Procoagulant Activity Of Cells Research Articles

Evaluation of tissue factor procoagulant activity on the surface of feline leukocytes in response to treatment with lipopolysaccharide and heat-inactivated fetal bovine serum

To use a chromogenic assay to measure tissue factor (TF) activity on the cell surface and in whole cell lysates of feline monocytes in response to treatment with lipopolysaccharide (LPS) and fetal bovine serum (FBS). 14 healthy cats. Peripheral blood monocytes were isolated via density gradient centrifugation followed by adhesion to plastic. Tissue factor procoagulant activity was measured by use of an assay that detects TF-activated factor X, on the basis of cleavage of a chromogenic TF-activated factor X-dependent substrate. Activity was quantified by comparison with a serially diluted human recombinant TF-activated factor x curve. The TF procoagulant activity assay was sensitive and specific for TF. Treatment with LPS stimulated TF procoagulant activity on the surface and in whole cell lysates of isolated feline leukocytes. The LPS response in intact cells was dose dependent and cell number dependent and was inhibited by FBS. Monocyte isolation was inefficient, with monocytes comprising a mean of 22% of the isolated cells. A TF-activated factor X-dependent chromogenic assay that uses human reagents successfully measured surface-expressed and intracellular TF activity of feline monocytes. Treatment with LPS induced TF expression on feline monocytes, but this response was inhibited by FBS. The chromogenic assay was a useful method for measuring TF procoagulant activity in feline cells in vitro and can be used as a research tool to investigate the role of cell-associated TF in thrombotic disorders in cats.

Phosphatidylserine exposure and procoagulant activity in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) frequently causes disseminated intravascular coagulation that can worsen with cytotoxic chemotherapy but improve with the therapeutic differentiating agents, all trans retinoic acid (ATRA) and arsenic trioxide (As(2)O(3)). APL cells display tissue factor but the relationship of tissue factor and other procoagulant activity to phosphatidylserine (PS) exposure is largely unknown. Lactadherin, a milk protein with stereospecific binding to phosphatidyl-L-serine, was used as a probe for PS exposure on an immortalized APL cell line (NB4) and on the cells of eight patients with APL. PS exposure was evaluated with flow cytometry, confocal microscopy, coagulation assays, and purified prothrombinase and factor (F) Xase assays. Plasma procoagulant activity of NB4 and APL cells increased approximately 15-fold after exposure to etoposide or daunorubicin and decreased 80% after treatment with ATRA or As(2)O(3). Procoagulant activity corresponded to exposed PS on viable APL cells. PS exposure decreased after treatment with ATRA or As(2)O(3) and increased after treatment with daunorubicin or etoposide. Excess lactadherin inhibited 80-85% of intrinsic FXase, FVIIa-tissue factor and prothrombinase activities on both NB4 cells and APL cells. Confocal microscopy identified membrane patches that stained with lactadherin, but not annexin V, demonstrating focal, low-level PS exposure. PS is exposed on viable APL cells and is necessary for approximately 80% of procoagulant activity.

Daunorubicin induces procoagulant activity of cultured endothelial cells through phosphatidylserine exposure and microparticles release

Administration of various chemotherapeutic agents is associated with an increased risk of thrombotic events. Although vascular endothelium plays a predominant role in blood coagulation and fibrinolysis, the effect of cytotoxic drugs on the procoagulant activity (PCA) of endothelial cells has not been well evaluated. Our study aims to investigate the possibility that daunorubicin, a chemotherapeutic agent, exerts prothrombotic effect on endothelial cells. We tested the impact of daunorubicin on phosphatidylserine (PS) exposure, endothelial microparticles (EMPs) release and consequent PCA. Human umbilical vein endothelial cells (HUVECs) were treated with daunorubicin (0.1, 0.2, 0.5, 1, 2 mM) for 24 hours. PCA of HUVECs was measured using clotting time and purified coagulation complex assays. Counts and PCA of EMPs were evaluated by flow cytometry and clotting time assay, respectively. Lactadherin was used as a novel probe for detection of PS exposure and EMPs release. We found that daunorubicin dose-dependently increased the PS exposure and consequent PCA of HUVECs. Moreover, daunorubicin treatment also enhanced the release of EMPs which were highly procoagulant. This increment was especially significant at 0.2 mM of daunorubicin or more. Blockade of PS with lactadherin inhibited over 90% of HUVECs and EMPs PCA. However, anti-TF antibody had no significant inhibition effect. Our results demonstrate that daunorubicin treatment enhanced PCA of HUVECs through PS exposure and shedding of procoagulant EMPs. Lactadherin acts as an efficient anticoagulant in this process.

Effect of Lead(IV) Acetate on Procoagulant Activity in Human Red Blood Cells.

Lead (Pb) is a ubiquitously occurring environmental heavy metal which is widely used in industry and human life. Possibly due to a global industrial expansion, recent studies have revealed the prevalent human exposure to Pb and increased risk of Pb toxicity. Once ingested by human, 95% of absorbed Pb is accumulated into erythrocytes and erythrocytes are known to be a prime target for Pb toxicity. Most of the studies were however, focused on Pb2+ whereas the effects of Pb4+, another major form of Pb on erythrocytes are poorly understood yet. In this study, we investigated and compared the effects of Pb4+, Pb2+ and other heavy metals on procoagulant activation of erythrocytes, an important factor for the participation of erythrocytes in thrombotic events in an effort to address the cardiovascular toxicity of Pb4+. Freshly isolated erythrocytes from human were incubated with Pb4+, Pb2+, Cd2+ and Ag+ and the exposure of phosphatidylserine (PS), key marker for procoagulant activation was measured using flow cytometry. As a result, while Cd2+ and Ag+ did not affect PS exposure, Pb4+ and Pb2+ induced significantly PS exposure in a dose-dependent manner. Of a particular note, Pb4+ induced PS exposure with a similar potency with Pb2+. PS bearing microvesicle (MV), another important contributor to procoagulant activation was also generated by Pb4+. These PS exposure and MV generation by Pb4+ were well in line with the shape change of erythrocyte from normal discocytes to MV shedding echinocytes following Pb4+ treatment. Meanwhile, nonspecific hemolysis was not observed suggesting the specificity of Pb4+-induced PS exposure and MV generation. These results indicated that Pb4+ could induce procoagulant activation of erythrocytes through PS exposure and MV generation, suggesting that Pb4+ exposure might ultimately lead to increased thrombotic events.

A Novel Mechanism of Action of Rivaroxaban: Inhibition of Monocyte and Macrophage Procoagulant Activity and Consequence On Inflammatory Process.

Abstract 3124Poster Board III-61 Aim of the studyTissue factor is normally absent from monocytes in circulating blood. It can be induced by inflammatory mediators leading to the formation of monocyte-associated prothrombinase activity, which participates to thrombin generation. This activity appears to be important in both thrombosis (venous and arterial) and in chronic inflammation by inducing the release of inflammatory cytokines. This could be attributed to the activation of PAR-1 and PAR-2 (protease-activated receptors) by factor Xa or by thrombin. In this study, we compared the action of Rivaroxaban and Fondaparinux, two specific factor Xa inhibitors, on the activation of coagulation and on the secretion of inflammatory cytokines in both activated monocytes and activated human monocyte/macrophage cell line THP1. Methods1-Monocytes were isolated from healthy volunteers and THP-1 cells were used as macrophages. 2- Activation of cells was performed by adding 1 μg/ml LPS for 2 hours at 37°C, in the presence of defibrinated human plasma which provides plasma coagulation factors, in the absence (control) or presence of Fondaparinux (500, 1000 and 1500 ng/ml) or Rivaroxaban (150, 250, 350 ng/ml, final concentrations). After incubation, the cells were isolated and their supernatants collected. 3- The procoagulant activity of cells was tested by measuring their effect on the clotting time of normal plasma in presence of calcium. 4- The release of cytokines was tested by antibody-cytokines array in the supernatants (RayBio®). 5-To test the neutralization of factor Xa bound to monocytes, activated monocytes were incubated with human purified factor Xa (100 ng/ml) for 10 min at room temperature. After isolation of the cells, 250 ng/ml Rivaroxaban or the appropriate solvent was added for 1, 10 and 30 min. The Xa activity associated to monocytes was measured by its amidolytic activity. Results1- The procoagulant activity of monocytes and macrophages was reduced by Rivaroxaban and not by Fondaparinux. Results of prothrombinase activity in the presence of Rivaroxaban at 150, 250, 350 ng/ml, expressed as percentage of the control value was: 30 ± 3, 16 ± 4 and 12 ± 2 % for monocytes and: 43 ± 2, 24 ±3, 15 ± 1 % for THP-1 cells. In contrast, Fondaparinux did not modify the prothrombinase activity of monocytes (105 ± 12%). It was also shown that in the conditions used, Rivaroxaban (250 ng/ml) inhibited completely the amidolytic activity of factor Xa bound to activated monocytes. 2- Both Rivaroxaban and Fondaparinux modify the profile of chemokines secretion by activated monocytes and THP-1. In monocytes and THP-1 cells, LPS induced an important increase in Il-8 and angiogenin and a moderate increase in MIP-1d (Macrophage Inflammatory Protein-1) and RANTES, a member of the Il-8 superfamily. Both Rivaroxaban and Fondaparinux decreased the secretion of these chemokines to the basal level of secretion by non activated monocytes or THP-1. The secretion of leptin was only induced by LPS- treated THP-1 and was strongly decreased by both FXa inhibitors. In contrast, secretion of EGF (epithelial growth factor) was only induced by activated monocytes and strongly decreased by Rivaroxaban and moderately by Fondaparinux. Discussion and conclusionThe results show that 1- Rivaroxaban induced a concentration-dependent inhibition of the procoagulant activity of activated monocytes and macrophages, whereas fondaparinux was devoid of this effect. This difference is attributed to a better access of Rivaroxaban to FXa bound to monocytes, as compared to the Fondaparinux-antithrombin complex. This inhibitory effect of Rivaroxaban could contribute to its antithrombotic activity. 2- Rivaroxaban inhibited the secretion of inflammatory chemokines by activated monocytes and THP-1. This decreased secretion was also observed with Fondaparinux, suggesting that it could be due to the decrease in thrombin generation in plasma, affecting the PAR-1 cell signaling system. As it was reported that that elevated IL-8 is associated with recurrent venous thrombosis and that these inflammatory cytokines are involved in plaque progression and rupture by recruitment of subpopulations of leukocytes and by potent angiogenic activity, this decrease in cytokines could also contribute to the antithrombotic efficacy of Rivaroxaban.This study received a support from Bayer Schering Pharma, France.Equal contribution of Marc Laurent and Rémi Varin DisclosuresNo relevant conflicts of interest to declare.

Cryptic Vs. Active Tissue Factor: Cystine186-Cystine 209 Disulfide Bond Switch Is Not the Answer.

Abstract 333▪▪This icon denotes an abstract that is clinically relevant.Tissue factor (TF) plays the key role in initiation of blood coagulation by allosterically activating coagulation factor VIIa (FVIIa). TF is essential for hemostasis but aberrant expression would lead to thrombotic disorders. TF on cell surfaces resides mostly in a cryptic state. At present it is not entirely clear how cryptic TF differs from procoagulant active TF and how de-encryption occurs. Recent studies have suggested that cryptic and active TF may have different conformations, i.e., cryptic TF contains unpaired cysteine thiols at Cys 186 and Cys 209 in the membrane proximal domain whereas procoagulant active TF contains the oxidized Cys186-Cys209 disulfide bond. It has been suggested that the catalytic switching of this disulfide bond between reduced and oxidized states by protein disulfide isomerase (PDI) might modulate the activity status of TF. Although the validity of this hypothesis has been questioned, more recent reports have supported the contention that PDI-catalyzed disulfide bond formation of Cys186-Cys209 in TF plays a role in regulation of TF activity both in vitro and in vivo. To reconcile the conflicting data in the recent literature, we re-examined in the present study the central dogma on which the hypothesis was originated, i.e., a TF mutant lacking Cys186-Cys 209 disulfide bond is functionally inactive (cryptic) and that TF activation requires formation of the Cys186-Cys209 disulfide bond. CHO cells were transfected with wild type TF or TF mutants that selectively preclude Cys186-Cys209 disulfide bond formation, i.e., TFC186S, TFC209S, or TFC186S/C209S, and TF procoagulant and antigen expression levels at the cell surface were quantified accurately by measuring TF procoagulant activity in a factor × activation assay and TF antigen levels in radioligand binding studies utilizing two different TF mAb (10H10 and 5G9) and FVIIa. CHO cells transfected with TFC186S, TFC209S or TFC186S/C209S expressed very little procoagulant activity (less than 1% of CHO cells transfected with wild-type TF under identical experimental conditions). However, TF mAb and FVIIa binding studies revealed that CHO cells transfected with TFC186S, TFC209S or TFC186S/C209S expressed very little TF protein on the cell surface (∼ 1 to 2% of CHO cells transfected with wild-type TF). Similar results were obtained whether we mutated Cys 186 or Cys 209 to Ser or Ala or whether we used transient or stable transfections. To confirm that the lower expression of TFC186S, TFC209S or TFC186S/C209S is neither an artifact nor limited to CHO cell model system, we extended these studies to endothelial cells. Human umbilical vein endothelial cells (HUVECs) were transduced with adenovirus particles (25 moi/cell) encoding wild-type TF, TFC186S, TFC209S or TFC186S/C209S and TF antigen levels at the cell surface and in cell lysates were measured using multiple methods - confocal microscopy, FACS, TF antigen assay, immuno blot analysis, TF mAb binding and FVIIa binding. HUVEC transduced with adenoviral particles encoding TFC186S, TFC209S or TFC186S/C209S expressed very low levels of TF antigen both at the cell surface ( 1 to 5%) as well as in total (10 to 15%) (TFC186S > TFC209S >TFC186S/C209S) compared to HUVEC transduced with the same number of adenovirus particles expressing wild-type TF. Cell surface TF activity measured in the presence of saturating concentrations of FVIIa revealed that the TF procoagulant activity of the mutants correlate well with the amount of TF antigen present on the cell surface, i.e., there were no significant differences in the specific activity of TF mutants and wild-type TF (TF procoagulant activity/amount of TF antigen, pM FXa formed/femto mole TF: wild-type TF, 68 ± 6; TFC186S, 55 ± 10; TFC209S, 57 ± 17; and TFC186S/C209S, 64 ± 0.2). More importantly, treatment of HUVEC with the thiol-oxidizing agent HgCl2 or with ionomycin increased the cell surface TF activity to the same extent (200 to 400% increase over HUVEC treated with control vehicle) in HUVEC expressing TFC186S/C209S or wild-type TF. In summary the present data provide clear evidence for that TF lacking Cys186-Cys209 bond is coagulantly active and a lower TF procoagulant activity in cells expressing the mutant TF that precludes Cys186-Cys209 disulfide bond formation is due to severe impairment in TF protein synthesis/processing and not because the mutant TF assumes the cryptic conformation. Our data also show that TF de-encryption does not require the formation of Cys186-Cys209 disulfide bond. Disclosures:No relevant conflicts of interest to declare.

Arsenic Trioxide (ATO) and All-Trans Retinoic Acid (ATRA) Differently Affect the Thrombin Generation Potential of Acute Promyelocytic Leukemia (APL) Cells.

Abstract 3986Poster Board III-922 IntroductionA deregulated expression of procoagulant and anticoagulant activities characterises the phenotype of APL cells and contributes to the coagulopathy of this disease. The APL molecular remission induced by differentiation therapy with ATRA or ATO significantly affects specific cellular hemostatic properties of APL blasts. It is not known whether these variations translate into a comparable reduction of the overall procoagulant activity of APL cells. In this study we characterize the overall APL cell hemostatic potential by the calibrated automated method (CAT), a standardized global assay which reflects the net results of pro- and anti-coagulant forces. The endogenous thrombin potential (ETP), measured as the area under the thrombin generation (TG) curve, is a good indicator of overall plasma prothrombotic and hemorrhagic tendency. We evaluated 1) the sensitivity of NB4 cell TG potential to treatment with ATRA or ATO; 2) the correlation of CAT parameters to the levels of two known procoagulants, i.e. tissue factor (TF) and cancer procoagulant (CP), and anticoagulants (i.e. thrombomodulin, TM) and 3) the association of global TG to cell differentiation, proliferation, and apoptosis/necrosis. MethodsNB4 cells were incubated 24h with either 0.1 μM ATO, or 1 μM ATRA, or the combination 0.1 μM ATO/1 μM ATRA, or the vehicle (control). The TG potential of NB4 cells was evaluated in normal pool plasma (NPP) by CAT assay; TF by chromogenic, ELISA, and cytofluorimetric assays; TFmRNA by RT-PCR; CP activity by chromogenic assay; TM by ELISA, cell differentiation by cytofluorimetric analysis of surface CD11b expression; and cell apoptosis/necrosis by annexin V/propidium iodide staining. ResultsTG potential of control NB4 cells (1350±70 nM*min) was significantly higher compared to normal granulocytes (p<0.05). Lower levels of ETP were generated when TG was induced in plasma deficient of factor VII (FVII), confirming a major role to global TG for TF, but also suggesting the contribution for other procoagulants (i.e. CP, phospholipids) to the remaining FVII-independent activity. The TG of NB4 cells treated with ATRA (980±100 nM*min) and ATRA/ATO (1090±90 nM*min) was significantly lower compared to control cells, whereas ATO alone had a minor effect (1200±120 nM*min). Results of specific assays to identify TF and CP confirmed a greater effect of ATRA and the combination ATO+ATRA in reducing both procoagulants compared to ATO alone. The observed reduction of TG potential by the two drugs paralleled the reduction of cell proliferation due to induction of apoptosis and cell differentiation (ATRA and ATO+ATRA) or to cell necrosis (ATO). ConclusionsATRA alone or in combination with ATO is more effective in reducing the NB4-associated TG potential compared to ATO alone. While the ATRA±ATO-induced reduction of NB4-TG potential is associated to a downregulation of procoagulant factors (i.e. TF and CP) and to a parallel increase of the anticoagulant TM, differently, the ATO-induced decrease of NB4-TG potential is associated with the downregulation of TF and CP, without TM modification. CAT is a reliable method to characterize the TG potential of APL cells, and is sensitive to cell pharmacological treatments. Studies are needed to confirm the utility of the CAT to understand the role of different TG phenotypes in predicting the APL coagulopathy. Disclosures:No relevant conflicts of interest to declare.

Daunorubicin induces procoagulant response through phosphatidylserine exposure in red blood cells

Introduction Cytotoxic chemotherapy induces or worsens hemostatic disorders in patients with acute myeloid leukemia. Procoagulant release and tissue factor expression on tumor cells are considered to contribute to chemotherapeutic agents-associated coagulopathy. The role of red blood cells during this process has not been determined; although they lack tissue factor, they may contribute suitable membranes for the amplification phase of blood coagulation. The present study aims to evaluate the possible impact of daunorubicin on phosphatidylserine exposure and consequent procoagulant property of red blood cells. Materials and methods Red blood cells from acute myeloid leukemia patients and healthy donors were treated with daunorubicin as well as all-trans-retinoic acid, arsenic trioxide or etoposide for 0-48 h. Procoagulant activity was assessed by measurement of a clotting time and by purified coagulation complex assays. Lactadherin was used as a probe for phosphatidylserine. Results Daunorubicin treatment increased procoagulant activity of red blood cells regardless of the cell origin. Moreover, coagulation complexes assays supported daunorubicin-evoked procoagulant response. The procoagulant property of red blood cells was not affected by the other three agents. The modulating effect of procoagulant activity was concomitant with and dependent on level of phosphatidylserine on the outer surface. Blockade of phosphatidylserine with lactadherin inhibited over 90% of tenase generation and prothrombinase activity and prolonged the coagulation time. Conclusions We conclude that daunorubicin interacts with red blood cells in a manner that increases phosphatidylserine exposure and consequent procoagulant activity. Lactadherin is an efficient anticoagulant of this process.

Procoagulant activity and phosphatidylserine of amniotic fluid cells

Amniotic fluid (AF) may induce disseminated intravascular coagulation (DIC) when it enters maternal circulation by breaching the placental-maternal circulation barrier. The precise mechanism of the procoagulant activity of AF is unclear, but tissue factor (TF) has been proposed to be the main cause. As one constituent of AF, AF cells accumulate and undergo apoptosis continuously. Therefore, we speculate that AF cells have procoagulant activity due to the externalisation of phosphatidylserine (PS). The present study aims to demonstrate that, in addition to TF, the PS that is externalised on AF cells is important for the procoagulant activity of AF. Ten AF samples from parturient women were analysed using lactadherin as the probe for PS. Anti-TF antibody also was used to identify TF and its associated coagulation functions in AF cells. Normal platelets, neutrophils, and lymphocytes were harvested as controls. Confocal microscopy and flow cytometry was used to assess PS expression on AF cells. The procoagulant activity of AF cells was demonstrated by a plasma coagulation assay and further confirmed by factor Xase/prothrombinase assays. PS and TF were present on most AF cells, providing substantial procoagulant activity. Furthermore, factor Xase and prothrombinase assays showed that AF cells substantially enforced the activation of factor X and prothrombin. PS on AF cells is an important procoagulant source for AF. Lactadherin is an ideal anticoagulant for inhibiting the procoagulant activity of AF cells.

Procoagulant Activity and Phosphatidylserine of Amniotic Fluid Cells

Amniotic fluid (AF) may induce disseminated intravascular coagulation when it enters maternal circulation by breaching the placental-maternal circulation barrier. The precise mechanism of the procoagulant activity of AF is unclear, we speculate that AF cells have procoagulant activity due to the externalization of phosphatidylserine (PS). The present study aims to demonstrate that, in addition to tissue factor (TF), the PS that is externalized on AF cells is important for the procoagulant activity of AF. Ten AF samples from parturient women were analyzed and normal platelets, neutrophils, and lymphocytes were harvested as controls. Lactadherin, a glycoprotein, binds to membranes containing PS, inhibits prothrombinase activity, factor Xase activity, and tissue factor-factor VIIa activity by blocking PS-containing membrane binding. Thus lactadherin was utilized as a PS probe for flow cytometry and confocal microscopy to enable comparison of PS distribution with TF and intrinsic factor Xase complex formation. Procoagulant activity of AF cells was first measured in plasma with AF cells serving as thromboplastin. Activity of AF cells supporting intrinsic and extrinsic factor Xase complexes was measured in purified systems. Lactadherin, as an agent to block exposed PS, inhibited 85% of intrinsic and extrinsic factor Xase activity. Competition binding studies indicated that lactadherin competed for 55% of factor VIII binding sites. However, binding of factor VIII was completely inhibited by PS-containing vesicles and by mAb that recognize the factor VIII C2 domain indicating that all fVIII binding was mediated by the membrane-binding motif or an overlapping epitope. Confocal microscopy identified patches and a rim-pattern indicating a diffuse PS exposure. Lactadherin binding sites and TF distributed to discreet, but overlapping regions of the cells. These results indicate that PS exposure parallels procoagulant activity on AF cells and is required for at least 85% of intrinsic and extrinsic factor Xase activities. However, the topographical pattern of PS exposure differs from the pattern of TF and the pattern of binding site distribution for intrinsic factor Xase complexes. Thus, the results imply that intrinsic factor Xase and extrinsic factor Xase activity are localized to small cell regions where PS exposure coincides with TF and intrinsic factor Xase binding sites, respectively.

Heme induces endothelial tissue factor expression: potential role in hemostatic activation in patients with hemolytic anemia.

We explored the possibility that heme, an inflammatory mediator and a product of intravascular hemolysis in patients with hemolytic anemia including sickle cell disease, could modulate hemostasis by an effect on endothelial tissue factor (TF) expression. Levels of TF mRNA, protein and procoagulant activity were measured in heme-treated endothelial cells. Heme induces TF expression on the surface of both macrovascular and microvascular endothelial cells in a concentration-dependent manner, with 12-fold to 50-fold induction being noted (enzyme-linked immunosorbent assay) between 1 and 100 microm heme (P < 0.05). Complementary flow cytometry studies showed that the heme-mediated endothelial TF expression was quantitatively similar to that of tumor necrosis factor-alpha (TNF-alpha). Heme also upregulated the expression of TF mRNA (8-fold to 26-fold), protein (20-fold to 39-fold) and procoagulant activity (5-fold to 13-fold) in endothelial cells in a time-dependent manner. The time-course of heme-mediated TF antigen expression paralleled the induction of procoagulant activity, with antibody blocking studies demonstrating specificity for TF protein. Interleukin (IL)-1alpha, and TNF-alpha are not involved in mediating the heme effect, as antibodies against these cytokines and IL-1-receptor antagonist failed to block heme-induced TF expression. Inhibition of heme-induced TF mRNA expression by sulfasalazine and curcumin suggested that the transcription factor nuclear factor kappaB is involved in mediating heme-induced TF expression in endothelial cells. Our results demonstrate that heme induces TF expression by directly activating endothelial cells, and that heme-induced endothelial TF expression may provide a pathophysiologic link between the intravascular hemolytic milieu and the hemostatic perturbations previously noted in patients with hemolytic anemia including sickle cell disease.

Increased procoagulant activity of red blood cells in the presence of cisplatin

Cisplatin based chemotherapy is a well recognized risk factor for coagulation disorders and thrombosis. The pathophysiological mechanisms by which cisplatin promote thrombosis are not well understood. Red blood cells (RBCs) were separated from peripheral blood of patients with breast cancer (n = 10) and healthy adults (n = 6) and treated with cisplatin. Coagulation time of RBCs was assessed by one step recalcification time and the productions of thrombin, intrinsic and extrinsic factor Xa were measured in the presence or absence of various concentrations of lactadherin. Exposed phosphatidylserine was stained with lactadherin and observed by confocal microscopy and flow cytometry. Neither fresh RBCs nor RBCs treated without cisplatin had potent procoagulant activity. Cisplatin treatment increased procoagulant activity of RBCs in a cell number- and concentration-dependent manner. Exposed phosphatidylserine was stained with lactadherin and after cisplatin treatment, strong fluorescence was revealed by confocal microscopy. Lactadherin bound RBCs from patients with breast cancer increased from (1.9 +/- 0.5)% on control RBCs to (68.0 +/- 3.5)% on RBCs treated with 10 micromol/L cisplatin for 24 hours. Cisplatin treatment increases procoagulant activity of RBCs, which have a strong association with exposure of phosphatidylserine. The increased procoagulant activity may contribute to the pathogenesis of thrombophilia during cisplatin based chemotherapy in breast cancer patients.

Aberrant NF-κB activity is critical in focal necrosis formation of human glioblastoma by regulation of the expression of tissue factor

Focal necrosis is a key pathologic feature that distinguishes glioblastoma from lower grade glioma. The presence of necrosis in a glioblastoma could promote its rapid growth and clinical progression. Focal necrosis of glioblastoma seems to be associated with thrombosis that result from hyper-coagulability. In the present study, we found that glioblastoma cells had a high level of constitutive nuclear factor (NF)-kappaB activity, which was directly correlated with necrosis in glioblastomas. We also found a direct correlation between NF-kappaB activity and the expression of tissue factor (TF), a potent procoagulant factor in gliomas. Inhibition of TF by an inhibitory antibody prevented the procoagulant activity of glioblastoma cells, indicating a TF-dependent mechanism. Blockade of NF-kappaB activation significantly inhibited TF expression and the procoagulant activity of glioblastoma cells in vitro. Blockade of NF-kappaB activation also significantly inhibited in vivo expression of TF, which was directly correlated with decreased necrosis formation and tumor growth of glioblastoma cells in nude mice. Collectively, these results suggest that elevated NF-kappaB activity in glioblastomas cells plays a critical role in necrosis formation of glioblastoma and that inhibition of NF-kappaB activity in glioblastoma can suppress necrosis formation and progressive growth.

Modulation of tissue factor and tissue factor pathway inhibitor-1 by neutrophil proteases

During systemic inflammation, neutrophil activation is accompanied by endothelial cell damage and hypercoagulability. Activated neutrophils release serine proteases that participate in tissue injury. We sought to investigate the effects of neutrophil proteases on proinflammatory and procoagulant changes in endothelial cells. The effects of elastase (HNE), cathepsin G (CG), and proteinase 3 (PR3) on expression of tissue factor (TF) and tissue factor pathway inhibitor-1 (TFPI) were examined in human umbilical vein endothelial cells. Flow cytometry demonstrated that these proteases proteolytically degraded endothelial cell-bound TFPI. TFPI mRNA expression was reduced by HNE and CG. PR3, but not HNE or CG, increased surface expression of TF and TF mRNA. Yet, increased TF expression did not enhance TF activity suggesting induction of encrypted TF. Using antibodies and siRNA to inhibit and silence PAR-1 and PAR-2, we observed that PR3 upregulation of TF is at least in part mediated by PAR-1. Although CG and HNE cleaved PAR-1, antibody reactivity to the PAR-1 hirudin-like sequence demonstrated inactivating cleavage, accounting for the selective ability of PR3 to induce PAR-1-mediated procoagulant effects. This was supported by induction of p42/44 MAPK by PR3. In conclusion, PR3 degradation of TFPI increases the procoagulant activity of endothelial cells. Release of PR3 after neutrophil activation may represent an important step in neutrophil-mediated vascular injury.

Staphylococcal peptidoglycan initiates an inflammatory response and procoagulant activity in human vascular endothelial cells: a comparison with highly purified lipoteichoic acid and TSST-1

Staphylococcus aureus is one of the most significant pathogens in human sepsis and endocarditis. A hallmark of these endovascular S. aureus infections is that the coagulation system is triggered by a tissue factor (TF)-dependent pathway. This study demonstrates that highly purified S. aureus peptidoglycan, lipoteichoic acid (LTA) and TSST-1 increase TF mRNA and TF surface protein in human umbilical vein endothelial cells (ECs). Concomitantly, peptidoglycan- and LTA-activated ECs express significant TF-dependent procoagulant activity (TF PCA). In addition peptidoglycan, but not LTA or TSST-1, induced surface expression of the EC inflammation markers ICAM-1 and VCAM-1, which supported the adhesion of monocytes to these ECs. During the coculture of peptidoglycan-activated ECs and adherent monocytes, a marked additional increase of TF PCA was observed. Marginal increases in TF PCA were observed in cocultures of monocytes with LTA- or TSST-1-activated ECs. This study defines in particular staphylococcal peptidoglycan, previously known as a potent initiator of TF PCA in monocytes, as also being an activator of a coagulant response in human ECs that is further intensified by the presence of surface-bound monocytes.

Procoagulant Function and Phosphatidylserine Distribution on Immortalized Acute Promyelocytic Leukemia Cells.

Acute promyelocytic leukemia (APL) causes diffuse intravascular coagulation that can worsen with chemotherapy. APL cells expose tissue factor (TF) that contributes to procoagulant activity but the relationship of procoagulant activity to phosphatidylserine (PS) exposure is largely unknown. Lactadherin, a milk protein, binds to membranes containing PS and the binding interaction is mediated by stereoselctive interaction with phosphatidyl-L-serine. As such, lactadherin can serve as a probe to detect low-level PS exposure on cell membranes when the quantity of PS remains below the threshold required for binding of annexin V. Lactadherin can also inhibit prothrombinase activity, factor Xase activity, and tissue factor-factor VIIa activity by blocking PS-containing membrane binding Lactadherin was utilized as a PS probe for flow cytometry and confocal microscopy to enable comparison of PS distribution with TF and intrinsic factor Xase complex formation. Procoagulant activity of NB4 cells was first measured in plasma with NB4 cells serving as thromboplastin. Activity of NB4 cells supporting intrinsic and extrinsic factor Xase complexes was measured in purified systems. Plasma procoagulant activity of NB4 cells increased approx. 15-fold after cells were exposed to 1 μM etoposide for 48 hr and decreased by 80% after 48 hr treatment with 1 μM all trans retinoic acid (ATRA) or As2O3, demonstrating that the procoagulant phenotype of NB4 cells reflects the clinical pattern of diffuse intravascular coagulation in APL. Increased plasma procoagulant activity corresponded to increased intrinsic and extrinsic factor Xase activity as well as increased PS exposure on cells that were not yet frankly apoptotic. ATRA and As2O3 treatment decreased PS exposure and activity of intrinsic and extrinsic enzyme complexes. Lactadherin inhibited 85% of intrinsic and extrinsic factor Xase activity. Competition binding studies indicated that lactadherin competed for ∼70% of factor VIII binding sites. However, binding of factor VIII was completely inhibited by PS-containing vesicles and by monoclonal Ab's that recognize the factor VIII C2 domain indicating that all fVIII binding was mediated by the membrane-binding motif or an overlapping epitope. Confocal microscopy identified patches that stained with lactadherin, but not annexin V, demonstrating focal, low-level PS exposure. Following treatment with etoposide, staining increased. Many cells exhibited a faint, rim-pattern indicating low level, diffuse PS exposure. Factor VIII binding sites and TF distributed to discreet, but overlapping regions of the cells. Fluorescein-Glu-Gly-Arg-chloromethyl ketone derivatized factor IXa bound to the cells in a factor VIIIa-dependent manner in a pattern that reflected the factor VIII(a) binding pattern but not the lactadherin distribution pattern. These results indicate that PS exposure parallels procoagulant activity on NB4 cells and is required for at least 85% of intrinsic and extrinsic factor Xasee activities. However, the topographical pattern of PS exposure differs from the pattern of TF and the pattern of binding site distribution for intrinsic factor Xase complexes. Thus, the results imply that intrinsic factor Xase and extrinsic factor Xase activity are localized to small cell regions where PS exposure coincides with TF and intrinsic factor Xase binding sites, respectively.

Contributions of Phosphatidylserine Exposure on Leukemic Cells to Unregulated Procoagulant Activity in Acute Promyelocytic Leukemia.

Acute promyelocytic leukemia (APL) causes coagulation, which can worsen following initiation of chemotherapy and is improved or corrected by the differentiating agents, all trans retinoic acid (ATRA) and arsenic trioxide (As2O3). The relationship of procoagulant activity to phosphatidylserine (PS) exposure on leukemic cells has not been clarified, although prior studies indicate that APL cells expose tissue factor (TF). Procoagulant activity of leukemic cells was measured in a modified prothrombin time in which leukemic cells replaced thromboplastin. The presence of phosphatidylserine (PS) in neutrophils and mononuclear cells (MNC) from healthy donors and APL cells from patients was investigated by flow-cytometry and confocal microscopy. The assembly of extrinsic tenase, intrinsic tenase and prothrombinase complexes on these cells was studied using enzymatic assays employing plasma or purified proteins. Lactadherin was utilized as a probe to track PS exposure and as an agent to block exposed PS. Isolated APL cells exhibited patches that stained with lactadherin, but neutrophils and MNC did not, indicating more PS exposure on APL cells than the other two cell types. Exclusion of propidium iodide by the leukemic cells indicated that PS exposure occurs in the absence of frank apoptosis. Coagulant activity increased approx 20-fold after cells were exposed to 0.1 μM daunorubicin for 24 hr with 70% of apoptosis and decreased by 85% after 24 hr treatment with 1 μM ATRA or As2O3 with partial differentiation. Inhibition of procoagulant activity by ATRA and As2O3 corresponded to decreased PS and decrease activity for all three enzyme complexes. Lactadherin, which blocks PS binding sites, inhibited Xase and prothrombin conversion by their respective APL-assembled activating complexes. APL cells support all procoagulant reactions leading to robust thrombin formation. This ability results from concomitant PS and TF exposure at the outer leaflet of cell membrane. In contrast, less PS on neutrophils and MNC do not support the coagulation.

Abstract 713: S 18886 Inhibits Thromboxane A 2 Receptor-dependent Tissue Factor Expression, Procoagulant Activity And NADP(H) Oxidase Activation In Stimulated Endothelial Cells

Background Tissue factor (TF) expression and surface exposure are key events in thrombosis, likely contributing to clinical events in vascular disease. Thromboxane (TX)A 2 , an unstable metabolite of arachidonic acid released from vaious cell types, is known for its pro-aggregating and vasoconstrictor properties. Cellular effects of TXA 2 are effected through the TP (TX-prostaglandin endoperoxide) receptor, also expressed in endothelial cells (EC). The TP receptor antagonist S 18886 (Terutroban) demonstrated antithrombotic and antiatherogenic effects in activated EC. As the underlying molecular mechanisms are largely unexplored, we studied the effects of TP agonism and of antagonism on TF expression and procoagulant activity in human umbilical vein endothelial cells (HUVEC), and signal transduction pathways involved. Methods and Results HUVEC ± 30 min pretreatment with the TP antagonist S 18886 were stimulated with the TP receptor agonist U 46619 or TNF-α for 6 hours. TF total expression and surface exposure were assessed by enzyme immunoassays, and TF-dependent procoagulant activity by the generation of Factor Xa. HUVEC exposed to U 46619 featured a concentration-dependent increase in TF total expression and surface exposure. These were associated with enhanced procoagulant activity. S 18886 (1 μmol/L) significantly reduced U 46619 (1 μM)-induced TF expression (−20% ± 7%, P&lt;0.05) and procoagulant activity (−32% ± 11%, P&lt;0.05). Interestingly, S 18886 (1 μmol/L) prevented the increase of TF expression after TNF-α (20 ng/mL) stimulation (−25% ± 9%, P&lt;0.05). Both U 46619- and TNF-α-induced TF expression were mediated by the increase of intracellular reactive oxygen species (ROS), and this was inhibited by S 18886 (−44% ± 6% and −24% ± 5% P&lt;0.05, respectively). S 18886 decreased the membrane association of p47-phox component of NADP(H) oxidase, accounting for the reduced production of ROS. Conclusions Our results show that endothelial TP receptor mediates TF expression, surface exposure and activity stimulated both by TP agonists and by TNF-α. This occurs through NADP(H) oxidase activation and the consequent generation of ROS. These procoagulant and oxidant pathways are inhibited by the TP receptor antagonist S 18886.

Irradiation modulates association of NF-Y with histone-modifying cofactors PCAF and HDAC

Post-irradiation complications including thrombus formation result from increased procoagulant activity of vascular endothelial cells and elevated levels of von Willebrand factor (VWF) contribute to this process. We have previously demonstrated that irradiation induction of the VWF is mediated through interaction of NF-Y transcription factor with its cognate binding site in the VWF promoter. We have also demonstrated that irradiation increases the association of NF-Y with histone acetyltransferase p300/CBP-associated factor (PCAF). We now report that irradiation decreases the association of NF-Y with histone deacetylase 1 (HDAC1). We demonstrate that irradiation-induced changes in association of NF-Y with HDAC1 and PCAF lead to increased PCAF recruitment to the VWF promoter, increased association of acetylated histone H4 with the VWF promoter and subsequently increased transcription. We also demonstrate that this process is correlated to dephosphorylation of HDAC1 and is inhibited by calyculin A, an inhibitor of protein phosphatase1.

Toll-like receptor 4 signal transduction inhibitor, M62812, suppresses endothelial cell and leukocyte activation and prevents lethal septic shock in mice

Sepsis occurs when microbes activate toll-like receptors (TLRs) stimulating widespread inflammation and activating coagulation cascades. TLR4 signal transduction has been recognized as a key pathway for lipopolysaccharide (LPS)-induced activation of various cells and an attractive target for treatment of sepsis. We found a new benzisothiazole derivative, M62812 that inhibits TLR4 signal transduction. This compound suppressed LPS-induced upregulation of inflammatory cytokines, adhesion molecules and procoagulant activity in human vascular endothelial cells and peripheral mononuclear cells. The half maximal inhibitory concentrations in these assays ranged from 1 to 3 μg/ml. Single intravenous administration of M62812 (10–20 mg/kg) protected mice from lethality and reduced inflammatory and coagulatory parameters in a murine d-galactosamine-sensitized endotoxin shock model. M62812 (20 mg/kg) also prevented mice from lethality in a murine cecal ligation and puncture model. These results suggest that inhibition of TLR4 signal transduction can suppress coagulation as well as inflammation during sepsis and may be clinically beneficial in sepsis treatment.