Tissue Factor Levels Research Articles

189 Altered platelet function and thrombosis in fetal growth restriction

BackgroundFetal growth restriction (FGR) affects 3%–8% of pregnancies and is associated with increased risk of stillbirth and perinatal morbidity, in addition to increased risk of cardiovascular disease later in life....

Evaluation of venous thrombosis and tissue factor in epithelial ovarian cancer

ObjectiveOvarian clear cell carcinoma (OCCC) and high grade serous ovarian cancer (HGSOC) are associated with the highest risk of VTE among patients with epithelial ovarian cancer (EOC). Tissue factor (TF) is a transmembrane glycoprotein which can trigger thrombosis. We sought to evaluate if there is an association between VTE and tumor expression of tissue factor (TF), plasma TF, and microvesicle TF (MV TF) activity in this high-risk population. MethodsWe performed a case-control study of OCCC and HGSOC patients with and without VTE. 105 patients who underwent surgery at a tertiary care center between January 1995 and October 2013 were included. Plasma TF was measured with an enzyme-linked immunosorbent assay. A TF-dependent Factor Xa generation assay was used to measure MV TF activity. Immunohistochemical (IHC) analysis was performed to evaluate tumor expression of TF. Results35 women with OCCC or HGSOC diagnosed with VTE within 9months of surgery were included in the case group. Those with VTE had a worse OS, p<0.0001, with a greater than three-fold increase in risk of death, HR 3.33 (CI 1.75–6.35). There was no significant difference in median plasma TF level or MV TF activity level between patients with and without VTE. OCCC patients had greater expression of TF in their tumors than patients with HGSOC, p<0.0001. ConclusionsTFMV activity and plasma TF level were not predictive of VTE in this patient population. Given the extensive expression of TF in OCCC tumors, it is unlikely IHC expression will be useful in risk stratification for VTE in this population.

Assessment of the Procoagulant Activity of Microparticles and the Protein Z System in Patients Undergoing Off-Pump Coronary Artery Bypass Surgery.

To understand the coagulation changes after off-pump coronary artery bypass (OPCAB) surgery, we evaluated the procoagulant activity of microparticles (MPs) and microparticles exposing tissue factor (MPs-TF), together with the levels of total tissue factor (TF), protein Z (PZ), protein Z-dependent protease inhibitor (ZPI), and factor X (FX) before (first day) and 1 week after surgery (seventh day) in plasma samples from 30 patients. Twenty healthy controls were also included. Compared to the controls, patients scheduled for surgery had significantly higher MPs-TF procoagulant activity and lower TF levels ( P = .0006, P = .02, respectively). In the whole cohort, median procoagulant activity of MPs-TF and median levels of TF and ZPI were significantly lower ( P = .02, P = .0003, and P = .004, respectively), while median levels of PZ and FX were significantly higher ( P = .02 and P = .002, respectively) on the seventh day compared to the first day. Our results suggest that OPCAB surgery has a significant effect on the procoagulant activity of MPs-TF and the PZ system.

Abstract 642: Specific Protein Kinase C Isoforms Are Critical Mediators of Physiologic Downregulation of Perivascular Tissue Factor

Tissue factor (TF) is a procoagulant and transmembrane receptor for FVII(a) that is upregulated in pathological conditions. We recently demonstrated that perivascular TF is downregulated around angiogenic vessels near a cutaneous wound. The goal of this study was to identify mechanisms that mediate TF loss. Primary cultures of human pericytes express high levels of TF. TF expression was lost during culture with phorbol-12 myristate 13-acetate (PMA) for 8 hours, and was maintained for 24 hours. This model recapitulates the pattern of downregulation observed in vivo . Using qRT-PCR we assessed changes in TF gene expression in response to PMA. TF mRNA decreased 4- and 6-fold at 8 and 12 hours after treatment (p&lt;0.01), and remained 2-fold lower in treated cells after 24 hours (p&lt;0.05). Inhibiting de novo transcription with actinomycin D showed that degradation of TF mRNA was similar in PMA- and vehicle-treated groups (p=ns). Thus, downregulation of TF mRNA occurs primarily through inhibition of its synthesis. We next identified a physiologic mediator of TF downregulation using specific inhibitors against PMA-responsive signaling proteins. Two different inhibitors against Protein Kinase C (PKC) were used: Go6983, which inhibits isoforms α, β, δ, ε, μ, and ζ, and GFX, which inhibits α, β, ε, and γ. Both inhibitors significantly attenuated PMA-mediated transcriptional downregulation (p&lt;0.001). Based on overlap of inhibited isoforms, this suggests a minimal role for ζ, μ, δ, and γ, while one or more of the α, β, and ε isoforms appear to be critical mediators of TF mRNA synthesis inhibition. Since the timing of TF protein loss is not fully explained by transcriptional inhibition, we investigated the role of protein degradation in TF loss. When protein synthesis was inhibited by cyclohexamide, addition of PMA shortened the half-life of pericyte TF from 11 hours to 5 hours (p&lt;0.001). This indicates that increased protein degradation contributes to PMA-induced loss of TF expression. Both Go6983 and GFX significantly attenuated TF protein degradation also. Taken together, our data show that TF downregulation is mediated by transcriptional inhibition and protein degradation, and PKC α, β, and ε have emerged as potential mediators of both mechanisms.

Effects of hyaluronic acid on bleeding following third molar extraction.

Objective To explore the effects of hyaluronic acid (HA) on bleeding and associated outcomes after third molar extraction.Methods Forty patients who had undergone molar extraction were randomly divided into two groups; 0.8% (w/v) HA was applied to the HA group (n=20) whereas a control group (n=20) was not treated. Salivary and gingival tissue factor (TF) levels, bleeding time, maximum interincisal opening (MIO), pain scored on a visual analog scale (VAS), and the swelling extent were compared between the two groups.Results HA did not significantly affect gingival TF levels. Salivary TF levels increased significantly 1 week after HA application but not in the control group. Neither the VAS pain level nor MIO differed significantly between the two groups. The swelling extent on day 3 and the bleeding time were greater in the HA group than in the control group.Conclusions Local injection of HA at 0.8% prolonged the bleeding time, and increased hemorrhage and swelling in the early postoperative period after third molar extractions.

Effect of Agkistrodon Acutus Venom PCA on HUVEC Activity

To investigate the effects of Agkistrodon acutus venom protein C activator(PCA) on ultrastructure of human umbilical vein endothelial cells(HUVEC), and the levels of tissue factor(TF), vascular von Willebrand factor (vWF) and endothelin-1 secreted by HUVEC and to clarify the anti-thrombotic mechanism of PCA. The experiments were divided into control group(DMEM), LPS group (LPS 0.1 µg/ml), PCA group(PCA 1 µg/ml) and PCA+LPS group (1 µg/ml PCA+ 0.1 µg/ml LPS). The morphology of endoplasmic reticulum, mitochondria and the number of autophagosome in HUVEC were observed by transmission electron microscopy. The TF, vWF and ET-1 were measured in the medium of each group by ELISA; RT-PCR was used to detect mRNA expression level of vWF and ET-1 in cells; and the protein expression level of TF in cells was detected by Western blot. Compared with the control group, the ultrastructural changes of HUVEC in the LPS group included the cell membrane getting rough, swelling of mitochondria and endoplasmic reticulum, and autophagosome increase, however, the ultrastructure differences between PCA and control group were not significant. Compared with the ultrastructure of HUVECs in LPS group, the swelling of mitochondria and endoplasmic reticulum disappeared in the LPS+PCA group, and the number of autophagosome decreased obviously. Compared with the control group, the content of ET-1, vWF and TF in cell culture supernatant, and the protein expression level of vWF, ET-1 gene and TF protein were significantly increased in LPS group (P<0.05); the expression levels of the 3 factors in the cell culture supernatant and cells in PCA group were not significantly different from the control group (P>0.05). The expression levels of TF, vWF and ET-1 in LPS group were significantly lower than those in LPS+PCA group (P<0.05). PCA(1 µg/ml) can reduce the ultrastructural changes of HUVEC induced by LPS, and inhibit the increase of TF, vWF and ET-1 secretion from HUVEC induced by LPS.

Tissue factor levels in type 2 diabetes mellitus.

Type 2 diabetes mellitus is a pandemic associated with disturbance in haemostasis that could contribute to the development of diabetic vascular disease and accelerated atherosclerosis. In this population, hypercoagulation is prevalent, as well as pathological changes to erythrocytes. This is mainly due to upregulated circulating inflammatory markers. Here we looked at tissue factor (TF) levels using ELISA, in a sample of diabetics, with and without cardiovascular complications. Diabetic subjects were recruited from the diabetic clinic at Steve Biko Academic Hospital, Pretoria, South Africa. 20 diabetics with cardiovascular disease and 22 without were enrolled to participate. TF levels were significantly elevated in both diabetic groups when compared to the controls. We suggest that pathologic plasma TF activity, as marker of increased propensity of clot pathology, should be investigated. Agents that might lower TF levels might also possibly lower thrombotic complications.

Orthostatic Challenge Shifts the Hemostatic System of Patients Recovered from Stroke toward Hypercoagulability

Aims: The objective of our study was to assess the effects of orthostatic challenge on the coagulation system in patients with a history of thromboembolic events and to assess how they compared with age-matched healthy controls.Methods: Twenty-two patients with histories of ischemic stroke and 22 healthy age-matched controls performed a sit-to-stand test. Blood was collected prior to- and at the end of- standing in the upright position for 6 min. Hemostatic profiling was performed by determining thrombelastometry and calibrated automated thrombogram values, indices of thrombin generation, standard coagulation times, markers of endothelial activation, plasma levels of coagulation factors and copeptin, and hematocrit.Results: Orthostatic challenge caused a significant endothelial and coagulation activation in patients (Group 1) and healthy controls (Group 2): Plasma levels of prothrombin fragment F1+2 were increased by approximately 35% and thrombin/antithrombin-complex (TAT) increased 5-fold. Several coagulation variables were significantly altered in Group 1 but not in Group 2: Coagulation times (CTs) were significantly shortened and alpha angles, peak rate of thrombin generation (VELINDEX), tissue factor (TF) and copeptin plasma levels were significantly increased (comparison between standing and baseline). Moreover, the shortening of CTs and the rise of copeptin plasma levels were significantly higher in Group 1 vs. Group 2 (comparison between groups).Conclusion: The coagulation system of patients with a history of ischemic stroke can be more easily shifted toward a hypercoagulable state than that of healthy controls. Attentive and long-term anticoagulant treatment is essential to keep patients from recurrence of vascular events.

Co-expression of tissue factor and IL-6 in immature endothelial cells of cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are congenital abnormal clusters of capillaries that are prone to leaking and thought to result from a disorder of endothelial cells. The underlying pathology of CCM is not fully understood. We analyzed the expression of tissue factor (TF) and interleukin-6 (IL-6) in CCMs to determine the association of TF and IL-6 with clinical and pathological findings. Thirteen cases of operative specimens of sporadic CCMs were included in this study. The expression of messenger RNA of TF and IL-6 was assayed and the association with clinical factors was investigated. Then, the distribution of TF and IL-6 was examined with immunofluorescence. The mRNA expression of TF of CCMs was significantly higher than that of the control (p=0.017), and was correlated with the number of hemorrhage appearances (p=0.190, ρ=0.62). The mRNA expression level of IL-6 was significantly correlated with the mRNA expression level of TF (p=0.034, ρ=0.58). Examination of immunostained sections indicated that TF+ cells were also positive for IL-6, and distributed around normal endothelial cells. Moreover, the TF+/IL-6+ cells expressed CD31 and VEGFR2. The expressions of IL-6 and TF were correlated, and both were present in the same immature endothelial cells. TF is elevated in CCM and might mediate progressive events. These factors may play a prognostic role in CCM.

Traumatic Hemothorax Blood Contains Elevated Levels of Microparticles that are Prothrombotic but Inhibit Platelet Aggregation.

Autotransfusion of shed blood from traumatic hemothorax is an attractive option for resuscitation of trauma patients in austere environments. However, previous analyses revealed that shed hemothorax (HX) blood is defibrinated, thrombocytopenic, and contains elevated levels of D-dimer. Mixing studies with normal pooled plasma demonstrated hypercoagulability, evoking concern for potentiation of acute traumatic coagulopathy. We hypothesized that induction of coagulopathic changes by shed HX blood may be due to increases in cellular microparticles (MP) and that these may also affect recipient platelet function. Shed HX blood was obtained from 17 adult trauma patients under an Institutional Review Board approved prospective observational protocol. Blood samples were collected every hour up to 4 h after thoracostomy tube placement. The corresponding plasma was isolated and frozen for analysis. The effects of shed HX frozen plasma (HFP) and isolated HX microparticles (HMP) on coagulation and platelet function were assessed through mixing studies with platelet-rich plasma at various dilutions followed by analysis with thromboelastometry (ROTEM), platelet aggregometry (Multiplate), enzyme-linked immunosorbent assays, and flow cytometry. Furthermore, HFP was assessed for von Willebrand factor antigen levels and multimer content, and plasma-free hemoglobin. ROTEM analysis demonstrated that diluted HFP and isolated HMP samples decreased clotting time, clotting formation time, and increased α angle, irrespective of sample concentrations, when compared with diluted control plasma. Isolated HMP inhibited platelet aggregation in response to adenosine diphosphate, arachidonic acid, and collagen. HFP contained elevated levels of fibrin-degradation products and tissue factor compared with control fresh frozen plasma samples. MP concentrations in HFP were significantly increased and enriched in events positive for phosphatidylserine, tissue factor, CD235, CD45, CD41a, and CD14. von Willebrand factor (vWF) multimer analysis revealed significant loss of high molecular weight multimers in HFP samples. Plasma-free hemoglobin levels were 8-fold higher in HFP compared with fresh frozen plasma. HFP induces plasma hypercoagulability that is likely related to increased tissue factor and phosphatidylserine expression originating from cell-derived MP. In contrast, platelet dysfunction is induced by HMP, potentially aggravated by depletion of high molecular weight multimers of vWF. Thus, autologous transfusion of shed traumatic hemothorax blood may induce a range of undesirable effects in patients with acute traumatic coagulopathy.

Perioperative assessment of changes in tissue factor and its inhibitor in patients with abdominal aortic aneurysm treated using classical and intravascular methods

Objectives: We examined plasma concentrations of essential regulators of coagulation cascade, tissue factor (TF) and tissue factor inhibitor pathway (TFPI) in patients with abdominal aortic aneurysm (AAA) during two types of surgical repair , open (OAR) and endovascular repair (EVAR). Background: The interplay between TF and TFPI is important for the proper functioning of coagulation cascade. Although abnormal coagulation is known to contribute to the formation of AAA, little is known about the prognostic value of TF and TFPI levels in the perioperative stage of AAA repair. Methods: We have selected the reference group of 53 healthy, first-time blood donors and 66 patients scheduled for AAA repair using EVAR or OAR and measured TF and TFPI in their blood plasma using ELISA (enzyme-linked immunosorbent assay) before surgery, immediately after and 24 hours after surgery. Results: Patients with AAA had elevated levels of TF (219.242 pg/ml) compared to the reference group (149.170 pg/ml). TF levels were higher in AAA patients with limb ischemia (p=0.0026) and mural thrombi >30 mm (p=0.0015) but decreased in patient with BMI >25 (p=0.027). No differences were observed in TF and TFPI levels during EVAR and OAR or de-pending on the stent graft type. However, there was a correlation between the TF level and occurrence of post-surgical complications in OAR type of surgery Conclusions: Our results suggest that monitoring levels of TF and TFPI in perioperative period would be useful in risk assessment of surgery complications, especially in AAA patients with ischemic disorders undergoing OAR surgery.

Procoagulant effects of lung cancer chemotherapy: impact on microparticles and cell-free DNA.

Lung cancer is the second leading type of cancer, with venous thromboembolism being the second leading cause of death. Studies have shown increased levels of microparticles and cell-free DNA (CFDNA) in cancer patients, which can activate coagulation through extrinsic and intrinsic pathways, respectively. However, the impact of lung cancer chemotherapy on microparticle and/or CFDNA generation is not completely understood. The aim of the study was to study the effects of platinum-based chemotherapeutic agents on generation of procoagulant microparticles and CFDNA in vitro and in vivo. Microparticles were isolated from chemotherapy-treated monocytes, human umbilical vein endothelial cells, or cancer cells. Tissue factor (TF) and phosphatidylserine levels were characterized and thrombin/factor Xa generation assays were used to determine microparticle procoagulant activity. CFDNA levels were isolated from cell supernatants and plasma. A murine xenograft model of human lung carcinoma was used to study the procoagulant effects of TF microparticles and CFDNA in vivo. In vitro, platinum-based chemotherapy induced TF/phosphatidylserine microparticle shedding from A549 and A427 lung cancers cells, which enhanced thrombin generation in plasma in a FVII-dependent manner. CFDNA levels were increased in supernatants of chemotherapy-treated neutrophils and plasma of chemotherapy-treated mice. TF microparticles were elevated in plasma of chemotherapy-treated tumour-bearing mice. Plasma CFDNA levels are increased in chemotherapy-treated tumour-free mice and correlate with increased thrombin generation. In tumour-bearing mice, chemotherapy increases plasma levels of CFDNA and TF/phosphatidylserine microparticles. Platinum-based chemotherapy induces the shedding of TF/phosphatidylserine microparticles from tumour cells and the release of CFDNA from host neutrophils.

A Pilot Study of Tissue Factor-Tissue Factor Pathway Inhibitor Axis and Other Selected Coagulation Parameters in Broiler Chickens Administered in Ovo with Selected Prebiotics*.

The tissue factor (TF) - tissue factor pathway inhibitor (TFPI) axis plays a major role in hemostasis. Disorders of the coagulation system are commonly diagnosed with the help of screening tests such as prothrombin time (PT), activated partial thromboplastin time (aPTT), and plasma fibrinogen concentration (PFC). However, the effect of prebiotics on the hemostasis system has not been characterized in poultry yet. This study was designed to determine the effect of in ovo administration ofprebiotics on blood coagulation parameters of broiler chickens depending on their age. The study was conducted with 180 broiler chick embryos, the air cells of which were injected on day 12 of incubation with prebiotics (experimental groups: Bi2tos, DiNovoo and RFO) or physiological saline solution (control group). At 1, 21 and 42 days of rearing, blood was sampled from 15 broiler chickens from each group. An enzyme immunoassay was performed to determine plasma TF and TFPI levels, and PT, aPTT and PFC were determined in the chicken blood. We demonstrated that: 1) total TF levels increased with age in the experimental groups, 2) prebiotics had no significant effect on TF levels between the groups at a particular age, 3) total TFPI levels differed between both the type of in ovo injected substance and the broiler chicken age, 4) in the control group, PT and aPTT were found to increase with age whilst fibrinogen concentration decreased. The main conclusion from this pilot study is that total TF and TFPI levels change with age, however no clear patterns regarding TFPI were detected yet. The levels of PT, aPTT and PFC varied with the prebiotics administered in ovo as well as with the age of broiler chickens.

Investigation for role of tissue factor and blood coagulation system in severe acute pancreatitis and associated liver injury

This study aims to investigate the molecular mechanisms underlying the pathogenesis of severe acute pancreatitis (SAP) and SAP-associated liver injury, we performed an association analysis of the functions of tissue factor (TF) and blood coagulation system in both SAP patients and mouse SAP model. Our results showed that serum TF and tissue factor-microparticle (TF-MP) levels were highly up-regulated in both SAP patients and SAP mouse model, which was accompanied by the dysfunction of blood coagulation system. Besides, TF expression was also highly up-regulated in the Kupffer cells (KCs) of SAP mouse model. After inhibiting KCs in SAP mouse model, the amelioration of blood coagulation system functions was associated with the decrease in serum TF and TF-MPs levels, and the reduction of SAP-associated liver injury was associated with the decrease of TF expression in KCs. In conclusion, the dis-regulated TF expression and associated dysfunction of blood coagulation system are critical factors for the pathogenesis of SAP and SAP-associated liver injury. TF may serve as a potential and effective target for treating SAP and SAP-associated liver injury.

Activated Factors XI and IX and Tissue Factor in Patients with Advanced Cancer

Background.Cancer patients often display procoagulant activity and are at a risk for developing venous thromboembolism (VTE). Multiple mechanisms may explain this procoagulant state, such as tumor cell expression of tissue factor (TF), host tissue response to tumor formation and also contributions from comorbid factors and treatment. We previously observed elevated levels of traditional biomarkers of coagulation (such as D-dimer, CRP, etc.) in patients with advanced cancer on therapy enrolled in a randomized trial comparing rosuvastatin versus placebo. In this study, we evaluated the frequency and concentration of endogenous plasma TF, Factor (F)XIa and FIXa in the cancer patient population analyzed for traditional coagulation biomarkers. The choice of new analytes was based on previous studies in which these 3 proteins were detected and quantitated in patients with cardiovascular diseases, inflammation, and trauma - often present over the course of several days to even weeks. By our knowledge, there are no published studies of the presence of FXIa and/or FIXa in cancer patients.Methods.Thirty-seven adult cancer patients receiving systemic therapy were originally enrolled in a randomized crossover trial comparing effects of rosuvastatin versus placebo on biomarkers of VTE. Inclusion criteria included locally-advanced or metastatic cancer, estimated survival time of greater than 6 months, and anticipated duration of therapy of at least 3 months. Patients on antithrombotic or statin therapy were excluded. Blood was collected on up to 4 occasions; at the start and end of each of 2 treatment periods corresponding to 2 cycles of systemic therapy with a 3-4 week washout period. Citrate plasma was prepared, frozen and stored at -80C. The assay, performed in previously unthawed and contact-pathway inhibited plasma, is based on a response of the lag phase of thrombin generation to corresponding monoclonal inhibitory antibodies (αFXIa-2, αFIXa-91 and αTF-5; all at 0.1 mg/mL). Concentrations of TF, FXIa and FIXa were calculated from corresponding calibration curves built by titrating purified proteins into multi-donor pooled plasma from healthy individuals.Results.A total of 116 blood samples were collected (not all patients were able to complete their regime) and analyzed for endogenous levels of TF, FXIa and FIXa (see table). Overall, rosuvastatin treatment did not have any significant impact on the levels and frequency of these 3 proteins. For the samples obtained from each patient’s first blood draw (n = 37) there was a weak correlation between FXIa and FIXa levels (R = 0.37, P = 0.02) and a moderate correlation between FXIa and TF levels (R = 0.57, P < 0.001), while no correlation between FIXa and TF was observed (R = 0.02, P = 0.91).Conclusions.1) A portion of patients with advanced cancer had active TF in their plasma while the majority, for the first time reported, had active FXIa and FIXa; 2) No pronounced differences were observed between the frequency and concentrations of these proteins at baseline versus later time-points; 3) There was a weak correlation between FXIa and FIXa and a moderate correlation between FXIa and TF, suggesting the TF-pathway is driving the majority of FXIa generation, although contribution from the contact pathway cannot be excluded; 4) Rosuvastatin treatment did not lead to any significant changes in the levels of TF, FXIa and/or FIXa. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cytoprotective Chaperone Proteins Are Novel Anti-Inflammatory Targets in Sickle Cell Disease

Sickle cell disease (SCD) is a pro-inflammatory condition. Levels of TNF-α, IL-6, IL-8, and IL-10 are elevated. There is clear evidence of endothelial cells (EC) dysfunction, and increased leukocyte, and erythrocyte adhesion in patients even in the non-crisis "steady state" condition. Additional insult, either via infection or vaso-occlusive ischemia, induce a dramatic increase in inflammation and EC dysfunction in SCD. Furthermore, there is a kindling of coagulation activation in patients with SCD. We, and others, have reported elevated levels of thrombin and monocyte tissue factor (TF) expression in patients. Both thrombin and monocyte TF expression increase during acute clinical events. In addition to the chronic impairment of lung function, acute chest syndrome (ACS) adds further insult to lung and cardiovascular impairment. In fact, ACS is the leading cause of sudden death in patients with SCD. Although there are multiple etiologies for ACS, infection/sepsis and the dramatic innate immune and coagulation response to it remain a major contributor to morbidity and mortality during ACS. Novel methods to reduce the inflammatory response during infection are needed as are methods that normalize the chronic pro-inflammatory state. Chaperone proteins, namely HSP90 and HSP70, are known agents that participate in inflammation and thus have significant potential to influence the inflammatory, pro-coagulant burden. Therefore, in this study, we wanted to evaluate the novel anti-inflammatory, anti-coagulatory properties of the chaperone proteins in SCD. We had previously determined that inhibition of HSP90 using the drug AUY-922 could block the bacterial toxin lipopolysaccharide (LPS) - induced TF expression and pro-inflammatory cytokine release from monocytes. Therefore, we used the Townes mouse model of SCD to evaluate AUY-922 in a pre-clinical study. Townes mice with SCD or without were administered AUY-922 intraperitoneal (IP) for 4 days prior to a 6 hour LPS-mediated induction of the inflammatory response and coagulation activation. Notably, the dose of LPS failed to induce any pro-inflammatory response in the AA mice (n=24). However, LPS-induced an exaggerated response in the SS mice. Levels of TNF-α, IL-6, IL-8, and IL-10 were elevated up to 40,000 fold over control treated SS mice. Pre-treatment with AUY-922 either completely ablated, or significantly attenuated the inflammatory cytokine response and normalized EC function. Furthermore, the treatment with AUY-922 doubled the amount of the anti-inflammatory chaperone molecule HSP70 in the livers of the SS mice. This particular result suggested that the function of HSP90 could be spared, and the induction of HSP70 was potentially sufficient to protect against the LPS-induced insult.Of note, the main function of HSP70 is cytoprotection in response to oxidative and febrile stress. Therefore, we next sought to determine, in a proof of principle in vitro study, whether induction of HSP70 alone was sufficient to block LPS-induced cytokine release and coagulation activation. We treated human monocytes with the HSP70 inducer, celastrol for 24h, followed by treatment with LPS (1µg/ml). We observed a significant release of the cytokines IL-6 and TNF-α with LPS treatment. However, induction of HSP70 via celastrol was sufficient to block this inflammatory response. Furthermore, we observed that celastrol blocked the LPS-induced, TF-specific clotting of plasma in vitro. Interestingly, we also observed that conditioned media from celastrol treated monocytes could block LPS-induced IL-6 release in an HSP70 dependent manner. Thus, secreted HSP70 was an active participant in cellular protection from LPS-induced insult. Initial studies suggest that secreted HSP70 levels may be lower in patients with SCD than in unaffected individuals. Therefore, replacement of this chaperone may be of significant benefit as therapeutic. Thus, taken together, our data demonstrate in both a pre-clinical and an in vitro proof of principle study, that the chaperone proteins HSP90 and HSP70 are attractive targets at reducing the inflammatory burden and associated acute lung injury in SCD. DisclosuresNo relevant conflicts of interest to declare.

123 - Inhibition of the Pro-Atherogenic Action of Serum Amyloid a (SAA) by Pharmacological Blockade of NFκB Pathway

The acute phase protein serum amyloid A (SAA) is a biomarker of inflammation. Elevated circulating SAA in chronic inflammatory disorders such as diabetes mellitus is associated with endothelial dysfunction and the earliest stages of atherosclerosis. Binding of SAA to cell receptors leads to increased production of mRNA encoding tumor necrosis factor (TNF), tissue factor (TF) and cytokines such as interleukin-6 (IL-6); the proteins produced by these genes display pro-inflammatory and pro-thrombotic activities that may be involved in promoting endothelial dysfunction. Activation of the transcription factor NFκB appears to be a central canonical pathway for SAA-mediated endothelial cell dysfunction. Targeting NFκB, using the specific inhibitor BAY11-7082, may be more effective at inhibiting SAA-mediated endothelial dysfunction than a pharmaceutical cocktail that targets its receptors. Human carotid artery endothelial (HCtAE) cells were cultured to confluence under normoglycaemic (5mM glucose) or hyperglycaemic conditions (25mM glucose) conditions and pre-incubated (1.5 h; 37°C) with 10µM BAY11-7082 or vehicle (control) followed by 10μg/mL SAA (4.5 h; 37°C). Notably, TF and TNF gene expression increased markedly in HCtAE cells after SAA stimulation whereas HCtAE cells pre-treated with BAY11-7082 (with SAA) showed TF and TNF mRNA levels similar to that determined in control cells (minus SAA). In the absence of BAY11-7082, both intracellular TNF and IL-6 protein increased following incubation with SAA as determined by immunofluorescence microscopy. This SAA activity was inhibited by BAY11-7082. ELISA analysis of HCtAE cell pellets and overlying supernatant confirmed the immunofluorescence data with SAA activity inhibited by BAY11-7082; IL-6 showed a greater sensitivity to the drug than TNF. In ex-vivo leukocyte adhesion studies of mice aorta, pre-treatment with BAY11-7082 showed a significant reduction in leukocyte adhesion compared with SAA-alone treated mice. Together these data suggest that inhibition of NFκB activation may protect the endothelium from the action of SAA by inhibiting pro-inflammatory and pro-thrombotic pathways.

Fasudil inhibits tissue factor and plasminogen activator Inhibitor-1 secretion by peripheral blood mononuclear cells in CAPD patients

Disturbances in hemostasis are common complications of kidney diseases and correlate well with cardiovascular mortality. Little is known about the effects of fasudil on tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) expression in peripheral blood mononuclear cells (PBMCs) in CAPD patients. PBMCs were isolated from 13 individuals with CAPD and 13 healthy subjects. After 4 h of incubation with or without LPS (10 ng/mL), TF and PAI-1 mRNA of PBMCs were detected by RT-PCR. The levels of TF and PAI-1 in culture supernatants of PBMCs were determined by ELISA. Compared with healthy controls, CAPD patients had increased TF, PAI-1 protein and mRNA expression by PBMCs at baseline and after stimulated by LPS (10 ng/mL) [p < 0.001]. The fasudil treatment resulted in a significant effect in decreasing TF and PAI-1 [p < 0.05] synthesis in PBMCs. TF and PAI-1 mRNA expression and activities in PBMCs were increased in CAPD patients. Fasudil reduced LPS-mediated TF and PAI-1 expression and activity in PBMCs. These effects may partially be relevant to the clinical benefits of fasudil in the treatment of CAPD patients.

PS2 - 203 Bone Marrow-Derived Mesenchymal Stem Cells Reverse Ionizing Radiation-Induced Active State of Endothelial Cells in Glioblastoma

As the central component of heightened vascularization in glioblastoma (GBM), endothelial cells (EC) are arguably the most responsive stromal cells in the tumour microenvironment during conventional treatment using ionizing radiation (IR). Although the inherent tumor tropism and angiogenic property of mesenchymal stem cells (MSC) has been documented in many cancer types including GBM, little is known about the function of MSC that are recruited to the GBM tumor microenvironment. The purpose of this study was to elucidate the effect of MSC on irradiated EC. Here we studied the influence of IR, MSC or MSC condition media (CM) on human umbilical vein endothelial cells (HUVECs). IR was found to up-regulate mRNA levels of CXCL5, CXCL10, ICAM1, VCAM1, VEGF-c and tissue factor (TF) in a dose-dependent manner whereas MSC co-culture boosted the expression of ICAM1, VCAM1, VEGF-c, TF, and MCP3. An additive effect of IR and MSC-culture was most detected with respect to ICAM1, TF and CXCL5 under 2Gy. MSC co-culture decreased IR-induced phospho-p53 in HUVECs at 15Gy. In addition, IR decreased the level of phospho-Akt in HUVECs as well as cell proliferation. Notably, both effects were countered by MSC CM. Interestingly, up-regulation of the same set of IR-driven genes in GBM was positively correlated with poor survival in the TCGA GBM database, a correlation that was lost if using gene list that was obtained from IR and MSC combine. These findings suggest that MSC promotes angiogenesis in GBM by overturning the IR-induced active state of endothelial cells and rendering them radio-resistant.

Increased circulating procoagulant and anticoagulant factors as TF and TFPI according to severity or infecting serotypes in human dengue infection

Tissue Factor (TF) is the initiator of coagulation and Tissue Factor Inhibitor (TFPI) is the physiological inhibitor of the TF/FVIIa complex. Circulating levels of TF and TFPI were quantified in dengue patients and the relationships with disease severity and infecting serotype analysed. A significant decrease in TF and TPFI plasma levels was observed in mild DF patients compared with severe dengue. Furthermore, both factors were associated with haemorrhagic manifestations. Finally, TF levels were significantly increased in DENV-1/2 infected patients as compared with DENV-4. These findings suggest that activation of TF-pathway is an important component of DENV -related coagulation disorders.