LPS-induced Tissue Factor Activity Research Articles

Procoagulant and proinflammatory effects of red blood cells on lipopolysaccharide‐stimulated monocytes

We aimed to evaluate the mechanisms underlying the effects of red blood cells (RBCs) on the reactivity of monocytes to lipopolysaccharide (LPS) stimulation. Measurements of tissue factor (TF) antigen and activity were performed on freshly isolated white blood cells (WBCs)/platelets resuspended in heparinized plasma, as well as cultured monocytic cells. In a dose-dependent manner, RBCs significantly enhanced LPS-induced TF activity and antigen levels in blood monocytes; potentiation of TF activity by both human and murine RBCs did not require the presence of neutrophils and/or platelets. We also measured the levels of monocyte chemotactic protein-1 (MCP-1), the key proinflammatory chemokine that binds to duffy antigen receptor for chemokines (DARC) on RBC surface, in plasma and RBC lysates after the incubation of RBCs with WBC/platelets; at the concentrations corresponding to normal blood counts, RBCs exerted a significant influence on the free plasma levels of MCP-1, with about two-thirds of detectable MCP-1 post-LPS stimulation being associated with RBCs. Critically, DARC-deficient murine RBCs failed to enhance LPS-induced TF activity, confirming the mechanistic significance of RBC-DARC. Our study reports a novel mechanism by which RBCs promote procoagulant and proinflammatory sequelae of WBC exposure to LPS, likely mediated by RBC-DARC in the microenvironment(s) that bring monocytes and RBCs in close proximity.

Endothelial cells but not platelets are the major source of Toll-like receptor 4 in the arterial thrombosis and tissue factor expression in mice.

It is known that Toll-like receptor (TLR)-4 plays an important role in myocardial infarction and atherothrombosis. The role of TLR-4 in arterial thrombosis is undefined. Both TLR-4-deficient (TLR-4(-/-)) and wild-type (WT) mice were subjected to FeCl3 carotid artery injury, and the time required to form an occlusive thrombus was measured. The mean time to occlusion in TLR-4(-/-) mice was significantly greater than that in WT mice after injury (303 ± 32 vs. 165 ± 34 s, P < 0.05). Furthermore, when we used a WT or TLR-4(-/-)-derived platelet reinfusion in a platelet depletion/reinfusion procedure, there was no significant change in the occlusion time and tissue factor (TF) activity in injured arteries between WT mice and platelet-depleted WT mice. Similarly, no significant difference was observed between TLR-4(-/-) mice and platelet-depleted TLR-4(-/-) mice for the WT or TLR-4(-/-)-derived platelet reinfusion. However, TF expression and activity were significantly reduced in the vascular wall of TLR-4(-/-) mice compared with WT mice. In vivo, lipopolysaccharide accelerated the occlusion time in WT mice but not TLR-4(-/-) mice. In vitro, LPS-induced TF activity was reduced in endothelial cells of TLR-4(-/-) mice relative to WT mice. The data demonstrate that TLR-4 contributes to arterial thrombosis formation in vivo and causes increased TF expression and activity in vitro. The results further suggest that the stimulation is mainly derived by endothelial cells but is not due to platelet-derived TLR-4.

Pro- and anti-inflammatory effects of histamine on tissue factor and TNFα expression in monocytes of human blood

Histamine is classified as an inflammatory mediator and has been reported to have anti- as well as pro-inflammatory properties. The aim of this study was to explore the role of histamine on the production of LPS-induced tissue factor (TF) activity and TNFα in monocytes of whole blood in the absence and presence of TNFα or PMA. Human blood anticoagulated with Fragmin was subjected to stimulation by LPS in the presence and absence of TNFα or PMA and various concentrations of histamine. Tissue factor (TF) activity was measured in lyzed cells after isolation of mononuclear cells whereas TNFα was quantified in plasma after centrifugation of cells. Histamine gave a dose dependent inhibitory effect on LPS-induced TF activity in monocytes of whole blood, with a 50% reduction at 0.033 μM. A similar effect was seen when the blood cells were stimulated with the combination of LPS and TNFα although TNFα enhanced LPS-induced TF activity almost two fold. In contrast, when blood was incubated with LPS and PMA in whole blood, histamine gave a significant rise in TF activity at 0.01 μM and 0.33 μM histamine. The effect of histamine was less at 0.1 μM or higher concentrations giving a biphasic profile. Contrary to the effect of histamine on LPS plus PMA induced TF activity, histamine caused a significant reduction in TNFα albeit less than in the absence of PMA. Intake of aspirin caused a significant rise in LPS-induced TF activity that was almost abolished by histamine at 0.033 μM. Our study shows that histamine has an anti-inflammatory effect on LPS and LPS/TNFα stimulated monocytes of whole blood. In contrast when blood cells are activated by a combination of LPS and PMA whereby PKC is activated, histamine has a procoagulant/pro-inflammatory effect through enhancement of TF activity expression.

Mechanical Stretch Inhibits Lipopolysaccharide-induced Keratinocyte-derived Chemokine and Tissue Factor Expression While Increasing Procoagulant Activity in Murine Lung Epithelial Cells

Previous studies have shown that the innate immune stimulant LPS augments mechanical ventilation-induced pulmonary coagulation and inflammation. Whether these effects are mediated by alveolar epithelial cells is unclear. The alveolar epithelium is a key regulator of the innate immune reaction to pathogens and can modulate both intra-alveolar inflammation and coagulation through up-regulation of proinflammatory cytokines and tissue factor (TF), the principal initiator of the extrinsic coagulation pathway. We hypothesized that cyclic mechanical stretch (MS) potentiates LPS-mediated alveolar epithelial cell (MLE-12) expression of the chemokine keratinocyte-derived cytokine (KC) and TF. Contrary to our hypothesis, MS significantly decreased LPS-induced KC and TF mRNA and protein expression. Investigation into potential mechanisms showed that stretch significantly reduced LPS-induced surface expression of TLR4 that was not a result of increased degradation. Decreased cell surface TLR4 expression was concomitant with reduced LPS-mediated NF-κB activation. Immunofluorescence staining showed that cyclic MS markedly altered LPS-induced organization of actin filaments. In contrast to expression, MS significantly increased LPS-induced cell surface TF activity independent of calcium signaling. These findings suggest that cyclic MS of lung epithelial cells down-regulates LPS-mediated inflammatory and procoagulant expression by modulating actin organization and reducing cell surface TLR4 expression and signaling. However, because LPS-induced surface TF activity was enhanced by stretch, these data demonstrate differential pathways regulating TF expression and activity. Ultimately, loss of LPS responsiveness in the epithelium induced by MS could result in increased susceptibility of the lung to bacterial infections in the setting of mechanical ventilation.

Thiopental Inhibits Lipopolysaccharide-Induced Tissue Factor Expression

During Gram-negative sepsis, lipopolysaccharide (LPS) stimulates toll-like receptor 4, resulting in an activation of the immune system and the expression of tissue factor on monocytes. As a consequence, intravascular coagulation, ischemia, and multiorgan dysfunction may occur. Because thiopental has been described to modulate the immune system, we tested the hypothesis that thiopental alters the LPS-induced tissue factor expression. (i) Citrated whole blood samples were incubated with thiopental (0, 0.25, 0.5, 1 mg/mL) and LPS (100 microg/mL) for 4 h. After recalcification, clotting time (CT) was determined by rotational thrombelastometry. (ii) The mechanism of the LPS-induced shortening of CT was investigated using the tissue factor blocker active-site inhibited factor VIIa and the protein synthesis inhibitor cycloheximide. (iii) A concentration response curve for the effect of tissue factor on CT was generated. LPS shortened CT from 618 +/- 122 s to 192 +/- 33 s (n = 6; P < 0.05). Shortening of CT was mediated by synthesis of tissue factor, because both inhibition of protein synthesis and blockade of tissue factor effects abolished this effect of LPS. Thiopental markedly inhibited the LPS-induced shortening of CT (372 +/- 86 s; n = 6; P < 0.001). Comparison of CT with a tissue factor standard curve demonstrated that thiopental reduced the LPS-induced tissue factor activity up to 86%. A direct effect of thiopental on coagulation was excluded, because tissue factor-induced CT was not affected by the barbiturate. Thiopental markedly inhibits the LPS-induced tissue factor expression in whole blood samples.

VASOACTIVE INTESTINAL PEPTIDE AND PITUARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE INHIBIT TISSUE FACTOR EXPRESSION IN MONOCYTE IN VITRO AND IN VIVO

Tissue factor (TF), which is expressed on the surface of activated monocytes, is the major procoagulant that initiates thrombus formation in sepsis. Two endogenous neuropeptides, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), are attractive candidates for the development of therapies against septic shock. The purpose of this study was to examine whether VIP or PACAP inhibit the LPS-induced TF expression in monocytes. Treatment of freshly isolated human monocytes or cultured monocytic THP-1 cells with VIP or PACAP leads to reduced LPS-induced TF protein, mRNA expression and activity, as demonstrated by Western blot, real-time polymerase chain reaction, and TF activity assay, respectively. In an endotoxemic model, VIP blunts the increase of LPS-induced TF expression in blood cells at the transcriptional level, as demonstrated by real-time polymerase chain reaction. However, neither neuropeptide affects the expression of TF pathway inhibitor in monocytes. In vitro, LPS increases the migration of c-Rel/p65 into the nucleus and the phosphorylation of p38 and JNK, all of which are essential for LPS-induced TF expression. In addition, interestingly, VIP and PACAP block both the migration of c-Rel/p65 and the phosphorylation of p38 and JNK, as demonstrated by Western blot analysis. These data indicate that VIP and PACAP inhibit LPS-induced TF expression in monocytes in vitro and in vivo, confirming these peptides as candidates for the multitarget therapy of septic shock.

Ochratoxin A inhibits the production of tissue factor and plasminogen activator inhibitor-2 by human blood mononuclear cells: Another potential mechanism of immune-suppression

The mycotoxin ochratoxin A (OTA), an ubiquitous contaminant of food products endowed with a wide spectrum of toxicity, affects several functions of mononuclear leukocytes. Monocytes/macrophages play a major role in fibrin accumulation associated with immune-inflammatory processes through the production of tissue factor (TF) and plasminogen activator inhibitor 2 (PAI-2). We studied the effect of OTA on TF and PAI-2 production by human blood mononuclear cells (MNC). The cells were incubated for 3 or 18 h at 37 °C with non toxic OTA concentrations in the absence and in the presence of lipopolysaccharide (LPS) or other inflammatory agents. TF activity was measured by a one-stage clotting test. Antigen assays were performed by specific ELISAs in cell extracts or conditioned media and specific mRNAs were assessed by RT-PCR. OTA had no direct effect on TF and PAI-2 production by MNC. However, OTA caused a dose-dependent reduction in LPS-induced TF (activity, antigen and mRNA) and PAI-2 (antigen and mRNA) production with > 85% inhibition at 1 μg/ml. Similar results were obtained when monocyte-enriched preparations were used instead of MNC. TF production was also impaired by OTA (1 μg/ml) when MNC were stimulated with phorbol myristate acetate (98% inhibition), IL-1β (83%) or TNF-α (62%). The inhibition of TF and PAI-2 induction might represent a hitherto unrecognized mechanism whereby OTA exerts immunosuppressant activity.

Heat shock inhibits lipopolysaccharide-induced tissue factor activity in human whole blood

BackgroundDuring gram-negative sepsis, lipopolysaccharide (LPS) induces tissue factor expression on monocytes. The resulting disseminated intravascular coagulation leads to tissue ischemia and worsens the prognosis of septic patients. There are indications, that fever reduces the mortality of sepsis, the effect on tissue factor activity on monocytes is unknown. Therefore, we investigated whether heat shock modulates LPS-induced tissue factor activity in human blood.MethodsWhole blood samples and leukocyte suspensions, respectively, from healthy probands (n = 12) were incubated with LPS for 2 hours under heat shock conditions (43°C) or control conditions (37°C), respectively. Subsequent to further 3 hours of incubation at 37°C the clotting time, a measure of tissue factor expression, was determined. Cell integrity was verified by trypan blue exclusion test and FACS analysis.ResultsIncubation of whole blood samples with LPS for 5 hours at normothermia resulted in a significant shortening of clotting time from 357 ± 108 sec to 82 ± 8 sec compared to samples incubated without LPS (n = 12; p < 0.05). This LPS effect was mediated by tissue factor, as inhibition with active site-inhibited factor VIIa (ASIS) abolished the effect of LPS on clotting time. Blockade of protein synthesis using cycloheximide demonstrated that LPS exerted its procoagulatory effect via an induction of tissue factor expression. Upon heat shock treatment, the LPS effect was blunted: clotting times were 312 ± 66 s in absence of LPS and 277 ± 65 s in presence of LPS (n = 8; p > 0.05). Similarly, heat shock treatment of leukocyte suspensions abolished the LPS-induced tissue factor activity. Clotting time was 73 ± 31 s, when cells were treated with LPS (100 ng/mL) under normothermic conditions, and 301 ± 118 s, when treated with LPS (100 ng/mL) and heat shock (n = 8, p < 0.05). Control experiments excluded cell damage as a potential cause of the observed heat shock effect.ConclusionHeat shock treatment inhibits LPS-induced tissue factor activity in human whole blood samples and isolated leukocytes.

The in vivo effect of melatonin on cellular activation processes in human blood during strenuous physical exercise

Melatonin has been reported to have anti- as well as pro-inflammatory properties. Because physical stress is associated with the activation of blood cells, the present study examines melatonin's role in exercise-induced cell activation processes. Eight healthy volunteers (aged 20-62 yr, mean = 31), exercised on an 'Ergometric' bike for 30 min at 80% of their calculated maximum pulse rate. Blood samples were taken just before melatonin administration, directly after exercise, and 2 hr after exercise completion. Cytokine and eicosanoid parameters were measured in plasma from blood stimulated with lipopolysaccharide (LPS) for 2 hr whereas tissue factor (TF) activity was measured in isolated monocytes. Melatonin significantly decreased LPS-induced TF activity by 48% (P < 0.01) directly after exercise, and a 44% reduction was seen 2 hr later (P < 0.02). Furthermore, melatonin significantly reduced the lymphocyte count rise produced directly after exercising by more than 30% (P < 0.01). A trend was also seen for melatonin suppressing the increase of WBC by around 10% and to strengthen the platelet increase by about 8% after physical stress. Melatonin also significantly lowered RBC and hemoglobin counts by 5 and 3-4% during exercise (P < 0.005 and <0.02 respectively). Two hours after exercise, melatonin tended to lower leukotriene B4 levels by 30%. Interleukin-8 and tumor necrosis factor-alpha levels tended to be lower in individuals who had taken melatonin following hard physical activity and a larger sample size may show significance. Thromboxane B2 production seemed unaffected by melatonin during exercise. In conclusion, in vivo intake of melatonin appears to suppress LPS-induced activation of monocytes in whole blood reactions associated with physical exercise and facilitates the down-regulation of inflammatory mediators.

Modulation of tissue factor expression by rapamycin and FK-506 in lipopolysaccharide-stimulated human mononuclear cells and serum-stimulated aortic smooth muscle cells

Inflammation is a key pathogenic component of atherosclerosis; it also promotes thrombosis, a process underlying acute coronary events and stroke. Cells present in atherosclerotic plaque show abnormal tissue factor (TF) expression. Macrolides, in addition to their antimicrobial properties, have antiinflammatory effects that might help prevent atherothrombosis. The aim of this study was to determine the effect of an immunosuppressant macrolide, rapamycin (Sirolimus), on the expression of TF and its inhibitor (TFPI) by monocytic cells (human blood mononuclear and THP-1 cells) and human aortic smooth muscle cells, in comparison with FK-506 and azithromycin. In monocytic cells, rapamycin and FK-506 inhibited LPS-induced TF activity, antigen and mRNA expression through a transcriptional mechanism involving NF-kappaB. In smooth muscle cells, rapamycin and azithromycin had no effect on serum-induced TF expression, while FK-506 increased serum-induced TF protein and mRNA expression. TFPI levels in the culture supernatants of serum-stimulated smooth muscle cells were not modified by any of the three macrolides. Rapamycin slightly inhibits TFPI induction by LPS in monocytic cells. In addition to its recently established efficacy in the prevention of stent restenosis, the inhibitory effect of rapamycin on the TF pathway might have interesting therapeutic implications.

Effect of Wine Phenolics and Their Analogs on Suppression of Tissue Factor Induction in Mononuclear Cells.

Several epidemiologic studies suggest that moderate consumption of alcohol, particularly red wine, lowers mortality rates from coronary heart diseases. Aberrant expression of tissue factor (TF), a cellular receptor of coagulation factor VIIa, is thought to contribute to the pathogenesis of coronary heart diseases (CHD) and acute coronary events associated with atherosclerosis. Therefore we hypothesized earlier that consumption of red wine provides protection against CHD by suppressing the aberrant expression of TF in vascular cells, and showed two red wine phenolics, resveratrol and quercetin, suppressed TF induction in endothelial cells and mononuclear cells. However these studies revealed that relatively high concentrations of these compounds (10 to 100 μM), which were unlikely to be achieved in vivo after moderate consumption of red wine, were needed to suppress TF induction. The present study was undertaken to investigate whether other red wine phenolics potentiate the inhibitory effects of resveratrol or quercetin and evaluate the effect of various resveratrol analogs, which were shown to be 10 to 1000 times more effective than resveratrol in modulating other cellular effects, on TF induction in mononuclear cells. Mononuclear cells isolated from healthy human blood donors were treated with various wine phenolics alone or in a combination for 1 h and the cells were stimulated with LPS (10 ng/ml for 6 h) to induce TF expression. TF activity in cell lysates was measured as its ability to support factor VIIa catalyzed activation of factor X. Studies of treating mononuclear cells with varying concentrations of resveratrol and quercetin alone or in a combination revealed that their inhibitory effects were additive and not synergistic. Treatment of cells with a cocktail of red wine phenolics (in concentrations that present in red wine) completely suppressed LPS-induced TF activity. Exclusion resveratrol, epicatechin, catechin or rutin from the cocktail did not diminish the inhibitory effect. In contrast, exclusion of quercetin from the cocktail fully attenuated the inhibitory effect of the cocktail. Further experiments showed that quercetin (55 μM) alone fully suppressed TF induction. Among various resveratrol analogs tested, the cis-form methylated derivative of resveratrol (3,5,4′-trimethoxystilbene, TMS) was 10-times more effective than resveratrol in inhibiting TF induction. A 2 μM of TMS inhibited the induction of TF by more than 75%. None of the wine phenolics or resveratrol analogs had a significant effect on the cell viability. Overall these data suggest that quercetin is the principal inhibitor of TF induction among red wine phenolics and no synergism exists among various wine phenolics in inhibiting TF induction. Comparison of our data on the effect of resveratrol analogs on TF induction with previously published studies on other cellular functions suggests that resveratrol analogs exhibit varying potencies in modulating different cellular activities.

Tissue factor in neutrophils: no

Tissue factor in neutrophils: no

Ex-vivo regulation of endotoxin-induced tissue factor in whole blood by eicosanoids.

The influence of several eicosanoids of the lipoxygenase pathway was examined in an ex vivo system of human whole blood subjected to stimulation by lipopolysaccharide (LPS). Exogenously added leukotriene B4 [5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4)] or 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) significantly (P<0.05) enhanced LPS-evoked expression of monocyte tissue factor (TF) activity in a concentration-dependent manner. 15(S)-HETE, on the other hand, exerted such activity only when added at certain concentrations, whereas 5(S)-HETE was devoid of any apparent activity. LPS-induced TF activity was inhibited by the lipoxygenase inhibitors nordihydroguaiaretic acid, CGS 23885 and ZM 230487, by 59, 32 and 88%, respectively. Furthermore, the production of LTB4 in LPS-stimulated whole blood was investigated, in the absence or presence of either tumor necrosis factor alpha (TNFalpha) or phorbol-12-myristate-13-acetate (PMA). LPS alone induced a moderate time-dependent and concentration-dependent release of LTB4, reaching the maximum concentration (1260 +/- 202 pg/ml) within 90 min at 5 ng/ml LPS. The prior and concurrent presence of PMA (5 ng/ml) or TNFalpha (10 ng/ml) further enhanced the LTB4 production approximately twofold (P < 0.05). TNFalpha added alone evoked approximately twice the LTB4 production seen when LPS (2200 +/- 243 versus 1260 +/- 203 pg/ml) was added alone. Considering these results, LPS and TNFalpha emerge as important agonists of LTB4 production in whole blood. LTB4 in turn appears to be of importance for the expression of TF in monocytes, potentially amplifying the thrombogenic potential of these cells.

The role of platelets in decrypting monocyte tissue factor.

Although about 80% of tissue factor (TF) extracellular domain antigen present in lipopolysaccharide (LPS)-stimulated monocytes is available at the cell surface, only 10% to 20% of the total extractable TF activity is expressed on the surface of intact monocytes. Thus, most of the TF activity is latent or encrypted in the cell membrane. When coincubated, leukocytes and platelets generate more TF activity than either cell type alone. We have shown that such platelet-promoted enhancement of LPS-induced TF activity in monocytes in whole blood depends on neutrophil involvement in a P-selectin/CD15 (a leukocyte membrane-bound carbohydrate)-dependent reaction. The effect was even more pronounced when both the phorbol ester, phorbol 12-myristate 13-acetate (PMA), and LPS were present during monocyte stimulation. We currently envisage that decryption is mediated through the secretion of TF-rich particles by monocytes. These particles express CD15 and bind P-selectin exposed on either activated platelets or platelet-derived microparticles. Interactions and fusion events, that typically occur between monocytes and platelets, would facilitate the generation of monocytes/monocyte microparticle and platelets/platelet microparticle hybrids, leading to particles rich in decrypted TF activity. In conclusion, platelets play a pivotal role in decrypting TF activity of monocytes, generating a hybrid TF terrain, which both triggers and favors thrombogenesis.

The role of platelets in decrypting monocyte tissue factor

Although about 80% of tissue factor (TF) extracellular domain antigen present in lipopolysaccharide ( (LP)-stimulated monocytes is available at the cell surface, only 10% to 20% of the total extractable TF activity is expressed on the surface of intact monocytes. Thus, most of the TF activity is latent or encrypted in the cell membrane. When coincubated, leukocytes and platelets generate more TF activity than either cell type alone. We have shown that such platelet-promoted enhancement of LPS-induced TF activity in monocytes in whole blood depends on neutrophil involvement in a P-selectin/CD15 (a leukocyte membrane-bound carbohydrate)-dependent reaction. The effect was even more pronounced when both the phorbol ester, phorbol 12-myristate 13-acetate (PMA), and LPS were present during monocyte stimulation. We currently envisage that decryption is mediated through the secretion of TF-rich particles by monocytes. These particles express CD15 and bind P-selectin exposed on either activated platelets or platelet-derived microparticles. Interactions and fusion events, that typically occur between monocytes and platelets, would facilitate the generation of monocytes/monocyte microparticle and platelets/platelet microparticle hybrids, leading to particles rich in decrypted TF activity. In conclusion, platelets play a pivotal role in decrypting TF activity of monocytes, generating a hybrid TF terrain, which both triggers and favors thrombogenesis.

The role of platelets in decrypting monocyte tissue factor

Although about 80% of tissue factor (TF) extracellular domain antigen present in lipopolysaccharide (LPS)-stimulated monocytes is available at the cell surface, only 10% to 20% of the total extractable TF activity is expressed on the surface of intact monocytes. Thus, most of the TF activity is latent or encrypted in the cell membrane. When coincubated, leukocytes and platelets generate more TF activity than either cell type alone. We have shown that such platelet-promoted enhancement of LPS-induced TF activity in monocytes in whole blood depends on neutrophil involvement in a P-selectin/CD15 (a leukocyte membrane-bound carbohydrate)-dependent reaction. The effect was even more pronounced when both the phorbol ester, phorbol 12-myristate 13-acetate (PMA), and LPS were present during monocyte stimulation. We currently envisage that decryption is mediated through the secretion of TF-rich particles by monocytes. These particles express CD15 and bind P-selectin exposed on either activated platelets or platelet-derived microparticles. Interactions and fusion events, that typically occur between monocytes and platelets, would facilitate the generation of monocytes/monocyte microparticle and platelets/platelet microparticle hybrids, leading to particles rich in decrypted TF activity. In conclusion, platelets play a pivotal role in decrypting TF activity of monocytes, generating a hybrid TF terrain, which both triggers and favors thrombogenesis.

The role of platelets in decrypting monocyte tissue factor

Although about 80% of tissue factor (TF) extracellular domain antigen present in lipopolysaccharide (LPS)-stimulated monocytes is available at the cell surface, only 10% to 20% of the total extractable TF activity is expressed on the surface of intact monocytes. Thus, most of the TF activity is latent or encrypted in the cell membrane. When coincubated, leukocytes and platelets generate more TF activity than either cell type alone. We have shown that such platelet-promoted enhancement of LPS-induced TF activity in monocytes in whole blood depends on neutrophil involvement in a P-selectin/CD15 (a leukocyte membrane-bound carbohydrate)-dependent reaction. The effect was even more pronounced when both the phorbol ester, phorbol 12-myrlstate 13-acetate (PMA), and LPS were present during monocyte stimulation. We currently envisage that decryption is mediated through the secretion of TF-rich particles by monocytes. These particles express CD15 and bind P-selectin exposed on either activated platelets or platelet-derived microparticles. Interactions and fusion events, that typically occur between monocytes and platelets, would facilitate the generation of monocytes/monocyte microparticle and platelets/platelet microparticle hybrids, leading to particles rich in decrypted TF activity. In conclusion, platelets play a pivotal role in decrypting TF activity of monocytes, generating a hybrid TF terrain, which both triggers and favors thrombogenesis.

Induction of Tissue Factor Expression in Whole Blood: Lack of Evidence for the Presence of Tissue Factor Expression in Granulocytes

The present investigation was undertaken to explore the effect of platelets, tumor necrosis factor (TNF) and phorbel ester [phorbol 12-myristate 13-acetate (PMA)] on lipopolysaccharide (LPS)-induced tissue factor (TF) activity and TF antigen by using Western blot and ELISA-techniques. LPS was found to induce correlating levels of TF antigen and the activity in monocytes. TNF and PMA, when used alone, failed to induce TF activity and the antigen in monocytes, but enhanced the LPS-induced TF activity and the antigen by 2 to 3-fold. Addition of platelet rich plasma to isolated blood cells enhanced the LPS-induced TF activity but not the antigen levels in monocytes. In contrast to whole platelets, platelet lysates enhanced both LPS-induced TF activity and the antigen. Granulocytes isolated from heparinized plasma incubated for 2 or 24 h with LPS alone or together with PMA, failed to generate TF antigen or the activity. Although granulocyte preparations isolated from whole blood that was incubated for 24 h with LPS and PMA apparently possessed a significant amount of TF activity and the antigen, this could be accounted for by trace levels of contaminating monocytes. Upregulation of LPS-induced TF activity but not the antigen by platelets in the presence of granulocytes suggests that the increased TF activity could be the result of PS enrichment of monocytes by fusion or platelets with activated monocytes.

TNF 41-62 and TNF 78-96 Have Distinct Effects on LPS-induced Tissue Factor Activity and the Production of Cytokines in Human Blood Cells

Biological activities of peptides representing two different regions in the TNF molecule were investigated. We have earlier reported that one of the peptides studied, TNF 36-62, induced chemotaxis in granulocytes and monocytes. TNF 41-62, a shorter analog of TNF 36-62, possessed similar chemotactic effects. Both peptides caused a weak enhancement of LPS -induced IL-6 production and tissue factor activity by monocytes in whole blood. The third peptide studied, TNF 78-96, was selected from a region located on the opposite side of the beta-sheet sandwich structure of the TNF molecule, and includes the loop 84-88 that has been shown to be involved in TNF receptor interaction. TNF 78-96 possessed properties quite different from TNF 36-62 and TNF 41-62. It amplified several fold PMA-induced secretion of elastase, and enhanced significantly PMA-induced secretion of cathepsin G from the neutrophils, activities which were effectively abolished by an anti-human TNF antibody. The TNF 78-96 peptide also inhibited LPS-induced TF activity in monocytes of whole blood, and it abolished the TNF enhancing effect of LPS-induced TF activity in a dose dependent manner. This suggests that the TNF 78-96 peptide may bind to the TNF receptor(s), without potentiating the same signals as native TNF. It may thereby prevent binding of the native TNF and the resultant activation effect of TNF. It also, at high concentrations, inhibited LPS-induced IL-6 production whereas it caused a doubling of LPS-induced IL-8 in monocytes and granulocytes in whole blood. These results clearly show that distinct TNF activities can be induced by peptide sequences taken from different regions of TNF. The TNF 78-96 peptide might be useful in downregulation of LPS-induced monocyte activations in vivo.

I. Suppression by compound 48/80 of bacterial endotoxin-inducible monocytic tissue factor activity: direct blockade of factor VII binding to THP-1 monocytes

Hypercoagulation with upregulated monocytic tissue factor (TF) activity often occurs under a variety of inflammatory conditions including endotoxemia. The antagonism to bacterial endotoxin (LPS) signaling often results in the depression in TF upregulation. We herein report that compound 48/80 (48/80) significantly depressed LPS-induced TF activity in human and cebus monkey peripheral blood monocytes. Employing a model monocyte-like cell line (THP-1), we explored the regulatory mechanism to identify the inhibitory site(s) of 48/80. We determine whether the inhibition results from the blockade of LPS signaling. 48/80 dose-dependently inhibited LPS-induced TF activity. Chase of LPS-challenged cells with 48/80 also significantly offset TF upregulation. In immunofluorescent approaches, FACScan analysis revealed that 48/80 had no effect on either LPS recognition or the expression of its receptors (CD14 and CD11b). Moreover, LPS-induced TF expression as well as synthesis remained unaffected in the presence of 48/80. Consistent with the independence of LPS action, 48/80 was also able to inhibit TF activity induced by A23187, ionomycin, or Quin-2 AM. Interestingly, 48/80 significantly decreased the FVII binding to either resting or LPS-challenged cells. In conclusion, our results elucidate that the inhibitory action of 48/80 was independent of LPS signaling including recognition, receptor expression, and the induced TF expression/synthesis. However, 48/80 was able to directly block FVII binding to monocytic TF, thereby resulting in such antagonism to LPS-induced TF-initiated extrinsic coagulation.