Bjarne Østerud

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.

The Inflammatory Properties of Contained and Noncontained Lumbar Disc Herniation

Study Design. The inflammatory properties of nucleus pulposus were assessed in biopsy samples from patients who underwent surgery for lumbar disc herniation. Objectives. To investigate the inflammatory properties of the different types of disc herniation. Background Data. High levels of phospholipase A2 previously have been demonstrated in a small number of patients undergoing lumbar disc surgery. Phospholipase A2 is the enzyme responsible for the liberation of arachidonic acid from cell membranes at the site of inflammation and is considered to be the limiting agent in the production of prostaglandins and leukotrienes, which are powerful mediators of inflammation. Cytokines are among the many agonists inducing phospholipase A2 activation. Several reports previously have demonstrated the difference in clinical appearance of different types of lumbar disc herniation. Methods. Thirty‐seven patients undergoing surgery for lumbar disc herniation were investigated. During surgery the disc pathology of each patient was classified into one of three groups: bulging disc, contained herniation, and noncontained disc herniation. Also during surgery, biopsy samples were taken from the nucleus, immediately frozen in liquid nitrogen, and subsequently stored at −70 C until analyzed. Results. No traces of interleukin‐6 or tumor necrosis factor alpha were found in the biopsy samples. There was a significant difference in the levels of leukotriene B4 and thromboxane B2 in contained versus noncontained disc herniation, and the highest concentration was found in the noncontained disc herniation group. Conclusion. The results support the theory that inflammatory mechanisms are involved in sciatica because of lumbar disc herniation and indicate that the different types of disc herniation have different inflammatory properties.

The effect of soluble β-1,3-glucan and lipopolysaccharide on cytokine production and coagulation activation in whole blood

Soluble beta-1,3-glucan has been demonstrated to protect against infection and shock in rats and mice, and clinical studies suggest that administration of soluble glucans to trauma/surgical patients decreases septic complications and improves survival. However, little is known about the precise mechanisms by which glucans influence the state of activation of blood cells, which are responsible for the fulminant cytokine production and the activation of the coagulation system observed in serious gram-negative infection. We studied therefore the effect of an underivatized, soluble yeast beta-1,3-glucan and lipopolysaccharide (LPS), either alone or in combination, on tumor necrosis factor-alpha (TNFalpha), interleukin-6 (IL-6), IL-8 and IL-10 secretion and monocyte tissue factor (TF) expression in human whole blood. As expected, LPS induced the secretion of substantial amounts of all measured parameters, whereas only minor amounts of TNFalpha, IL-6, and IL-10 were induced by beta-glucan itself. However, beta-glucan itself induced the production of significant amounts of IL-8 and TF. Soluble beta-1,3-glucan had a strong synergistic effect on the LPS-induced secretion of IL-8, IL-10, and on monocyte TF activity, but not on TNFalpha and 1L-6 production. On the other hand, soluble beta-glucan strongly primed LPS stimulation of all parameters, including TNFalpha and IL-6. beta-Glucan also induced detectable neutrophil degranulation within 15 min, whereas a response to LPS was first detected after 90 min. In conclusion, soluble beta-1,3-glucan upregulated leukocyte activity, both on its own and in concert with LPS.

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.

Tissue factor: (patho)physiology and cellular biology.

The transmembrane glycoprotein tissue factor (TF) is the initiator of the coagulation cascade in vivo. When TF is exposed to blood, it forms a high-affinity complex with the coagulation factors factor VII/activated factor VIIa (FVII/VIIa), activating factor IX and factor X, and ultimately leading to the formation of an insoluble fibrin clot. TF plays an essential role in hemostasis by restraining hemorrhage after vessel wall injury. An overview of biological and physiological aspects of TF, covering aspects consequential for thrombosis and hemostasis such as TF cell biology and biochemistry, blood-borne (circulating) TF, TF associated with microparticles, TF encryption–decryption, and regulation of TF activity and expression is presented. However, the emerging role of TF in the pathogenesis of diseases such as sepsis, atherosclerosis, certain cancers and diseases characterized by pathological fibrin deposition such as disseminated intravascular coagulation and thrombosis, has directed attention to the development of novel inhibitors of tissue factor for use as antithrombotic drugs. The main advantage of inhibitors of the TF•FVIIa pathway is that such inhibitors have the potential of inhibiting the coagulation cascade at its earliest stage. Thus, such therapeutics exert minimal disturbance of systemic hemostasis since they act locally at the site of vascular injury.

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.

Macrophages produce blood coagulation factors

2 .l . Preparation of macrophage cultures Macrophages were obtained from hybrid C3DZF1 (C3H/Tifo9 X DBA/2 d) mice by peritoneal washing with phosphate-buffered saline without anticoagulant. Cells were routinely cultured in MEM (minimum essential medium with Earle’s salts, Gibco Bio-cult, Paisley, Scotland) supplemented with 0.05% (w/v) heat inactivated (lOO’C, 10 min) la&albumin hydrolysate (Nutritional Biochemicals Corp., Cleveland, OH) and 100 IU/ml of penicillin and streptomycin (Gibco Biocult). The cells were either seeded in Costar plastic plates (Costar, Broadway, Cambridge, MA) in 16 mm circular wells on glass coverslips (0.7 X lo6 cells seeded/well) or in 100 mm Falcon plastic dishes (20 X lo6 cells seeded/dish). After 2 h in culture non-adherent cells were washed away and new medium containing lactalbumin hydrolysate was added. The incubation took place at 37OC in an atmosphere of 5% CO2 in air. The medium collected after 24 h in culture was centrifuged at 500 X g and the supernatant tested for coagulation factors or subjected to chromatography on dextran sulphateSepharose for partial separation of the factors. In the experiments with [35S]methionine, cells were cultures in medium supplemented with 50 /.&i [35S]methionine/ml. After 24 h incubation the

Effects of dietary marine oils and olive oil on fatty acid composition, platelet membrane fluidity, platelet responses, and serum lipids in healthy humans

The influence of various dietary marine oils and olive oil on fatty acid composition of serum and platelets and effects on platelets and serum lipids were investigated as part of an extensive study of the effects of these oils on parameters associated with cardiovascular/thrombotic diseases. Healthy volunteers (266) consumed 15 mL/d of cod liver oil (CLO); whale blubber oil (refined or unrefined); mixtures of seal blubber oil and CLO; or olive oil/CLO for 12 wk. In the CLO, seal oil/CLO, and whale oil groups, serum levels of eicosapentaenoic acid (EPA) were increased. In platelets, EPA was increased in the CLO, seal/CLO, and olive oil/CLO groups. The localization of n-3 polyunsaturated fatty acids in the triacylglycerols did not seem to influence their absorption. Intake of oleic acid is poorly reflected in serum and platelets. No significant differences in triacylglycerols (IG), total cholesterol, or high density lipoprotein cholesterol were observed, even though TG were reduced in the CLO, CLO/seal oil, and whale oil groups. Mean platelet volume increased significantly in both whale oil groups and the CLO/olive oil group. Platelet count was significantly reduced in the refined whale oil group only. Lipopolysaccharide-stimulated blood tended to generate less thromboxane B2 in CLO, CLO/seal, and CLO/olive groups. The whale oils tended to reduce in vivo release of β-thromboglobulin. In conclusion, intake of various marine oils causes changes in platelet membranes that are favorably antithrombotic. The combination of CLO and olive oil may produce better effects than these oils given separately. The changes in platelet function are directly associated with alterations of fatty acid composition in platelet membranes.

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.

Tissue factor expression in blood cells

The popular concept of TF serving predominantly as a hemostatic envelope encapsulating the vascular bed, has recently been challenged by the observation that blood of healthy individuals may form TF-induced thrombus under conditions entailing shear stress and activated platelets, corroborating the notion of blood borne TF. Accordingly, small amounts of TF activity is detected in calcium ionophore-stimulated monocytes, whereas it is questionable whether neutrophils and eosinophils express TF. Still there are contradicting reports on TF synthesis and expression in activated platelets, but when using a very sensitive and specific assay for TF activity measurements, we fail to detect TF activity associated with platelets activated with various agonists. However, activated platelets may play a role in decrypting monocyte TF activity in a process entailing transfer of TF to activated platelets in a P-selectin-PSGL-1 reaction whereby inactive TF (encrypted) becomes active through the availability of clusters of phosphatidylserine. Microparticles from plasma of healthy subjects possess weak TF-like activity which is not inactivated by anti-TF antibody. Endothelial cells are well documented to synthesize TF by several agonists in vitro. In contrast, there is little evidence that these cells are capable of synthesizing TF in vivo, and a recent report fails to show that TF on the endothelium may play any role in thrombin generation in a murine endotoxemia model. It may be concluded that monocytes are the only blood cells that synthesize and express TF and which may be the only source for TF-induced thrombosis when the endothelium is intact.