Anti-tissue Factor Pathway Inhibitor Antibody Research Articles

Health-Related Quality of Life in Patients with Hemophilia A or B with Inhibitors on Concizumab Prophylaxis: Results from the Phase 3 explorer7 Study

Introduction: Concizumab is currently in clinical development as a humanized, monoclonal anti-tissue factor pathway inhibitor antibody that enhances the initiation phase of coagulation. In the phase 3 explorer7 study (NCT04083781), patients with hemophilia A or B with inhibitors received once-daily subcutaneous prophylactic treatment with concizumab for the prevention of bleeding episodes. Patients with hemophilia generally report impaired health-related quality of life (HRQoL), particularly in the areas of pain and physical functioning. Here, we present an analysis of changes in reported HRQoL for patients treated with concizumab for 24 weeks in the explorer7 study. Methods: A total of 133 male patients aged ≥12 years were randomized to no prophylaxis (arm 1) or concizumab prophylaxis (arm 2) or assigned to non-randomized concizumab prophylaxis (arms 3 or 4) depending on the treatment they received before entering the study. All patients were asked to complete the 36-item Short Form Health Survey (SF-36v2; Ware & Sherbourne. Med Care 1992; 30:473-83) and patients aged ≥17 years could complete the Hemophilia Quality of Life Questionnaire for Adults (Haem-A-QoL; von Mackensen & Gringeri, Handbook of Disease Burdens and Quality of Life 2010:1896-920; von Mackensen et al.Haemophilia 2017;23(3):383-391; Wyrwich et al.Haemophilia 2015;(5):578-84). Both questionnaires could be completed at baseline (Week 0), and at Weeks 4, 8, 16 and 24. The SF-36v2 health scales "bodily pain" (BP) and "physical functioning" (PF) were analyzed as key secondary endpoints. Changes in Haem-A-QoL total score and "physical health" score, as well as the remaining SF-36v2 health scales and component summary scores, were analyzed as exploratory endpoints. Unfortunately, due to technical issues, several patient-reported outcomes recordings could not be collected for statistical analysis. As the preplanned statistical analysis could not be implemented, a post-hoc mixed model for repeated measures (MMRM) was applied after the randomization code was broken. The MMRM analysis included patients from arms 1 and 2 who had completed the questionnaire at baseline and had at least one post-baseline visit on the new dosing regimen. Results: The mean estimated change in score from baseline to Week 24 on the SF-36v2 BP scale was 2.2 points (95% CI: -5.1; 9.5) for patients not on prophylaxis (n=9), and 9.2 points (95% CI: 5.1; 13.3) for those on concizumab (n=23), which yielded an estimated treatment difference (ETD) of 7.0 points (95% CI: -1.6; 15.6) (Figure 1). For the SF-36v2 PF scale, the mean change in score from baseline to Week 24 was 1.2 points (95% CI: -4.8; 7.1) in the no prophylaxis arm (n=9) versus 4.5 points (95% CI: 0.8; 8.2) in the concizumab treatment arm (n=23), which yielded an ETD of 3.3 points (95% CI: -3.8; 10.4) (Figure 1). While BP and PF scores were not significantly different between both arms, several other health scales, including "general health", "mental health", "role-emotional" and "vitality", showed statistically significant differences (Figure 1). The total score from the Haem-A-QoL questionnaire showed a significant improvement for patients on concizumab prophylaxis (n=13) compared to no prophylaxis (n=4) (ETD: -22.6 points [95% CI: -42.5; -2.7]; Figure 2). A significant difference was not observed for "physical health", but there were significant improvements in "feeling", "treatment", "view of yourself", and "sport and leisure" (Figure 2). Conclusion: Patients on daily subcutaneous concizumab prophylaxis generally reported better scores on generic (SF-36v2) and disease-specific (Haem-A-QoL) questionnaires when compared with patients treated on-demand. The differences were mostly in favor of concizumab, with the largest ETDs being related to mental and general health. SF-36v2 and Haem-A-QoL are being investigated as part of the explorer8 concizumab phase 3 study in patients with hemophilia A or B without inhibitors. These data reflect the potential benefit of concizumab prophylaxis to improve HRQoL in patients with hemophilia A and B with inhibitors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

COVID-19 Is Associated with Endothelial Dysfunction and Enhanced Plasma Thrombin Generation Despite Pharmacological Thromboprophylaxis

BackgroundHospitalised patients with severe COVID-19 (requiring critical care level support) appear to be at increased risk of thrombosis despite standard pharmacological thromboprophylaxis. The magnitude of thrombotic risk in patients with COVID-19 of moderate severity (not requiring critical care) is less clear. The optimal approach to thromboprophylaxis (and the role of intensified thromboprophylaxis) remains to be determined.Evidence of endothelial dysfunction has been widely reported in COVID-19 (particularly in severe COVID) and this may contribute to hypercoagulability.AimTo assess differences in patterns of hypercoagulability and endothelial dysfunction between a group of patients with moderate COVID-19 and a group of age-matched hospitalized patients (SARS-CoV-2 PCR negative) receiving low molecular weight heparin (LMWH) thromboprophylaxis.MethodsBlood was collected from individuals admitted to hospital with COVID-19 of moderate severity (not requiring critical care level support) and a group of age-matched patients admitted with infective/inflammatory illness (SARS-CoV-2 PCR negative). All subjects received standard-dose LMWH thromboprophylaxis, with blood drawn at 12 hours post-dose (and with measurement of anti-FXa activity levels). Circulating levels of endothelial & fibrinolytic markers including ICAM, PAI-1, VCAM, soluble thrombomodulin (sTM), and tissue plasminogen activator (tPA) were determined by ELISA. Thrombin generation (TG) in platelet-poor plasma was assessed by calibrated automated thrombography in the presence of tissue factor (Final concentration, 1pM & 5pM), thrombomodulin (TM) (Final concentration, 6.25nM), and an inhibitory anti-tissue factor pathway inhibitor antibody (anti-TFPI; Final concentration 100μg/mL).Results14 COVID-19 positive subjects and 11 hospitalized controls were recruited. There were no differences in mean age (69.7±4.5 vs 61.6±4.7 years; p= 0.2) or mean Body mass index (25.7±1.1 vs 22.7±1.2 Kg/m2; p=0.1) between groups. No COVID-19 patient or control required critical care support. In the COVID group, radiological evidence of pneumonitis [diffuse (n=3) or peripheral infiltrates (n=7)] was present in the majority of cases. None of the COVID-19 cases were requiring supplemental oxygen at the time of recruitment.All controls were admitted with either respiratory or urinary infection [radiological evidence of pneumonia in 4/11; supplemental oxygen requirement in 2/11, (28-36% FiO2 via nasal cannula)].Plasma levels of sTM, ICAM, PAI-1 & VCAM were similar in both groups. Levels of t-PA were significantly higher in the COVID group (8.31±4.35 vs 4.91±2.37 ng/mL; p= 0.005). Despite similar plasma anti-Xa activity in both groups (0.06 vs 0.04 IU/mL; p=0.2), mean endogenous thrombin potential (ETP) was significantly higher in the COVID group (1929±119.7 vs 1528±138.9 nM*min; p=0.02), although peak thrombin was similar (173.6±26 vs 161.5±31nM). ETP-TM ratio was similar between groups (0.3±0.1 vs 0.2±0.1; p=0.3).Despite increased ETP, the lag time to thrombin generation was significantly prolonged in the COVID group (8.3±0.6 vs 5.8±0.5 mins, p= 0.006). This pattern has previously been observed in vascular diseases associated with altered plasma tissue factor pathway inhibitor (TFPI) activity. In the presence of an anti-TFPI antibody, the difference in lagtime between groups was attenuated (4.7±0.2 vs 3.5±0.1 mins; p= 0.002) and the difference in overall thrombin generation (delta TG) between both groups became significantly increased (Fig.1).ConclusionPlasma thrombin generation is enhanced in patients with non-severe COVID-19 despite pharmacological thromboprophylaxis. Endothelial dysfunction is also observed in this group and appears to modulate parameters of plasma thrombin generation. The clinical implications of these observations are not known although clinical studies of intensified thromboprophylaxis in attenuating thrombotic risk and other complications are ongoing.Fig 1. Inhibition of TFPI activity enhances thrombin generation in COVID-19.In the presence of an inhibitory anti-TFPI antibody, peak plasma thrombin generation was enhanced in COVID-19 in contrast to that observed among SARS-CoV-2 PCR negative hospitalised patients (339.6+25.2 vs 247.4+10.1, p=0.01). [Display omitted] DisclosuresMaguire: Actelion: Research Funding; Bayer Pharma: Research Funding. Ni Ainle: Daiichi-Sankyo: Research Funding; Actelion: Research Funding; Leo Pharma: Research Funding; Bayer Pharma: Research Funding. Kevane: Leo Pharma: Research Funding.

Current developments in hemostatic treatment for patients with hemophilia

The supplementation of factor (F-) VIII or F-IX products for hemophilia treatment has helped in preventing arthropathy and considerably improving the quality of life (QOL) of patients. However, serious issues related to hemostatic treatment of hemophilia include the need for repeated intravenous infusion of products, inhibitor development, and hemostatic treatment for patients with inhibitors. To overcome these issues, some extended half-life products and nonclotting factor products were developed. An anti-F-IX/F-X bispecific antibody, emicizumab, which has a longer half life and can be administered subcutaneously, has achieved significant reduction in bleeding in patients with severe hemophilia, irrespective of the inhibitor. Furthermore, clinical trials of si-RNA anti-antithrombin therapy and antitissue factor pathway inhibitor antibody therapy, based on the concept of rebalancing coagulation, have been ongoing. Moreover, gene therapy has been currently developed by the improvement of vector. These newly developed therapies for hemophilia, as a paradigm shift in hemophilia treatment, could provide further improvement in the QOL of patients.

MG1113, a specific anti–tissue factor pathway inhibitor antibody, rebalances the coagulation system and promotes hemostasis in hemophilia

BackgroundReplacement therapy is the most common treatment for reduction of bleeding and control of episodic bleeding in individuals with hemophilia. Despite the proven effectiveness of factor replacement therapy, repeated intravenous administration is a heavy burden to individuals with hemophilia. ObjectivesTo reduce the burden, therapeutic agents that can be subcutaneously administered need to be developed, and an anti–tissue factor pathway inhibitor (TFPI) antibody may be a suitable candidate for this purpose. MethodsMG1113 is an IgG4 monoclonal antibody that binds to Kunitz‐2 domain (KD2) of TFPI. To confirm the coagulation potential of MG1113, several tests were conducted using factor VIII (FVIII)‐ or IX (FIX)‐deficient plasma. For the ex vivo spiking test, platelet‐poor plasma samples from 14 individuals with hemophilia were spiked with MG1113. The in vivo efficacy was determined using blood loss tests, modified prothrombin time (mPT), and free TFPI quantification after intravenous or subcutaneous administration of MG1113 into hemophilia A (HA)‐induced rabbits. ResultsRadiographic crystallography demonstrated the specific binding site between MG1113 and KD2. In FVIII‐deficient plasma and the plasma of individuals with hemophilia, peak thrombin and endogenous thrombin levels were increased by MG1113 in a concentration‐dependent manner. Rotational thromboelastometry assay revealed that clotting time, clot formation time, and maximum clot firmness were normalized in MG1113‐treated blood of patients. Intravenous or subcutaneous injection of MG1113 into HA‐induced rabbits resulted in rebalancing of blood loss, mPT, and free TFPI levels. ConclusionsThese results indicate that subcutaneous administration of MG1113 neutralizes the function of TFPI and regulates bleeding in individuals with hemophilia.

Laboratory monitoring of hemophilia A treatments: new challenges

AbstractMonitoring factor VIII (FVIII) activity has traditionally been complicated by discrepancies between assays for the various sorts of FVIII molecules. The advent of novel nonfactor therapies (emicizumab, fitusiran, and anti-tissue factor pathway inhibitor antibodies) in hemophilia A poses a new level of difficulty on the laboratory monitoring of these patients. To use the correct assays and for a proper interpretation of their results, it is pertinent to understand the mode of action of these nonfactor agents. Furthermore, the biochemical consequences for the different types of activity assays (whether it be specific FVIII activity assays or global coagulation assays) should be taken into account as well. In this review, these aspects will be discussed. In addition, the use of various animal models to estimate FVIII-equivalence of the nonfactor therapies will be presented.

Correction of bleeding in experimental severe hemophilia A by systemic delivery of factor VIII-encoding mRNA.

The treatment or prevention of bleeding in patients with hemophilia A relies on replacement therapy with different factor VIII (FVIII)-containing products or on the use of by-passing agents, i.e., activated prothrombin complex concentrates or recombinant activated factor VII. Emerging approaches include the use of bispecific anti-factor IXa/factor X antibodies, anti-tissue factor pathway inhibitor antibodies, interfering RNA to antithrombin, and activated protein C-specific serpins or gene therapy. The latter strategies are, however, hampered by the short clinical experience and potential adverse effects including the absence of tight temporal and spatial control of coagulation and the risk of uncontrolled insertional mutagenesis. Systemic delivery of mRNA allows endogenous production of the corresponding encoded protein. Thus, injection of erythropoietin-encoding mRNA in a lipid nanoparticle formulation resulted in increased erythropoiesis in mice and macaques. Here, we demonstrate that a single injection of in vitro transcribed B domain-deleted FVIII-encoding mRNA to FVIII-deficient mice enables endogenous production of pro-coagulant FVIII. Circulating FVIII:C levels above 5% of normal levels were maintained for up to 72 h, with an estimated half-life of FVIII production of 17.9 h, and corrected the bleeding phenotype in a tail clipping assay. The endogenously produced FVIII did however exhibit low specific activity and induced a potent neutralizing IgG response upon repeated administration of the mRNA. Our results suggest that the administration of mRNA is a plausible strategy for the endogenous production of proteins characterized by poor translational efficacy. The use of alternative mRNA delivery systems and improved FVIII-encoding mRNA should foster the production of functional molecules and reduce their immunogenicity.

GlycoPEGylated recombinant factor IX for hemophilia B in context

Decisions over hemophilia treatment selection and switching involve balancing many clinical and patient-related factors. The current standard of care for patients with hemophilia B is prophylaxis with plasma-derived or recombinant factor IX (rFIX) concentrates. However, several extended half-life (EHL) rFIX products have recently been developed to improve treatment convenience and clinical outcomes for these patients. Nonacog beta pegol, an rFIX product that combines the FIX protein with a 40 kDa polyethylene glycol moiety, has been evaluated in 115 previously treated patients with hemophilia B (including 25 children) in the paradigm clinical trial program. FIX activity levels and pharmacokinetics were monitored throughout these trials and showed that nonacog beta pegol offers significant pharmacological improvements over standard FIX products. Once-weekly prophylaxis with nonacog beta pegol 40 IU/kg resulted in fewer bleeds in all patients (median annualized bleeding rate of 1.0 across all ages), resolved 90% of target joints, and improved health-related quality of life. No patients developed FIX inhibitors, and there were no thromboembolic events or unexpected safety concerns. Nonacog beta pegol was also safe and effective in the perioperative setting. These findings show that nonacog beta pegol is highly effective, while also offering more convenient dosing than standard FIX products. Nonacog beta pegol represents a significant advance in the current context of treatment for hemophilia B, offering effective management across several treatment modalities and settings, and potentially easing the treatment burden for patients of all ages. Meanwhile, the development of novel treatment strategies, such as gene therapy, anti-tissue factor pathway inhibitor antibodies, and RNA interference therapy, may provide patients with additional therapeutic options, which would require reassessment of the role of EHL products in the future.

Translational Pharmacokinetic/Pharmacodynamic Characterization and Target-Mediated Drug Disposition Modeling of an Anti–Tissue Factor Pathway Inhibitor Antibody, PF-06741086

Tissue factor pathway inhibitor (TFPI) exhibits multiple isoforms, which are known to present in multiple locations such as plasma, endothelium, and platelets. TFPI is an endogenous negative modulator of the coagulation pathway, and therefore, neutralization of TFPI function can potentially increase coagulation activity. A human monoclonal antibody, PF-06741086, which interacts with all isoforms of TFPI is currently being tested in clinic for treating hemophilia patients with and without inhibitors. To support clinical development of PF-06741086, pharmacokinetics (PK) and pharmacodynamics of PF-06741086 were characterized in monkeys. In addition, a mechanistic model approach was used to estimate PK parameters in monkeys and simulate PK profiles in human. The results show that PF-06741086 exhibited target-mediated drug disposition and had specific effects on various hemostatic markers including diluted prothrombin time, thrombin generation, and thrombin-antithrombin complex in monkeys after administration. The model-predicted and observed human exposures were compared retrospectively, and the result indicates that the exposure prediction was reasonable within less than 2-fold deviation. This study demonstrated in vivo efficacy of PF-06741086 in monkeys and the utility of a rational mechanistic approach to describe PK for a monoclonal antibody with complex target binding.

The effect of corn trypsin inhibitor, anti-tissue factor pathway inhibitor antibodies and phospholipids on microvesicle-associated thrombin generation in patients with pancreatic cancer and healthy controls.

Circulating microvesicles (MVs) are suggested to be important contributors to cancer-associated thrombosis due to the presence of surface-bound procoagulant molecules like tissue factor (TF) and phosphatidylserine (PS). Pancreatic cancer is considered to be one of the most prothrombotic malignancies. The aim of this study was to describe the impact of analytical variables on MV-associated thrombin generation in patients with pancreatic cancer and in healthy controls. MVs were isolated from citrated plasma and added to pooled normal plasma (PNP). Thrombin generation was measured by the calibrated automated thrombogram. The impact of corn trypsin inhibitor (CTI), anti-tissue factor pathway inhibitor (TFPI) antibodies and phospholipids was described. Antibodies against TF were used to assess TF-dependency, and MV-bound PS activity was measured with the Zymuphen MP-activity kit. MVs from the pancreatic cancer patients displayed higher thrombin generation and higher PS-activity than MVs from the healthy control group, while TF-dependency was observed in only 1 out of 13 patient samples. Adequate thrombin generation-curves were only achieved when CTI was omitted and anti-TFPI antibodies were added to PNP prepared in low contact-activating tubes. Addition of phospholipids reduced the significant differences between the two groups, and should be omitted. This modified thrombin generation assay could be useful for measurement of procoagulant circulating MVs, allowing the contribution from MVs affecting both the intrinsic and the extrinsic pathway to be measured.

Acute Efficacy of a Novel Anti-Tissue Factor Pathway Inhibitor Antibody BAY 1093884 in Hemophilia A Mouse Severe Tail Bleeding

Tissue factor pathway inhibitor (TFPI) is the major inhibitor of the tissue factor initiated extrinsic coagulation pathway in blood and is intact in patients with hemophilia. The inhibition of TFPI may restore hemostasis in patients with hemophilia. BAY 1093884 is a fully human monoclonal antibody against TFPI developed as a bypass agent for hemophilia patients with or without inhibitors. It restores thrombin burst for stable clot formation in hemophilic conditions in vitro. The goal of these studies was to determine the in vivo acute efficacy of BAY 1093884 in the hemophilia A (HemA) mouse.In the first study, the acute efficacy of BAY 1093884 (3−100 mg/kg) was demonstrated and compared with full-length recombinant factor VIII (rFVIII; 10−100 IU/kg) by a HemA mouse tail clip model, in which blood loss from a severed tail tip was measured over 45 minutes after injury (n=12−27 mice/group). Naive C57/BL6 and HemA mice were used as positive and negative controls, respectively. Whereas isotype control antibody−treated HemA mice had median blood loss of 870 μL, increasing doses of BAY 1093884 to 50 and 100 mg/kg significantly reduced blood loss to a median of 55 and 5 μL. The dose required to reduce blood loss by 50% was 18 mg/kg, approximately equivalent to the efficacy of 20 IU/kg rFVIII.In a second study, we characterized the combined action of BAY 1093884 and activated recombinant factor VII (rFVIIa; n=10−25 mice/group). Low doses of BAY 1093884 (2.5 mg/kg) and rFVIIa (0.5 and 1.0 mg/kg) with minimal efficacies were tested. Untreated HemA mice had median blood loss of 860 μL. As stand-alone treatments, 2.5 mg/kg BAY 1093884, 0.5 mg/kg rFVIIa, and 1 mg/kg rFVIIa provided minimal blood loss protection, with bleeding volume reduced to 675, 830, and 770 μL, respectively. In comparison, the combination of 2.5 mg/kg BAY 1093884 with 0.5 mg/kg rFVIIa or 1.0 mg/kg rFVIIa reduced median blood loss to 215 and 35 μL, respectively. These results showed a combination effect of BAY 1093884 and rFVIIa in this severe acute efficacy model.These studies demonstrate that BAY 1093884 could potently reduce acute blood loss in HemA mice and may offer a new treatment option for hemophilia patients. DisclosuresXu:Bayer HealthCare LLC: Employment. Koellenberger:Bayer Pharma AG: Employment. Laux:Bayer Pharma AG: Employment. Kauser:Bayer HealthCare LLC: Employment. Sim:Bayer HealthCare LLC: Employment.

Tissue Factor Pathway Inhibitor Isoforms of Macro- and Microvascular Endothelial Cells

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that inhibits both FXa and TF-FVIIa and is an important physiological inhibitor of the extrinsic coagulation pathway. The main portion (~80%) of TFPI in humans is reportedly associated with endothelial cells (ECs). At least 2 different isoforms of TFPI exist in humans, namely, TFPI alpha (α) and TFPI beta (β). In contrast to TFPIα, which consists of 3 Kunitz domains (KD) and a basic C-terminal part, TFPIβ lacks the third KD (KD3) and the basic C–terminal region. In TFPIβ, these 2 domains are replaced, due to alternative splicing, by a sequence that adds a glycosylphosphatidylinositol (GPI) anchor to the protein, linking it to the cell membrane. The present study aimed to identify possible differences in the TFPI level of macro- and microvascular ECs of various tissues to obtain further insight into the relevance of TFPI isoforms α and β at the cellular level.Primary macro- and microvascular ECs of various tissues were treated with phosphatidylinositol phospholipase C (PI-PLC) to remove the GPI-anchored TFPI isoform β from the cell surface. After detachment with an enzyme-free cell dissociation solution, living cells were washed and stained with a polyclonal anti human TFPI antibody and subsequently used for fluorescence activated cell sorting (FACS). TFPIα, the non-GPI anchored TFPI isoform, is not affected by PI-PLC treatment and therefore remains on the cell surface. Measuring the fluorescence intensities of these cells by FACS allowed us to determine the relative amount of cell surface TFPIβ. The supernatants and similarly treated cells were used in two different ELISA assays to quantify TFPI antigen. The first ELISA quantifies total TFPI, consisting of both TFPIα and β, whereas the second ELISA specifically determines the TFPIα content using another capture antibody. Subtraction of TFPIα from the total TFPI amount enabled us to determine the concentration of TFPIβ.Three macrovascular (HUVEC, HAoEC and HPAEC) and four microvascular (HDMEC, HDBEC, HCMEC and HPMEC) cell types were used for TFPI analysis. Based on availability, ECs from more than one donor were analyzed to explore individual differences in TFPI expression. FACS results of living cells revealed that ~85% of the cell surface TFPI of all analyzed ECs represents TFPIβ. ELISA measurements of supernatants and cell lysates showed that TFPIα was not affected by PI-PLC treatment, whereas TFPIβ increased in the supernatant and decreased in the total cell lysate after PI-PLC treatment. These results were obtained with all tested cell types. Differences in the absolute TFPI level of individual donors were also detected and microvascular cells were shown to exhibit more total TFPI than macrovascular cells.In conclusion, both TFPIα and β appear to be present on micro- and macrovascular ECs and ~85% of cell surface TFPI to represent TFPIβ. Furthermore, TFPI levels in the various vasculatures were shown to be dependent on the size of the blood vessel and on the individual donor. DisclosuresDockal:Baxter Innovations GmbH, Vienna, Austria: Employment. Pachlinger:Baxter Innovations GmbH, Vienna, Austria: Employment. Baldin-Stoyanova:Baxter Innovations GmbH, Vienna, Austria: Employment. Knofl:Baxter Innovations GmbH, Vienna, Austria: Employment. Ullrich:Baxter Innovations GmbH, Vienna, Austria: Employment. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.

Focal Procoagulant Activity in Space Between TNF-α Treated Endothelial Cells Mediates Focal Fibrin Accumulation and Anti-Streptococcal Function

AbstractAbstract 3307 Background:Stimulated endothelial cells withdraw from the junctions with neighboring cells and develop a mounded morphology. These cells have mixed procoagulant and anticoagulant function, in contrast to the dominant anticoagulant function of quiescent cells. Recent findings from our laboratory indicate that tumor necrosis factor-a (TNFα) treated endothelial cells support localized assembly of the prothrombinase complex on the margins of cells and on filopodia that span the inter-cellular space. The prothrombinase binding sites are characterized by exposed phosphatidylserine and high convex curvature. We asked whether this inter-cellular space is a focal procoagulant environment while the body of the endothelial cell maintains an overall anticoagulant function. We also asked whether focal procoagulant activity has a function in innate immunity. Methods:Human umbilical vein endothelial cells (HUVECs) were grown to a confluent monolayer on coverslips coated with 1% gelatin or Sigmacote. Cells were treated with 5 nM TNFα or an equal volume of saline for 12 hr. Rinsed cells were then overlaid with re-calcified plasma, diluted 1:4 with buffered saline and placed on a shaker plate at 37 °C at 1000 rpm. Plasma was removed from cells at various times and the cells and fibrin were assessed by confocal microscopy. Fluorescein-labeled fibrinogen was added to plasma to enable imaging fibrin deposition. Alternatively, fibrin derived from native fibrinogen was imaged with fluorescein-labeled mAb 59D8, specific for the beta-chain of fibrin. Streptococci pyogenes strain 700294 was obtained from ATCC and grown and plated using standard techniques. Results:Quiescent endothelial cells responded to TNFα by retracting from the cell-cell junctions, as previously described. As we previously reported, the filopodia and localized regions on the cell margins stained with lactadherin, indicating phosphatidylserine exposure and convex curvature, while the cell bodies did not. The plasma-saline mixtures incubated over endothelial cells did not clot over a 2 hr time span, but plasma incubated over control gelatin-coated coverslips clotted within 10 min. Confocal microscopy indicated that fibrin strands were deposited between TNFa-treated endothelial cells but were largely absent from cover slips with untreated cells. Soluble, fluorescent fibrin bound to the margins of TNFα-treated endothelial cells but not to quiescent endothelial cells or to the Sigmacote-treated cover slips. Fibrin deposition was greatly diminished by reducing Ca++ to < 1 mM, by an anti-tissue factor mAb, and by hirudin. The quantity of fibrin was increased by an anti-tissue factor pathway inhibitor antibody but not by corn trypsin inhibitor, anti-activated protein C mAb, anti-thrombomodulin mAb, or anti-factor VIII mAb. Thus, focal procoagulant activity is mediated by the extrinsic coagulation pathway. To determine whether the fibrin deposition has a function in innate immunity, Streptococcus pyogenes, was added to plasma at a concentration of 106cfu/ml. The streptococci concentration was decreased to <40% in three hours in the presence of TNFa treated endothelial cells, but increased more than 3-fold in the presence of control cells. Addition of hirudin to the TNFα-treated cells enabled Streptococci to grow at the same rate as in citrated plasma alone or re-calcified plasma incubated over quiescent endothelial cells. Confocal microscopy indicated that streptococci decorated the fibrin strands in the inter-endothelial space but not the intercellular matrix or the endothelial cell bodies. Conclusion:TNFα treated HUVECs support focal procoagulant activity via the extrinsic coagulation pathway while maintaining anticoagulant function toward bulk plasma. Focal endothelial procoagulant activity leads to cell-bound fibrin and anti-streptococcal activity, independent of leukocytes and platelets. Discussion:Focal support of procoagulant activity by endothelial cells may have a role in innate immunity without clotting of bulk plasma and independent of leukocytes or platelets. The conditions under which vascular beds react in this manner, the breadth of anti-bacterial activity, and the mechanisms of coagulation-dependent anti-bacterial activity are promising areas for further investigation. Disclosures:No relevant conflicts of interest to declare.

Shiga toxin enhances functional tissue factor on human glomerular endothelial cells: implications for the pathophysiology of hemolytic uremic syndrome

The pathogenesis of Shiga toxin (Stx)-mediated childhood hemolytic uremic syndrome (HUS) is not fully delineated, although current evidence implicates a prothrombotic state. We hypothesized that the tissue factor (TF) pathway plays a major role in the pathophysiology of HUS. We measured cell surface TF activity in response to tumor necrosis factor-alpha (TNF-alpha) (20 ng mL(-1), 2-144 h), Stx-1 (10(-11) mol L(-1), 4-144 h), or their combination (TNF-alpha 22 h and Stx-1 for the last 0.5-4 h of TNF-alpha incubation) on human glomerular (microvascular) endothelial cells (HGECs) and human umbilical vein (macrovascular) endothelial cells (HUVECs). We observed that while TNF-alpha caused an increase in cell surface TF activity on both cell types, the combination of TNF-alpha and Stx-1 differentially affected HGECs. On these cells, TF activity was increased further by 2.67 +/- 0.38-fold (n = 38, P < 0.001), consistent with our parallel observation that Stx-1 binds to HGECs but not to HUVECs. Anti-TF antibody abolished functional TF while anti-tissue factor pathway inhibitor antibody enhanced TF activity. Stx-1 alone did not induce TF activity on either cell type. Measurement of TF antigen levels and quantitative real-time polymerase chain reaction demonstrated that exposure to TNF-alpha markedly increased TF protein and TF mRNA for HGECs, but the exposure to the combination of TNF-alpha and Stx-1 did not increase further the amount of either TF protein or TF mRNA. We conclude that cytokine-activated HGECs, but not HUVECs, undergo a significant augmentation of cell surface TF activity following exposure to Stx, suggesting an important role for TF in the coagulopathy observed in HUS.

Detached endothelial cells and microparticles as sources of tissue factor activity

Introduction Cytokine activation of endothelial cell monolayers is associated with cell detachment, microparticle shedding from plasma membranes, and phosphatidylserine appearance in the plasma membrane outer leaflets. While tissue factor expression on activated endothelial cells and microparticles is well documented, the contribution of detached endothelial cells to tissue factor activity is less clear. We studied tissue factor expression and the role of tissue factor pathway inhibitor on adherent and detached endothelial cells and on microparticles following endothelial cell activation with TNF-α. Materials and methods Detached endothelial cells and microparticles were obtained from cultures of human umbilical vein endothelial cells by differential centrifugation of cell culture supernatant. For microparticle capture, an antibody directed against CD146 was used. Functional tissue factor activity was measured by chromogenic assay and tissue factor antigen by ELISA. Endothelial cell and microparticle morphology was examined by light and transmission electron microscopy. Results After cell activation for 22 h, functional tissue factor activity was distributed as follows: 60%, adherent endothelial cells; 35%, detached cells; and 5%, microparticles. Tissue factor protein followed a similar distribution. Cell detachment was 47%. Electron microscopy demonstrated shedding of microparticles with a diameter of 0.1–0.6 μm. Cy3–annexin V revealed increased phosphatidylserine on activated adherent endothelial cells and microparticles. Pre-incubation of adherent and detached endothelial cells and microparticles with anti-tissue factor antibody blocked factor Xa production. Pre-incubation with anti-tissue factor pathway inhibitor antibody increased tissue factor activity of adherent endothelial cells 2.8-fold, detached cells 1.4-fold, and microparticles 45-fold. Conclusions Detached endothelial cells as well as microparticles from activated endothelial cell monolayers express tissue factor activity, and this activity on microparticles is markedly inhibited by microparticle-associated tissue factor pathway inhibitor.

Sepsis-Induced Coagulation in the Baboon Lung Is Associated with Decreased Endothelial Tissue Factor Pathway Inhibitor.

Activation of tissue factor (TF)-dependent coagulation is an early event in the pathogenesis of sepsis, responsible for microvascular thrombosis and consequent organ injury. We hypothesized that sepsis-induced TF procoagulant activity may be paralleled by a decreased expression or function of its natural inhibitor, tissue factor pathway inhibitor (TFPI). To test this hypothesis we used a non-human primate sepsis model in which adult baboons were infused with live E. coli at 109 CFU/kg (sublethal dose), 1010 CFU/kg (lethal dose) or saline (control group) and the animals were sacrified after 2, 8, or 24 hours. Lung tissue was snap frozen for protein and mRNA extraction or fixed and processed for light and electron microscopy. Tissue cryosections were immunostained using double/multiple fluorescence labeling approaches for TF, TFPI, factor VII/FVIIa and fibrin, in conjunction with cell markers for endothelial cells (CD31 or von Willebrand factor), leukocytes (PSGL-1), macrophages (CD68), PMN (myeloperoxidase) and platelets (gpIIb/IIIa). Large amounts of TF were detected in leukocytes, endothelial cells and platelet-rich microthrombi, starting from 2 hours and throughout the examined period. Concomitantly, confocal and electron microscopy analysis revealed increased leukocyte infiltration, platelet aggregates and fibrin deposition in the intravascular and interstitial compartments. In addition, TF induction was documented by semiquantitative RT-PCR, ELISA, western blot and factor Xa activation assays. Whereas TFPI mRNA showed only a modest increase, tissue-associated TFPI protein was found considerably decreased, especially during the first eight hours post E. coli infusion. Moreover, TFPI inhibitory activity of lung extracts from septic animals was 6–8 fold lower comparing to controls. TF activity measurements in the presence of inhibitory anti TFPI antibodies showed that only a very small fraction of endogenous TF was inhibited by tissue-associated TFPI, suggesting that most of the active TFPI available in the vascular compartment was depleted. The decrease of TFPI inhibitory potency cannot be exclusively explained by its proteolytic degradation, as we did not find significant amounts of truncated TFPI on western blots. In conclusion, our studies demonstrate that the exposure to septic and inflammatory stimuli lead to a decrease of TFPI-dependent endothelial anticoagulant potential, simultaneous with a strong TF-dependent procoagulant response. Activation of TF-dependent coagulation pathway not adequately countered by TFPI may have important roles in the pathogenesis of sepsis-associated disseminated intravascular coagulation. Strategies aimed to restore the physiological anticoagulant function of TFPI may help preventing sepsis-induced multiple organ dysfunction syndrome and death.