The key roles of complement and tissue factor in Escherichia coli-induced coagulation in human whole blood.

The complement system and the Toll-like (TLR) co-receptor CD14 play important roles in innate immunity and sepsis. Tissue factor (TF) is a key initiating component in intravascular coagulation in sepsis, and long pentraxin 3 (PTX3) enhances the lipopolysaccharide (LPS)-induced transcription of TF. The aim of this study was to study the mechanism by which complement and CD14 affects LPS- and Escherichia coli (E. coli)-induced coagulation in human blood. Fresh whole blood was anti-coagulated with lepirudin, and incubated with ultra-purified LPS (100 ng/ml) or with E. coli (1 × 10(7) /ml). Inhibitors and controls included the C3 blocking peptide compstatin, an anti-CD14 F(ab')2 antibody and a control F(ab')2 . TF mRNA was measured using quantitative polymerase chain reaction (qPCR) and monocyte TF surface expression by flow cytometry. TF functional activity in plasma microparticles was measured using an amidolytic assay. Prothrombin fragment F 1+2 (PTF1.2) and PTX3 were measured by enzyme-linked immunosorbent assay (ELISA). The effect of TF was examined using an anti-TF blocking antibody. E. coli increased plasma PTF1.2 and PTX3 levels markedly. This increase was reduced by 84->99% with compstatin, 55-97% with anti-CD14 and > 99% with combined inhibition (P < 0·05 for all). The combined inhibition was significantly (P < 0·05) more efficient than compstatin and anti-CD14 alone. The LPS- and E. coli-induced TF mRNA levels, monocyte TF surface expression and TF functional activity were reduced by > 99% (P < 0·05) with combined C3 and CD14 inhibition. LPS- and E. coli-induced PTF1.2 was reduced by 76-81% (P < 0·05) with anti-TF antibody. LPS and E. coli activated the coagulation system by a complement- and CD14-dependent up-regulation of TF, leading subsequently to prothrombin activation.

Circadian rhythms of hemostatic factors in tetraplegia: a double-blind, randomized, placebo-controlled cross-over study of melatonin.

STUDY DESIGN: This is a double-blind, randomized, placebo-controlled cross-over study of melatonin in complete tetraplegia. OBJECTIVES: Tetraplegic patients have an increased risk of venous thrombosis despite prophylaxis, blunted variations in melatonin and altered circadian variation of several hemostatic markers. To examine whether melatonin could modify the regulation of hemostasis, we measured plasma melatonin and several markers of hemostasis in tetraplegic subjects with or without melatonin supplement. SETTING: The study was conducted in the Section for Spinal Cord Injury, Sunnaas Hospital, Nesoddtangen, Norway. METHODS: Six subjects with long-standing complete tetraplegia were included in this cross-over study with 2 mg of melatonin or placebo given 4 days before sampling. We also included six able-bodied men without any intervention. Plasma samples were then collected frequently during a 24-h awake/sleep cycle. The plasma concentrations of melatonin and the various markers were analyzed using linear mixed models. RESULTS: The 24-h profiles of prothrombin fragment 1+2 and von Willebrand factor, but not D-dimer, activated FVII, tissue factor pathway inhibitor and plasminogen activator inhibitor type 1, differed (P<0.05) between tetraplegic patients and able-bodied subjects. The absolute plasma concentration of activated FVII was higher (P<0.05) among the able-bodied compared with the tetraplegic groups. Supplementation of melatonin had no impact on these findings. CONCLUSIONS: We found differences in circadian variation of several hemostatic markers between able-bodied and tetraplegics. These differences were apparently unrelated to fluctuations in the melatonin concentrations, suggesting little or no role of melatonin in the regulation of hemostasis in tetraplegia. SPONSORSHIP: Financial support was provided from the Throne Holst Foundation.

C1-inhibitor efficiently inhibits Escherichia coli-induced tissue factor mRNA up-regulation, monocyte tissue factor expression and coagulation activation in human whole blood.

Both the complement system and tissue factor (TF), a key initiating component of coagulation, are activated in sepsis, and cross-talk occurs between the complement and coagulation systems. C1-inhibitor (C1-INH) can act as a regulator in both systems. Our aim in this study was to examine this cross-talk by investigating the effects of C1-INH on Escherichia coli-induced haemostasis and inflammation. Fresh human whole blood collected in lepirudin was incubated with E. coli or ultrapurified E. coli lipopolysaccharide (LPS) in the absence or presence of C1-INH or protease-inactivated C1-INH. C3 activation was blocked by compstatin, a specific C3 convertase inhibitor. TF mRNA was measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and TF surface expression was measured by flow cytometry. In plasma, the terminal complement complex, prothrombin F1·2 (PTF1·2) and long pentraxin 3 (PTX3) were measured by enzyme-linked immunosorbent assay (ELISA). Cytokines were analysed using a multiplex kit. C1-INH (1·25-5 mg/ml) reduced both LPS- and E. coli-induced coagulation, measured as a reduction of PTF1·2 in plasma, efficiently and dose-dependently (P < 0·05). Both LPS and E. coli induced marked up-regulation of TF mRNA levels and surface expression on whole blood monocytes. This up-regulation was reduced efficiently by treatment with C1-INH (P < 0·05). C1-INH reduced the release of PTX3 (P < 0·05) and virtually all cytokines measured (P < 0·05). Complement activation was inhibited more efficiently with compstatin than with C1-INH. C1-INH inhibited most of the other readouts more efficiently, consistent with additional non-complement-dependent effects. These results indicate that complement plays a role in activating coagulation during sepsis and that C1-INH is a broad-spectrum attenuator of the inflammatory and haemostatic responses.

The effects of selective complement and CD14 inhibition on the E. coli-induced tissue factor mRNA upregulation, monocyte tissue factor expression, and tissue factor functional activity in human whole blood.

BACKGROUND: The complement pathway and CD14 play essential roles in inflammation, but little is known about the relative roles of complement and CD14 in E. coli-induced tissue factor (TF) mRNA upregulation, expression by monocytes, and functional activity in human whole blood. METHODS: Whole E. coli bacteria were incubated for up to 4 h in human whole blood containing the anticoagulant lepirudin, which does not affect complement activation. TF mRNA levels were analyzed using reverse transcription, quantitative real-time PCR (RT-qPCR), and the expression of TF on the cell surface was analyzed using flow cytometry. Complement was selectively inhibited using the C3 convertase inhibitor compstatin or a C5a receptor antagonist (C5aRa), while CD14 was blocked by an anti-CD14 F(ab')2 monoclonal antibody. RESULTS: The E. coli-induced TF mRNA upregulation was reduced to virtually background levels by compstatin, whereas anti-CD14 had no effect. Monocyte TF expression and TF activity in plasma microparticles were significantly reduced by C5aRa. Anti-CD14 alone only slightly reduced E. coli-induced monocyte TF expression but showed a modest additive effect when combined with the complement inhibitors. Inhibiting complement and CD14 efficiently reduced the expression of the E. coli-induced cytokines IL-1beta, IL-6, IL-8, and platelet-derived growth factor bb. CONCLUSION: Our results indicate that E. coli-induced TF mRNA upregulation is mainly dependent on complement activation, while CDI4 plays a modest role in monocyte TF expression and the plasma TF activity in human whole blood.

Activation of human factor VII in plasma and in purified systems: roles of activated factor IX, kallikrein, and activated factor XII.

Factor VII can be activated, to a molecule giving shorter clotting times with tissue factor, by incubating plasma with kaolin or by clotting plasma. The mechanisms of activation differ. With kaolin, activated Factor XII (XII(a)) was the apparent principal activator. Thus, Factor VII was not activated in Factor XII-deficient plasma, was partially activated in prekallikrein and high-molecular weight kininogen (HMW kininogen)-deficient plasmas, but was activated in other deficient plasmas. After clotting, activated Factor IX (IX(a)) was the apparent principal activator. Thus, Factor VII was not activated in Factor XII-,HMW kininogen-, XI-, and IX-deficient plasmas, but was activated in Factor VIII-, X-, and V-deficient plasmas. In further studies, purified small-fragment Factor XII(a) (beta-XII(a)), kallikrein, and Factor IX(a) were added to partially purified Factor VII and to plasma. High concentrations of beta-XII(a) activated Factor VII in a purified system; much lower concentrations of beta-XII(a) activated Factor VII in normal plasma but not in prekallikrein or HWM kininogen-deficient plasmas. Kallikrein alone failed to activate partially purified Factor VII but did so when purified Factor IX was added. Kallikrein also activated Factor VII in normal, Factor XII-, and Factor IX-deficient plasmas. Purified Factor IX(a) activated partially purified Factor VII and had no additional indirect activating effect in the presence of plasma. These results demonstrate that both Factor XII(a) and Factor IX(a) directly activate human Factor VII, whereas kallikrein, through generation of Factor XII(a) and Factor IX(a), functions as an indirect activator of Factor VII.

High tissue factor-expressing human monocytes carry low surface CD36: application to intersubject variability.

BACKGROUND: The 'high and low responder' phenomenon describes an intersubject variability in mononuclear cell (MNC) prothrombotic reactivity to lipopolysaccharide (LPS) stimulation. Because alterations in surface CD36 expression in monocytes were associated with impaired monocyte function, we studied the relationship between the levels of surface CD36 presentation and the prothrombotic reactivity of monocytes from high-responder (HR) and low-responder (LR) individuals. METHODS AND RESULTS: The relationship between levels of tissue factor (TF) expression and surface CD36 presentation in MNCs from HR individuals (n = 7) and LR individuals (n = 8) was investigated. Resting MNCs from HR individuals contained significantly more TF mRNA but levels of TF antigen and procoagulant activity similar to MNCs from LR individuals. Resting CD14+ MNCs from HR individuals expressed significantly lower surface CD36, as mean fluorescence intensities (MFIs) were 70.4 +/- 6.3 vs. 132.0 +/- 14.5 arbitrary units (AU) in HR and LR individuals, respectively. MFI from surface TF negatively correlated with surface CD36 in the population of resting (r = -0.598, P = 0.031) and LPS-stimulated (r = -0.672, P = 0.009) CD14+ cells. LPS-stimulated MNCs from HR individuals contained significantly more TF in a surface pool (2079 +/- 199 vs. 786 +/- 57 AU) along with higher TF procoagulant activity (57.3 +/- 15.2 vs. 21.1 +/- 4.5 mU 10(6) cells(-1)) as compared with LR individuals. CD14+ MNCs from HR individuals expressed less surface CD36 during a 2-h LPS challenge. CONCLUSIONS: A novel phenotype of monocytes characterized by high TF and low CD36 presentation could be further developed for use as a marker for detection of HR individuals prone to developing prothrombotic conditions.

In-cell Western assay: a new approach to visualize tissue factor in human monocytes.

BACKGROUND: Tissue factor (TF) is an integral membrane protein essential for the initiation of the extrinsic pathway of hemostasis. A precise understanding of the TF regulation is still limited and dependent on the availability of methodological tools. Here, we describe a new approach for assessing TF amounts in human mononuclear cells (MNCs) by using the whole blood experimental conditions. AIM: In order to study TF antigen levels in human MNCs, we applied a quantitative immunostaining technique-- in-cell Western (ICW) assay using an Odyssey Infrared Imaging System. METHODS AND RESULTS: The ICW assay of TF in resting or lipopolysaccharide (LPS)-stimulated human MNCs was performed. Several sample preparation conditions were tested, namely the plating of MNCs prior to immunostaining, paraformaldehyde fixation, and an adequate cell number was used in the assay. By the use of recombinant human TF standards, it was possible, for the first time, to measure TF amounts in LPS-stimulated MNCs as 0.09 +/- 0.02 ng and 0.43 +/- 0.15 ng 10(-6) cells of surface and total TF, respectively. The concentrations of TF in resting MNCs, however, were below the detection limit. CONCLUSIONS: A novel TF ICW assay is a reproducible, time- and cost-saving method, which could become useful for studies in the fields of physiology and pathophysiology of human hemostasis.

Platelet activating factor enhancement of lipopolysaccharide-induced tissue factor activity in monocytes: requirement of platelets and granulocytes.

Incubation of platelet activating factor (PAF) with heparinized blood caused no induction of tissue factor activity in monocytes. However, when PAF was added in addition to weak lipopolysaccharide (LPS), it amplified the LPS effect by 80%. By using separated fresh cell populations resuspended in plasma, PAF was shown to have no enhancing effect when added to mononuclear cells incubated with platelet-rich plasma in the presence of LPS. In contrast, when granulocytes also were included, PAF caused an almost 3-fold increase in LPS-induced tissue factor activity. A PAF antagonist blocked this effect and also reduced LPS-induced tissue factor activity of monocytes in whole blood in a dose-dependent manner. In the recombined cell incubation system, the maximal inhibition by the antagonist was observed in the presence of granulocytes. These data provide evidence for an effect of PAF on granulocytes that probably generates a product that, together with platelets, enhances LPS-induced tissue factor activity in monocytes.

Granulocytes enhance LPS-induced tissue factor activity in monocytes via an interaction with platelets.

In the present study we have investigated the effect of platelets and granulocytes on bacterial lipopolysaccharide (LPS)-induced tissue factor (TF) activity in monocytes. Experiments were performed on freshly isolated cells resuspended in heparinized plasma and recombined with platelet-poor or platelet-rich plasma. In a platelet-dependent reaction the granulocytes enhanced LPS-induced TF activity by an average of 100%. The effect was dose dependent with regard to the number of both granulocytes and platelets, respectively. Granulocytes and/or platelets did not affect LPS-induced tumor necrosis factor (TNF) secretion from monocytes. Phorbol myristate acetate (PMA) per se was not able to induce TF activity in our system. In contrast, the agonist caused a substantial increase in TF activity induced by LPS. The effect was totally dependent on the presence of platelets and was shown to be due to stimulation of both granulocytes and monocytes (the activity rose from 30 +/- 7 to 83 +/- 12 mU/10(6) cells in the presence of platelets and from 69 +/- 8 to 143 +/- 22 mU/10(6) cells in the presence of platelets and granulocytes). Effects similar to those observed with PMA were obtained with physiological concentrations (10 ng/ml) of TNF. A combination of these two agonists gave no further amplification of LPS-induced TF activity compared with the effect of the agonists separately. Low concentrations of a monoclonal anti-CD15 antibody abolished the stimulatory effects of platelets and granulocytes. Furthermore, the anti-CD15 antibody neutralized the effect of TNF, whereas the PMA effect was reduced by almost 75%. These results were confirmed in a whole-blood system. The inhibitory effect of the antibody may be associated with CD15's role as a complementary ligand for PADGEM. Our study indicates that a close interaction between granulocytes, platelets, and monocytes is essential for optimal TF activity induced by LPS. It is hypothesized that the effect of granulocytes is related to their ability to activate platelets. We propose that upon activation granulocytes secrete a product that enhances the capacity of platelets to stimulate TF activity in monocytes.

A novel biological effect of platelet factor 4 (PF4): enhancement of LPS-induced tissue factor activity in monocytes.

In a previous study we have shown that granulocytes enhance lipopolysaccharide (LPS)-induced tissue factor (TF) activity in monocytes in a platelet-dependent reaction. The present investigation was undertaken to examine the role of a platelet activation product, platelet factor 4 (PF4), in LPS-induced TF activity in monocytes. Platelet lysate supernatant, purified PF4, and the COOH-terminal tridecapeptide of PF4, termed PF4(58-70), enhanced LPS-induced TF activity in monocytes of whole blood dose dependently. A monoclonal antibody against P-selectin eliminated the enhancing effect of PF4(58-70) on LPS-induced TF activity in monocytes, and PF4(58-70) was shown to act synergistically with tumor necrosis factor alpha (TNF-alpha). However, PF4(58-70) did not enhance TNF-alpha secretion in LPS-stimulated whole blood. The major effect of PF4(58-70) was granulocyte dependent. Our results suggest that PF4 might play an important role in LPS-stimulated monocyte TF activity of whole blood.

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

BACKGROUND: We aimed to evaluate the mechanisms underlying the effects of red blood cells (RBCs) on the reactivity of monocytes to lipopolysaccharide (LPS) stimulation. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Alterations in markers of coagulation and fibrinolysis in patients with Paroxysmal Nocturnal Hemoglobinuria before and during treatment with eculizumab.

BACKGROUND: Paroxysmal Nocturnal Hemoglobinuria is characterized by complement-mediated hemolysis and an increased thrombosis risk. Eculizumab, an antibody to complement factor C5, reduces thrombotic risk via unknown mechanisms. Clinical observations suggest that eculizumab has an immediate effect. OBJECTIVES: A better understanding of the mechanism via which eculizumab reduces thrombotic risk by studying its pharmacodynamic effect on coagulation and fibrinolysis. METHODS: We measured microparticles (MP), tissue factor (TF) activity, prothrombin fragment 1+2 (F1+2), D-dimer and simultaneously thrombin and plasmin generation in 55 PNH patients. In 20 patients, parameters were compared before and during eculizumab treatment (at 1 and 2hours, 1, 4 and≥12weeks after commencement). RESULTS: Patients with a history of thrombosis had elevated D-dimers (p=0.02) but not MP. Among patients on anticoagulants, those with thrombosis had higher F1+2 concentrations (p=0.003). TF activity was undetectable in plasma MP. Unexpectedly, thrombin peak height and thrombin potential were significantly lower in PNH patients than in healthy controls. Fibrinolysis parameters were normal. During eculizumab treatment D-dimer levels significantly decreased after 1hour (p=0.008) and remained decreased at≥12weeks (p=0.03). F1+2 (p=0.03) and thrombin peak height (p=0.02) in patients not on anticoagulants significantly decreased at≥week 12. MP remained unchanged. CONCLUSIONS: Eculizumab induces an immediate decrease of D-dimer levels but not of other markers. The decrease in thrombin peak height and F1+2 suggests that eculizumab reduces thrombin generation. Elevated D-dimer levels in untreated PNH patients with a history of thrombosis suggest possible value in predicting thrombotic risk.

Evidence for exclusive role of the p55 tumor necrosis factor (TNF) receptor in mediating the TNF-induced collagenase expression by human dermal fibroblasts.

The aim of this study was to examine the roles of the TNF receptors p55 and p75 in the TNF-enhanced expression of collagenase by human dermal fibroblasts. The agonistic p55 monoclonal antibody Htr9 and TNF induced production of similar amounts of collagenase. Polyclonal or monoclonal agonistic p75 antibodies failed to enhance collagenase production, and the antagonistic p75 antibody 5E12 did not inhibit TNF-enhanced expression of collagenase. This strongly suggests that p55, but not p75, is involved in TNF-induced production of collagenase. Cells continued to produce an elevated level of collagenase after the removal of TNF or Htr9. These data suggest that it may be useful to use specific inhibitors of collagenase rather than to block cytokine action directly in the treatment of diseases with chronic enhanced collagenolytic activity. A peptide of residues 36-62 of TNF previously reported to be chemotactic to leukocytes was also able to enhance the expression of collagenase activity by dermal fibroblasts. Thus, design of peptides with specific TNF effects may offer a novel approach for treatment of fibrotic disorders.

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 mandatory role of complement in the endotoxin-induced synthesis of tissue thromboplastin in blood monocytes.

Fresh isolated blood cells recombined with normal heparinized plasma and then incubated with endotoxin, induced a 100-fold increase in monocyte tissue thromboplastin synthesis. In contrast, recombination of these cells with heat inactivated plasma, cobra venom factor-treated plasma, Ca2+-free plasma, or BioRex 70-treated plasma (plasma free of Clq and D) before incubation with endotoxin, failed to induce monocyte synthesis of tissue thromboplastin. These results strongly support the hypothesis that complement is required for endotoxin stimulation of blood monocyte synthesis of tissue thromboplastin.

The promoting effect of epinephrine on lipopolysaccharide-induced interleukin-8 production in whole blood may be mediated by thromboxane A2.

Epinephrine is known to enhance lipopolysaccharide (LPS)-induced interleukin (IL)-8 secretion in a platelet dependent manner. To determine whether thromboxane A2 (TxA2; a product from activated platelets) is involved in this process, blood samples drawn either before or 2 h after oral administration of 440 mg acetylsalicylic acid (ASA) were stimulated with LPS (5 ng mL(-1)) and different concentrations of epinephrine were added (0.1-100.0 micromol L(-1)). ASA ingestion significantly (global P < 0.05) reduced the enhancing effect of epinephrine on LPS-induced IL-8 release by 15-28%. To further explore whether TxA2 may be involved in this process, a TxA2 agonist (U46619) was added to whole blood together with LPS instead of epinephrine. U46619 mimicked the epinephrine effect: 20 ng mL(-1) U46619 enhanced LPS-induced IL-8 release by 39% (P < 0.05). Furthermore, preincubation of whole blood with 75 micro mol L-1 or 150 micromol L(-1) SQ29548, a TxA2 receptor antagonist, completely blocked epinephrine's promoting effect on LPS-induced IL-8 release. Since thrombin-activated platelets have been reported to be important in the production of IL-8 in monocytes through the activation of monocytes by exposed RANTES in a P-selectin-dependent reaction, we suggest that the epinephrine effect is mediated by enhanced TxA2 production and subsequent rise in the exposure of RANTES and P-selectin on the platelets of whole blood.

Thromboxane production in the blood of women increases after menopause whereas tumor necrosis factor is reduced in women compared with men.

Previously we have shown that incubation of heparinized blood with a low dose of lipopolysaccharides (5 ng/ml) resulted in a 60% higher generation of TxB2 in the blood of young men as compared with that of young women. In the present study, we investigated a group consisting of 38 healthy men and 38 healthy postmenopausal women aged 50-73 years with no drug use and no known chronic disease. In contrast to our earlier observation that young men produce more TxB2 than young women, no significant difference was observed between the men and women when all the participants above 50 years of age were included (5.7 +/- 0.6 ng/l for men versus 5.2 +/- 0.7 ng/l for women). However, a strong correlation was found with simple regression analysis when increasing TxB2 generation was compared with years after menopause (P < 0.0001). No such correlation was observed for increasing age of men and their TxB2 production. The LPS stimulation system of whole blood was also used to evaluate the production of tumor necrosis factor (TNF-alpha) in older people. Men were found to generate 60% more TNF-alpha than women, but no correlation was found between increasing age of women and TNF-alpha production as observed with TxB2. Risk factors such as SDS-cholesterol, fibrinogen and factor VII were the same in men and women, whereas total cholesterol was higher in women than in men (P < 0.05). Since TxA2 is known to be a mediator of atherosclerotic-induced lesions and TNF-alpha is a well-established indicator of inflammatory reactions, we propose that the reduced production of TxB2 and TNF-alpha in women in our model system may partially explain the lower incidence of atherosclerosis in women as compared with men, and the phenomenon of increased incidence of this disease after menopause.

Increased tissue thromboplastin activity in monocytes of patients with meningococcal infection: related to an unfavourable prognosis.

In 16 patients, 13 with meningococcal infection and 3 suspected to have this infection, 8 patients were found to possess significant higher level of tissue thromboplastin activity of their monocytes isolated from the blood at the admission to the hospital than normal. Five of those 8 patients had an extremely high concentration, greater than 60-300 fold increase, and all these patients died. The exposed tissue thromboplastin activity on the surface of the endotoxin stimulated monocytes is probably the direct inducer of disseminated intravascular coagulation (DIC) in meningococcal infection.

Physical exercise enhances plasma levels of extrinsic pathway inhibitor (EPI).

The risk of cardiovascular complications is reported to be several times higher in severe exercise than in daily activity. EPI-mediated inhibition of factor VIIa/thromboplastin enzymatic activity is believed to be an important modulator of blood coagulation during hemostasis. The plasma concentration of EPI, corrected for changes in plasma volume, was determined in healthy male subjects prior to, immediately after and at various time intervals after strenuous exercise of different duration. A slight, but significant decrease in EPI (7.5 +/- 2.8%, p less than 0.03) was found after a short term run (1.7 km), whereas no significant change was seen after a middle term run (4.8 km). In contrast, we observed a marked increase in EPI after a long term run (20.3 +/- 6.9%, p less than 0.03) and in a second group of athletes participating in the Norwegian championship of 30 km cross country skiing (39.9 +/- 10.3%, p less than 0.02). A peak value was reached 2 h after the run, and after that the curve started to approach baseline values. The rise in EPI might reflect a significant release of EPI from the endothelium that is greater than eventually any consumption. Another explanation for this enhancement in EPI might be that the increase in lipoprotein lipase activity after physical exercise causes a rise in the availability of EPI since EPI is known to be associated with lipoproteins in the circulation. It is hypothesized that mobilization of EPI during extensive physical exercise may suppress activation of the clotting system.

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.