Growing evidence supports a role for extracellular vesicles (EVs) in haemostasis and thrombosis due to exposure of negatively charged procoagulant phospholipids (PPL). Current commercial ...PPL-dependent clotting assays use chemically phospholipid depleted plasma to measure PPL activity. The purpose of our study was to modify the PPL assay by substituting the chemically phospholipid depleted plasma with PPL depleted plasma obtained by ultracentrifugation This in order to get readily access to a sensitive and reliable assay to measure PPL activity in human plasma and cell supernatants. The performance of the assay was tested, including the influence of individual coagulation factors and postprandial lipoproteins and compared to a commercial PPL assay (STA-Procoag-PPL). The two PPL assays displayed similar sensitivity to exogenously added standardized phospholipids. The PPL activity measured by the modified assay strongly correlates with the results from the commercial assay. The intraday- and between-days coefficients of variation ranged from 2-4% depending on the PPL activity in the sample. The modified PPL assay was insensitive to postprandial lipoprotein levels in plasma, as well as to tissue factor (TF) positive EVs from stimulated whole blood. Our findings showed that the modified assay performed equal to the comparator, and was insensitive to postprandial lipoproteins and TF
EVs.
Seven healthy male volunteers participated in short- (STR, 1.7 km), middle- (MTR, 4.8 km) and long- (LTR, 10.5 km) term runs at a speed close to their maximum. A prompt mobilization of white cells, ...and lymphocytes in particular, appeared following the exercise. The initial increase in the number of lymphocytes was succeeded by a significant decrease (P less than 0.03) lymphopenial, which on average was 32%-39% of the pre-exercise values in all groups. A close correlation was found between the initial increase in plasma cortisol concentration after exercise and the subsequent lymphopenia. A modest enhancement in the number of granulocytes immediately after the exercise was accompanied by a comprehensive increase in polymorphonuclear (PMN) elastase concentration accounting for 78.6%, SEM 16.3%, 140.7%, SEM 31.8% and 241.3%, SEM 48.1% in the STR, MTR and LTR groups. No correlation was found between granulocyte number and the plasma PMN elastase concentration. A delayed granulocytosis was noted in all subjects, reaching a peak between 2 and 4 h after the exercise. The magnitude of the granulocytosis varied among subjects and peak values of the number of circulating granulocytes were found to be 5.7 x 10(9) cells.l-1, SEM 0.5, 6.7 x 10(9) cells.l-1, SEM 0.6 and 8.8 x 10(9) cells.l-1, SEM 0.5 in STR, MTR and LTR respectively, whereas the mean baseline value was 3.6 x 10(9) cells.l-1, SEM 0.4. The neutrophilic granulocytosis was not accompanied by a corresponding enhancement in PMN elastase concentration. The plasma cortisol concentration reached a peak 30 min after exercise and declined below the control level in 4 h. Neither the initial increase, nor the subsequent decrease in plasma cortisol concentration were found to be essential for the magnitude of the delayed leukocytosis.
Lipopolysaccharide (LPS) stimulation of human monocytes in heparinized whole blood in vitro as expressed by induced activity of thromboplastin, has been studied. An essential role of arachidonic acid ...(20:4) release was found. 2,4'-Dibromoacetophenone, a phospholipase A2 inhibitor, totally blocked the induced synthesis of thromboplastin activity. Furthermore, nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, had an effect on the LPS-induced thromboplastin synthesis which varied from no inhibition in individuals insensitive to LPS ('low responders'), up to 80% inhibition in the person with the highest response ('high responder') to LPS. Platelets were found to be partially responsible for this difference. Thus, monocytes from high responders cross-combined with platelets from low responders were much less prone to LPS stimulation than they were in the presence of high responder platelets. Intake of acetylsalicylic acid caused a 50% increment of LPS-induced thromboplastin synthesis, and this effect was mediated by platelets.
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.
Seven healthy male volunteers were subjected to exercise of short (STR; 1.7 km), middle (MTR; 4.8 km) and long (LTR; 10.5 km) term runs at a speed close to maximal capacity. Blood samples were drawn ...before, immediately after exercise and at intervals over the next 10 h. FVIIIR:Ag (von Willebrand factor) rose 2.2-3.2 fold and persisted at higher levels than baseline during the observation time. A spontaneous drop in FVII (p less than 0.03) was found immediately after STR (13.5 +/- 2.5%) and LTR (18.3 +/- 2.4%), whereas only a minor decrease (7.5 +/- 6.5%) occurred in MTR. The procoagulant activity of monocytes isolated from whole blood exposed to LPS showed a striking enhancement in STR and MTR. An immediate enhancement in fibrinolytic activity was found in all groups (p less than 0.03) assessed by increased plasma levels of t-PA and shortened whole blood clot lysis time (WBCLT). The transient shortening of WBCLT was succeeded by a tendency to prolongation of the lysis time. A 45-year old male differed markedly from the others by demonstrating an extreme and consistent prolongation of WBCLT. Thus, it has been speculated that strenuous exercise possibly makes a subject more susceptible to a thrombotic event.
Nine healthy male volunteers (mean age 24.3 years, range 21-27 years) ingested 25 ml cod liver oil (CLO) daily for 8 weeks. Blood samples were collected before and after the intervention period. A ...slight increase in total serum cholesterol (+ 12.2%, P less than 0.01) and high density lipoprotein cholesterol (+ 16.9%, P less than 0.01) were calculated to have a non-significant effect on the high/low density lipoprotein ratio. Polyunsaturated fatty acids in serum were displaced from the n-6 family to the n-3 family, reflected by an eight-fold increase of the eicosapentaenoic (20:5 n-3)/arachidonic (20:4n-6) acid ratio. Human umbilical vein endothelial cells were grown in a medium to which 30% sterile filtered serum, collected before and after CLO supplementation for 72 h, was added. After 2 h exposure to lipopolysaccharides the thromboplastin activity in endothelial cultures incubated with serum enriched with n-3 fatty acids was 43% lower than in cultures containing serum collected before the intervention (1.86 +/- 0.48 10(-3)/10(6) cells vs 3.26 +/- 0.85 10(-3)/10(6) cells). It is suggested that serum prepared from subjects given n-3 fatty acids may support the thromboresistence of endothelial cells.
Conflicting results have been reported about the absorption of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) either as an ethyl ester (EE) or in a triglyceride (TG) formula. We decided ...to conduct a randomized double-blind study to compare the effects of EE and TG on plasma fatty acids, platelet function and haemostasis. Thirty-one healthy normolipaemic men were allocated to receive fish oil concentrate either as EE or TG with equal amounts of EPA (2.2 g and 2.2 g, respectively) and DHA (1.2 g and 1.4 g, respectively) or placebo daily for 7 weeks. Total cholesterol and the triglyceride level were not influenced differently by the two compounds. Repeated measurement ANOVA revealed a difference between TG and EE regarding incorporation of arachidonic acid (P = 0.034) and EPA (P = 0.007) into plasma cholesteryl esters. A discrimination not observed within plasma phospholipids. Both formulas had equal inhibitory effects on collagen-induced platelet aggregation and thromboxane B2 (TxB2) production in whole blood. Fibrinogen decreased 16% in EE (P = 0.034) and 12% in the placebo group (P = 0.11), but variance analysis of delta change during intervention did not indicate differences between groups. It is concluded that TG and EE fish oils are well incorporated into plasma lipids and have similarly beneficial influence one platelet function in men.
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.
Heparinized blood sample taken from 81 healthy persons, 40 men and 41 women of whom 17 were on combined oral contraceptives, were incubated with 2 ng lipopolysaccharides (LPS)/ml blood. Quantitation ...of thromboxane B2 (TxB2) in the resultant plasma revealed a significantly (p less than 0.001) higher generation of TxB2 in men than in women. No sexual differences were observed in thromboplastin activity in unstimulated and LPS stimulated monocytes. Addition of liposomes together with LPS enhanced the synthesis of thromboplastin 2-10 fold and unveiled a higher synthesis of thromboplastin among men. No differences were observed in TxB2 and thromboplastin synthesis in blood collected from females or females using oral contraceptives. A significant correlation between TxB2 and the thromboplastin activity exists within the male (r = 0.46, p = 0.003) and the oral contraceptive (r = 0.57, p = 0.016) group after exposure to LPS. This study suggests that males exposed to LPS may generate more thromboxane A2 than females and thus be at higher risk of developing thrombosis.
PDF
