Functional Tissue Factor Research Articles

Do Atrial Differences in Endothelial Damage, Leukocyte and Platelet Activation, or Tissue Factor Activity Contribute to Chamber‐Specific Thrombogenic Status in Patients with Atrial Fibrillation?

Thrombi form mainly in the left rather than the right atria of patients with atrial fibrillation (AF), the reason of this predilection being unknown. The purpose of this study was to investigate whether atrial-specific differences in endothelial damage, leukocyte activation, platelet stimulation, and tissue factor activity occur in patients with AF. Twenty-two patients (16 men, 6 women; age 56 ± 8 years; 16 paroxysmal AF, 6 persistent AF) with AF undergoing pulmonary vein isolation were investigated. Blood samples from the left and the right atrium were obtained at the start of the procedure. Microparticles (MPs) released by apoptotic/stimulated cells were measured by capture assays. Their procoagulant abilities were quantified by functional prothrombinase and tissue factor assays and their cellular origin were determined (endothelium, platelet, leukocyte). Platelet reactivity was evaluated by whole blood flow cytometry for expression of platelet P-selectin (CD62P), active glycoprotein IIb/IIIa receptor (PAC-1). Platelet aggregation was evaluated using ADP, TRAP and collagen-induced whole blood aggregometry. There were no atrial-specific differences in the levels of total procoagulant MPs, leukocyte-derived-MPs or platelet-derived MPs. Conversely, endothelial-derived MPs and tissue factor activity and collagen-induced platelet aggregation were slightly elevated in the right atrium (P < 0.05). Our data show no evidence for increased thrombogenic status in the left atrium that would account for its greater propensity for thrombus formation in patients with AF.

Thrombotic Biomarker Profiling Of Plasma Samples From Patients Undergoing Bypass Surgery Using Protein Chip Array

IntroductionCardiovascular bypass surgical procedures, such as the coronary bypass procedure (CAP), patients are exposed to large doses of heparin and protamine sulfate. There is a high prevalence of anti-heparin platelet factor-4 antibodies in these patients. Although a fraction of these patients develop HIT syndrome, the role of circulating anti-heparin platelet factor-4 antibodies is not clear. Some of these patients may be at a high risk for post-surgical thrombotic complications. Such thrombogenic mediators as the microparticle and tissue factor, may also be up regulated in these patients. Inflammatory processes may also contribute to the overall post-thrombotic complications. The relevance of thrombotic mediators and inflammatory processes remains to be further explored in these patients. Recently protein chip array approach using SELDI/TOF mass spectrometry methods have been employed to identify the unique biomarker in various diseases. The purpose of this study was to determine the protein chip array profile and quantification of various mediators of thrombotic activation in patients who have undergone bypass surgery. Materials & MethodsPlasma samples from 79 patients were collected immediately prior to and two weeks after the CAP. Protein chip array profiling was carried out on a SELDI/TOF mass spectrometric method (PCS4000, BioRad, Richmond, CA) employing a gold chip array in the molecular weight range of 3000-150,000. The intensity of unique peaks was also calculated in terms of relative intensities. Microparticles were measured using a functional method (Hyphen Biomedical, France) and tissue factor antigen levels were measured using an ELISA method. The anti-heparin platelet factor-4 antibodies were also measured using a commercially available ELISA method (Genprobe, Wisconsin). ResultsOf the 79 patients, 20 showed a unique biomarker peak around 11-12kDa in the pre-op samples, which was absent from normal controls. 77 of the patients showed this unique biomarker peak at one week, whereas only 48 patients exhibited at two weeks after surgery. The relative intensity of the 11.6kDa biomarker was much higher at one week (6-fold) and was decreased at two weeks (3-fold). In addition to this unique peak, other biomarker peaks were noted at 15.1 and 15.8kDa. However, these peaks were not changed at different time points. In comparison to the normal, the microparticle levels were higher at the baseline sample (10.1±3.2nM) and increased to 19.3±6.1 and 24.5±8.1nM. Similarly, the tissue factor levels were increased at three weeks' time period. The anti-heparin platelet factor-4 titer rose 28% from the baseline at week one and 33% at week two. ConclusionsThe results on the biomarker profile are consistent to the earlier finding, where the presence of a unique biomarker in the range of 11-12kDa have been reported in patients with high prevalence of anti-heparin platelet factor-4 antibody. The increased level of microparticles and tissue factor at post-surgical periods of one week and two weeks suggest endogenous activation of thrombogenic mechanisms, which appears proportional to the up regulation of the anti-heparin platelet factor-4 antibodies. Thus, this data indicates that the non-functional anti-heparin platelet factor-4 antibodies may result in the activation of cellular processes leading to thrombogenesis. Further characterization of the unique biomarkers identified in these patients may be useful in understanding of the pathogenesis of inflammatory processes and their relevance to thrombogenesis in CABG patients. Clinical ImplicationsThese results indicate that the nonfunctional anti-heparin platelet factor 4 antibodies are capable of mediating inflammatory and thrombotic responses without symptomatic thrombocytopenia. Therefore, the measurement of these antibodies along with inflammatory and thrombogenic mediators may be helpful in the diagnostic and prognostic management of these patients. Disclosures:No relevant conflicts of interest to declare.

Microparticle-associated tissue factor activity is reduced by inhibition of the complement protein 5 in Neisseria meningitidis-exposed whole blood

Neisseria meningitidis causes fulminant meningococcal sepsis with a massive activation of the coagulation and complement cascades. Bacterial cell envelope molecules from N. meningitidis, particularly lipopolysaccharide (LPS), induce tissue factor (TF) expression. In meningococcal sepsis, TF can be detected on circulating monocytes and microparticles (MPs) within the bloodstream. During infection, Nm activates C5 and C5a, which also is able to induce TF. We evaluated the effect of eculizumab, a C5-blocking monoclonal antibodies (mAb), on cell- and MP-associated TF. Using a lepirudin-anticoagulated whole blood model, we activated the coagulation and complement cascades by N. meningitidis, and investigated the interaction between the cascade systems with special focus on cell-associated TF-expression (mRNA and protein) and MP-associated TF-dependent thrombin and fibrin generation in platelet-free plasma. We also examined the ability of TF-positive MPs to support clot formation in whole blood. In addition, the effect of corn trypsin inhibitor and time-dependent changes on MP-associated functional TF activity was examined. Inhibition of C5 reduced cell-associated TF expression at both gene and protein level, and reduced MP-associated TF-dependent thrombin and fibrin generation in platelet-poor plasma, MP-induced TF-dependent clot formation in whole blood, implying that the complement and coagulation cascades are interplayers in N. meningitidis-mediated activation of these cascades.

Intercellular transfer of tissue factor via the uptake of tumor-derived microvesicles

Coagulation proteins play a critical role in numerous aspects of tumor biology. Cancer cells express tissue factor (TF), the protein that initiates blood clotting, which frequently correlates with processes related to cell aggressiveness, including primary tumor growth, invasion, and metastasis. It has been demonstrated that TF gets incorporated into tumor-derived microvesicles (MVs), a process that has been correlated with cancer-associated thrombosis. Here, we describe the exchange of TF-bearing MVs between breast cancer cell lines with different aggressiveness potential. The highly invasive and metastatic MDA-MB-231 cells displayed higher surface levels of functional TF compared with the less aggressive MCF-7 cells. MVs derived from MDA-MB-231 cells were enriched in TF and accelerated plasma coagulation, but MCF-7 cell-derived MVs expressed very low levels of TF. Incubating MCF-7 cells with MDA-MB-231 MVs significantly increased the TF activity. This phenomenon was not observed upon pretreatment of MVs with anti-TF or annexin-V, which blocks phosphatidylserine sites on the surface of MVs. Our data indicated that TF-bearing MVs can be transferred between different populations of cancer cells and may therefore contribute to the propagation of a TF-related aggressive phenotype among heterogeneous subsets of cells in a tumor.

Circulating Levels of Tissue Factor Microparticle Procoagulant Activity Are Reduced With Antiretroviral Therapy and Are Associated With Persistent Inflammation and Coagulation Activation Among HIV-Positive Patients

Activation of coagulation pathways may contribute to risk for non-AIDS-related conditions among HIV-positive patients. Tissue factor (TF)-dependent procoagulant activity on circulating microparticles (MP-TF) in the plasma of 163 HIV-positive participants, both untreated and treated, with viral suppression was measured. MP-TF activity was 39% lower among treated versus untreated participants (P < 0.001), which persisted in adjusted models (-36%, P = 0.03). Among treated participants, MP-TF activity correlated modestly with D-dimer (r = 0.24, P = 0.01), von Willebrand factor (r = 0.36, P < 0.001), and interleukin-6 (r = 0.20, P = 0.04) levels. Future research should focus on mechanisms driving residual functional TF activity and whether these alterations have clinical consequences for non-AIDS-defining complications.

THU0053 Anti-TNF Treatment Results in a Lower Activation State and Prothrombotic Profile of Platelets from Patients with Rheumatoid Arthritis

BackgroundCardiovascular disease is the major cause of mortality in patients with rheumatoid arthritis (RA) and it is not fully explained by traditional risk factors. Some evidence exists that RA treatment,...

Shiga toxin downregulates tissue factor pathway inhibitor, modulating an increase in the expression of functional tissue factor on endothelium

IntroductionEndothelial expression of tissue factor (TF) may play a major role in (Stx)-related hemolytic uremic syndrome. We examined human umbilical vein endothelial cell (HUVEC) monolayers to determine the interaction between TF and TF pathway inhibitor (TFPI), hypothesizing that changes in TFPI modulate TF expression. Materials and MethodsWe studied 1) cell surface expression of globotriasylceramide (Gb3, the receptor for Stx) with Stx-1 (10 pM), TNFα (20Ng/ml), or Stx-1 plus TNFα compared to control, 2) gene expression of TF and TFPI, 3) total cellular and cell surface antigenic TF and TFPI, 4) TFPI secretion into supernatant, and 5) factor Xa production. Results and ConclusionsGb3 expression, negligible with control and Stx-1 alone, increased significantly with TNFα and with Stx-1 plus TNFα. TF mRNA increased 1.25±0.32- fold (N=9; p=0.041) with Stx-1 alone vs. 2.82±0.92-fold (N=13; p<0.0005) with TNFα alone. However, Stx-1 plus TNFα yielded a 6.51±3.48-fold increase (N=17; p<0.0005). TFPI mRNA decreased with TNFα (p<0.001) and Stx-1 plus TNFα (p<0.0005). Total cellular and cell surface TF antigen increased significantly with TNFα, but no further with Stx-1 plus TNFα. Total TFPI cellular and cell surface antigen levels, and TFPI secretion decreased significantly with Stx-1 plus TNFα. Median factor Xa production for Stx-1 plus TNFα vs TNFα alone increased (p<0.001) 3.24-fold. Our results indicate that a subinhibitory concentration of Stx-1 plus TNFα impairs TFPI gene expression, synthesis, cell-surface association, and secretion, leading to augmented functional TF.

Shiga Toxin Upregulates Tissue Factor Gene Expression in and Functional Tissue Factor On Endothelium in the Presence of Complement Activation

Abstract 1075The pathophysiology of Shiga toxin (Stx)-related hemolytic uremic syndrome remains poorly understood. We hypothesize that tissue factor (TF) expression on vascular endothelium is central to the genesis of this disorder and is driven both by direct effects of Stx on endothelium activated by inflammatory cytokines derived from the action of Stx in the gastrointestinal tract, and by Stx-induced complement activation, which further augments TF expression. We used human umbilical vein endothelial cell monolayers to examine 1) gene expression of TF and TF pathway inhibitor (TFPI) with TNFα (20 ng/ml) ± Stx-1 (10 pM) compared to control, 2) total cellular and cell surface antigenic TF and TFPI, 3) TFPI secretion into supernatant, 4) factor Xa production, and 5) soluble levels of C5b-9 in complete media in response to 3–30 pM Stx-1 in the absence of cells. TF mRNA increased 2.82 ± 0.92-fold (N=13; p <0.0005) with TNFα alone vs. 1.25 ± 0.32-fold (N= 9; p =0.041) for Stx-1 alone (Fig. 1). TNFα plus Stx-1 yielded a 6.51 ± 3.48-fold increase (N=17; p <0.0005; Fig. 1). TFPI mRNA decreased with TNFα (p <0.001) andTNFα plus Stx-1 (p < 0.0005). Total cellular (p = 0.048) and cell surface (p = 0.027) TF antigen increased with TNFα, and no further with TNFα plus Stx-1. Total TFPI cellular (p = 0.0017) and cell surface (p = 0.030) antigen levels, and secretion of TFPI (p = 0.018) decreased with TNFα plus Stx-1. Median factor Xa production for TNFα plus Stx-1 vs. TNFα alone increased (p < 0.001) 3.24-fold. In the absence of cells, M199 with 10% human serum to which was added Stx-1 yielded levels of C5b-9 of 648 and 1102 ng/ml vs. 106 and 313 ng/ml in the presence of 25 mM EDTA (to block the classical and alternative pathways of complement), and intermediate levels in the presence of 15 mM EGTA and 7.5 mM Mg++ (to block the classical pathway). Similar results were found with M199 with 10% human plasma, collected using 0.2 U/ml FC in media of dalteparin. C5b-9 levels increased between 3 and 10 pM Stx-1, but did not increase further at 30 pM Stx-1 (Fig. 2). Finally, by means of immunofluorescence we also detected and measured C5b-9 on fixed cell monolayers, whether treated prior to fixation with TNFα alone or TNFα plus Stx-1. We conclude that Stx-1 augments functional TF on endothelium by increasing TF mRNA, and impairing synthesis, cell-surface association, and secretion of TFPI, while at the same time activating both the alternative and classical complement pathways. These findings support the development of therapeutic approaches to epidemic HUS that incorporate inhibitors of the TF pathway, inhibitors of the alternative and classical complement pathways, or both. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Potential factors influencing the development of thrombocytopenia and consumptive coagulopathy after genetically modified pig liver xenotransplantation

Upregulation of tissue factor (TF) expression on activated donor endothelial cells (ECs) triggered by the immune response (IR) has been considered the main initiator of consumptive coagulopathy (CC). In this study, we aimed to identify potential factors in the development of thrombocytopenia and CC after genetically engineered pig liver transplantation in baboons. Baboons received a liver from either an α1,3-galactosyltransferase gene-knockout (GTKO) pig (n = 1) or a GTKO pig transgenic for CD46 (n = 5) with immunosuppressive therapy. TF exposure on recipient platelets and peripheral blood mononuclear cell (PBMCs), activation of donor ECs, platelet and EC microparticles, and the IR were monitored. Profound thrombocytopenia and thrombin formation occurred within minutes of liver reperfusion. Within 2 h, circulating platelets and PBMCs expressed functional TF, with evidence of aggregation in the graft. Porcine ECs were negative for expression of P- and E-selectin, CD106, and TF. The measurable IR was minimal, and the severity and rapidity of thrombocytopenia were not alleviated by prior manipulation of the IR. We suggest that the development of thrombocytopenia/CC may be associated with TF exposure on recipient platelets and PBMCs (but possibly not with activation of donor ECs). Recipient TF appears to initiate thrombocytopenia/CC by a mechanism that may be independent of the IR.

Eculizumab therapy results in rapid and sustained decreases in markers of thrombin generation and inflammation in patients with PNH independent of its effects on hemolysis and microparticle formation

Paroxysmal Nocturnal Hemoglobinuria (PNH) is a clonal bone marrow disorder which results in the loss of glycosylphosphatidyl inositol (GPI) anchors from cell membranes. As a consequence, membrane inhibitors of complement are lost rendering the cells more susceptible to complement mediated destruction. This results in hemolysis, leukopenia, thrombocytopenia and thrombophilia. Eculizumab, a monoclonal antibody to complement protein 5, has been approved for the treatment of PNH and is associated with a significant reduction in hemolysis, thromboembolic events and fatigue. We prospectively studied the effect of Eculizumab therapy on plasma markers of thrombin generation (D-Dimers, TAT), inflammation (IL-6), soluble P-selectin (sP-selectin), antigenic (TFMP) and functional (fTFMP) tissue factor bearing microparticles and total plasma microparticle ex vivo factor Xa generation (MPFXa) in eleven Eculizumab naive PNH patients. Blood sampling occurred day 1, prior to Eculizumab treatment, then on days 8,15,22,29, 43, 90. Our results demonstrate a statistically significant reduction in D-Dimer, TAT, IL-6, sP-selectin, and TFMP during the induction phase of treatment (day 1–29) which was sustained during the maintenance treatment (day 29–90). Although the serum LDH levels decreased rapidly, there was no correlation between the change in LDH and the markers of thrombin generation and inflammation. Although there was a statistically significant decrease in TFMP, this decrease did not correlate with changes in markers of thrombin generation or inflammation. Ex vivo MPFXa generation did not decrease with Eculizumab treatment suggesting continued microparticle formation despite inhibition of hemolysis. Ex vivo total microparticle FXa generation was found to have an inverse correlation with markers of thrombin generation, suggesting that in PNH patients in vivo thrombin generation occurs by a pathway independent of hemolysis and microparticle generation.

Tissue factor over-expression by human pancreatic cancer cells BXPC3 is related to higher prothrombotic potential as compared to breast cancer cells MCF7

Cancer histology influences the risk of venous thromboembolism and tissue factor (TF) is the key molecule in cancer-induced hypercoagulability. We investigated the relation between TF expression by pancreatic and breast cancer cells (BXPC3 and MCF7 respectively) and their capacity to trigger in vitro thrombin generation in normal human plasma. Flow cytometry and Western blot analysis for TF expression were performed using murine IgG1 monoclonal antibody against human TF. Real-time PCR for TFmRNA was also performed. Activity of TF expressed by cancer cells was measured with a specific chromogenic assay. Thrombin generation in PPP was assessed using calibrated automated thrombogram. Cancer cells were added to platelet poor plasma from healthy volunteers. In separate experiments cells were incubated with the anti-TF antibody at concentration that completely neutralized the activity of recombinant human TF on thrombin generation. BXPC3 cells expressed significantly higher amounts of functional TF as compared to MCF7 cells. Incubation of BXPC3 and MCF7 cells with PPP resulted in acceleration of the initiation phase of thrombin generation. BXPC3 cells manifested higher procoagulant potential than MCF7 cells. The incubation of BXPC3 or MCF7 cells with the anti-TF monoclonal antibody which resulted in reversal of their effect on thrombin generation.The present study establishes a link between the amount of TF expressed by cancer cells with their procoagulant activity. Both studied types of cancer cells trigger thrombin generation but they have different procoagulant potential. The procoagulant activity of BXPC3 and MCF7 cells is related to the amount of TF expressed. Kinetic parameters of thrombogram are the most relevant for the detection of the TF-dependent procoagulant activity of cancer cells. TF expression is one of the mechanisms by which cancer cells manifest their procoagulant potential but it is not the unique one. The present experimental model will allow the characterization the procoagulant fingerprint of cell lines from the same or different histological types of cancer.

Evaluation of tissue factor expression in canine tumor cells

To determine whether canine tumor cell lines express functional tissue factor and shed tissue factor-containing microparticles. Cell lines derived from tumors of the canine mammary gland (CMT12 and CMT25), pancreas (P404), lung (BACA), prostate gland (Ace-1), bone (HMPOS, D-17, and OS2.4), and soft tissue (A72); from normal canine renal epithelium (MDCK); and from a malignant human mammary tumor (MDA-MB-231). Tissue factor mRNA and antigen expression were evaluated in cells by use of canine-specific primers in a reverse transcriptase PCR assay and a rabbit polyclonal anti-human tissue factor antibody in flow cytometric and immunofluorescent microscopic assays, respectively. Tissue factor procoagulant activity on cell surfaces, in whole cell lysates, and in microparticle pellets was measured by use of an activated factor X-dependent chromogenic assay. Canine tissue factor mRNA was identified in all canine tumor cells. All canine tumor cells expressed intracellular tissue factor; however, the HMPOS and D-17 osteosarcoma cells lacked surface tissue factor expression and activity. The highest tissue factor expression and activity were observed in canine mammary tumor cells and pulmonary carcinoma cells (BACA). These 3 tumors also shed tissue factor-bearing microparticles into tissue culture supernatants. Tissue factor was constitutively highly expressed in canine tumor cell lines, particularly those derived from epithelial tumors. Because tumor-associated tissue factor can promote tumor growth and metastasis in human patients, high tissue factor expression could affect the in vivo biological behavior of these tumors in dogs.

Tissue Factor and Tissue Factor Pathway Inhibitor as Key Regulators of Global Hemostasis: Measurement of Their Levels in Coagulation Assays

The tissue factor (TF)/factor (F)VIIa complex is the primary initiator of coagulation in vivo. Tissue factor pathway inhibitor (TFPI) is the physiological inhibitor of the TF/FVIIa complex. Deficiencies of either TF or TFPI have not been reported in humans, and a complete absence of either of these two proteins in mice is embryonically lethal. To maintain normal hemostasis, levels of TF and TFPI need to be balanced. Increased levels of TF can overwhelm the inhibitory capacity of TFPI, resulting in thrombosis. Decreased levels of TF are associated with bleeding. Global assays of coagulation are defined as tests capable of evaluating all components of the clotting cascade that are present in plasma. In these tests the thrombogenic surface is either provided by platelets or exogenous phospholipids. Clotting assays currently used in clinical practice are not designed to measure endogenous levels of TF and TFPI. Therefore, there is a need to develop sensitive and specific assays for measuring levels of functional TF and TFPI in whole blood and plasma. These assays could be useful in patient management in many scenarios.

Atorvastatin or transgenic expression of TFPI inhibits coagulation initiated by anti-nonGal IgG binding to porcine aortic endothelial cells.

Intravascular thrombosis remains a barrier to successful xenotransplantation. Tissue factor (TF) expression on porcine aortic endothelial cells (PAECs), which results from their activation by xenoreactive antibodies (Abs) to Galα1,3Gal (Gal) and subsequent complement activation, plays an important role. The present study aimed to clarify the role of Abs directed against nonGal antigens in the activation of PAECs to express functional TF and to investigate selected methods of inhibiting TF activity. PAECs from wild-type (WT), α1,3-galactosyltransferase gene-knockout (GT-KO) pigs, or pigs transgenic for CD46 or tissue factor pathway inhibitor (TFPI), were incubated with naïve baboon serum (BS) or sensitized BS (with high anti-nonGal Ab levels). TF activity of PAECs was assessed. Only fresh, but not heat-inactivated (HI), naïve BS activated WT PAECs to express functional TF. Similarly, PAECs from CD46 pigs were resistant to activation by naïve BS, but not to activation by fresh or HI sensitized BS. HI sensitized BS also activated GT-KO PAECs to induce TF activity. TF expression on PAECs induced by anti-nonGal Abs was inhibited if serum was pretreated with (i) an anti-IgG Fab Ab or (ii) atorvastatin, or (iii) when PAECs were transgenic for TFPI. Anti-nonGal IgG Abs activated PAECs to induce TF activity through a complement-independent pathway. This implies that GT-KO pigs expressing a complement-regulatory protein may be insufficient to prevent the activation of PAECs. Genetic modification with an 'anticoagulant' gene (e.g. TFPI) or a therapeutic approach (e.g. atorvastatin) will be required to prevent coagulation dysregulation after pig-to-primate organ transplantation.

Elevated Levels of Tissue Factor Bearing Microparticles in Patients with Antiphospholipid Antibodies: Analysis of Fresh, Unfrozen Samples.

Abstract 148 Introduction:The antiphospholipid syndrome (APS) is characterized by venous or arterial thrombosis, and/or recurrent fetal loss in the presence of circulating antiphospholipid antibodies (APL). The pathogenesis of this disorder may involve, in part, the activation of vascular endothelial cells, platelets, and monocytes. Upon activation, these cells shed components of their plasma membranes as microparticles (MP), and procoagulant MPs are associated with an increased risk for thromboemboli. The goal of these studies was to characterize, for the first time, the frequency by which increased levels of endothelial cell, platelet and monocyte-derived microparticles are observed in freshly-obtained, unfrozen plasma from patients with APL. Patients and Methods:Whole citrated (3.2%) blood samples from patients/healthy donors were centrifuged at 1500xg for 15 min, and the supernatant further centrifuged at 13000xg for 2 min to obtain platelet free plasma (PFP). To detect microparticles, and their origin, PFP was labeled with the following conjugated monoclonal antibodies and analyzed by flow cytometry using an LSR II flow cytometer (BD): Annexin V-APC; CD105-PE and CD144-PE (endothelial cells); CD41-PECy5 (platelets); CD14-PE (monocytes); and anti-tissue factor mAb-Alexa Fluor 647 (a kind gift from Dr. Kenneth Mann, Univ of VT). The binding of control antibodies labeled with identical fluorophores as the monoclonal antibodies was subtracted to adjust for nonspecific binding. We assessed microparticles in 47 patients with APL and 144 healthy controls. ResultsLevels of annexin-V, CD105, CD144, CD41 and tissue factor positive microparticles were significantly higher in patients (47,000±7900, 10,140±2164, 46,690±8783, 127,200±28,050 and 7270±2810 microparticles/ml, respectively) versus controls (17,000±1800, 3,824±374, 17,320±2636, 54,910±7132 and 2,537±747 microparticles/ml, respectively). These differences were statistically significant. In contrast, there was no significant difference in CD14 positive microparticle levels (1035±167 in APL patients versus 1409±465 in controls; p=0.655). Conclusions:Our results demonstrate elevation of annexin V+, CD105+, CD144+, CD41+ and tissue factor-bearing microparticles in freshly-obtained, unfrozen plasma obtained from patients with APL. These results suggest that ongoing activation of platelets and endothelial cells occurs in many patients with APL even in the absence of active thrombosis, and may contribute to the prothrombotic phenotype of these patients. Data is currently being analyzed for associations of microparticle levels with APL serologies, as well as the expression of functional microparticle-associated tissue factor activity. Disclosures:No relevant conflicts of interest to declare.

Shiga Toxin and Lipopolysaccharide Induce Platelet-Leukocyte Aggregates and Tissue Factor Release, a Thrombotic Mechanism in Hemolytic Uremic Syndrome

BackgroundAggregates formed between leukocytes and platelets in the circulation lead to release of tissue factor (TF)–bearing microparticles contributing to a prothrombotic state. As enterohemorrhagic Escherichia coli (EHEC) may cause hemolytic uremic syndrome (HUS), in which microthrombi cause tissue damage, this study investigated whether the interaction between blood cells and EHEC virulence factors Shiga toxin (Stx) and lipopolysaccharide (LPS) led to release of TF.Methodology/Principal FindingsThe interaction between Stx or LPS and blood cells induced platelet-leukocyte aggregate formation and tissue factor (TF) release, as detected by flow cytometry in whole blood. O157LPS was more potent than other LPS serotypes. Aggregates formed mainly between monocytes and platelets and less so between neutrophils and platelets. Stimulated blood cells in complex expressed activation markers, and microparticles were released. Microparticles originated mainly from platelets and monocytes and expressed TF. TF–expressing microparticles, and functional TF in plasma, increased when blood cells were simultaneously exposed to the EHEC virulence factors and high shear stress. Stx and LPS in combination had a more pronounced effect on platelet-monocyte aggregate formation, and TF expression on these aggregates, than each virulence factor alone. Whole blood and plasma from HUS patients (n = 4) were analyzed. All patients had an increase in leukocyte-platelet aggregates, mainly between monocytes and platelets, on which TF was expressed during the acute phase of disease. Patients also exhibited an increase in microparticles, mainly originating from platelets and monocytes, bearing surface-bound TF, and functional TF was detected in their plasma. Blood cell aggregates, microparticles, and TF decreased upon recovery.Conclusions/SignificanceBy triggering TF release in the circulation, Stx and LPS can induce a prothrombotic state contributing to the pathogenesis of HUS.

TF Independent Potentiation of FVIIa Activity in CAD Plasma: An Assessment Using Two Chromogenic Assays.

Background. Tissue Factor (TF), the regulatory cofactor of FVII(a), triggers blood coagulation as it converts FX and FIX to their active forms FXa and FIXa, respectively. In 2003 we showed that level of decryptable circulating TF activity in diabetic, smoker, and hyperlipidemic populations is a functionally labile entity. Existence of such form of circulating TF has generated the concept of vulnerable or hyperreactive blood. Recently, heightened TF activity was noted by many groups in various types of clinical samples, including human plasma, via utilization of a commercial TF activity assay termed ACTICHROME® whose approach to measure plasma TF activity is direct and distinct from that developed by us.Objective. To assess levels of plasma TF activity in patients with coronary artery disease (CAD), by the ACTICHROME® as well as our assay.Results. Suitability of the ACTICHROME® assay for direct assessment of TF activity in plasma was compared with our technique of plasma TF immunocapture followed by TF relipidation. We assessed TF activity in plasma aliquots pre-incubated with FVIIai, an inactive form of FVIIa that retains binding affinity to TF yet lacks protease activity, thereby serving as a highly effective and specific TF inhibitor. The ACTICHROME® assay was not able to detect bona fide TF activity: the tested plasma exhibited activity that could not be inhibited by pre-incubation with FVIIai. Thus, under the conditions of the ACTICHROME® assay, FX to FXa conversion in human platelet rich plasma is achieved by FVIIa in a TF independent manner. In contrast to the ACTICHROME® assay, in our TF assay pre-incubation of samples with FVIIai fully precluded FX activation. We therefore refer to plasma TF activity levels obtained using our TF assay as tTFp (total TF potential), and to the FVIIa activity levels obtained using the ACTICHROME® assay – ACTI-FVIIa. Assessment of tTFp in CAD plasma samples (n=28) revealed that in a subset of patients, tTFp was lower than that found in normal plasma (58.5%-69.6%, p=0.0001 NL, n=6 vs CAD tTFp <70%, n=4), while another subset of patients presented with tTFp that was higher than that of normal plasma (134.2% – 233.6%, p=0.003 NL, n=6 vs CAD tTFp >130%, n=7). The tTFp values in age matched healthy subjects (n=6) were in the close range of 75.2%-109.5%. We also assessed each healthy control and CAD patient sample for ACTI-FVIIa. To our surprise, ACTI-FVIIa values in all CAD samples were high, ranging between 15.8–132.1 pM of the TF standard, whereas ACTI-FVIIa values in the healthy subject group were much lower, in the close range of 3.2–8.8 pM. Interestingly, ACTI-VIIa of the three CAD samples exhibiting the highest tTFp was significantly lower than that of the three CAD samples exhibiting the lowest tTFp (highest tTFp: ACTI-VIIa = 39.2 ± 17.3, lowest tTFp: ACTI-VIIa = 97.4 ± 30.1, p=0.043). Heightened ACTI-FVIIa values in CAD samples prompted us to ascertain whether circulating MP and/or platelets may account for this phenomenon. Conventional size MP and platelets were removed from plasma by ultracentrifugation. Approximately 50% of ACTI-FVIIa in normal plasma and ≥95% of ACTI-FVIIa in CAD plasma was attributable to conventional size MP; in contrast, tTFp values in plasma samples pre- and post-spin were fairly similar: ultracentrifugation of CAD plasma resulted in only ~24.5% reduction of TF activity whereas in normal plasma, it resulted in ~42% reduction in TF activity, indicating that similarly to CAD plasma, the majority of functional TF species in normal plasma are not physically associated with conventional size MP, yet a somewhat larger proportion of functional TF is indeed carried by them compared to CAD plasma.Conclusions. We show that in CAD plasma, circulating MP of the conventional size do not carry most of functional TF yet, interestingly, CAD plasma strongly potentiates FVIIa activity in a TF-independent manner. We also show that the ACTICHROME® assay, when used to measure TF activity in human plasma, yields results that do not reflect bona fide TF activity. The majority of functional TF in human plasma appears to circulate in very small MP and in true solution; lastly, all examined plasma samples carried TF activity, underscoring the fact that potentially active TF protein is always present in circulation. These observations highlight the therapeutic possibilities of TF pathway inhibitors for treatment and prevention of atherothrombotic diseases.

Immunomodulatory Drugs (Thalidomide and Lenalidomide) Up-Regulate Endothelial Tissue Factor in Vitro Resulting in An Acquired Hypercoagulable State.

Background: The immunomodulatory drugs (IMIDs), thalidomide and lenalidomide, are considered first line treatments for multiple myeloma and are being investigated for an expanding range of indications including myelodysplastic syndrome, chronic lymphocytic leukaemia, and other B-Cell malignancies. One of the adverse effects of IMIDs are venous thromboembolic events (VTE). Tissue factor (TF) is the cellular receptor for clotting factor VII/VIIa and plays a central role in the initiation of coagulation in vivo. Presently there is no consensus regarding the optimal thromboprophylaxis strategy in IMID treated patients. We hypothesize that in the presence of an inflammatory stimulus IMIDs induce a hypercoagulable state through increased endothelial TF expression and activity.Materials and Methods: Commercially available endothelial cells (HUVEC) were cultured to subconfluent conditions and incubated with thalidomide, lenalidomide or vector control (DMSO) under inflammatory conditions induced by TNFα (10ng/ml). Drug concentrations used were similar to therapeutic human plasma levels. The following parameters were evaluated: TF procoagulant surface activity (colorimetric assay), TF antigen in whole cell lysates (ELISA), and TF mRNA (real time-PCR). Each experiment was repeated at least 3 times. Neutralizing anti TF antibodies were used as a quality control for the functional TF assay. To investigate endothelial cell derived “micro”particles culture supernatants were first centrifuged for 10 minutes at 300g to remove detached whole cells. The resulting supernatant was centrifuged twice at 17000 g for 30min to precipitate potential “micro”particles. Size and surface markers of resuspended particles were investigated by flow cytometry.Results: In the presence of inflammatory conditions (TNFα 10 ng/ml) IMIDs, thalidomide and lenalidomide (both tested at 0.5μM), increase surface related TF activity by 31% and 16% respectively (p<0.01, Wilcoxon test) compared to inflammatory baseline levels. IMID induced increased TF activity could be specifically inhibited by a cocktail of two monoclonal anti TF antibodies. These findings suggest that TF protein accounts for >95% of the TF activity we observed. TF antigen estimated in whole cell lysates was upregulated by 26% by thalidomide and 24% by lenalidomide (vs TNFα treated cells). To evaluate whether the observed TF surface activity and antigen up-regulation was due to a possible effect of IMIDs on TF gene transcription, TF mRNA was measured by RT-PCR after one and six hours. Compared to TNFα treated cells thalidomide and lenalidomide increased TF mRNA by 53 and 33% at one hour while at six hours the corresponding values were 17 and 18% respectively. Cell culture media from TNF, thalidomide and lenalidomide treated cells contained precipitable particles that when resuspended showed TF activity while media from control cells lacked such activity.Conclusions: Under inflammatory conditions thalidomide and lenalidomide lead to increased endothelial cell TF synthesis, increased TF antigen and increased activity of surface bound TF. We furthermore detected increased endothelial derived “micro”particles with TF activity indicating an overall increased turnover and shedding of endothelial TF. Our findings need to be reproduced in vivo but may provide insights into the hypercoaglubale state observed in patients treated with IMIDs und thus may be utile in designing efficient thromboprophylaxis strategies.

Circulating microparticles in renal diseases

well-regulated process generating MP that present specific features, with significant implications on their functional activity. Indeed, MP behave as vectors of bioactive molecules able to disseminate biological information in the vascular compartment. Due to the expression of both membrane PhtdSer and functional tissue factor (TF), MP are catalytic procoagulant surfaces involved in thrombogenesis. They also harbour inflammatory components (LpA, IL-1 and chemokine receptor CCR5) [4,5], growth factors (TGF-beta and VEGF) and proteases (uPA and MMP) that are related to their involvement in inflammation, immune response, angiogenesis and cancer [6–8].

C5a and TNF-alpha up-regulate the expression of tissue factor in intra-alveolar neutrophils of patients with the acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is characterized by the presence of fibrin-rich inflammatory exudates in the intra-alveolar spaces and the extensive migration of neutrophils into alveoli of the lungs. Tissue factor (TF)-dependent procoagulant properties of bronchoalveaolar lavage fluid (BALF) obtained from ARDS patients favor fibrin deposition, and are likely the result of cross-talk between inflammatory mediators and hemostatic mechanisms. However, the regulation of these interactions remains elusive. Prompted by previous findings suggesting that neutrophils, under certain inflammatory conditions, can express functional TF, we investigated the contribution of intra-alveolar neutrophils to the procoagulant properties of BALF from patients with ARDS. Our results confirm that the procoagulant properties of BALF from ARDS patients are the result of TF induction, and further indicate that BALF neutrophils are a main source of TF in intra-alveolar fluid. We also found that BALF neutrophils in these patients express significantly higher levels of TF than peripheral blood neutrophils. These results suggest that the alveolar microenvironment contributes to TF induction in ARDS. Additional experiments indicated that the ability of BALF to induce TF expression in neutrophils from healthy donors can be abolished by inhibiting C5a or TNF-alpha signaling, suggesting a primary role for these inflammatory mediators in the up-regulation of TF in alveolar neutrophils in ARDS. This cross-talk between inflammatory mediators and the induction of TF expression in intra-alveolar neutrophils may be a potential target for novel therapeutic strategies to limit ARDS-associated disturbances of coagulation.