Jan Ole Olsen

Intracellular and Surface Distribution of Monocyte Tissue Factor: Application to Intersubject Variability

Objective—The high and low responder phenomenon describes individual differences in lipopolysaccharide (LPS)-induced monocyte tissue factor (TF) activity. We characterized patterns of intracellular accumulation, externalization, and shedding of TF in response to LPS in mononuclear cells (MNCs) from high responders (HRs) and low responders (LRs). Methods and Results—After 2 hours of LPS stimulation of whole blood, flow cytometry analyses revealed a larger population of TF-positive monocytes in HRs (32.0±3.5%) versus LRs (11.2±1.2%; P≤0.05), along with a stronger mean fluorescence intensity of TF signal in HRs (7.1±0.5 arbitrary units [AU]) compared with LRs (5.4±0.4 AU; P≤0.05). The LPS-treated blood of the HR group contained 2-fold more TF-positive microparticles than LRs. In-cell Western assay demonstrated higher intracellular accumulation of TF in mononuclear cells (MNCs) from LRs because LPS induced a 3.7-fold increase of total TF levels in LRs versus a 1.5-fold increase in HRs. In contrast, in response to LPS stimulation, MNCs from HRs exhibited a 4-fold induction of surface TF, whereas MNCs from LRs only had a minor increase in surface TF levels. Conclusions—The higher availability of surface TF antigen on MNCs from HRs and TF-containing microparticles might make these individuals more susceptible to hypercoagulation.

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 lipopoly‐ saccharide (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/106 cells in the presence of platelets and from 69 ± 8 to 143 ± 22 mU/106 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‐GDI 5 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 CD15s 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.

Enhanced Incorporation of n-3 Fatty Acids from Fish Compared with Fish Oils

This work was undertaken to study the impact of the source of n−3 FA on their incorporation in serum, on blood lipid composition, and on cellular activation. A clinical trial comprising 71 volunteers, divided into five groups, was performed. Three groups were given 400 g smoked salmon (n=14), cooked salmon (n=15), or cooked cod (n=13) per week for 8 wk. A fourth group was given 15 mL/d of cod liver oil (CLO) (n=15), and a fifth group served as control (n=14) without supplementation. The serum content of EPA and DHA before and after intervention revealed a higher rise in EPA and DHA in the cooked salmon group (129% rise in EPA and 45% rise in DHA) as compared with CLO (106 and 25%, respectively) despite an intake of EPA and DHA in the CLO group of 3.0 g/d compared with 1.2 g/d in the cooked salmon group. No significant changes were observed in blood lipids, fibrinogen, fibrinolysis, or lipopolysaccharide (LPS)-induced tissue factor (TF) activity, tumor necrosis factor-α (TNFα), interleukin-8 (IL-8), leukotriene B4 (LTB4), and thromboxane B2 (TxB2) in whole blood. EPA and DHA were negatively correlated with LPS-induced TNFα, IL-8, LTB4, TxB2, and TF in whole blood. In conclusion, fish consumption is more effective in increasing serum EPA and DHA than supplementing the diet with fish oil. Since the n−3 FA are predominantly in TAG in fish as well as CLO, it is suggested that the larger uptake from fish than CLO is due to differences in physiochemical structure of the lipids.

Seafood diets: hypolipidemic and antiatherogenic effects of taurine and n-3 fatty acids.

BACKGROUND Health aspects of seafood have primarily been linked to n-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Although animal studies have suggested beneficial contributions from taurine, highly abundant in seafood, its effect in humans is obscure. This study evaluates the combined effects of n-3 PUFA and taurine. METHODS Healthy volunteers (n=80) were recruited to a 7-week double-blind and parallel intervention trial. One group (n=39) received fish pâté (36g/day) enriched in n-3 (1.1gEPA+DHA/day) and the second (n=41) an identical pâté enriched both in n-3 and taurine (425mg/day). RESULTS Total cholesterol (TC) (-5%, P<0.001), low-density lipoprotein (LDL)-cholesterol (-8%, P<0.001) and Apo B (-4%, P<0.001) decreased more in the n-3+taurine compared to the n-3 group. A significant within-group enhancement of high-density lipoprotein (HDL)-cholesterol was demonstrated in the n-3+taurine group (6%, P<0.0001). Reductions in triacylglycerol (TG) (-16%, P<0.05 in n-3; -14%, P<0.05 in n-3+taurine), thromboxane B(2) (TxB(2)) (-21%, P<0.001 in n-3; -15%, P<0.05 in n-3+taurine), tumor necrosis factor (TNFalpha) (-24%, P<0.001 in n-3; -12%, P<0.05 in n-3+taurine) and monocyte chemotactic protein (MCP-1) (-12%, P<0.05 in n-3; -6%, P<0.0001 in n-3+taurine) were evident in both groups. Reductions in interleukin (IL)-6 (-16%, P<0.05) and LTB(4) (-18%, P<0.05) were only significant in the n-3 group. CONCLUSIONS The effects, particularly on blood lipids, of combining n-3 PUFAs and taurine proved superior to those of n-3 alone.

EFFECT OF MARINE OILS SUPPLEMENTATION ON COAGULATION AND CELLULAR ACTIVATION IN WHOLE BLOOD

A study was performed to explore the effects of supplemental intake of various marine oils known to be part of the Eskimo diet. Healthy men and women (134) were randomly selected to consume 15 mL/d of oil from blubber of seal, cod liver, seal/cod liver, blubber of Minke whale, or no oil for ten weeks. Total cholesterol was unchanged in the oil groups, whereas high density lipoprotein cholesterol increased 7% in the seal/cod liver oil (CLO) group (P<0.05) and 11% in the whale oil group (P<0.005). Triacylglycerol was significantly reduced in the CLO group only. The concentration of prothrombin fragment 1+2 was reduced 25% (P<0.05) after whale oil supplementation. No change in fibrinogen or factor VIIc was detected. Tumor necrosis factor generation in lipopolysaccharide (LPS)-stimulated blood was 30% reduced after whale oil (P<0.05), but was unaffected by intake of seal or CLO. The LPS-induced tissue factor activity in monocytes was reduced to a significant degree only in the seal/CLO group (34%) and whale oil group (35%) (P<0.05). The most dramatic change in thromboxane B2 in LPS-stimulated blood was seen after whale oil intake with 44% reduction (P<0.01). Supplementation of a regular diet with a combination of seal oil and CLO and especially with whale oil seems to have beneficial effects on several products thought to be associated with cardiovascular and thrombotic diseases.

Human platelets do not express tissue factor.

BACKGROUND The controversy about the expression of tissue factor (TF) in platelet after de novo synthesis prevail despite many groups recognize that platelet isolation, assays and reagents, particularly non-specific antibodies, may account for the diversity. In this study the potential of TF expression was evaluated using immune-purified human platelets and employing a very sensitive and highly specific TF activity assay. METHODS Isolated platelets in plasma anti-coagulated with Fragmin were subjected to stimulation by LPS plus PMA, IgG antibody or TRAP and tested for TF activity. RESULTS Platelets stimulated with LPS plus PMA for 4 hours expressed trace amounts of TF like activity (PCA), not inhibited by anti-TF antibody (0.2±0.1 mU/ml blood). Platelets, not immune-adsorbed to remove monocytes, showed significant TF activity (2.0±0.9 mU/ml blood) that was nearly abolished by anti-TF antibody. IgG antibody from patient with lupus anticoagulant failed to enhance the trace amount of PCA as compared to the control in contrast to high TF activity induced in monocytes (0.4±0.1 mU/ml blood versus 27.5±10.5 mU/10(6) cells) showing that activation of complement is not mediating TF expression. Platelet subjected to TRAP activation for 10 min possessed only trace amounts of PCA that was not inhibited by anti-TF antibody and slightly enhanced by anti-TFPI antibody. CONCLUSIONS It is concluded that platelets free of monocytes do not express TF activity when stimulated by LPS or activated complement factors, implying no role for Toll like receptor (TLR4) as suggested recently. There is no evidence of TF activity associated with platelets as a result of rapid and dynamic process.

Differential ability of tissue factor antibody clones on detection of tissue factor in blood cells and microparticles.

INTRODUCTION Tissue factor (TF), the primary initiator of coagulation in vivo, plays a major role in both thrombosis and hemostasis. The expression of TF in monocytes is well documented, but its presence in other blood cells has been disputed, possibly due to methodological variations among different studies. MATERIALS AND METHODS We studied TF expression on platelets, monocytes, lymphocytes and microparticles (MPs) by flow cytometry (FCM) with five commercially available mouse anti-human TF antibodies (HTF-1, TF9-10H10, CLB/TF-5, VIC7 and VD8). The ability of different TF antibodies to inhibit cell surface TF activity was explored by incubating LPS-stimulated monocytes and MPs derived from LPS-stimulated monocytes (MMPs) with TF antibodies followed by measuring TF activity. RESULTS HTF-1 detected TF only on LPS-stimulated monocytes, whereas, TF9-10H10 and VD8 detected TF associated with MPs and MMPs in addition to LPS stimulated monocytes. Surprisingly, CLB/TF-5 and VIC7 detected TF on platelets, monocytes even under unstimulated conditions, in addition to MPs and MMPs. CLB/TF-5 also detected TF on unstimulated lymphocytes. Inhibitory studies showed that at a final concentration of 10 μg/mL, HTF-1, CLB/TF-5 and VD8 inhibited monocyte TF activity by 81-84% and MMP TF activity by 92-96%; whereas TF9-10H10 had no inhibitory effect on TF activity in monocytes and MMPs. CONCLUSIONS Our results suggest non-specific binding by the CLB/TF-5 and VIC7 antibodies in a FCM test system and explain at least some of the reports on TF presence in blood cells, particularly TF associated with platelets and MPs. TF9-10H10 and VD8 are more suitable to detect TF on MPs by FCM.

Effect of strenuous exercise on blood monocytes and their relation to coagulation

Changes were explored in the behavior of circulating monocytes and their potential association with the activation of the coagulation system as assessed following strenuous exercise. Twelve men and nine women from the Norwegian national cross country skiing team and 19 men and six women from a level just below that of the national team were studied before and after ski race competition. Mononuclear cells were isolated after incubation of heparinized blood with lipopolysaccharides (LPS; 3 ng.ml-1) for 2 h. After a 50 km race for men, the specific thromboplastin activity of the stimulated monocytes rose from 3.5 x 10(-3)/10(6) cells to 21.4 x 10(-3)/10(6) cells. This probably reflects the mobilization of a new population of monocytes that are more sensitive to such stimuli. Resting top-athlete skiers had monocytes which were significantly less responsive to the LPS stimulus compared to nontrained people. There was an inverse correlation of plasma factor VII and the monocyte responsiveness to in vitro stimulation (r = 0.814; P less than 0.002) from blood drawn after a race. Furthermore, factor VII was significantly reduced after a 50 km race, and a modest decline in the fibrinogen level was also observed (P less than 0.05). It is concluded that endurance ski racing causes white cell mobilization and more active white cells that may induce activation of the coagulation system and account for the involvement of factor VII and fibrinogen.

Haemostatic parameters related to lipids and adhesion molecules.

Several components of blood, e.g. lipids, coagulation and fibrinolytic factors, are thought to be important risk factors in cardiovascular diseases. The aim of this study was to correlate these risk factors and the soluble adhesion proteins, soluble P-selection (sP-selectin) and soluble vascular cell adhesion molecule (sVCAM-1), in healthy men and women as well as to unravel any effects of smoking. One hundred and forty-two fasting men (median age 36 years) including 39 smokers, and 124 women (median age 34 years) including 35 smokers, were tested between 0800 h and 1000 h. Fibrinogen correlated positively with white blood cells (WBC) (r = 0.25), prothrombin fragment 1.2 (F1.2) (r = 0.21), cholesterol (r = 0.27), beta-thromboglobulin (r = 0.29), Factor VII clotting activity (FVIIc) (r = 0.27) (all P < 0.0001), tissue plasminogen activator antigen (t-PAag) (r = 0.22, P < 0.0005), plasminogen activator inhibitor-1 antigen (PAI-1ag) (r= 0.20) and VCAM-1 (r= 0.19) (both P< 0.002). Cholesterol and triacylglycerol (TG) correlated positively with t-PA antigen (t-PAag) (r = 0.36 and r = 0.38), PAI-1 antigen (PAI-1ag) (r = 0.35 and r = 0.50), P-selectin (r = 0.26 and r = 0.27) (all P < 0.0001) and WBC (r = 0.17, P < 0.007 and r = 0.18, P < 0.004). Cholesterol correlated also with F1.2 (r = 0.29) and TG (r= 0.44) (P< 0.0001). In addition to cholesterol and TG, sP-selectin correlated postively with PAI-1ag (r= 0.39), t-PAag (r= 0.27) and WBC (r = 0.25) (all P < 0.0001). Comparing the various test parameters in men and women, it was found that women had significantly higher levels of F 1.2 and high-density lipoprotein-cholesterol than men, whereas men had higher levels of t-PAag, PAI-lag and P-selectin than women. Smoking was associated with a rise in several of the test parameters. It can be concluded that there are correlations between several risk factors. Of particular interest is the positive correlation between sP-selectin and a number of established risk factors of cardiovascular diseases.

Increased lipopolysaccharide-induced tissue factor activity and tumour necrosis factor production in monocytes after intake of aspirin: possible role of prostaglandin E2.

To determine how aspirin intake might influence lipopolysaccharide (LPS)-induced tissue factor (TF) activity and tumour necrosis factor (TNF) in human blood monocytes, we collected blood before and at various times after intake of 300 mg aspirin in 25 healthy volunteers. Aspirin intake reduced LPS-induced thromboxane B2 and PGE2 production in whole blood by 50% and 65% respectively, measured 1 h after aspirin intake. Subsequently, a 95% rise in LPS-induced TF activity in monocytes was seen as compared to a 26% rise in TNF. The rise in TF activity was maximal within 1 h after aspirin intake and no further rise was observed 3, 4 or 24 h after aspirin intake. In contrast, TF activity induced by incubating whole blood in the absence of LPS fell rapidly after the intake of aspirin. In separate experiments, a dose-dependent inhibition by PGE2 was observed in LPS-induced TF activity in monocytes. It is proposed that the increased LPS-induced TF activity and TNF production following aspirin intake may be due to suppressed PGE2 formation. The more pronounced rise in TF activity compared to TNF production may be due to an enhancement of the platelet lipoxygenase pathway that has been shown to be important for LPS-induced TF activity in monocytes.