Surface Expression Of Tissue Factor Research Articles

The fluorochrome-to-protein ratio is crucial for the flow cytometric detection of tissue factor on extracellular vesicles

Extracellular vesicles (EVs) have crucial roles in hemostasis and coagulation. They sustain coagulation by exposing phosphatidylserine and initiate clotting by surface expression of tissue factor (TF) under inflammatory conditions. As their relevance as biomarkers of coagulopathy is increasingly recognized, there is a need for the sensitive and reliable detection of TF+ EVs, but their flow cytometric analysis is challenging and has yielded controversial findings for TF expression on EVs in the vascular system. We investigated the effect of different fluorochrome-to-protein (F/P) ratios of anti-TF-fluorochrome conjugates on the flow cytometric detection of TF+ EVs from activated monocytes, mesenchymal stem cells (MSCs), and in COVID-19 plasma. Using a FITC-labeled anti-TF antibody (clone VD8), we show that the percentage of TF+ EVs declined with decreasing F/P ratios. TF was detected on 7.6%, 5.4%, and 1.1% of all EVs derived from activated monocytes at F/P ratios of 7.7:1, 6.6:1, and 5.2:1. A similar decline was observed for EVs from MSCs and for EVs in plasma, whereas the detection of TF on cells remained unaffected by different F/P ratios. We provide clear evidence that next to the antibody clone, the F/P ratio affects the flow cytometric detection of TF+ EVs and should be carefully controlled.

Montelukast Inhibits Platelet Activation Induced by Plasma From COVID-19 Patients.

Leukotrienes are important pro-inflammatory lipid mediators derived from the arachidonic acid metabolism. In particular, cysteinyl leukotrienes, namely LTC4, LTD4, and LTE4 are involved in many of the principal features of asthma, while more recently they have also been implicated in cardiovascular diseases. COVID-19 is characterized by an overwhelming state of inflammation, sometimes resulting in an acute respiratory distress syndrome. Furthermore, severe COVID-19 patients present an endothelial cell damage characterized by a hyperinflammatory/procoagulant state and a widespread thrombotic disease. Leukotriene receptor antagonists, such as montelukast, have long been proven to have an efficacy in asthma, while more recently they have been suggested to have a protective role also in cardiovascular diseases. As elevated levels of LTE4 have been detected in bronchoalveolar lavage of COVID-19 patients, and montelukast, in addition to its anti-inflammatory properties, has been suggested to have a protective role in cardiovascular diseases, we decided to investigate whether this drug could also affect the platelet activation characteristic of COVID-19 syndrome. In this contribution, we demonstrate that montelukast inhibits platelet activation induced by plasma from COVID-19 patients by preventing the surface expression of tissue factor (TF) and P-selectin, reducing the formation of circulating monocyte– and granulocyte–platelet aggregates, and, finally, in completely inhibiting the release of TFpos-circulating microvesicles. These data suggest the repurposing of montelukast as a possible auxiliary treatment for COVID-19 syndrome.

Effects of colchicine on tissue factor in oxLDL-activated T-lymphocytes.

Several studies have shown that T-cells might be involved in pathophysiology of acute coronary syndromes (ACS). Tissue factor (TF) plays a key role in ACS. Many evidences have indicated that some statins reduce TF expression in several cell types. However, literature about rosuvastatin and TF and about statins effects on T-cells is still scanty. Colchicine is an anti-inflammatory drug recently proven to have beneficial effects in ACS via unknown mechanisms. This study investigates the effects of colchicine and rosuvastatin on TF expression in oxLDL-activated T-cells. T-cells, isolated from buffy coats of healthy volunteers, were stimulated with oxLDL (50µg/dL). T-cells were pre-incubated with colchicine (10µM) or rosuvastatin (5µM) for 1h and then stimulated with oxLDL (50μg/mL). TF gene (RT-PCR), protein (western blot), surface expression (FACS) and procoagulant activity (FXa generation assay) were measured. NF-κB/IκB axis was examined by western blot analysis and translocation assay. Colchicine and rosuvastatin significantly reduced TF gene, and protein expression and procoagulant activity in oxLDL stimulated T-cells. This effect was associated with a significant reduction in TF surface expression as well as its procoagulant activity. These phenomena appear modulated by drug effects on the transcription factor NF-kB. Rosuvastatin and colchicine prevent TF expression in oxLDL-stimulated T-cells by modulating the NF-κB/IκB axis. Thus, we speculate that this might be another mechanism by which these drugs exert benefic cardiovascular effects.

The procoagulant activity of tissue factor expressed on fibroblasts is increased by tissue factor-negative extracellular vesicles.

Tissue factor (TF) is critical for the activation of blood coagulation. TF function is regulated by the amount of externalised phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the surface of the cell in which it is expressed. We investigated the role PS and PE in fibroblast TF function. Fibroblasts expressed 6-9 x 104 TF molecules/cell but had low specific activity for FXa generation. We confirmed that this was associated with minimal externalized PS and PE and characterised for the first time the molecular species of PS/PE demonstrating that these differed from those found in platelets. Mechanical damage of fibroblasts, used to simulate vascular injury, increased externalized PS/PE and led to a 7-fold increase in FXa generation that was inhibited by annexin V and an anti-TF antibody. Platelet-derived extracellular vesicles (EVs), that did not express TF, supported minimal FVIIa-dependent FXa generation but substantially increased fibroblast TF activity. This enhancement in fibroblast TF activity could also be achieved using synthetic liposomes comprising 10% PS without TF. In conclusion, despite high levels of surface TF expression, healthy fibroblasts express low levels of external-facing PS and PE limiting their ability to generate FXa. Addition of platelet-derived TF-negative EVs or artificial liposomes enhanced fibroblast TF activity in a PS dependent manner. These findings contribute information about the mechanisms that control TF function in the fibroblast membrane.

Induction of tissue factor expression by anti-β2-glycoprotein I is mediated by tumor necrosis factor α.

Antiphospholipid antibodies (aPL) are heterogeneous and there is evidence that binding specificity determines which cellular effects they can trigger. We have therefore hypothesised that the induction of tissue factor (TF) in monocytes and endothelial cells by aPL depends on their binding specificity. To further investigate this, we have analyzed the ability of three human monoclonal aPL with distinctly different binding specificities to induce transcription and cell surface expression of TF in monocytes and endothelial cells. Results with human monoclonal aPL were validated with IgG-fractions obtained from patients with APS. We confirmed previous results that a lipid reactive human monoclonal aPL rapidly induced TF transcription and cell surface expression in monocytes and endothelial cells. A monoclonal aPL reactive against β2 glycoprotein I (β2GPI) induced TF with a delayed time course. This was fully dependent on the induction of tumor necrosis factor alpha (TNFα) secretion as capture of TNFα by adalimumab prevented TF induction. This pattern was confirmed with patient IgG fractions. Both lipid reactive and anti-β2GPI induced TF transcription. Unexpectedly, this activity of anti-β2GPI was mediated fully by TNFα which was secreted in response to incubation with anti-β2GPI. The role of TNFα in mediating TF induction by anti-β2GPI may have wider implications for APS pathogenesis.

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.

ACL/β2GPI and aPS/PT show synergic thrombogenic effects in suppressing anticoagulant activity of APC and stimulating tissue factor expression and TNF-α secretion by mononuclear cells

IntroductionPatients with systemic lupus erythematosus (SLE) possessing anti-phospholipid antibodies (aPLs) are often complicated by thrombotic vascular events. aPLs commonly associated with the complications are anti-cardiolipin/β2-glycoprotein I antibodies (aCL/β2GPI) and anti-phosphatidylserine/prothrombin antibodies (aPS/PT). However, the pathological mechanisms leading to thrombosis remain unclear. We explored clinical features of SLE patients with aCL/β2GPI and aPS/PT and investigated thrombogenic effects of their IgG fractions. Materials and methodsWe enrolled 97 SLE patients and 38 healthy control volunteers and performed activated protein C (APC) resistance screening test using their plasma samples. To detect the direct effect of aPLs IgG on APC, we developed an APC sensitivity ratio assay. Effects of aPLs IgG on monocytes were studied by measuring the surface expression of tissue factor (TF) and excretion of TNF-α from peripheral blood mononuclear cell culture. Results and conclusionThrombotic complications among SLE patients were closely associated with aCL/β2GPI or aPS/PT, with higher prevalence in patients with both antibodies. Addition of aPLs(+)-IgG to the APC sensitivity ratio assay led to significant suppression of the anticoagulant activity of APC. The suppression was more pronounced in double-positive cases. TF expression on monocytes and concentration of TNF-α in culture medium were increased by aPLs, again more pronounced in double-positive cases. These results indicate that the effects of aCL/β2GPI and aPS/PT are synergic both for APC anticoagulant activity and for production of TF and TNF-α from mononuclear cells. These modes of thrombogenic action of aPLs could be an important target for developing specific measures to prevent complications of SLE.

Staphylococcus aureus‐induced complement activation promotes tissue factor‐mediated coagulation

Background There is extensive cross-talk between the complement system, the Toll-like receptors (TLRs), and hemostasis. Consumptive coagulopathy is a hallmark of sepsis, and is often mediated through increased tissue factor (TF) expression. Objectives To study the relative roles of complement, TLRs and TF in Staphylococcus aureus-induced coagulation. Methods Lepirudin-anticoagulated human whole blood was incubated with the three S. aureus strains Cowan, Wood, and Newman. C3 was inhibited with compstatin, C5 with eculizumab, C5a receptor 1 (C5aR1) and activated factor XII with peptide inhibitors, CD14, TLR2 and TF with neutralizing antibodies, and TLR4 with eritoran. Complement activation was measured by ELISA. Coagulation was measured according to prothrombin fragment 1 + 2 (PTF1 + 2 ) determined with ELISA, and TF mRNA, monocyte surface expression and functional activity were measured with quantitative PCR, flow cytometry, and ELISA, respectively. Results All three strains generated substantial and statistically significant amounts of C5a, terminal complement complex, PTF1 + 2 , and TF mRNA, and showed substantial TF surface expression on monocytes and TF functional activity. Inhibition of C5 cleavage most efficiently and significantly inhibited all six markers in strains Cowan and Wood, and five markers in Newman. The effect of complement inhibition was shown to be completely dependent on C5aR1. The C5 blocking effect was equally potentiated when combined with blocking of CD14 or TLR2, but not TLR4. TF blocking significantly reduced PTF1 + 2 levels to baseline levels. Conclusions S. aureus-induced coagulation in human whole blood was mainly attributable to C5a-induced mRNA upregulation, monocyte TF expression, and plasma TF activity, thus underscoring complement as a key player in S. aureus-induced coagulation.

Differing mechanisms of thrombin generation in live haematological and solid cancer cells determined by calibrated automated thrombography.

: Calibrated automated thrombography (CAT) is emerging as a reliable tool for real-time estimation of thrombin generation potential. There is a clinical need for knowledge about the pathways underlying the thrombotic phenotype of different malignancies. Cells from solid (e.g. pancreatic cancer; n = 7) and malignant haematological cell lines (e.g. multiple myeloma; n = 5) were evaluated for thrombin generation, using CAT, with the addition of control plasma (NormTrol; Helena Biosciences, Gateshead, UK)) or plasma deficient in coagulation factors VII and XII. In addition, tissue factor (TF) cell surface expression was determined by flow cytometry. In platelet-free plasma, thrombin generation in all cancer cell lines was cell concentration dependent, with the pancreatic cancer line CFPAC-1 producing the highest thrombin of 220 nmol/l at 5 × 10-cells/ml concentration. Lag times and times to peak reflected most significant differences out of all thrombin generation parameters measured and were inversely correlated with cell surface TF surface expression. Solid tumour cell lines had higher thrombin peaks, faster lag times, and a thrombin generation profile of overall greater magnitude than haematological cell lines. In the absence of factor VII in platelet-free plasma, thrombin generation in solid pancreatic cancer cell lines was significantly reduced unlike in haematological cell lines. However, in the absence of factor XII, thrombin generation was reduced more in haematological cells but had little or no effect on solid cell lines. The CAT assay identified characteristic differences in thrombin generation kinetics between solid tumour and haematological cancer cell lines, of which lag time and time to peak correlated with TF cell surface expression.

Complement C5, phagocytosis and Toll-like receptor 4 play key roles in Escherichia coli-induced surface expression of tissue factor on human monocytes

Complement C5, phagocytosis and Toll-like receptor 4 play key roles in Escherichia coli-induced surface expression of tissue factor on human monocytes

Cancer microvesicles induce tissue factor-related procoagulant activity in endothelial cells in vitro.

: Microvesicles associated with tissue factor (TF) may play a role in cancer-related venous thromboembolism; however, not much is known about their interaction with the tumour stroma, especially the endothelium or any procoagulant changes seen because of this interaction. Using a head and neck squamous cell carcinoma line (UMSCC81B) and human umbilical vein endothelial cells (HUVECs), this study explored the interaction of cancer microvesicles released into cell culture media with endothelial cells in vitro, and assessed the procoagulant activity resulting from this interaction. Cell-free media containing UMSCC81B cancer microvesicles supported coagulation in a concentration-dependent manner, suggesting TF and microvesicle presence, this media was then added to HUVECs and flow cytometry analysis showed a subpopulation of HUVECs that had acquired a significantly high expression of TF, which was dependent upon the concentration of UMSCC81B media containing microvesicles present and confocal microscopy confirmed HUVECs associated with labelled microvesicles. The range of TF-positive HUVECs was determined to be 0, 4.2(±1.4), 12.5(±3.72), and 45.9(±18.7)% for microvesicle-positive media concentration of 0, 25, 50, and 100%, respectively, which resulted in decreasing prothrombin values of more than 600 (no clot), 126.4, 65.8, and 47.8 s. Our results demonstrate that procoagulant microvesicles shed by UMSCC81B induced a procoagulant effect in HUVECs through increased clotting activity and cell membrane surface expression of TF.

Anti-thrombotic and pro-fibrinolytic effects of levosimendan in human endothelial cells in vitro

AimsLevosimendan is an inodilator for the treatment of acute decompensated heart failure (HF). Data from clinical studies suggest that levosimendan is particularly effective in HF due to myocardial infarction. After acute revascularization, no reflow-phenomenon is a common complication that may lead to pump failure and cardiogenic shock. Our aim was to examine whether levosimendan interferes with the pro-thrombotic phenotype of activated endothelial cells in vitro. MethodsHuman heart microvascular endothelial cells (HHMEC) and human umbilical vein endothelial cells (HUVEC) were treated with interleukin-1β (IL-1β) (200U/mL) or thrombin (5U/mL) and co-treated with or without levosimendan (0.1–10μM) for 2–24h. In addition, flow experiments were performed. Effects on plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) expression and activity were measured by rt-PCR, specific ELISA and flow cytometry. ResultsTreatment with IL-1β or thrombin significantly increased the expression of PAI-1 and TF in endothelial cells. Co-treatment with levosimendan strongly attenuated the effects of IL-1β and thrombin on PAI-1 and TF mRNA by up to 50% and 45%, in a dose- and time-dependent manner. Similar results were obtained under flow conditions. Furthermore, co-treatment with levosimendan dampened the antigen production of PAI-1 and the surface expression of TF by 35% and 45%, respectively. Additionally, levosimendan diminished both TF and PAI-1 activity. ConclusionLevosimendan down-regulates the expression of the pro-thrombotic and anti-fibrinolytic biomolecules TF and PAI-1 in activated human endothelial cells. Our findings may, at least in part, explain some of the beneficial effects of levosimendan after myocardial reperfusion.

Abstract 13445: Thrombin-Inhibiting Nanoparticle Treatment Focally Reduces NF-kB Activation and Tissue Factor Expression in Atherosclerotic Plaques

Introduction: Thrombin plays a significant role in atherogenesis through PAR-1 signaling on various cell types and downstream activation of NF-kB, a potent driver of inflammation and hypercoagulability via tissue factor (TF) production in plaques. Herein, we demonstrate the use of perfluorocarbon nanoparticles (NP) bound to the direct thrombin inhibitor D-phenylalanyl-L-prolyl-L-chloromethylketone (PPACK: 13650 per NP) for downregulation of NF-kB activity and inhibition of tissue factor (TF) production. Methods and Results: Cell surface TF expression was assessed in vitro using a functional assay of Xa generation on human aortic endothelial cells (HAECs) and THP-1 monocytes following a six hour incubation with 1 U/ml and 4 U/ml thrombin and either saline, free PPACK, control NP or PPACK NP. Treatment with PPACK NP in both cell types at the two thrombin concentrations completely suppressed excess surface tissue factor expression over baseline levels, in contrast to cells treated with saline or control NP. Thrombin’s ability to activate NF-kB in HAECs via degradation of the cytoplasmic regulatory complex IkB was fully inhibited by PPACK NP as well. PPACK NP were evaluated in vivo in ApoE-/- mice fed a Western diet for a total of 4 months. The mice received 1 ml/kg saline, control NP or PPACK NP intravenously 3 times/week for the final month of feeding. Following treatment, aortas were sectioned for histological analysis of tissue factor expression and NF-kB activation. PPACK NP treatment resulted in a 25.95% decrease in plaque associated tissue factor (Figure, p = 0.027) and diminished phosphorylated-p65 staining compared to saline and control NP treatment. Clotting parameters and bleeding times normalized within 60 min of i.v. delivery of PPACK NP. Conclusion: Inhibition of thrombin and PAR-1 signaling with PPACK NP exhibits a sustained reduction in local NF-kB activation and tissue factor expression in atherosclerotic plaques without causing a bleeding diathesis.

The key roles of complement and tissue factor in Escherichia coli-induced coagulation in human whole blood.

The complement system and the Toll-like (TLR) co-receptor CD14 play important roles in innate immunity and sepsis. Tissue factor (TF) is a key initiating component in intravascular coagulation in sepsis, and long pentraxin 3 (PTX3) enhances the lipopolysaccharide (LPS)-induced transcription of TF. The aim of this study was to study the mechanism by which complement and CD14 affects LPS- and Escherichia coli (E. coli)-induced coagulation in human blood. Fresh whole blood was anti-coagulated with lepirudin, and incubated with ultra-purified LPS (100 ng/ml) or with E. coli (1 × 10(7) /ml). Inhibitors and controls included the C3 blocking peptide compstatin, an anti-CD14 F(ab')2 antibody and a control F(ab')2 . TF mRNA was measured using quantitative polymerase chain reaction (qPCR) and monocyte TF surface expression by flow cytometry. TF functional activity in plasma microparticles was measured using an amidolytic assay. Prothrombin fragment F 1+2 (PTF1.2) and PTX3 were measured by enzyme-linked immunosorbent assay (ELISA). The effect of TF was examined using an anti-TF blocking antibody. E. coli increased plasma PTF1.2 and PTX3 levels markedly. This increase was reduced by 84->99% with compstatin, 55-97% with anti-CD14 and > 99% with combined inhibition (P < 0·05 for all). The combined inhibition was significantly (P < 0·05) more efficient than compstatin and anti-CD14 alone. The LPS- and E. coli-induced TF mRNA levels, monocyte TF surface expression and TF functional activity were reduced by > 99% (P < 0·05) with combined C3 and CD14 inhibition. LPS- and E. coli-induced PTF1.2 was reduced by 76-81% (P < 0·05) with anti-TF antibody. LPS and E. coli activated the coagulation system by a complement- and CD14-dependent up-regulation of TF, leading subsequently to prothrombin activation.

Fructose induces prothrombotic phenotype in human endothelial cells : A new role for "added sugar" in cardio-metabolic risk.

Intake of large amounts of added sweeteners has been associated with the pathogenesis of cardiometabolic risk. Several studies have shown that fructose increases the cardiovascular risk by modulating endothelial dysfunction and promoting atherosclerosis. Recently, a potential role for fructose in cardiovascular thrombosis has been suggested but with controversial results. Tissue factor (TF) plays a pivotal role in the pathophysiology of cardiovascular thrombosis by triggering the formation of intracoronary thrombi following endothelial injury. This study investigates the effects of fructose, in a concentration range usually observed in the plasma of patients with increased cardiovascular risk, on TF in human umbilical endothelial cells (HUVECs). Cells were stimulated with increasing concentrations of fructose (0.25, 1 and 2.5mM) and then processed to evaluate TF-mRNA levels by real-time PCR as well as TF expression/activity by FACS analysis and procoagulant activity. Finally, a potential molecular pathway involved in modulating this phenomenon was investigated. We demonstrate that fructose induces transcription of mRNA for TF. In addition, we show that this monosaccharide promotes surface expression of TF that is functionally active. Fructose effects on TF appear modulated by the oxygen free radicals through activation of the transcription factor NF-κB since superoxide dismutase and NF-κB inhibitors suppressed TF expression. Data of the present study, although in vitro, indicate that fructose, besides promoting atherosclerosis, induces a prothrombotic phenotype in HUVECs, thus indicating one the mechanism(s) by which this sweetener might increase cardiometabolic risk.

The adipokine apelin-13 induces expression of prothrombotic tissue factor.

Adipocytes are cells able to produce and secrete several active substances (adipokines) with direct effects on vascular cells. Apelin, one of the most recently identified adipokines has been studied in cardiovascular system physiology in regard to vessel vasodilation and myocardial contraction, but it has not yet completely characterised for its pathophysiological role in cardiovascular disease and especially in acute coronary syndromes (ACS). Several studies have indicated that tissue factor (TF) plays a pivotal role in the pathophysiology of ACS by triggering the formation of intracoronary thrombi following endothelial injury. This study investigates the effects of apelin 12 and apelin 13 on TF in human umbilical endothelial cells (HUVECs) and monocytes. Cells were stimulated with increasing concentrations of apelin 12 or apelin 13 and then processed to evaluate TF-mRNA levels by real-time PCR as well as TF expression/activity by FACS analysis and pro-coagulant activity. Finally, a potential molecular pathway involved in modulating this phenomenon was investigated. We demonstrate that apelin 13 but not apelin 12 induces transcription of mRNA for TF. In addition, we show that this adipokine promotes surface expression of TF that is functionally active. Apelin 13 effects on TF appear modulated by the activation of the G-protein-transcription factor nuclear factor (NF)-κB axis since G-protein inhibitors suppressed NF-κB mediated TF expression. Data of the present study, although in vitro, indicate that apelin-13, induces a procoagulant phenotype in HUVECs and monocytes by promoting TF expression. These observations support the hypothesis that this adipokine might play a relevant role as an active partaker in athero-thrombotic disease.

Abstract 19260: Prothrombotic Activity and Reduction in Coronary Flow Reserve Associated With Therapeutic Intracoronary Delivery of Bone Marrow-Derived Mesenchymal Stem Cells is Ameliorated by Heparin Co-Administration in a Porcine Myocardial Infarction Model

Cell therapy remains an evolving therapeutic option for cardiac repair in the case of acute myocardial infarction (AMI) associated with significant cardiomyocyte death. Mesenchymal stem cells (MSCs) are promising candidates for promoting myocardial salvage post AMI, but concerns regarding the safety of intracoronary MSC delivery remain. Surface expression of tissue factor (TF), a key initiator of the soluble coagulation cascade, has recently been implicated as a possible cause of MSC procoagulant activity. This study aimed to characterize the prothrombotic activity of MSC and identify means of managing any thrombotic side effects during therapeutic intracoronary delivery post AMI. Expression of TF on MSC was detected by immunoflourescence, flow cytometry and immunoblotting. TF antigen was catalytically active (252.4 ± 8.3 pM/2 x 105 MSC) and capable of supporting thrombin generation in vitro. Addition of MSCs to whole citrated blood enhanced platelet-driven thrombus deposition on collagen at arterial shear flow (12.67 ± 1.0% untreated vs 18.9 ± 0.9% surface coverage with MSC, p&gt;0.001), an effect abolished by heparin co-administration (p&lt;0.01 vs MSC alone). Infusion of 25 x 106 MSC via an intracoronary route in a porcine AMI model was associated with a decrease in coronary flow reserve when delivered during reperfusion (-0.7 ± 0.05) but not when coadministered with heparin (0.1 ± 0.02, p&lt;0.01). Heparin also reduced MSC-associated thrombosis incorporating platelets and vWF in the microvasculature (p&lt;0.05). Therapeutic MSC delivery was able to reduce apoptosis in the infarct border zone 24 post AMI (p&lt;0.01). Procoagulant activity associated with therapeutic MSCs is independently associated with reduction in myocardial perfusion when delivered I.C. which may be successfully managed with heparin co-administration. This study highlights an important mechanistic insight into safety concerns associated with therapeutic intracoronary MSC delivery for AMI.

Pivotal Role Of Heat Shock Proteins In Monocyte and Endothelial Cell Pathology In Sickle Cell Disease

BackgroundIt is well established that sickle cell disease (SCD) manifests global perturbations of hemostasis. Vaso-occlusion, inflammation and coagulopathy all likely contribute to the protean complications of SCD. Central to both inflammation and coagulation is the monocyte. These cells can be profoundly pro-inflammatory and can express tissue factor on their surface and thus influence both inflammation and coagulation. Monocytosis is common in SCD, as is steady state monocyte activation. Exaggerated monocytic response to stimulus may also contribute to the severity of acute events. Thus, agents that regulate monocyte function are potentially of significant relevance in SCD.To this end, we have discovered that the heat shock proteins (HSPs) are potential master regulators of these cells. We previously demonstrated that inhibition of one such HSP, HSP90, could completely ablate the profound and hyper-responsive monocytic inflammatory release upon lipopolysacchiride (LPS) challenge. Inhibition of HSP90 also blocked LPS induced NFk-B translocation to the nucleus. Thus, these results suggested a potent role for HSP90 in LPS-induced monocyte based inflammation. However, the role of HSP90 is cytokine-induced monocyte activation was speculative. The role of HSP90 in monocyte tissue factor expression, or reactive oxygen species (ROS) generation remained unknown. ObjectivesWe sought to determine the role of HSP90 in regulating the pro-inflammatory, pro-coagulant, and ROS generating potential in monocytes. We then wanted to establish a potential role for HSP90 inhibition. MethodsPeripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll density separation. Flow cytometry was employed to measure the LPS- or cytokine-induced monocyte tissue factor expression and ROS generation. Monocyte ROS generation was detected using L012 based chemiluminescence or visualized in individual cells using flow cytometry with CELLROX. Inflammatory cytokines and tissue factor gene expression was evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). NFkB translocation to the nucleus was detected via cell fractionation followed by western blotting or indirect immunofluorescence. PBMCs or THP-1 cells were pre-treated with the HSP90 inhibitors 17-DMAG or AUY922 prior to assay. Monocyte- induced endothelial cell permeability was measured using endothelial cell-substrate impedance sensing (ECIS). Unless otherwise stated p<0.05. ResultsAt baseline, PBMCs from patients with SCD demonstrated elevated monocyte ROS generation and tissue factor expression than those from healthy controls. Inhibition of significantly reduced these measures of steady state monocyte activation. HSP90 inhibition also inhibited both LPS and cytokine induced tissue mRNA accumulation and subsequent cell surface expression of tissue factor in monocytes. Cytokine- induced ROS generation was significantly interrupted in monocytes upon inhibition of HSP90. A panel of monocyte pro-inflammatory genes could be inhibited with AUY922, whereas the anti-inflammatory cytokine, IL-10, was induced upon HSP90 inhibition with AUY922. Mechanistically, we also noted a profound translocation of NFkB to the monocyte nucleus upon cytokine stimulation. Consistent with the effects on tissue factor expression, pro-inflammatory potential, and ROS generation, this translocation could be completely ablated with HSP90 inhibition. Importantly, HSP90 inhibitors significantly attenuated the LPS-activated monocyte -induced lung microvascular endothelial permeability. ConclusionOur data suggest that Hsp90 inhibitors significantly reduced the both pro-inflammatory and pro-coagulatory potential of PBMCs from patients with SCD. These results thus position HSP90 as a potential master regulator of hemostasis, endothelial cell permeability, and thus suggest that HSP90 is an attractive therapeutic target in patients with SCD. Disclosures:Kutlar:Celgene Corporation: Research Funding. Meiler:Celgene Corporation: Research Funding.

Phosphatidylserine index as a marker of the procoagulant phenotype of acute myelogenous leukemia cells

Patients with acute myelogenous leukemia (AML) are at risk for thrombotic complications. Risk to develop thrombosis is closely tied to leukemia subtype, and studies have shown an association between leukocytosis and thrombosis in AML M3. We evaluated the relative roles of cell count and the surface expression of tissue factor (TF) and phosphatidylserine (PS) in the procoagulant phenotype of AML cell lines. The TF-positive AML M3 cell lines, NB4 and HL60, and AML M2 cell line, AML14, exhibited both extrinsic tenase and prothrombinase activity in a purified system and promoted experimental thrombus formation. In contrast, the TF-negative AML cell line, HEL, exhibited only prothrombinase activity and did not affect the rate of occlusive thrombus formation. In plasma, NB4, HL60 and AML14 shortened clotting times in a cell-count, PS- and TF-dependent manner. Exposure of cultured NB4, HL60, and AML14 cells to the chemotherapeutic agent daunorubicin increased their extrinsic tenase activity and PS expression. Clot initiation time inversely correlated with logarithm of PS index, defined as the product of multiplying leukocyte count with cell surface PS exposure. We propose that leukemia cell PS index may serve as a biomarker for procoagulant activity.

C1-inhibitor efficiently inhibitsEscherichia coli-induced tissue factor mRNA up-regulation, monocyte tissue factor expression and coagulation activation in human whole blood

Both the complement system and tissue factor (TF), a key initiating component of coagulation, are activated in sepsis, and cross-talk occurs between the complement and coagulation systems. C1-inhibitor (C1-INH) can act as a regulator in both systems. Our aim in this study was to examine this cross-talk by investigating the effects of C1-INH on Escherichia coli-induced haemostasis and inflammation. Fresh human whole blood collected in lepirudin was incubated with E. coli or ultrapurified E. coli lipopolysaccharide (LPS) in the absence or presence of C1-INH or protease-inactivated C1-INH. C3 activation was blocked by compstatin, a specific C3 convertase inhibitor. TF mRNA was measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and TF surface expression was measured by flow cytometry. In plasma, the terminal complement complex, prothrombin F1·2 (PTF1·2) and long pentraxin 3 (PTX3) were measured by enzyme-linked immunosorbent assay (ELISA). Cytokines were analysed using a multiplex kit. C1-INH (1·25-5 mg/ml) reduced both LPS- and E. coli-induced coagulation, measured as a reduction of PTF1·2 in plasma, efficiently and dose-dependently (P < 0·05). Both LPS and E. coli induced marked up-regulation of TF mRNA levels and surface expression on whole blood monocytes. This up-regulation was reduced efficiently by treatment with C1-INH (P < 0·05). C1-INH reduced the release of PTX3 (P < 0·05) and virtually all cytokines measured (P < 0·05). Complement activation was inhibited more efficiently with compstatin than with C1-INH. C1-INH inhibited most of the other readouts more efficiently, consistent with additional non-complement-dependent effects. These results indicate that complement plays a role in activating coagulation during sepsis and that C1-INH is a broad-spectrum attenuator of the inflammatory and haemostatic responses.