Functional Tissue Factor Research Articles

E-Cigarettes induce expression of procoagulant tissue factor in cultivated human endothelial cells.

E-cigarettes (ECIG) are proposed as an alternative for regular tobacco users with less dangerous effects for health. Several studies demonstrated that ECIG exert deleterious cardiovascular effects and promote platelet dependent thrombosis. However, ECIG role on Tissue Factor-dependent thrombosis is still unknown. Dysfunctional endothelial cells (ECs) are known to express Tissue Factor (TF) on their surface. Aim of the present study was to investigate whether ECIG might promote TF expression in ECs, shifting them to a pro thrombotic phenotype. Human Umbilical Vein Endothelial Cells (HUVEC) were incubated with increasing doses of ECIG (commercially available and mix of propylene glycol/vegetable glycerine/nicotine 18mg/mL) up to 1.8mg/mL. TF gene expression and protein levels were assessed at different time points by Real Time PCR and Western Blot, respectively. TF surface expression and activity were also measured by FACS analysis and coagulation assay. Finally, NF-kB translocation was investigated as possible mechanism of action. Potential protective effects by Rosuvastatin were also investigated. ECIG significantly increased TF expression at both gene and protein levels in a time and dose dependent manner. Surface expression and procoagulant activity were increased as well. These phenomena appeared modulated by the NF-κB pathway. Rosuvastatin reduced ECIG effects on TF-mRNA. Although in vitro, we indicate that ECIG promote a pro thrombotic phenotype in ECs via expression of functional TF. Data of the present study permit to shed a brighter light on the still partially unresolved issue about the role of ECIG in development of cardiovascular diseases suggesting that they might represent a potential risk factor for thrombotic cardiovascular events.

Production of functional recombinant human tissue factor for the prothrombin time test

Production of functional recombinant human tissue factor for the prothrombin time test

Idiopathic multicentric Castleman disease - TAFRO results in high levels of mTOR activator SVEP1, tissue factor, and endotheliopathy

AbstractIdiopathic multicentric Castleman disease (iMCD) is an inflammatory disease associated with a cytokine storm, activation of the PI3K/AKT/mTOR pathway, coagulopathy, and increased risk of thrombosis. The mechanisms underlying these pathologic processes remain elusive. We studied novel markers of mTOR activation and thrombosis in 1 patient with typical features of iMCD with TAFRO (thrombocytopenia, anasarca, fevers, reticulin myelofibrosis, and organomegaly) syndrome (iMCD-TAFRO). Plasma levels of SVEP1 (Sushi, von Willebrand factor type A, epidermal growth factor, and pentraxin domain–containing 1 protein), a newly identified mTOR activator associated with cardiovascular diseases and dementia, in addition to cytokines, chemokines and components of the coagulation cascade and complement system were evaluated by enzyme-linked immunosorbent assay (ELISA) and arrays. Compared with healthy controls, a 15-fold increase in SVEP1 was observed. High levels of factor VIIa/antithrombin and microparticles expressing functional tissue factor (TF) were detected. The anticoagulants thrombomodulin and soluble endothelial protein C receptor were elevated, indicating shedding from endothelial cells. Plasminogen activator inhibitor 1 was increased, consistent with hypofibrinolysis, whereas high levels of C3b and C5a are in keeping with complement activation. Furthermore, markers of endothelial cell activation (e.g. von Willebrand factor, angiopoietin-2), cell adhesion molecules, and angiogenesis mediators were upregulated. SVEP1 emerges as a potential mechanism of mTOR activation in iMCD-TAFRO, while multiple pathways influence coagulopathy. Immunothrombosis emerges as a potential therapeutic target for iMCD.

Idiopathic Multicentric Castleman Disease Results in Endothelium Activation and Coagulation Dysregulation Via Tissue Factor and PAI-1

Idiopathic Multicentric Castleman disease (iMCD) is an inflammatory disease which may be associated with thrombosis. Diagnosis of this condition may be delayed with increase in morbidity and mortality. We investigated the cause of hypercoagulability in one patient with iMCD, who presented with shortness of breath, anasarca, abdominal distension and severe pulmonary hypertension with clinicopathologic findings consistent with iMCD with some features of TAFRO (thrombocytopenia, anasarca, fever, reticulin myelofibrosis, organomegaly) variant. Lab results detected high levels of inflammatory markers IL-6, CRP and ferritin. During the patient's hospital course, our patient developed evidence of consumptive coagulopathy, based on prolonged PT, aPTT, thrombocytopenia, high D-dimers and reduced fibrinogen levels, with associated microvascular thrombosis, limb gangrene, and biopsy-proven vascular fibrin formation. We examined several markers of coagulation activation in this patient to characterize the pattern of hypercoagulability in mCD. Cytokine and chemokine arrays and ELISA for selected coagulation factors were employed. We found an elevation in FVIIa/AT suggestive of in vivo upregulation of Tissue Factor (TF) as well as an increase in microparticles expressing functional TF (inhibited by Ixolaris). Our patient also demonstrated a marked increase in C5a and C3b. With respect to anticoagulants associated with the endothelium, both thrombomodulin and sEPCR were augmented in our patient, indicating endothelial cell injury and decreased protein C activation (APC). High levels of PAI-1, the main fibrinolysis inhibitor was evident. In addition, an increase in the chemoattractants for monocytes MCP-1, MCP-4 was noted. Similarly, we noted an increase in angiopoietin-2 and other markers associated with endothelial cell activation including adhesion molecules E-selectin, sICAM-1, sICAM-3 and sVCAM-1. Molecules affecting angiogenesis such as endoglin, IL-8, VEGF-A were also increased. Most markers were at normal levels when the patient was in remission. Our findings suggest that multiple pathways influence hypercoagulability in iMCD, including direct activation of coagulation through TF and complement, increase in thromboinflammation via monocytes, and endotheliopathy. Mechanistically, it is likely that the cytokine storm in iMCD, characterized by endothelial activation, components of immunothrombosis and complement activation result in TF-mediated coagulation dysregulation, shifting hemostasis towards a prothrombotic and antifibrinolytic state. Components of immunothrombosis, including inhibitors of TF, emerge as a therapeutic target in iMCD.

PB1067 Quantification of Functional Tissue Factor Activity on Microvesicles during the Peripartum Period

PB1067 Quantification of Functional Tissue Factor Activity on Microvesicles during the Peripartum Period

Efficient refolding and reconstitution of tissue factor into nanodiscs facilitates structural investigation of a multicomponent system on a lipid bilayer

Structural data on membrane proteins in a lipid membrane environment is challenging to obtain but needed to provide information on the, often essential, protein-lipid interplay. A common experimental bottleneck in obtaining such data is providing samples in sufficient amounts and quality required for structural studies. We developed a new production protocol for the single-pass transmembrane protein (SPTMP) tissue factor (TF), exploiting the high expression level in E. coli inclusion bodies and subsequent refolding. This provided more than 5 mg of functional TF per liter bacterial culture. This is substantially more than what was obtained by the classical approaches for expressing TF in the membrane-anchored configuration. We optimized reconstitution into circularized nanodiscs enabling the formation of stable, TF loaded nanodiscs with different lipid compositions and with a limited material waste. The blood coagulation cascade is initiated by the complex formation between TF and Factor VIIa (FVIIa), and we probed this interaction by a functional assay and SPR measurements, which revealed similar activity and binding kinetics as TF produced by other protocols, demonstrating that high-yield production does not compromise TF function. Furthermore, the amounts of sample produced permitted initial small angle X-ray scattering studies providing the first structural information about TF and its binding to FVIIa in a lipid environment. This strategy possibly allows for probing the multicomponent complex TF:FVIIa together with its substrate Factor X on a lipid bilayer, but may also be relevant as a production strategy for other SPTMP for which structural information, in general, is limited.

Murine tissue factor disulfide mutation causes a bleeding phenotype with sex specific organ pathology and lethality.

Tissue factor (TF) is highly expressed in sub-endothelial tissue. The extracellular allosteric disulfide bond Cys186–Cys209 of human TF shows high evolutionary conservation and in vitro evidence suggests that it significantly contributes to TF procoagulant activity. To investigate the role of this allosteric disulfide bond in vivo, we generated a C213G mutant Tf mouse by replacing Cys213 of the corresponding disulfide Cys190-Cys213 in murine Tf. A bleeding phenotype was prominent in homozygous C213G Tf mice. Pre-natal lethality of one third of homozygous offspring was observed between embryonic (E) day E9.5 and E14.5 associated with placental hemorrhages. After birth, homozygous mice suffered from bleedings in different organs and reduced survival. Homozygous C213G Tf male mice showed higher incidence of lung bleedings and lower survival rates than females. In both sexes, C213G mutation evoked a reduced protein expression (about 10- fold) and severely reduced pro-coagulant activity (at least 100-fold). Protein glycosylation was impaired and cell membrane exposure decreased in macrophages in vivo. Single housing of homozygous C213G Tf males reduced the occurrence of severe bleeding and significantly improved survival, suggesting that inter-male aggressiveness might significantly account for the sex differences. These experiments show that the TF allosteric disulfide bond is of crucial importance for normal in vivo expression, post-translational processing and activity of murine TF. Although C213G Tf mice do not display the severe embryonic lethality of Tf knock-out mice, their postnatal bleeding phenotype emphasizes the importance of fully functional TF for hemostasis.

Oxidized low-density lipoproteins induce tissue factor expression in T-lymphocytes via activation of lectin-like oxidized low-density lipoprotein receptor-1.

T-lymphocytes plays an important role in the pathophysiology of acute coronary syndromes. T-cell activation in vitro by pro-inflammatory cytokines may lead to functional tissue factor (TF) expression, indicating a possible contribution of immunity to thrombosis. Oxidized low-density lipoproteins (oxLDLs) are found abundantly in atherosclerotic plaques. We aimed at evaluating the effects of oxLDLs on TF expression in T cells and the role of the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). CD3+ cells were isolated from healthy volunteers. Gene, protein, and surface expression of TF, as well as of LOX-1, were assessed at different time-points after oxLDL stimulation. To determine whether oxLDL-induced TF was LOX-1 dependent, T cells were pre-incubated with an LOX-1 inhibiting peptide (L-RBP) or with an anti-LOX-1 blocking antibody. To exclude that TF expression was mediated by reactive oxygen species (ROS) generation, oxLDL-stimulated T cells were pre-incubated with superoxide dismutase + catalase or with 4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol), an intracellular free radical scavenger. Finally, to determine if the observed findings in vitro may have a biological relevance, the presence of CD3+/TF+/LOX-1+ cells was evaluated by immunofluorescence in human carotid atherosclerotic lesions. oxLDLs induced functionally active TF expression in T cells in a dose- and time-dependent manner, independently on ROS generation. No effect was observed in native LDL-treated T cells. LOX-1 expression was also induced by oxLDLs in a time- and dose-dependent manner. Pre-incubation with L-RBP or anti-LOX-1 antibody almost completely inhibited oxLDL-mediated TF expression. Interestingly, human carotid plaques showed significant infiltration of CD3+ cells (mainly CD8+ cells), some of which were positive for both TF and LOX-1. oxLDLs induce functional TF expression in T-lymphocytes in vitro via interaction of oxLDLs with LOX-1. Human carotid atherosclerotic plaques contain CD3+/CD8+cells that express both TF and LOX-1, indicating that also in patients these mechanisms may play an important role.

Different Potential of Extracellular Vesicles to Support Thrombin Generation: Contributions of Phosphatidylserine, Tissue Factor, and Cellular Origin

Cells release diverse types of vesicles constitutively or in response to proliferation, injury, inflammation, or stress. Extracellular vesicles (EVs) are crucial in intercellular communication, and there is emerging evidence for their roles in inflammation, cancer, and thrombosis. We investigated the thrombogenicity of platelet-derived EVs, which constitute the majority of circulating EVs in human blood, and assessed the contributions of phosphatidylserine and tissue factor exposure on thrombin generation. Addition of platelet EVs to vesicle-free human plasma induced thrombin generation in a dose-dependent manner, which was efficiently inhibited by annexin V, but not by anti-tissue factor antibodies, indicating that it was primarily due to the exposure of phosphatidylserine on platelet EVs. Platelet EVs exhibited higher thrombogenicity than EVs from unstimulated monocytic THP-1 cells, but blockade of contact activation significantly reduced thrombin generation by platelet EVs. Stimulation of monocytic cells with lipopolysaccharide enhanced their thrombogenicity both in the presence and in the absence of contact activation, and thrombin generation was efficiently blocked by anti-tissue factor antibodies. Our study provides evidence that irrespective of their cellular origin, EVs support the propagation of coagulation via the exposure of phosphatidylserine, while the expression of functional tissue factor on EVs appears to be limited to pathological conditions.

Extracellular Vesicles in the Antiphospholipid Syndrome.

Antiphospholipid antibodies (aPL), particularly those directed against β2-glycoprotein I, cause activation of vascular cells (endothelial cells, platelets, monocytes) and release of extracellular vesicles (EVs), which include exosomes and microparticles (MPs). MPs, particularly endothelial MPs, have been most extensively studied in antiphospholipid syndrome (APS). Compared with healthy controls, patients with aPL have significantly higher levels of circulating endothelial and platelet MPs, including MPs expressing immunological and functional tissue factor. Although a consistent relationship of EVs with APS-related thrombosis and obstetric events has not yet been demonstrated, elevated levels of MPs occurring remote from thrombotic events suggest a chronic state of vascular activation in APS. In addition to being a marker of cellular activation, EVs express bioactive lipids, proteins, and nucleic acids, particularly microribonucleic acid (microRNA). EVs may potentially play a pathogenic role in APS by stimulating thrombosis through tissue factor-dependent and independent mechanisms and by promoting vascular activation. Further research is needed to understand these mechanisms and to determine whether EVs may be a useful biomarker to identify patients with aPL at highest risk of clinical events.

Continuous Thrombin Generation in Whole Blood: New Applications

Background.Hemostatic tests have been utilized to clarify the blood coagulation potential of both healthy and diseased individuals. They include tests for whole blood and plasma clotting times, coagulation and fibrinolytic factors and, recently, thrombin generation (TG). TG assays provide explicit information and remain the most physiologically-relevant hemostatic tests ex vivo. We have currently been using a TG assay in whole blood, described by Ninivaggi et al. (2012), for the evaluation of several hemostatic agents and disorders of blood coagulation.Methods.Whole blood from either healthy donors or trauma patients was drawn into 3.2% sodium citrate [± 0.1 mg/mL corn trypsin inhibitor (CTI) prior to the assay; prevents contact pathway activation] or into 3.2% sodium citrate/0.1 mg/mL CTI. Selected agents were added to blood prior to recalcification and TG was monitored in a fluorogenic assay.Packed red blood cells (pRBC) from healthy donors were reconstituted with platelet-poor plasma (PPP) and treated with 0.1 mg/mL CTI. TG was monitored in the absence of any exogenous initiation, in the presence of 5 pM TF initiation and also in the presence of synthetic phospholipids.Results.Whole BloodA. Blood from 10 healthy donors was collected into both citrate (with CTI added prior to the assay) and CTI/citrate and TG was monitored ± 5 pM relipidated tissue factor (TF) initiation. In the absence of TF, blood collected into citrate led to a lag phase ~3-fold longer than that in the presence of TF and peak thrombin ~1.4-fold lower. Blood collected into CTI/citrate, in the absence of TF, led to a lag phase ~6.4-fold longer than that in the presence of TF and peak thrombin ~4.3-fold lower.B. Titrations of relipidated TF and FXIa into citrated blood containing CTI led to concentration-dependent changes in the duration of the lag phase.C. Several TG antagonists were evaluated in citrated blood containing CTI triggered with 5 pM TF.a. 0.5 U/mL unfractionated heparin prolonged the lag phase ~2-fold and suppressed the peak thrombin ~3-fold.b. Hemophilia A and B were "induced" via the addition of inhibitory antibodies to FVIII and FIX, respectively, and although the lag phase was not altered both additions led to significant TG suppression with peak thrombin dropping ~3-fold.c. Several thrombin and FXa inhibitors were evaluated at their pharmacologic concentrations. Fondaparinux slightly prolonged the lag phase, whereas dabigatran increased it by 2.6-fold. Rivaroxaban doubled the lag phase while bivalirudin was slightly less efficient. None of these inhibitors had a pronounced effect on the rate and peak value of TG.d. Activated protein C (APC) at 10 nM slightly prolonged the lag phase and suppressed the peak thrombin.D. Citrate blood from a trauma patient was tested for endogenous activity. In the presence of CTI and absence of an initiator, α-TF antibody did not alter TG, indicating the absence of functional TF. α-FXIa antibody prolonged the lag phase and suppressed TG and α-FIXa antibody completely abolished TG, indicating the presence of active FXIa and FIXa.Packed Red Blood Cells (pRBC)In the absence of an initiator, no TG was observed in pRBC/CTI reconstituted with PPP. The addition of 5 pM TF led to TG after ~8 min with a peak value slightly lower than in blood (described in A).The addition of phospholipids did not change the TG profile.Conclusions.This continuous whole blood TG assay requires 15 µL of blood for a triplicate analysis of a single condition and has the potential for the evaluation of TG in disorders relevant to blood coagulation and for the monitoring of treatments administered in response to these disorders. DisclosuresMann:Haematologic Technologies, Inc.: Employment, Equity Ownership; Baxter: Consultancy; Bayer: Consultancy; Biogen IDEC: Consultancy; CSL Behring: Consultancy; Merck: Consultancy; Pfizer: Consultancy; The Medicines Company: Consultancy; XO1: Consultancy; Vascular Solutions: Consultancy; Stago: Consultancy.

Expression of functional tissue factor in activated T-lymphocytes in vitro and in vivo: A possible contribution of immunity to thrombosis?

ObjectiveT-lymphocyte activation plays an important role in the pathophysiology of acute coronary syndromes (ACS). Plaques from ACS patients show a selective oligoclonal expansion of T-cells, indicating a specific, antigen-driven recruitment of T-lymphocytes within the unstable lesions. At present, however, it is not known whether T-cells may contribute directly to thrombosis by expressing functional tissue factor (TF). Accordingly, the aim of the present study was to investigate whether T-cells are able to express functional TF in their activated status. MethodsIn vitro, CD3+-cells, isolated from buffy coats, were stimulated with anti-CD3/CD28 beads, IL-6, TNF-α, IL-17, INF-γ or PMA/ionomycin. Following stimulation, TF expression on cell-surface, at gene and protein levels, as well as its procoagulant activity in whole cells and microparticles was measured. In vivo, TF expression was evaluated in CD3+-cells isolated from the aorta and the coronary sinus of ACS-NSTEMI and stable coronary artery disease (SCAD) patients. The presence of CD3+-TF+cells was also evaluated by immunohistochemistry in thrombi aspirated from ACS-STEMI patients. ResultsPMA/ionomycin and IL-17 plus INF-γ stimulation resulted in a significant TF increase at gene and protein levels as well as at cell-surface expression. This was accompanied by a parallel increase in FXa generation, both in whole cells and in microparticles, indicating that the induced membrane-bound TF was active. Furthermore, transcardiac TF gradient was significantly higher in CD3+-cells obtained from ACS-patients compared to SCAD-patients. Interestingly, thrombi from ACS-STEMI patients resulted enriched in CD3+-cells, most of them expressing TF. ConclusionsOur data demonstrate that activated T-lymphocytes in vitro express functional TF on their membranes, suggesting a direct pathophysiological role of these cells in the thrombotic process; this hypothesis is further supported by the observations in vivo that CD3+-cells from coronary circulation of ACS-NSTEMI patients show increased TF levels and that coronary thrombi from ACS-STEMI patients are enriched in CD3+-cells expressing TF.

Particulate matter induces prothrombotic microparticle shedding by human mononuclear and endothelial cells

Particulate airborne pollution is associated with increased cardiopulmonary morbidity. Microparticles are extracellular vesicles shed by cells upon activation or apoptosis involved in physiological processes such as coagulation and inflammation, including airway inflammation. We investigated the hypothesis that particulate matter causes the shedding of microparticles by human mononuclear and endothelial cells.Cells, isolated from the blood and the umbilical cords of normal donors, were cultured in the presence of particulate from a standard reference. Microparticles were assessed in the supernatant as phosphatidylserine concentration. Microparticle-associated tissue factor was assessed by an one-stage clotting assay. Nanosight technology was used to evaluate microparticle size distribution.Particulate matter induces a dose- and time- dependent, rapid (1h) increase in microparticle generation in both cells. These microparticles express functional tissue factor. Particulate matter increases intracellular calcium concentration and phospholipase C inhibition reduces microparticle generation. Nanosight analysis confirmed that upon exposure to particulate matter both cells express particles with a size range consistent with the definition of microparticles (50–1000nm).Exposure of mononuclear and endothelial cells to particulate matter upregulates the generation of microparticles at least partially mediated by calcium mobilization. This observation might provide a further link between airborne pollution and cardiopulmonary morbidity.

Abstract 14366: T-lymphocytes May Contribute to Thrombus Formation in Patients With Acute Coronary Syndrome via Expression of Tissue Factor

Background: Activation of T-cells plays an important role in the pathophysiology of acute coronary syndromes (ACS). We have previously shown that plaques from ACS patients are characterized by a selective oligoclonal expansion of T-cells, indicating a specific, antigen-mediated recruitment of T-cells within the unstable lesions. We have also previously reported that activated T-cells in vitro express functional Tissue Factor (TF) on their surface. At the moment, however it is not known whether expression of TF by T-cells may contribute to thrombus formation in vivo. Methods: Blood was collected from the aorta and the coronary sinus of 13 NSTEMI and 10 stable CAD patients. CD3+ cells were selectively isolated and TF gene expression (real time PCR)and protein levels (western blot) were evaluated. In additional 7 STEMI patients, thrombotic formation material was obtained from the occluded coronary artery by catheter aspiration during primary PCI. TF expression in CD3+ cells was evaluated by immunohistochemistry and confocal microscopy. Results: Transcardiac TF expression in CD3+ cells was significantly higher in NSTEMI patients as compared to CD3+ cells obtained from stable CAD patients. Interestingly, thrombi aspirated from STEMI patients resulted enriched in CD3+cells, which expressed TF on their surface as shown in the figure. Conclusions: Our data demonstrate that in patients with ACS, T-lymphocytes may express surface TF, thus contributing to the process of thrombus formation. This finding may shed new light into the pathophysiologyof ACS.

The Aryl Hydrocarbon Receptor is a Critical Regulator of Tissue Factor Stability and an Antithrombotic Target in Uremia.

Patients with CKD suffer high rates of thrombosis, particularly after endovascular interventions, yet few options are available to improve management and reduce thrombotic risk. We recently demonstrated that indoxyl sulfate (IS) is a potent CKD-specific prothrombotic metabolite that induces tissue factor (TF) in vascular smooth muscle cells (vSMCs), although the precise mechanism and treatment implications remain unclear. Because IS is an agonist of the aryl hydrocarbon receptor (AHR), we first examined the relationship between IS levels and AHR-inducing activity in sera of patients with ESRD. IS levels correlated significantly with both vSMC AHR activity and TF activity. Mechanistically, we demonstrated that IS activates the AHR pathway in primary human aortic vSMCs, and further, that AHR interacts directly with and stabilizes functional TF. Antagonists directly targeting AHR enhanced TF ubiquitination and degradation and suppressed thrombosis in a postinterventional model of CKD and endovascular injury. Furthermore, AHR antagonists inhibited TF in a manner dependent on circulating IS levels. In conclusion, we demonstrated that IS regulates TF stability through AHR signaling and uncovered AHR as an antithrombotic target and AHR antagonists as a novel class of antithrombotics. Together, IS and AHR have potential as uremia-specific biomarkers and targets that may be leveraged as a promising theranostic platform to better manage the elevated thrombosis rates in patients with CKD.

Expression of functional tissue factor by neutrophil extracellular traps in culprit artery of acute myocardial infarction.

Neutrophil extracellular traps (NETs) are chromatin filaments released by activated polymorphonuclear neutrophils (PMNs) and decorated with granule proteins with various properties. Several lines of evidence implicate NETs in thrombosis. The functional significance and the in vivo relevance of NETs during atherothrombosis in humans have not been addressed until now. Selective sampling of thrombotic material and surrounding blood from the infarct-related coronary artery (IRA) and the non-IRA was performed during primary percutaneous revascularization in 18 patients with ST-segment elevation acute myocardial infarction (STEMI). Thrombi isolated from IRA contained PMNs and NETs decorated with tissue factor (TF). Although TF was expressed intracellularly in circulating PMNs of STEMI patients, active TF was specifically exposed by NETs obtained from the site of plaque rupture. Treatment of NET structures with DNase I abolished TF functionality measurement. In vitro treatment of control PMNs with plasma obtained from IRA and non-IRA was further shown to induce intracellular up-regulation of TF but not NET formation. A second step consisting of the interaction between PMNs and thrombin-activated platelets was required for NET generation and subsequent TF exposure. The interaction of thrombin-activated platelets with PMNs at the site of plaque rupture during acute STEMI results in local NET formation and delivery of active TF. The notion that NETs represent a mechanism by which PMNs release thrombogenic signals during atherothrombosis may offer novel therapeutic targets.

Diverse packaging of TF into platelets.

Tissue factor (TF) plays a central role in haemostasis serving as protective envelope around vascularised organs. Whether TF expressed by intravascular cells and by circulating or de novo generated microvesicles serve haemostasis or primarily cause thrombosis remains an active area of research. Vessel wall TF contributes to arterial thrombosis and myeloid cell TF is important for both, arterial and venous, thrombus formation in mice. In contrast, the pathophysiological roles of TF expressed by platelets remain uncertain or have been disputed altogether. Platelet TF mRNA and antigen expression was demonstrated in isolated platelets. Because TF was not only detected in platelets, but also on microvesicles, a passive transfer of leukocyte-derived TF (1) remained a possibility. Another important advance was the demonstration of incompletely spliced platelet TF pre-mRNA that is processed following agonist stimulation into functional TF (2). The delayed protein expression documented in several studies (3) indicated that platelet TF may primarily serve to stabilise thrombi, rather than initiate TF-dependent clot formation and hemostasis. Brambilla and colleagues (4) now studied in detail the transfer of TF from megakaryocytes to platelets. By employing a myeloblastic cell line and non-transformed bone marrow progenitors differentiated into mature megakaryocytes, these experiments minimised the transfer of premRNA or TF on microvesicles from other bone marrow cells during platelet production. While both fully processed as well as the previously demonstrated pre-mRNA was detected in megakaryocytes, unspliced TF mRNA was selectively transferred into newly generated platelets. Intriguingly, a subpopulation of platelets expressed TF protein, but low to undetectable unspliced TF mRNA. While the experimental setup of platelet release under static conditions in vitro likely fails to completely recapitulate shear induced homeostatic platelet production or megakaryocyte rupture under inflammatory stress conditions, the demonstrated heterogeneity of platelet TF cargo raises important new questions on prethrombotic states in humans. Future studies aiming to assess the inter-individual variability in platelet procoagulant potential should simultaneously evaluate TF mRNA and protein cargo in platelets.

Interaction of Human Platelets with Enterohemorrhagic E. coli O111, Induces an Increase in the Procoagulant Activity, Thrombin Generation and Adhesion to Endothelial Cells

Acute inflammation in response to severe bacterial infection, results in hemostatic abnormalities ranging from subclinical to sustained systemic clotting activation leading to massive thrombin and fibrin formation and microvascular thrombosis. Endothelial activation and dysfunction are critical determinants of the host response and provide an explanation for the different abnormalities involved in the pathophysiology of sepsis.Infection with pathogenic E. coli may present with a wide spectrum of clinical manifestations, from no symptoms or mild non-bloody diarrhea to severe cases, such as hemolytic uremic syndrome or thrombotic thrombocytopenic purpura, which is characterized by hemolytic anemia and low platelet counts.Although the understanding of the mechanisms that are involved in blood coagulation abnormalities in sepsis has gradually progressed, the role of platelets (Plts) on the procoagulant state during a severe infection remains to be addressed. Human platelets contain functional tissue factor (TF) (Panes et al. 2007) and TFPIa, but it is unknown if bacteria-platelet interaction affects platelet-TF procoagulant or platelet TFPI anticoagulant activities. Moreover, the effects of bacterial activation of platelets on thrombin generation (TG) in platelet rich plasma (PRP) or adhesion to endothelial cells have not been explored.Aims: We assessed the effect of platelet-E. coli interaction on platelet TF-dependent procoagulant activity (PCA), the changes induced by this interaction on platelet TFPI, in TG in PRP and in the adhesive capacity of platelets on cultured HUVEC.Plts activation by E. coli was demonstrated by a significant increase of p-selectin exposure on platelet surface compared to control Plts after 30 min of interaction with this microorganism harvested at exponential phase of growth and incubated in a ratio Plts/bacteria 1:10.Platelet TF-dependent PCA was assessed by FXa generation in washed Plts exposed to E. coli, with addition of exogenous FVIIa and FX, with no extra source of TF. Using the same ratio Plts/bacteria, we observed an increase in FXa after 30 min of incubation, compared with control platelets (p=0.0002, n=12). This enhancement in TF-PCA was concomitant with a decreased expression of TFPI in Plts surface after exposure of PRP to E. coli for 30 min. (p=0.0012, n=9).TG was measured in PRP, previously stimulated by E. coli for 30 min. We observed a shortening in the Lag time and time to peak and a higher thrombin peak in stimulated than in control PRP (p=0.0001, p=0.005 and p=0.0342, respectively, n=12). The reduction in lag time and time to peak was more pronounced than that obtained after eliciting platelet activation with Ristocetin. Preincubation of Plts with E. coli also increased the velocity index of TG compared to PRP alone (p=0,005; n=12).Static adhesion of Pts to endothelium was studied by stimulating fresh washed Plts with E. coli for 30min and then co-incubating them with HUVEC. After 20 min, an increased number of bacterial-activated Plts were adhered to HUVEC, compared with unstimulated Pts. Moreover, visible Plts aggregates were observed, which were positive for fibrin immunostaining, suggesting clot formation during the interaction of Plts with E. coli O111.Our findings show that Plts activated by bacteria results in an enhanced platelet procoagulant activity and adhesion to endothelium. By extension, these in vitro results suggest that platelets play an important role in the prothrombotic state associated with bacterial infections.This work was supported by FONDECYT-Chile 1130835 DisclosuresNo relevant conflicts of interest to declare.

Abstract 17: Cyclooxygenase-2 Deletion Favors Deep Vein Thrombosis in Mice

Deep vein thrombosis (DVT) is a common condition that often leads to pulmonary thromboembolism and death. Cyclooxygenase (COX)-1 and -2 catalyze the formation of prostaglandins and thromboxane (TX)A2 from arachidonic acid and play a critical role in the thrombotic events, however, the impact of COX-2 deletion (COX-2KO) in the DVT remains unclear. In this study, traditional (thromboelastomethry, mass weight and histology) and innovative (venous ultrasonography) approaches were used to assess the effect of COX-2 ablation on DVT in mice. In particular, ligation of the inferior vena cava to induce thrombus formation in COX-2KO and WT mice was carried out. Thromboelastometry analyses performed in whole blood of COX-2KO revealed alterations of clotting factors, and of platelet activity, with consequent greater clot firmness and clot elasticity. COX-2KO mice have increased plasma levels of functional fibrinogen, factor VIII and tissue factor (TF), and higher TXA2 serum than WTs. Finally, PAI-1 activity was the highest in COX-2KO, whereas tPA activity was similar in the two groups. The propensity to thrombosis of COX-2KO mice was also confirmed by ultrasonography. Genetic deletion of COX-2 was associated with bigger thrombi already at early time point. At 48 hours COX-2KO thrombi showed a greater content of connective tissue and fibrin, and increased number of infiltrated monocytes compared to WT. Remarkably, Annexin A2 (ANXA2), a fibrinolytic receptor, was significantly increased in cells present in COX-2KO thrombi. In addition, peritoneal macrophages from COX-2KO mice showed increased expression of ANXA2 with different distribution at cellular level compared to WTs. In particular, ANXA2 was expressed in the cytoplasm and in the nucleus of COX-2KO macrophages, whereas in control cells the protein was found mainly localized on cell membrane/periphery. In conclusion, the increased basal activation of haemostatic system observed in COX-2KO mice may partly explain the predisposition of these mice to thrombosis. In addition, the different localization of ANXA2 in monocytes from COX-2KO mice with respect to wild type mice suggests that this protein may display additional protective, as yet unknown, mechanisms influencing the stability of venous thrombi.

Extracellular haemoglobin upregulates and binds to tissue factor on macrophages: Implications for coagulation and oxidative stress

The mechanisms of crosstalk between haemolysis, coagulation and innate immunity are evolutionarily conserved from the invertebrate haemocyanin to the vertebrate haemoglobin (Hb). In vertebrates, extracellular Hb resulting from haemolytic infections binds bacterial lipopolysaccharide (LPS) to unleash the antimicrobial redox activity of Hb. Because bacterial invasion also upregulates tissue factor (TF), the vertebrate coagulation initiator, we asked whether there may be functional interplay between the redox activity of Hb and the procoagulant activity of TF. Using real-time PCR, TF-specific ELISA, flow cytometry and TF activity assay, we found that Hb upregulated the expression of functional TF in macrophages. ELISA, flow cytometry and immunofluorescence microscopy showed binding between Hb and TF, in isolation and in situ. Bioinformatic analysis of Hb and TF protein sequences showed co-evolution across species, suggesting that Hbβ binds TF. Empirically, TF suppressed the LPS-induced activation of Hb redox activity. Furthermore, Hb desensitised TF to the effects of antioxidants like glutathione or serum. This bi-directional regulation between Hb and TF constitutes a novel link between coagulation and innate immunity. In addition, induction of TF by Hb is a potentially central mechanism for haemolysis to trigger coagulation.