT and LV17/18tg, respectively, peak thrombin was 216 ± 16 and 195 ± 13 nM and endogenous thrombin potential was 1718 ± 41 and 1642 ± 56 nM. Clotting time determined by ROTEM in WT and LV17/18tg, respectively, was 424 ± 20 and 705 ± 37 seconds and maximum clot firmness was 51.3 ± 1.4 and 54.0 ± 1.7 mm. Fibrinogen and D-Dimer levels were similar in WT and LV17/18tg mice. While TAT levels were significantly higher in LV17/18tg (11.6 ± 1.1 ng/ml) than in WT mice (6.0 ± 0.5 ng/ml), interestingly this increase was also observed in FVIIInull mice (11.0 ± 1.2 ng/ml). Therefore, we attributed the elevation of TAT levels to the FVIII deficient background. Why the FVIII deficient mice have elevated TAT levels is not clear and may reflect an abnormality in the FVIII deficient line that we are further investigating. When we monitored time to occlusion in an in vivo thrombus formation model using ferric chloride induced carotid artery injury, vessels in LV17/18tg mice did not occlude faster than in WT mice (time to occlusion: 14.5 ± 0.5 versus 5.9 ± 0.5 min, respectively). Thus, under steady state, platelet-FVIII did not seem to be pro-thrombotic. This led us to evaluate the thrombosis risk under pro-thrombotic conditions. Because inflammation induces a pro-thrombotic state, we challenged mice with 40mg/kg LPS and analyzed the animals 16 hours later. WT and LV17/18tg mice treated with LPS showed a similar significant increase in fibrinogen, D-Dimer and TAT levels over PBS treated control mice. While no fibrin deposition was observed in the liver of WT or LV17/18tg mice, LPS challenge did induce fibrin deposition, but there was no difference between WT and LV17/18tg mice.In conclusion, 24-fold higher platelet-FVIII levels than in our originally described mouse line in which hemostasis was fully restored, did not show any signs of thrombosis in steady state or under inflammatory conditions. Therefore, in this regard, platelet targeted FVIII gene therapy can be considered a safe therapy with a relatively wide therapeutic window that is in excess of 24-fold. DisclosuresBaumgartner:Novo Nordisk: Research Funding. Shi:BloodCenter of Wisconsin: Patents & Royalties: METHOD OF INDUCING IMMUNE TOLERANCE THROUGH TARGETED GENE EXPRESSION. . Montgomery:Biogen: Consultancy; Bayer: Consultancy; CSL Behring: Consultancy; Baxter: Consultancy; Octapharma: Consultancy; Grifols: Consultancy.

Association Between Necropsy Evidence of Disseminated Intravascular Coagulation and Hemostatic Variables Before Death in Horses With Colic.

BackgroundDisseminated intravascular coagulation (DIC) is frequent in horses with severe gastrointestinal disorders. Postmortem studies have found fibrin microthrombi in tissues of these horses, but studies relating these histopathological findings with antemortem hemostatic data are lacking.HypothesisAntemortem classification of coagulopathy is related to the presence and severity of fibrin deposits observed postmortem in horses with severe gastrointestinal disorders.AnimalsAntemortem hemostatic profile data and postmortem tissue samples (kidney, lung, liver) from 48 horses with colic.MethodsTissue samples were stained with phosphotungstic acid hematoxylin and immunohistochemical methods for histological examination. A fibrin score (grades 0–4) was assigned for each technique, tissue and horse, as well as the presence or absence of DIC at postmortem examination. D‐dimer concentration, prothrombin time (PT), activated partial thromboplastin time (aPTT), and antithrombin (AT) activity, as well as the clinicopathological evidence of coagulopathy, were determined from plasma samples collected 0–24 hours before death or euthanasia. Histologic and clinicopathologic data from the same horses were compared retrospectively.ResultsNo association was found between antemortem classification of coagulopathy and postmortem diagnosis of DIC based on tissue fibrin deposition. None of the hemostatic parameters was significantly different between horses with or without postmortem diagnosis of DIC. There was no association between horses with fibrin in tissues or different cut‐offs for D‐dimer concentration and postmortem evidence of DIC.Conclusions and Clinical ImportanceAbnormalities of the routine clotting profile, including D‐dimer concentration, were not useful in predicting histologic evidence of DIC at necropsy in horses with severe gastrointestinal disorders.

The Antifibrinolytic Drug Tranexamic Acid Reduces Liver Injury and Fibrosis in a Mouse Model of Chronic Bile Duct Injury

Hepatic fibrin deposition has been shown to inhibit hepatocellular injury in mice exposed to the bile duct toxicant α-naphthylisothiocyanate (ANIT). Degradation of fibrin clots by fibrinolysis controls the duration and extent of tissue fibrin deposition. Thus, we sought to determine the effect of treatment with the antifibrinolytic drug tranexamic acid (TA) and plasminogen activator inhibitor-1 (PAI-1) deficiency on ANIT-induced liver injury and fibrosis in mice. Plasmin-dependent lysis of fibrin clots was impaired in plasma from mice treated with TA (1200 mg/kg i.p., administered twice daily). Prophylactic TA administration reduced hepatic inflammation and hepatocellular necrosis in mice fed a diet containing 0.025% ANIT for 2 weeks. Hepatic type 1 collagen mRNA expression and deposition increased markedly in livers of mice fed ANIT diet for 4 weeks. To determine whether TA treatment could inhibit this progression of liver fibrosis, mice were fed ANIT diet for 4 weeks and treated with TA for the last 2 weeks. Interestingly, TA treatment largely prevented increased deposition of type 1 collagen in livers of mice fed ANIT diet for 4 weeks. In contrast, biliary hyperplasia/inflammation and liver fibrosis were significantly increased in PAI-1(-/-) mice fed ANIT diet for 4 weeks. Overall, the results indicate that fibrinolytic activity contributes to ANIT diet-induced liver injury and fibrosis in mice. In addition, these proof-of-principle studies suggest the possibility that therapeutic intervention with an antifibrinolytic drug could form a novel strategy to prevent or reduce liver injury and fibrosis in patients with liver disease.

Activation of coagulation and tissue fibrin deposition in experimental influenza in ferrets.

BackgroundEpidemiological studies relate influenza infection with vascular diseases like myocardial infarction. The hypothesis that influenza infection has procoagulant effects on humans has been investigated by experimental animal models. However, these studies often made use of animal models only susceptible to adapted influenza viruses (mouse adapted influenza strains) or remained inconclusive. Therefore, we decided to study the influence of infection with human influenza virus isolates on coagulation in the well-established ferret influenza model.ResultsAfter infection with either a seasonal-, pandemic- or highly pathogenic avian influenza (HPAI-H5N1) virus strain infected animals showed alterations in hemostasis compared to the control animals. Specifically on day 4 post infection, a four second rise in both PT and aPTT was observed. D-dimer concentrations increased in all 3 influenza groups with the highest concentrations in the pandemic influenza group. Von Willebrand factor activity levels increased early in infection suggesting endothelial cell activation. Mean thrombin-antithrombin complex levels increased in both pandemic and HPAI-H5N1 virus infected ferrets. At tissue level, fibrin staining showed intracapillary fibrin deposition especially in HPAI-H5N1 virus infected ferrets.ConclusionThis study showed hemostatic alterations both at the circulatory and at the tissue level upon infection with different influenza viruses in an animal model closely mimicking human influenza virus infection. Alterations largely correlated with the severity of the respective influenza virus infections.

Acute and severe coagulopathy in adult mice following silencing of hepatic antithrombin and protein C production

AbstractMice deficient in the anticoagulants antithrombin (Serpinc1) or protein C (Proc) display premature death due to thrombosis-related coagulopathy, thereby precluding their use in gene function studies and thrombosis models. We used RNA interference to silence Serpinc1 and/or Proc in normal adult mice. The severe coagulopathy that followed combined “knockdown” of these genes is reported. Two days after siRNA injection, thrombi (occlusive) were observed in vessels (large and medium-sized) in multiple tissues, and hemorrhages were prominent in the ocular, mandibular, and maxillary areas. Tissue fibrin deposition and reduction of plasma fibrinogen accompanied this phenotype. The coagulopathy was prevented by dabigatran etexilate treatment. Silencing of Serpinc1 alone yielded a comparable but milder phenotype with later onset. The phenotype was absent when Proc was targeted alone. We conclude that RNA interference of Serpinc1 and/or Proc allows for evaluation of the function of these genes in vivo and provides a novel, controlled mouse model for spontaneous venous thrombosis.

Mast Cell Restricted Mouse and Human Tryptase·Heparin Complexes Hinder Thrombin-induced Coagulation of Plasma and the Generation of Fibrin by Proteolytically Destroying Fibrinogen

The mouse and human TPSB2 and TPSAB1 genes encode tetramer-forming tryptases stored in the secretory granules of mast cells (MCs) ionically bound to heparin-containing serglycin proteoglycans. In mice these genes encode mouse MC protease-6 (mMCP-6) and mMCP-7. The corresponding human genes encode a family of serine proteases that collectively are called hTryptase-β. We previously showed that the α chain of fibrinogen is a preferred substrate of mMCP-7. We now show that this plasma protein also is highly susceptible to degradation by hTryptase-β· and mMCP-6·heparin complexes and that Lys(575) is a preferred cleavage site in the protein α chain. Because cutaneous mouse MCs store substantial amounts of mMCP-6·heparin complexes in their secretory granules, the passive cutaneous anaphylaxis reaction was induced in the skin of mMCP-6(+)/mMCP-7(-) and mMCP-6(-)/mMCP-7(-) C57BL/6 mice. In support of the in vitro data, fibrin deposits were markedly increased in the skin of the double-deficient mice 6 h after IgE-sensitized animals were given the relevant antigen. Fibrinogen is a major constituent of the edema fluid that accumulates in tissues when MCs degranulate. Our discovery that mouse and human tetramer-forming tryptases destroy fibrinogen before this circulating protein can be converted to fibrin changes the paradigm of how MCs hinder fibrin deposition and blood coagulation internally. Because of the adverse consequences of fibrin deposits in tissues, our data explain why mice and humans lack a circulating protease inhibitor that rapidly inactivates MC tryptases and why mammals have two genes that encode tetramer-forming serine proteases that preferentially degrade fibrinogen.

Pathogenesis of Marburg Hemorrhagic Fever in Cynomolgus Macaques

Marburg virus (MARV) infection causes a severe and often fatal hemorrhagic disease in primates; however, little is known about the development of MARV hemorrhagic fever. In this study we evaluated the progression of MARV infection in nonhuman primates. Eighteen cynomolgus monkeys were infected with MARV; blood and tissues were examined sequentially over an 8-day period to investigate disease pathogenesis. Disease caused by MARV in cynomolgus macaques was very similar to disease previously described for Ebola virus-infected macaques. Monocytes, macrophages, Kupffer cells, and dendritic cells (DCs) were identified as the initial targets of MARV infection. Bystander lymphocyte apoptosis occurred at early stages in the disease course in intravascular and extravascular locations. The loss of splenic and lymph node DCs or downregulation of dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) on DCs as early as day 2 and continuing through day 8 after MARV infection was a prominent finding. Evidence of disseminated intravascular coagulation was noted; however, the degree of fibrin deposition in tissues was less prominent than was reported in Ebola-infected macaques. The sequence of pathogenic events identified in this study provides an understanding of the development of disease processes and also may provide new targets for rational prophylactic and chemotherapeutic interventions.

Pathogenesis of lassa fever in cynomolgus macaques

BackgroundLassa virus (LASV) infection causes an acute and sometimes fatal hemorrhagic disease in humans and nonhuman primates; however, little is known about the development of Lassa fever. Here, we performed a pilot study to begin to understand the progression of LASV infection in nonhuman primates.MethodsSix cynomolgus monkeys were experimentally infected with LASV. Tissues from three animals were examined at an early- to mid-stage of disease and compared with tissues from three animals collected at terminal stages of disease.ResultsDendritic cells were identified as a prominent target of LASV infection in a variety of tissues in all animals at day 7 while Kupffer cells, hepatocytes, adrenal cortical cells, and endothelial cells were more frequently infected with LASV in tissues of terminal animals (days 13.5-17). Meningoencephalitis and neuronal necrosis were noteworthy findings in terminal animals. Evidence of coagulopathy was noted; however, the degree of fibrin deposition in tissues was less prominent than has been reported in other viral hemorrhagic fevers.ConclusionThe sequence of pathogenic events identified in this study begins to shed light on the development of disease processes during Lassa fever and also may provide new targets for rational prophylactic and chemotherapeutic interventions.

Synergistic effect of deficiency in thrombosis-related genes

To investigate the interaction of deficiency in thrombosis-related gene in a mouse model. To generate mice carrying mutations in alpha-galactosidase A (Gla) and factor V Leiden (Fvl) and analyze the phenotypes, namely, tissue fibrin deposition and thrombus formation in organs. Fibrin deposition in organs of mice carrying both mutations in Gla and Fvl was significantly increased compared with that in mice with single mutaton: [Gla(-/0) Fv(Q/Q)+Gla(-/-)Fv(Q/Q)] vs.[Gla(-/0)Fv(+/+)]=(0.28+/-0.03)% vs.(0.07+/-0.007)%, P<0.01; [Gla(-/0)Fv(Q/Q)+Gla(-/-)Fv(Q/Q)] vs.[Gla(+/0)Fv(Q/Q)+Gla(+/+)Fv(Q/Q)]=(0.28+/-0.03)% vs.(0.11+/-0.02)%, P< 0.01. Meanwhile, the number of thrombi on organ sections of mice carrying both mutations in Gla and Fvl was significantly increased compared with the single mutation carrier: [Gla(-/0)Fv(Q/Q)+Gla(-/-)Fv(Q/Q)] vs.[Gla(-/0)Fv(+/+)]=1.9+/-0.7 vs. 0.0+/-0.0, P<0.05; [Gla(-/0)Fv(Q/Q)+Gla(-/-)Fv(Q/Q)] vs. [Gla(+/0)Fv(Q/Q)+Gla(+/+)Fv(Q/Q)]=1.9+/-0.7 vs. 0.3+/-0.1, P<0.05. These observations demonstrated that there was synergistic effect in Gla and Fvl deficiency in mice. It suggested that there could be a combination of GLA deficiency and FVL or other thrombosis-related gene defect in patients with genetic severe early-onset thrombosis.

Abstract 5484: Activation of Endothelial NF- κ B Signaling Promotes Coagulation by Inhibiting Protein C Anticoagulant Mechanism in Endotoxemic Mice

Although the role of systemic activation of NF- κ B pathway in septic coagulation has been well documented, little is known about the mechanistic contribution of endothelial intrinsic NF- κ B signaling in this pathological process. This study defined the causative role of endothelial NF- κ B signaling in septic coagulation. Wild type (WT) and transgenic mice (TG) that conditionally overexpress a mutant I- κ B β , an inhibitor of NF- κ B, selectively on endothelium, were injected with saline (1 ml/kg, i.p.) or E Coli LPS (5 mg/kg, i.p.). Tissue fibrin deposition, plasma markers of coagulation, plasma levels of protein C and plasminogen activator inhibitor-1 (PAI-1) were measured at 6 hours after LPS injection. Immunohistochemical staining of frozen tissue sections of heart, liver and kidney from WT-LPS mice showed a widespread tissue fibrin deposition. Fibrin deposition was not evident in tissue sections of the three organs from TG-LPS mice. Compared with WT-Con and TG-Con mice, WT-LPS mice showed a 79% reduction in plasma fibrinogen level, which was restored to control level in TG-LPS mice. WT-LPS displayed 13.7- and 3.2-fold increase in plasma levels of thrombin-antithrombin and D-dimer, which were reduced by approximately 69% and 37% in TG-LPS mice. Plasma levels of total and activated protein C (mO.D. units) for WT-Con, TG-Con, WT-LPS and TG-LPS groups were: 73.4±2.4, 72.0±3.2, 26.0±6.2 and 58.5±4.7 (p &lt; 0.01, WT-LPS vs other 3 groups), and 45.6±1.6, 43.0±1.2, 2.8±1.8 and 29.9±4.1 (p &lt; 0.01, WT-LPS vs other 3 groups). Plasma level of PAI-1 (ng/ml) for WT-Con, TG-Con, WT-LPS and TG-LPS groups was 0.06±0.05, 0.0±0.0, 31.3±4.8 and 18.1±3.3 (p &lt; 0.05, WT-LPS vs other 3 groups). These results indicate that endothelial intrinsic NF- κ B signaling plays a pivotal role in the activation of coagulation in endotoxemic mice, and suggest that activation of endothelial NF- κ B pathway promotes coagulation by impairing the protein C anticoagulant mechanism and by inhibiting the fibrinolytic system. (Supported by NIH R01GM063907).

Activation of endothelial intrinsic NF-κB pathway impairs protein C anticoagulation mechanism and promotes coagulation in endotoxemic mice

AbstractAlthough the role of systemic activation of the nuclear factor κB (NF-κB) pathway in septic coagulation has been well documented, little is known about the contribution of endothelial-specific NF-κB signaling in this pathologic process. Here, we used transgenic mice that conditionally overexpress a mutant I-κBα, an inhibitor of NF-κB, selectively on endothelium, and their wild-type littermates to define the role of endothelial-specific NF-κB in septic coagulation. In wild-type mice, lipopolysaccharide (LPS) challenge (5 mg/kg intraperitoneally) caused markedly increased plasma markers of coagulation, decreased plasma fibrinogen level, and widespread tissue fibrin deposition, which were abrogated by endothelial NF-κB blockade in transgenic mice. Endothelial NF-κB blockade inhibited tissue factor expression in endothelial cells, but not in leukocytes. Endothelial NF-κB blockade did not inhibit LPS-induced tissue factor expression in heart, kidney, and liver. Endothelial NF-κB blockade prevented LPS down-regulation of endothelial protein C receptor (EPCR) and thrombomodulin protein expressions, inhibited tissue tumor necrosis factor-α converting enzyme activity, reduced EPCR shedding, and restored plasma protein C level. Our data demonstrate that endothelial intrinsic NF-κB signaling plays a pivotal role in septic coagulation and suggests a link between endothelial-specific NF-κB activation and the impairment of the thrombomodulin-protein C-EPCR anticoagulation pathway.

A key role for Toll-like receptor-3 in disrupting the hemostasis balance on endothelial cells

AbstractVarious virus infections cause dysfunctional hemostasis and in some instances lead to the development of viral hemorrhagic fever syndrome. How do diverse viruses induce the expression of tissue factor on vascular cells? We hypothesize that a direct stimulation of pattern recognition receptors (PRR) by viral nucleic acids may be the key. Double-stranded RNA (dsRNA) is produced by many viruses and is recognized by various PRR, including Toll-like receptor-3 (TLR3). We have investigated whether poly I:C, a model for viral dsRNA, can influence cellular hemostasis. Poly I:C could up-regulate tissue factor and down-regulate thrombomodulin expression on endothelial cells but not on monocytes. The response to poly I:C was diminished upon small interfering RNA (siRNA)–mediated inhibition of TLR3, but not other PRR. In vivo, application of poly I:C induced similar changes in the aortic endothelium of mice as determined by enface microscopy. D-dimer, a circulating marker for enhanced coagulation and fibrinolysis, and tissue fibrin deposition was elevated. All the hemostasis-related responses to poly I:C, but not cytokine secretion, were blunted in TLR3−/− mice. Hence, the activation of TLR3 can induce the procoagulant state in the endothelium, and this could be relevant for understanding the mechanisms of viral stimulation of hemostasis.

Combined Tissue Factor Pathway Inhibitor (TFPI) and Thrombomodulin (TM) Deficiency Produces an Augmented Hypercoagulable State with Tissue Specific Fibrin Deposition.

Background: TFPI and TM are endothelial associated anticoagulant proteins thought to control hemostasis in specific vascular beds. Mice lacking either TFPI or TM have an intrauterine lethal phenotype. A prothrombotic phenotype has not previously been described in TFPI+/− mice. TMpro mice have a Glu to Pro mutation between the fourth and fifth epidermal growth factor domains. This mutation reduces TM expression and disrupts TM dependent activation of protein C. TMpro/pro mice are viable and fertile and do not exhibit spontaneous thrombosis on the C57/Bl6 background. TFPI+/−/TMpro/pro mice were generated to examine the consequences of TFPI deficiency in the presence of a compounding procoagulant state caused by reduced TM function. TFPI and TM are located 37 centimorgans apart of chromosome 2 resulting in allele segregation in about 40% of meioses. Therefore, breeding studies were performed without defining haplotypes of individual mice.Methods and Results: TFPI+/−/TMpro/pro mice were generated in three crosses in which the mother had a different prothrombotic phenotype. Embryonic lethality was observed in two of the crosses. TFPI+/−/TMpro/pro mice are born at less than expected frequency in either TFPI+/−/TMpro/+ female with TMpro/pro male crosses (7/151 or 4.6%) or TMpro/pro female with TFPI+/−/TMpro/+ male crosses (11/129 or 8.5%). Timed matings revealed similar reduced numbers at E18.5, but not E13.5, demonstrating that the mice die in utero during embryonic development. TMpro/+ are the least prothrombotic mothers that can produce TFPI+/−/TMpro/pro mice. In contrast to the other crosses, TFPI+/−/TMpro/pro mice are born at near the expected frequency in TMpro/+ mothers crossed with TFPI+/−/TMpro/pro fathers (15/75 or 20%). Our results show that both maternal and fetal genotypes contribute to embryonic survival. Adult TFPI+/−/TMpro/pro mice have elevated thrombin-antithrombin complex in plasma (11.4±8.12 ng/ml for TFPI+/−/TMpro/pro n=16; 4.82±3.05 ng/ml for TMpro/pro n=10, p<0.03; wild type and TFPI+/− had no detectable TAT complex) and increased thrombus volume in an electrical injury model of venous thrombosis (mean thrombus volume of wild type is 0.042±0.03 mm3; of TFPI+/−/TMpro/pro 0.125±0.063 mm3 p<0.005). Interestingly, TFPI+/− mice also have increased thrombus volume in this model (0.092±0.045 p<0.02), demonstrating for the first time a thrombotic phenotype in these mice. A fluorescently labeled cyclic peptide (CGLIIQKNEC) that specifically binds to fibrin-fibronectin complexes was used to detect tissue fibrin deposition (Pilch et al. PNAS 2006; 103:2800). In striking contrast to TFPI+/− and TMpro/pro mice, TFPI+/−/TMpro/pro mice exhibit augmented fibrin deposition in the liver and in the cerebral microvasculature.Conclusions: TFPI+/−/TMpro/pro mice display a prothrombotic phenotype with tissue specific fibrin deposition in embryonic development and as adults. They exhibit partial intrauterine lethality when carried by mothers with an underlying prothrombotic state, demonstrating for the first time that TFPI-dependent control of hemostasis in the vascular bed of the placenta fulfills a critical role for successful pregnancy outcome. In addition to the placenta, partial TFPI deficiency interacts with decreased TM function in an organ selective manner within adult mice to produce fibrin deposition in a second specific vascular bed, the brain microcirculation.

Detection of Fibrin Deposits in Tissues from Horses with Severe Gastrointestinal Disorders

Background:In humans and experimental animals, disseminated intravascular coagulation (DIC) causes fibrin deposition in several organs, which eventually leads to ischemia and multiorgan failure.Hypothesis:Horses who died or were euthanized for severe gastrointestinal disorders could have fibrin deposits in different tissues.Animals:Tissue‐organ samples collected during postmortem examinations on 66 colic horses with poor prognoses (eg, severe intestinal ischemia, enteritis, peritonitis), from 11 colic horses with good prognoses (eg, large‐colon obstruction or displacement), and from 16 slaughter horses.Methods:Tissue samples (kidney, lung, liver) were stained with hematoxylin and eosin, and phosphotungstic acid hematoxylin for a blinded histologic examination. A fibrin score (grades 0 to 4) was established for each tissue sample and for each horse.Results:Fibrin deposits were found in tissue specimens of 11 of 27 of horses (40.7%) in the ischemic group, 8 of 21 in the enteritis group (38.1%), and 7 of 18 in the peritonitis group (39.0%), whereas none of the horses in the obstructive group (n = 11) and only 1 horse in the slaughter group (n = 16) had fibrin deposits in their tissues. In addition, the mean fibrin score values for the ischemic, enteritis, and peritonitis groups (1.3 ± 1.7, 1.1 ± 1.6, and 0.9 ± 1.3, respectively) were statistically higher than those for the obstructive and slaughter groups (0.0 ± 0.0 and 0.1 ± 0.5, respectively). The largest fibrin deposits were found in the lungs.Conclusions and Clinical Importance: Horses with severe gastrointestinal disorders have fibrin deposits that are consistent with capillary microthrombosis, multiorgan failure, and DIC.

Detection of Fibrin Deposits in Tissues from Horses with Severe Gastrointestinal Disorders

In humans and experimental animals, disseminated intravascular coagulation (DIC) causes fibrin deposition in several organs, which eventually leads to ischemia and multiorgan failure. Horses who died or were euthanized for severe gastrointestinal disorders could have fibrin deposits in different tissues. Tissue-organ samples collected during postmortem examinations on 66 colic horses with poor prognoses (eg, severe intestinal ischemia, enteritis, peritonitis), from 11 colic horses with good prognoses (eg, large-colon obstruction or displacement), and from 16 slaughter horses. Tissue samples (kidney, lung, liver) were stained with hematoxylin and eosin, and phosphotungstic acid hematoxylin for a blinded histologic examination. A fibrin score (grades 0 to 4) was established for each tissue sample and for each horse. Fibrin deposits were found in tissue specimens of 11 of 27 of horses (40.7%) in the ischemic group, 8 of 21 in the enteritis group (38.1%), and 7 of 18 in the peritonitis group (39.0%), whereas none of the horses in the obstructive group (n = 11) and only 1 horse in the slaughter group (n = 16) had fibrin deposits in their tissues. In addition, the mean fibrin score values for the ischemic, enteritis, and peritonitis groups (1.3 +/- 1.7, 1.1 +/- 1.6, and 0.9 +/- 1.3, respectively) were statistically higher than those for the obstructive and slaughter groups (0.0 +/- 0.0 and 0.1 +/- 0.5, respectively). The largest fibrin deposits were found in the lungs. Horses with severe gastrointestinal disorders have fibrin deposits that are consistent with capillary microthrombosis, multiorgan failure, and DIC.

α-Galactosidase A Deficiency Leads to Increased Tissue Fibrin Deposition and Thrombosis in Mice Homozygous for the Factor V Leiden Mutation

Factor V Leiden (FVL) is a common genetic risk factor for vascular thrombosis in humans. Fabry disease, an X-linked lysosomal storage disorder attributable to alpha-galactosidase A (GLA) deficiency, is associated with premature vascular events that may be thrombotic in nature. To examine a potential interaction between FvL and Gla deficiency in vivo, we analyzed tissue fibrin deposition in mice carrying combined mutations in FvL and Gla. Gla deficiency markedly increased tissue fibrin deposition in mice carrying the FvL mutation (0.33+/-0.03%; n=7) compared with FvL mutation (0.14+/-0.02%; n=10; P<0.0005). These observations demonstrate a synergistic interaction between Gla deficiency and FvL toward tissue fibrin deposition in mice. Concomitant mutations in these genes may increase the penetrance of vascular thrombotic events in humans.