Expression Of Procoagulant Activity Research Articles

Defective Ca(2+)-induced microvesiculation and deficient expression of procoagulant activity in erythrocytes from a patient with a bleeding disorder: a study of the red blood cells of Scott syndrome

The erythrocytes from a patient with Scott syndrome, a bleeding disorder characterized by an isolated defect in expression of platelet procoagulant activity, have been studied. When incubated with the calcium ionophore A23187, Scott syndrome red blood cells (RBCs) expressed less than 10% of the prothrombinase (enzyme complex of coagulation factors Va and Xa) activity of A23187-treated RBCs obtained from normal controls. Consistent with the results from enzyme assay, the ionophore-treated Scott syndrome erythrocytes exhibited diminished membrane vesiculation and decreased exposure of membrane binding sites for factor Va compared with identically treated controls. When examined by scanning electron microscopy, untreated Scott syndrome RBCs were indistinguishable from normal cells. After incubation with A23187, however, the morphology of Scott syndrome RBCs contrasted markedly from normal erythrocytes. Whereas the Ca2+ ionophore induced marked echinocytosis and spiculation of normal RBCs, Scott syndrome RBCs remained mostly discoid under these conditions, with only an occasional echinocyte-like cell observed. These aberrant responses to intracellular Ca2+ were also observed for resealed ghosts prepared from Scott syndrome erythrocytes, indicating that they are related to a defect in the membrane or membrane-associated cytoskeleton. The finding that the erythrocytes of this patient share many of the membrane abnormalities reported previously for Scott syndrome platelets suggests that this defect is common to both cell lines and involves a membrane component required for vesicle formation and for expression of prothrombinase sites.

Defective Ca2+-Induced Microvesiculation and Deficient Expression of Procoagulant Activity in Erythrocytes From a Patient With a Bleeding Disorder: A Study of the Red Blood Cells of Scott Syndrome

The erythrocytes from a patient with Scott syndrome, a bleeding disorder characterized by an isolated defect in expression of platelet procoagulant activity, have been studied. When incubated with the calcium ionophore A23187, Scott syndrome red blood cells (RBCs) expressed less than 10% of the prothrombinase (enzyme complex of coagulation factors Va and Xa) activity of A23187-treated RBCs obtained from normal controls. Consistent with the results from enzyme assay, the ionophore-treated Scott syndrome erythrocytes exhibited diminished membrane vesiculation and decreased exposure of membrane binding sites for factor Va compared with identically treated controls. When examined by scanning electron microscopy, untreated Scott syndrome RBCs were indistinguishable from normal cells. After incubation with A23187, however, the morphology of Scott syndrome RBCs contrasted markedly from normal erythrocytes. Whereas the Ca2+ ionophore induced marked echinocytosis and spiculation of normal RBCs, Scott syndrome RBCs remained mostly discoid under these conditions, with only an occasional echinocyte-like cell observed. These aberrant responses to intracellular Ca2+ were also observed for resealed ghosts prepared from Scott syndrome erythrocytes, indicating that they are related to a defect in the membrane or membrane-associated cytoskeleton. The finding that the erythrocytes of this patient share many of the membrane abnormalities reported previously for Scott syndrome platelets suggests that this defect is common to both cell lines and involves a membrane component required for vesicle formation and for expression of prothrombinase sites.

Expression of procoagulant activity by equine lung macrophages: stimulation by blood lymphocytes

Increases in procoagulant activities ( pca) in equine lung macrophages were induced by non-adherent blood lymphocytes which were prestimulated with phytohaemagglutinin for 48 to 72 hours or by supernatants harvested from prestimulated blood lymphocyte cultures. However, prestimulated lymphocyte suspensions themselves expressed pca which was most probably derived from contaminating monocytes. Because non-adherent cells from lymphocyte suspensions may have attached to adherent macrophages, cells within lymphocyte suspensions might have contributed to the pcas expressed by lymphocyte-stimulated lung macrophages. Stimulation of lung macrophages for 24 hours by supernatants of phytohaemagglutinin-prestimulated blood lymphocytes induced a significantly greater pca increase than stimulation by phytohaemagglutin alone. Thus, cytokines from lymphocyte cultures might have triggered or enhanced pca induction. Direct stimulation of lung cell preparations with phytohaemagglutinin for 48 hours resulted in a progressive increase of pca in only two of five specimens tested. The failure to induce pca in three specimens could be due to the absence of sufficient numbers of T cells within the adherent lung cell preparations. In conclusion, pca response of equine lung macrophages might be lymphocyte-stimulated in which case pca might be a useful tool for monitoring the processes of cell-mediated immunity in horses.

Spontaneous and lipopolysaccharide-induced expression of procoagulant activity by equine lung macrophages in comparison with blood monocytes and blood neutrophils

The procoagulant activity (PCA) associated with equine bronchoalveolar lavage cells was determined and compared with that expressed by peripheral blood mononuclear cells and neutrophils. Lung cell preparations from horses affected with chronic pulmonary disease were included in all experiments and there was no difference in the qualitative type of response compared with lung cells which were obtained from healthy horses. Significant amounts of PCA were expressed by cells freshly procured from bronchoalveolar lavages of healthy and diseased horses. When adherent lung cells were kept in culture for some time, cell-associated PCA slightly decreased within 4 h, reached its lowest point after approximately 24 h and rose again during the second week of culture. In contrast, freshly isolated blood mononuclear cells or neutrophils expressed little PCA. Following culture for 24 h, mononuclear cells began to express increased PCA levels. Both cultivated lung cells (comprised mainly on alveolar macrophages) and blood mononuclear cells responded to LPS by dramatically increased PCA expression, whereas neutrophils showed a small augmentation of PCA on LPS stimulation. Fresh mononuclear cells and cultivated lung cells differed in their PCA response to LPS in several respects. Blood mononuclear cells were more sensitive to LPS than lung macrophages and responded to a 100-fold lower LPS concentration than the latter. Mononuclear cell-associated PCA peaked 4 h after stimulation whereas that of cultured macrophages continued to increase up to 24 h after stimulation. Lung macrophages cultured in adherence responded to LPS stimulation with a much higher PCA increase than macrophages cultured in suspension, in teflon containers. However, the culture vessel did not influence the PCA expressed by unstimulated cells. PCA expression depended to a large extent on transcription and translation, as evidenced by a 60–85% reduction of PCA in cycloheximide- or actinomycin D-treated, LPS-stimulated lung macrophages. PCA was largely cell-associated; only a small proportion of cell-associated PCA was shed into the medium. The PCA associated with mononuclear cells and with lung macrophages was tissue factor because of its dependence on clotting factor VII and its independence from clotting factor VIII. The expression of PCA by freshly isolated cells, the lower sensitivity to LPS, and the loss of PCA in the first 24 h of cultivation are indicative of in vivo activation of lung macrophages.

Lipopolysaccharide-Dependent and Lipopolysaccharide-Independent Pathways of Monocyte Desensitisation to Lipopolysaccharides

The present study demonstrates that with time in culture blood monocytes (MO) lose their ability to express procoagulant activity (PCA) and secrete tumor necrosis factor-alpha (TNF alpha) in culture medium in response to lipopolysaccharide (LPS) stimulation. Thus, upon 10 micrograms/ml LPS stimulation for 4 hours 2-day-old MO produced lower levels of PCA and TNF alpha than fresh MO. The decrease in responsiveness was not caused by cell death, since in the case of TNF alpha it was fully reversible by interferon-gamma (IFN-gamma). Compared with cells pre-incubated in medium alone, the responsiveness of MO pre-incubated in LPS was further decreased. Thus, in MO LPS pre-incubation was followed by an LPS refractory state. It was expected that the decrease in responsiveness induced by cultivation in medium alone was mediated by LPS contamination of culture medium. However, as we were unable to prevent this decrease by neutralizing LPS contamination of the culture medium with polymyxin B, the loss in LPS-induced activities of cultured MO is likely to be mediated by culture conditions other than LPS contamination. Taken together the present data demonstrate that LPS-dependent as well as LPS-independent pathways of MO desensitization to LPS exist.

Generation of procoagulant (thromboplastin) and plasminogen activator activities in peripheral blood monocytes after total hip replacement surgery. Effects of high doses of corticosteroids

Development of thromboplastin (tpl) and plasminogen activator (PA) activity in monocytes and the effects of high doses of corticosteroids (HCD) on these activities were studied in patients undergoing a standardized surgical trauma. Twelve patients who received uncemented total hip prostheses were divided into a nonsteroid group (n=6) and a steroid group (n=6). We found no significant differences between the two patient groups regarding tpl or PA activities of peripheral blood mononuclear cells (PBM) isolated during the postoperative phase. However, in the nonsteroid group there was a tendency for increased expression of procoagulant activity and decreased fibrinolytic activity on the 1st postoperative day, favoring the formation of fibrin in the monocyte microenvironment. Further, PBM isolated on the 1st and 2nd day after surgery were significantly less capable of generating tpl activity on endotoxin stimulation than cells isolated preoperatively. This was not the case in the steroid group. These patients had also a tendency for decreased fibrinolysis at the end of the 1st postoperative week, indicating increased imbalance towards a more thrombotic stage after surgery.

Lack of Effect of Granulocyte-Macrophage and Granulocyte Colony-Stimulating Factors on Cultured Human Endothelial Cells

AbstractThe hematopoietic growth factors, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF), enhance the effector functions of mature myeloid cells, including the interaction with vascular endothelium. We examined the direct effect of recombinant human GM-CSF (rhGM-CSF) and recombinant human G-CSF (rhG-CSF) on the growth and function of cultured human umbilical vein endothelial cells (HUVEC). Endothelial cell growth supplement (ECGS) increased the proliferation of passaged and primary cells by 305% ± 45% (mean ± SEM, n = 5, P < .01) over control cells at 4 days; GM-CSF and G-CSF had no effect. Endothelial cell procoagulant activity was increased after 4-hour incubation with recombinant interleukin-1 β (IL-1β) 10 U/mL and recombinant tumor necrosis factor (TNF) 10 U/mL to 1,721% ± 376% (n = 7, P < .005) and 247% ± 71% (n = 4) of control levels, respectively. γ-Interferon (γ-IFN) 50 U/mL had no direct effect of its own but was able to prime the response to IL-1β. There was no direct or priming effect of GM-CSF (1 ng to 1 μg/mL) on the expression of procoagulant activity in endothelial cells. GM-CSF and G-CSF (1 ng/mL to 1 μg/mL) had no effect on the expression of either tissue plasminogen activator (tPA) or plasminogen activator inhibitor-1 (PAI-1) by endothelial cells. The secretion of tPA by endothelial cells was increased, however, after 24-hour incubation with thrombin 4 U/mL (314% ± 72% of control levels, n = 5, P < .025). The production of PAI-1 was increased by TNF 200 U/mL (241% ± 44% of control, n = 3, P < .005), thrombin 4 U/mL (180% ± 12% of control, n = 5,P < .0005) and IL-1 β 10 U/mL (275% ± 44% of controls, n = 5, P < .0005). In four experiments, endothelial cells showed no specific binding of 125I-GM-CSF, whereas peripheral blood (PB) neutrophils demonstrated the presence of 802 ± 78 high-affinity receptors for GM-CSF. Thus, we found no effect of rhGM-CSF or rhG-CSF on the proliferation of HUVEC or the expression of procoagulant and fibrinolytic activities by these cells. These findings are in accordance with the lack of demonstrable receptors for GM-CSF on cultured HUVEC.

Lack of effect of granulocyte-macrophage and granulocyte colony- stimulating factors on cultured human endothelial cells [see comments

The hematopoietic growth factors, granulocyte-macrophage colony- stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF), enhance the effector functions of mature myeloid cells, including the interaction with vascular endothelium. We examined the direct effect of recombinant human GM-CSF (rhGM-CSF) and recombinant human G-CSF (rhG-CSF) on the growth and function of cultured human umbilical vein endothelial cells (HUVEC). Endothelial cell growth supplement (ECGS) increased the proliferation of passaged and primary cells by 305% +/- 45% (mean +/- SEM, n = 5, P less than .01) over control cells at 4 days; GM-CSF and G-CSF had no effect. Endothelial cell procoagulant activity was increased after 4-hour incubation with recombinant interleukin-1 beta (IL-1 beta) 10 U/mL and recombinant tumor necrosis factor (TNF) 10 U/mL to 1,721% +/- 376% (n = 7, P less than .005) and 247% +/- 71% (n = 4) of control levels, respectively. gamma-Interferon (gamma-IFN) 50 U/mL had no direct effect of its own but was able to prime the response to IL-1 beta. There was no direct or priming effect of GM-CSF (1 ng to 1 microgram/mL) on the expression of procoagulant activity in endothelial cells. GM-CSF and G-CSF (1 ng/mL to 1 microgram/mL) had no effect on the expression of either tissue plasminogen activator (tPA) or plasminogen activator inhibitor-1 (PAI- 1) by endothelial cells. The secretion of tPA by endothelial cells was increased, however, after 24-hour incubation with thrombin 4 U/mL (314% +/- 72% of control levels, n = 5, P less than .025). The production of PAI-1 was increased by TNF 200 U/mL (241% +/- 44% of control, n = 3, P less than .005), thrombin 4 U/mL (180% +/- 12% of control, n = 5, P less than .0005) and IL-1 beta 10 U/mL (275% +/- 44% of controls, n = 5, P less than .0005). In four experiments, endothelial cells showed no specific binding of 125I-GM-CSF, whereas peripheral blood (PB) neutrophils demonstrated the presence of 802 +/- 78 high-affinity receptors for GM-CSF. Thus, we found no effect of rhGM-CSF or rhG-CSF on the proliferation activities by these cells. These findings are in accordance with the lack of demonstrable receptors for GM-CSF on cultured HUVEC.

The role of endothelium in the expression of procoagulant activity

University of Vermont Department of Medicine, College of Medicine, Given Medical Building, Burlington, VT, USA.

Clinical relevance of monocyte procoagulant activity in horses with colic

Summary Endotoxin-activated monocytes express a thromboplastin-like procoagulant activity on the cell surface that may serve as a focal point for formation of microvascular thrombi. Because coagulopathy is a common sequela to endotoxemia in the equine species, we investigated the ability of monocytes, isolated from horses with colic, to express procoagulant activity. On the day of admission, and on the third and fifth day of hospitalization, monocytes were isolated from 30 adult horses with colic. A coagulation profile, including prothrombin time, activated partial thromboplastin time, thrombin time, and plasma fibrinogen and serum fibrin degradation products concentrations, was determined at each sample collection. The concentration of endotoxin in the plasma was quantitated at the time of admission. Ten clinically normal adult horses served as controls. The procoagulant activity of monocytes isolated from horses with colic was significantly (P &lt; 0.05) greater than that of the monocytes isolated from clinically normal horses. On the first and third day of hospitalization, the mean prothrombin time was significantly (P &lt; 0.05) longer in horses with colic, compared with clinically normal horses, and was the most common abnormality in the coagulation profile on the day of admission (25/30; 83%). Mean fibrin degradation products concentration was significantly (P &lt; 0.05) greater in horses with colic on the day of admission and was the second most common abnormality in the coagulation profile on day 1 (23/30; 77%). In horses with colic, the mean prothrombin and activated partial thromboplastin times were significantly (P &lt; 0.05) longer in horses that did not survive, compared with horses that survived. Of the 30 horses with colic, 9 had detectable endotoxin in the plasma at the time of admission. The presence of endotoxin in the plasma was useful in predicting survival in horses with colic; all horses with plasma endotoxin concentration ≥ 10 pg/ml (6/6) did not survive.

Effects of simulated extracorporeal circulation on human leukocyte elastase release, superoxide generation, and procoagulant activity

Activated leukocytes are thought to contribute to respiratory dysfunction, alterations in microvascular permeability, disseminated intravascular coagulation, and thrombosis, all of which can complicate extracorporeal circulation. The purpose of this work was to determine the effects of extracorporeal circulation on leukocyte functions likely to mediate organ damage. White blood cell counts in the bubble circuits (n = 5) fell to 51% +/- 7% (mean +/- standard error of the mean; p less than 0.05) of initial levels within 2 hours of recirculation. In contrast, counts from both the spiral coil (n = 5) and hollow-fiber (n = 5) groups remained at 91% +/- 12% and 100%, respectively. Plasma levels of human neutrophil elastase rose from 0.28 +/- 0.06 micrograms/ml to 3.14 +/- 0.36 micrograms/ml (p less than 0.05) and 0.20 +/- 0.02 micrograms/ml to 1.61 +/- 0.35 micrograms/ml (p less than 0.05) in bubble and spiral coil circuits, respectively, but from only 0.20 +/- 0.03 micrograms/ml to 0.96 +/- 0.42 micrograms/ml in the hollow-fiber circuit despite 2 hours of recirculation. Consistently, in response to N-formyl-L-methionyl-L-leucyl-L-phenylalanine, a chemotactic peptide, cells from spiral coil and bubble circuits released and generated significantly less elastase and superoxide anion, respectively. In contrast, neutrophils from the hollow-fiber circuits demonstrated enhancement of N-formyl-L-methionyl-L-leucyl-L-phenylalanine-induced elastase release and superoxide generation. Finally, mixed leukocytes from all circuits expressed procoagulant activity reaching statistical significance in bubble circuits. In conclusion, extracorporeal circulation has pronounced effects on neutrophil elastase release, superoxide anion generation, and leukocyte procoagulant activity. Spiral coil and bubble oxygenators cause granule release and, subsequently, reduced sensitivity to soluble agonists. In contrast, hollow-fiber oxygenators "prime" cells, actually enhancing reactivity. Recirculation through all circuits induces leukocyte procoagulant activity that is likely to contribute to surface-induced thromboses and excessive bleeding.

The role of calpain in stimulus-response coupling: evidence that calpain mediates agonist-induced expression of procoagulant activity in platelets

Although calpain (the Ca2(+)-dependent protease) is widely distributed, its function is poorly understood. One cell in which it becomes activated as a consequence of activation of the cell is the blood platelet. The aim of the present study was to determine whether activation of calpain was responsible for any of the responses of platelets to stimulation. Platelets were incubated with calpeptin, a membrane-penetrating inhibitor of calpain, before being exposed to an agonist. Concentrations of calpeptin that totally inhibited the agonist- induced hydrolysis of actin-binding protein (ABP) by calpain had no effect on many other responses associated with platelet activation: phosphorylation of myosin light chain, phosphorylation of P47, platelet shape change, aggregation of platelets, secretion of granule contents, or retraction of fibrin clots. However, these concentrations of inhibitor decreased the agonist-induced generation of procoagulant activity (assayed as the ability of platelets to catalyze the conversion of prothrombin to thrombin in the presence of factor Va and factor Xa). When thrombin was the agonist, the amount of ABP that was hydrolyzed was small; only a small component of the total agonist- induced procoagulant activity was inhibited by calpeptin. When collagen was the agonist, more ABP was hydrolyzed and the amount of procoagulant activity generated was greater; calpeptin decreased the collagen- induced procoagulant activity to levels comparable with those induced by thrombin in the presence of the inhibitor. We suggest that there are at least two mechanisms by which procoagulant activity is generated on activated platelets and that the agonist-induced activation of calpain mediates one of these mechanisms. These results show that activation of calpain is a component of the stimulus-response pathway in platelets.

The Role of Calpain in Stimulus-Response Coupling: Evidence That Calpain Mediates Agonist-Induced Expression of Procoagulant Activity in Platelets

AbstractAlthough calpain (the Ca2+-dependent protease) is widely distributed, its function is poorly understood. One cell in which it becomes activated as a consequence of activation of the cell is the blood platelet. The aim of the present study was to determine whether activation of calpain was responsible for any of the responses of platelets to stimulation. Platelets were incubated with calpeptin, a membrane-penetrating inhibitor of calpain, before being exposed to an agonist. Concentrations of calpeptin that totally inhibited the agonist-induced hydrolysis of actin-binding protein (ABP) by calpain had no effect on many other responses associated with platelet activation: phosphorylation of myosin light chain, phosphorylation of P47, platelet shape change, aggregation of platelets, secretion of granule contents, or retraction of fibrin clots. However, these concentrations of inhibitor decreased the agonist-induced generation of procoagulant activity (assayed as the ability of platelets to catalyze the conversion of prothrombin to thrombin in the presence of factor Va and factor Xa). When thrombin was the agonist, the amount of ABP that was hydrolyzed was small; only a small component of the total agonist-induced procoagulant activity was inhibited by calpeptin. When collagen was the agonist, more ABP was hydrolyzed and the amount of procoagulant activity generated was greater; calpeptin decreased the collagen-induced procoagulant activity to levels comparable with those induced by thrombin in the presence of the inhibitor. We suggest that there are at least two mechanisms by which procoagulant activity is generated on activated platelets and that the agonist-induced activation of calpain mediates one of these mechanisms. These results show that activation of calpain is a component of the stimulus-response pathway in platelets.

Separation of equine bronchopulmonary lavage cells by density gradient centrifugation and expression of procoagulant activity in unpurified cells and cell subpopulations

Bronchopulmonary lavage was performed in 10 healthy horses and in 39 horses with chronic pulmonary disease. The predominant cell types were macrophages in healthy horses and neutrophils in severely diseased horses. Procoagulant activity (PCA) was detected in all 32 cell-free supernatants examined and in all 49 unpurified cell suspensions. Cells were separated by centrifugation on discontinuous gradients prepared either with Percoll or with Metrizamide. Macrophages were enriched in subpopulations of low density. Neutrophils could not be purified by density gradient centrifugation using either gradient medium. PCAs of cell subpopulations were plotted against their respective macrophage, neutrophil, and lymphocyte content. PCA was positively correlated with macrophage content (P less than 0.001) and negatively correlated with neutrophil (P less than 0.02) and lymphocyte (P less than 0.001) content. Therefore, PCA of equine lung cells most likely originates from macrophages as shown in other species. The density shift of lung neutrophils requires further investigation.

Asbestos Exposure Results in Increased Lung Procoagulant Activity in Vivo and in Vitro

Enhanced fibrin deposition is a common histologic finding in fibrotic lung disorders including asbestosis and may be an important mechanism by which fibroblast proliferation is modulated. Asbestos-induced activation of lung interstitial cells may result in enhanced expression of procoagulant activity which contributes to the inflammatory response resulting in subsequent fibrin deposition. The current study examines procoagulant activity in bronchoalveolar lavage fluid from patients with clinically diagnosed asbestosis, patients with asbestos exposure without asbestosis, and normal, control subjects. Results indicated that asbestos exposure resulted in increased lung procoagulant expression in vivo, and furthermore, suggested that both endothelial cells and alveolar macrophages represented lung parenchymal cells which may contribute to this activity. This imbalance in coagulation homeostasis may be important in the regulation of fibrotic responses observed in asbestosis.

A polypeptide factor produced by fibrosarcoma cells that induces endothelial tissue factor and enhances the procoagulant response to tumor necrosis factor/cachectin.

Intravascular clot formation, localized to the neoplasm, is an early component of the vascular response to tumor necrosis factor (TNF)/cachectin. Fibrin is closely associated with the endothelial cell surface, and multiple microthromboses lead to reduced blood flow in the tumor. We have identified a tumor-derived mediator which enhances endothelial procoagulant activity and the cellular response to TNF using cultured cells derived from a murine methylcholanthrene A (meth A)-induced fibrosarcoma as a model system. A heat-stable protease K-sensitive polypeptide, Mr approximately 44,000 on nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr approximately 56,000 reduced), was purified approximately 500,000-fold from serum-free culture supernatants of meth A cells by sequential Q-Sepharose, Mono S, reversed phase, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based on immunologic criteria, biologic activity, and other molecular properties, meth A factor appears to be distinct from other cytokines and growth factors. Purified meth A factor induced transcription of the tissue factor gene and expression of procoagulant activity by cultured human endothelium (half-maximal effect for the latter at approximately 6-8 pM). Furthermore, co-incubation of endothelium with meth A factor together with TNF enhanced induction of tissue factor in a more than additive manner. These data indicate that certain tumors elaborate an apparently unique molecule which can alter hemostatic properties of the vessel wall, potentially modulating reactivity of the tumor vasculature to host response mediators.

Levels of three hemostatic factors in relation to serum lipids. Monocyte procoagulant activity, tissue plasminogen activator, and type-1 plasminogen activator inhibitor.

To explore the relationship between blood lipid levels and a predisposition to thrombosis, levels of three hemostatic factors were measured in 41 human subjects and correlated with serum lipids. Procoagulant activity associated with peripheral blood monocytes isolated and purified after a 2-hour incubation in whole blood was not significantly related to lipid levels. However, activity in monocytes incubated with 100 ng/ml of bacterial endotoxin was significantly correlated with high density lipoprotein (HDL) cholesterol (r = 0.55, p less than 0.005), while net procoagulant activity (endotoxin-challenged minus basal) was significantly correlated with both HDL cholesterol (r = 0.61, p less than 0.005) and total cholesterol (r = 0.50, p less than 0.01). Plasma levels of the fibrinolytic factor, tissue plasminogen activator, were significantly correlated with total cholesterol (r = 0.41, p less than 0.01), while those of the type-1 plasminogen activator inhibitor were significantly correlated with both total cholesterol (r = 0.46, p less than 0.01) and total triglycerides (r = 0.31, p less than 0.05). The balance between the fibrinolytic factors was not significantly related to serum lipids. These results suggest that the expression of procoagulant activity by peripheral blood monocytes exposed to endotoxin may be enhanced in cases where HDL cholesterol levels are high. In addition, these results suggest that hypertriglyceridemia may be associated with a decreased fibrinolytic capacity due to elevated secretion of plasminogen activator inhibitor.

Thrombogenicity of total parenteral nutrition solutions: II. Effect on induction of endothelial cell procoagulant activity

Thrombosis is a common sequela of total parenteral nutrition. We have recently demonstrated in vitro that hypertonic total parenteral nutrition solutions are potent inducers of a tissue factor monocyte procoagulant activity, the initiating cofactor of the extrinsic clotting cascade. We have further studied, in vitro, the effects of the component solutions of total parenteral nutrition on the induction and modulation of endothelial cell procoagulant activity. Cultured porcine aortic endothelial cells were incubated with (a) 200 μl of dextrose solution (5%, 10%, 20%, 25%, and 50%), (b) 200 μl of amino acid solution [full strength (N), one-fourth strength, and one-half strength], and (c) 200 μl of 10% lipid emulsion. Cocultures of lipid emulsion and 20% dextrose, lipid emulsion and full-strength 10% amino acid solution (N-amino acid), and lipid emulsion and bacterial lipopolysaccharide also were studied. Cells were incubated for intervals of 3–108 h, washed and frozen, harvested, and assayed for endothelial cell procoagulant activity. Units of endothelial cell procoagulant activity were derived from a standard thromboplastin curve. Our results show that amino acid and hypertonic dextrose total parenteral nutrition solutions are able to strongly induce endothelial cell procoagulant activity expression in vitro. In contrast, lipid emulsion significantly inhibited the induction of endothelial cell procoagulant activity by 20% dextrose, N-amino acid, and lipopolysaccharide. These results provide further evidence for the role of the cellular pathways of coagulation in total parenteral nutritioninduced thrombosis. Furthermore, the inhibitory properties of lipid emulsion may be of practical advantage in reducing total parenteral nutritioninduced thrombosis.

Procoagulant activity expression by macrophages from atheromatous vascular plaques.

Embolization of thrombi from ulcerated plaques is an important cause of morbidity from atherosclerotic carotid artery disease. Factors controlling thrombus formation on these lesions are not well understood. Macrophages were isolated from atherosclerotic plaques to assess their potential to promote local fibrin deposition. Plaques were collected from 11 patients undergoing carotid endarterectomy and 9 patients undergoing reconstructive procedures for atherosclerotic disease of their distal aorta or femoral arteries. Blood was also collected concurrently to isolate monocytes. Procoagulant activity (PCA) of carotid macrophages (8.6 +/- 4.1 mU/10(6) cells) was significantly higher than that of macrophages from non-carotid lesions (0.35 +/- 0.20 mU/10(6) cells; P less than 0.05) or blood monocytes from either group of patients. The PCA of carotid plaque macrophages from patients with recent emboli was 16.1 +/- 8.4 mU/10(6) cells (n = 5) compared to 2.4 +/- 0.8 mU/10(6) cells (n = 6) for plaque macrophages from assymptomatic carotid endarterectomy patients. Carotid macrophage PCA was factor V and factor VII dependent. Its functional activity was inhibited by an anti-tissue factor antibody, and immunohistochemical studies on tissue sections from carotid plaques showed tissue factor in areas where macrophages were abundant. These studies demonstrate that macrophages within carotid artery plaques have augmented procoagulant activity compared with blood monocytes and macrophages from other atherosclerotic lesions and indicate that carotid plaque macrophages are activated. Augmented macrophage PCA may contribute to thrombus formation on ulcerated plaques.

Studies on leukemic cell tissue factor

Apoprotein part of tissue factor of human placenta was purified 871 fold from the starting material with 4.2% yield by concanavalin A-Sepharose affinity chromatography and SDS-PAGE. The molecular weight of purified apoprotein was 45,000 in non-reduced condition and 49,000 in reduced condition. Tissue factor of human leukemia cells ( FAB classification:M2 and M3 ) and cultured leukemia cell lines (HL-60 and Molt-4) was analyzed using specific rabbit anti-tissue factor IgG raised against purified material. Endotoxin stimulated HL-60 and Molt-4 also expressed procoagulant activity which was inhibited by tissue factor immune IgG. By immunostaining of the purified material, the lysate of leukemia cells ( M2 and M3 ) and cultured leukemia cells (HL-60 and MOLT-4 ) revealed a major band of the same apparent molecular weight. Immuno-electron microscopic study on tissue factor of HL-60 cells produced the following findings: stimulation by endotoxin resulted in the formation of pseudopods of the cell membrane, and immunogold particles accumulated mainly on these pseudopods and cisternal spaces of rough endoplasmic reticulum, indicating exposure of the tissue factor to the surface of perturbed cell membrane with concurrent increase in tissue factor synthesis.