Evaluation of Optimal Sample Processing Conditions for Accurate Measurement of Protein S Activity.

OBJECTIVE: Protein S(PS) activity, especially PS-specific activity calculated by total PS activity (tPSAct) divided by total PS antigen (tPSAg), is important in the diagnosis of hereditary PS deficiency (PSD). The cleavage of PS at a thrombin-sensitive region (TSR) by proteases reduces the anticoagulant activity of PS. Therefore, we investigated the effect of sample processing and storage on tPSAct and PS cleavage. METHODS: Blood samples were collected from ten healthy subjects, and tPSAg and tPSAct were measured in whole blood or plasma stored at room temperature (RT) or 4°C. The cleaved PS was detected by western blotting, and the relationship between decreases in PS-specific activity and increase rates of cleaved PS was evaluated. Furthermore, the stability of tPSAg and tPSAct on the long-term storage of plasma was also evaluated. RESULTS: Both whole blood and plasma stored at RT and whole blood stored at 4°C showed decreased tPSAct (50-80%) after 24 hours (P<0.05). PS-specific activity levels negatively correlated with the increase rate of cleaved PS (r =-0.84, P<0.001). The tPSAct was decreased to 60% after three days in plasma stored at 4°C (P<0.05) but was stable for about one month when stored at -20°C or below. CONCLUSION: Inappropriate processing and storage result in falsely low PS-specific activity due to the cleavage of PS in the blood collection tubes, which may lead to misdiagnosis of PSD. Samples should be centrifuged immediately after collection, and the plasma should be frozen.

Edoxaban improves acute venous thromboembolism while preserving protein C and protein S levels.

BACKGROUND: It is well known that warfarin inhibits the synthesis of vitamin K-dependent anticoagulants, including thrombin, protein C and S, and factor Xa, leading, paradoxically, to an initial hypercoagulable state. Edoxaban, a direct inhibitor of activated factor X is widely used for the treatment of acute venous thromboembolism (VTE). However, the effect of edoxaban on circulating coagulation factors, in patients with acute VTE, remains unknown. METHODS AND RESULTS: We enrolled 57 patients with acute VTE with/without pulmonary embolism treated with edoxaban (n=37) or warfarin (n=20) in a clinical setting. Before treatment and 2 weeks after treatment, we evaluated thrombotic burden using ultrasound or computed tomography angiography. We also evaluated thrombin generation, represented by prothrombin fragment F1+2; thrombus degradation, represented by D-dimer; and levels of anticoagulants, including protein C, protein S, and antithrombin III. Both edoxaban and warfarin treatment improved thrombotic burden and decreased prothrombin fragment F1+2, and D-dimer. Edoxaban treatment preserved protein C and protein S levels. In contrast, warfarin decreased protein C and protein S levels. Neither treatment affected antithrombin III. CONCLUSIONS: Edoxaban improves VTE while preserving protein C and protein S levels, thereby indicating that edoxaban improves thrombotic burden while maintaining levels of anticoagulants.

Hemostatic effects of two desogestrel-containing combined oral contraceptive regimens: a multinational, multicenter, randomized, open-label study.

PURPOSE OF INVESTIGATION: To compare the effects of desogestrel (DSG) 150 mcg/ethinyl estradiol (EE) 20 mcg for 21 days followed by either seven days of EE ten mcg (21/7-active) or no treatment (DSG/EE+no Tx) on hemostatic markers. MATERIALS AND METHODS: This was a randomized, multicenter, open-label study that enrolled healthy premenopausal women. Non-inferiority of 21/7-active to DSG/EE+no Tx was determined if the upper limit of the two-sided 95% CI of the mean treatment difference in prothrombin fragment 1+2 (F1+2) over 24 weeks between groups was < 130 pmol/L. RESULTS: 246 subjects (n = 125, 21/7-active; n = 121, DSG/EE+no Tx) comprised the primary analysis. Mean F1+2 levels increased in both 21/7-active and DSG/EE+no Tx regimens (least square [LS] mean changes +45 pmol/L and +56.8 pmol/L, respectively). LS mean treatment difference was -11.8 pmol/L (95% CI: -54.8, 31.2). CONCLUSION: The effect of adding EE ten mcg to the seven-day hormone-free interval of DSG/EE on F1+2 levels was non-inferior to traditional DSG/EE.

Impact on hepatic estrogen-sensitive proteins by a 1-year contraceptive vaginal ring delivering Nestorone® and ethinyl estradiol.

OBJECTIVES: Estrogen-sensitive hepatic proteins were assessed in women using a contraceptive vaginal ring (CVR) delivering 150mcg Nestorone® (NES) and 15mcg ethinyl estradiol (EE). STUDY DESIGN: A substudy of the Contraceptive Clinical Trials Network of the National Institute of Child Health and Human Development enrolled 129 participants, with assessments of factor VIII, fibrinogen, protein S (PS) and sex hormone binding globulin (SHBG). Thirty-six participants had used combined hormonal contraceptives (CHCs) in the cycle preceding first CVR use (recent users) and 70 had no history of recent use (nonusers). RESULTS: Mean values at baseline were within the normal range for all four proteins but were higher for factor VIII, fibrinogen and SHBG and significantly lower for PS in recent compared to nonusers. During NES/EE CVR use, factor VIII, fibrinogen and PS were within the normal range; however, SHBG levels were increased by nearly 100% at Cycle 13. The change from baseline to final evaluation was statistically significant for all proteins in nonusers. The change in recent users was significant for factor VIII at Cycle 6 and for SHBG at Cycles 6 and 13, but not for PS or fibrinogen. CONCLUSION: NES/EE CVR for up to 13cycles was associated with changes from baseline in plasma levels of factor VIII, fibrinogen and PS that were within the normal range, with SHBG levels above the normal range by Cycle 6. Nonusers of CHC before CVR showed wider changes in values versus recent users whose baseline values were increased by previous EE exposure. IMPLICATIONS: Recent use of CHCs demonstrated significant changes in all four measured hepatic proteins at baseline compared to nonusers. Use of the NES/EE CVR further changed these hepatic protein markers, but values remained within the normal range. Prebaseline exposure to estrogen can obscure interpretation of hepatic proteins changes associated with a second CHC.

Reactive oxygen species-quenching and anti-apoptotic effect of polaprezinc on indomethacin-induced small intestinal epithelial cell injury.

BACKGROUND: To protect the small intestine from mucosal injury induced by nonsteroidal anti-inflammatory drugs is one of the critical issues in the field of gastroenterology. Polaprezinc (PZ), a gastric muco-protecting agent, has been widely used for the treatment of gastric ulcer and gastritis for its unique effects, such as its strong reactive oxygen species (ROS)-quenching effect. The aim of this study was to clarify the mechanism by which indomethacin-induced small intestinal mucosal injury occurs, by using a rat intestinal epithelial cell line (RIE-1). In addition, the protective role of PZ and the possible mechanism of its effect on indomethacin-induced small intestinal injury were investigated. METHODS: Cell death was evaluated by methyl thiazolyl tetrazolium (MTT) assay and a double-staining method with Hoechst33342 dye and propidium iodide. Indomethacin-induced ROS production was evaluated by detecting the oxidation of a redox-sensitive fluorogenic probe, RedoxSensor, and the oxidation of cysteine residues of proteins (protein S oxidation). The activation of cytochrome c, smac/DIABLO, and caspase-3 was assessed by western blotting. In some experiments, PZ or its components, L: -carnosine and zinc, were used. RESULTS: We found that indomethacin caused apoptosis in RIE-1 cells in a dose- and time-dependent manner. Indomethacin also induced ROS production and an increase in the protein S oxidation of RIE-1. Pretreatment of RIE-1 with PZ or zinc sulfate, but not L: -carnosine, significantly reduced the indomethacin-induced apoptosis. PZ prevented ROS production and the increase in protein S-oxidation. PZ inhibited indomethacin-induced cytochrome c and smac/DIABLO release and subsequent caspase-3 activation. CONCLUSIONS: The protective effect of PZ on indomethacin-induced small intestinal injury may be dependent on its ROS-quenching effect.

Effect of phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine on the protein C/protein S anticoagulation system.

Phosphatidylserine is known to significantly accelerate the blood coagulation reaction. In a previous communication submitted for publication, we demonstrated that phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine showed effects on the blood coagulation reaction using the factor Xa-prothrombin reaction system, and discuss a new function of membrane phospholipids. The present study examined the role of phospholipids in the blood coagulation regulatory reaction (anticoagulation system), by studying the effects of phospholipids on the protein C/protein S reaction. We have established quantitative methods for measuring activated protein C activity and protein S activity, and used them to measure their activity after the addition of liposomes with different phospholipid compositions. We found that phosphatidylcholine inhibited activated protein C and protein S activities in a dose-dependent manner, as in the factor Xa-prothrombin reaction system. On the other hand, phosphatidylethanolamine and lysophosphatidylcholine showed no effect on activated protein C activity. Phosphatidylethanolamine inhibited and lysophosphatidylcholine accelerated coagulation activity in the factor Xa-prothrombin system, but such effects were not observed in the protein C/protein S reaction system. The coagulation and anticoagulation reactions are exquisitely balanced by thrombin, with a role both as a procoagulant and anticoagulant. Therefore, it is understandable that phosphatidylethanolamine and lysophosphatidylcholine show different effects in the factor Xa-prothrombin and protein C/protein S reaction systems. It appears that coagulation and anticoagulation reactions are co-ordinated and controlled by changes in phospholipid composition of the cellular membrane where the coagulation reaction takes place.

Effect of intravenous N-acetylcysteine infusion on haemostatic parameters in healthy subjects.

BACKGROUND AND AIMS: N-acetylcysteine is used to treat paracetamol overdose but depresses the activity of plasma coagulation factors II, VII, and X, which are often used to assess liver injury. The aim of this study was to investigate the effect of N-acetylcysteine on haemostasis in normal volunteers. METHODS: Haemostatic parameters in 10 healthy subjects were analysed before and following intravenous infusion of therapeutic doses of N-acetylcysteine, as well as in vitro. RESULTS: N-acetylcysteine induced significant decreases in plasma levels of vitamin K dependent haemostatic proteins in vivo, being maximal at one hour following the start of infusion, with maximal decreases from 1.00 to 0.73 (0.67-0.79) (mean (95% confidence interval)), 0.66 (0.58-0.73), 0.81 (0.73-0.90), 0.64 (0.57-0.70), 0.74 (0.65-0.82), and 0.61 (0.54-0.67) for factor II, VII, IX, and X activities, protein C activity, and free protein S reactivity, respectively. These data suggest that N-acetylcysteine induces protein modifications affecting activity. Five subjects developed an adverse reaction to infusion of N-acetylcysteine and these were associated with a rapid increase in levels of factor VIII and its carrier protein von Willebrand factor (vWf) from 1.0 to 1.85 (1.08-2.62) and 1.77 (0.83-2.71), respectively, which suggests that the allergic reaction induced release of vWf from endothelial cells. N-acetylcysteine did not affect factor VIII or vWf in subjects without adverse reactions, and nor did it affect factor V or antithrombin in any of the subjects. CONCLUSION: Therapeutic doses of N-acetylcysteine cause abnormal haemostatic activity, and this should be taken into account when using haemostatic function tests as an indicator of hepatic injury.

Effects of raloxifene therapy on the anticoagulant system in postmenopausal women.

BACKGROUND: Raloxifene therapy is associated with a three-fold increase in the risk for venous thromboembolism; however, its effects on the hemostatic system in postmenopausal women have not been well defined. OBJECTIVE: To determine the effects of raloxifene therapy on the levels of natural anticoagulant proteins in postmenopausal women. METHODS: Sixteen healthy postmenopausal women were enrolled in this prospective longitudinal study. The patients were treated with raloxifene hydrochloride (60 mg/day) for a period of 6 months. Antithrombin and protein C activities and protein S antigen levels were measured in all users at baseline, and after 1, 3 and 6 months of treatment. Statistical analysis included one-way analysis of variance (ANOVA) and the Bonferroni test for multiple comparisons among the study periods. RESULTS: Statistically significant 5.1% and 6.5% reductions of plasma antithrombin activity were observed at 3 and 6 months of therapy, respectively (p < 0.05). Compared with baseline, raloxifene did not significantly affect protein C activity or protein S level. CONCLUSIONS: The results of this prospective study show for the first time that raloxifene use is associated with a significant reduction in plasma antithrombin activity. This effect may contribute to a procoagulant state and partly explain the increased risk of venous thromboembolism in raloxifene users.

Coagulation factor content of solvent/detergent plasma compared with fresh frozen plasma.

Solvent/detergent (S/D) plasma is being increasingly widely used in clinical practice, as it carries significantly lower risk of lipid-enveloped viral transmission than standard fresh frozen plasma (FFP). However, previous reports have suggested that S/D processing might influence the coagulation factor content of plasma. We have investigated this question by measuring procoagulant factors (fibrinogen, factor V and factor VIII), anticoagulant factors (protein C and protein S) and routine coagulation screening tests (prothrombin time and activated partial thromboplastin time) in 48 single-donor units of FFP, and in 16 units of S/D plasma (Octaplas). All routine coagulation screening tests, factor VII and protein C levels were within the normal reference range for both S/D plasma and FFP. However, we found significant reductions in factor V (31%), factor VIII (28%) and protein S (50%) in S/D plasma. The observed quantitative differences in coagulation factor levels may be further exacerbated by the lower volume of solvent detergent plasma units (200 ml) compared with units of standard fresh frozen plasma (250 ml). These findings are of potential clinical significance, particularly in those patients with liver disease, constitutional factor V deficiency and congenital or acquired protein S deficiency.

Reversal of protein S-glutathiolation by glutaredoxin in the retinal pigment epithelium.

Protein cysteines can serve both sensory and activation roles in the regulation of protein function. The modulation of mixed disulfides with glutathione may promise to be a broad mechanism of redox signalling. Using both protein extract and intact RPE cells, we have generated covalent adduction of glutathione to protein cysteines and further show that glutaredoxin (Grx-1) is able to remove glutathione from protein S-glutathiolated substrates. Our data demonstrate that glutathione can modify a wide range of RPE proteins in intact cells, but that the reversal of this process--deglutathiolation and thiol bond restoration--may require a specific catalytic reaction with glutaredoxin. More generally, our experiments support the hypothesis that glutathione can non-specifically become adducted to protein cysteines during oxidative stress, but that the specific, functional reconstitution of protein thiols depends on recognition by an oxidoreductase such as glutaredoxin. This concept offers the idea that redox signalling involves both adduction of a non-specific non-protein reducing equivalent such as glutathione and specific protein based removal by glutaredoxin.

Prothrombotic effects and clinical implications of third-generation oral contraceptives use.

Although the use of oral contraceptives has been frequently associated with an increased risk of thromboembolic events, definitive prothrombotic mechanisms have not so far been fully elucidated. The aim of our investigation was the evaluation of the activities of antithrombin, protein C, protein S and the resistance to activated protein C in 137 healthy users of third-generation oral contraceptives and in 170 healthy women who were not consuming oral contraceptives. Women on oral contraceptives showed a marked prothrombotic pattern, characterized by reduced activities of antithrombin and protein S, and increased resistance to activated protein C. Nearby 50% of oral contraceptive users displayed activities of protein S below the lower value of the reference range (controls, 10%; P < 0.001). No significant differences were observed between two progestagens (desogestrel or gestodene) on the coagulation parameters tested. We believe that, due to the adverse effect on haemostasis, the administration of third-generation oral contraceptives should be carefully considered in women carrying prothrombotic abnormalities.

C4b-binding protein inhibits the factor V-dependent but not the factor V-independent cofactor activity of protein S in the activated protein C-mediated inactivation of factor VIIIa.

Activated protein C (APC) is an important inactivator of coagulation factors Va and VIIIa. In the inactivation of factors Va and VIIIa, protein S serves as a cofactor to APC. Protein S can bind to C4b-binding protein (C4BP), and thereby loses its cofactor activity to APC. By modulating free protein S levels, C4BP is an important regulator of protein S cofactor activity. In the factor VIIIa inactivation, protein S and factor V act as synergistic cofactors to APC. We investigated the effect of C4BP on both the factor V-independent and factor V-dependent cofactor activity of protein S in the factor VIIIa inactivation using a purified system. Protein S increased the APC-mediated inactivation of factor VIIIa to 60% and in synergy with protein S, factor V at equimolar concentrations increased this effect further to 90%. The protein S/factor V synergistic effect was inhibited by preincubation of protein S and factor V with a four-fold molar excess of C4BP. However, C4BP did not inhibit the factor V-independent protein S cofactor activity in the purified system whereas it inhibited the cofactor activity in plasma. We conclude that C4BP-bound protein S retains its cofactor activity to APC in the factor VIIIa inactivation.

[Effects of second and third generation oral contraceptives on hemostasis]. / Effecten van orale anticonceptiva van de tweede en de derde generatie op de hemostase.

Use of oral contraceptives induce changes in haemostatic parameters: changes occur in the procoagulant, anticoagulant, and fibrinolytic systems. The increased risk of venous thromboembolism with use of third, as compared with second generation oral contraceptives, found in epidemiological studies, has stimulated new research in haemostatic changes induced by both generations of oral contraceptives. A randomized crossover study showed that use of the third generation pill caused a greater increase of factor VII and prothrombin and a more pronounced decrease of factor V than the second generation pill. Acquired resistance to activated protein C (APC) was induced more strongly by preparations of the third than by those of the second generation. The concentration of protein S decreased markedly exclusively during use of the third generation pill, while it did not change during use of the second generation pill. The oral contraception-related effects on the anticoagulant system strongly resemble those of some forms of hereditary thrombophilia. If a woman with hereditary APC resistance (caused by factor V Leiden) uses oral contraceptives as well, and especially when she uses those of the third generation, she is subject to a considerable increase of the risk of venous thrombosis and becomes even more resistant to the anticoagulant action of protein C. In view of the epidemiological backgrounds of the difference in risk of thrombosis between second and third generation contraceptives, the second generation pill is recommended as the first choice for oral contraception.

Correlation between different intensities of anti-vitamin K treatment and coagulation parameters.

In order to study the effect of different intensities of anti-vitamin K treatment on coagulation parameters, 23 patients with venous thromboembolism were given, after the initial treatment period, warfarin at doses giving an International Normalised Ratio of 1.3-2.0 for 4 weeks, and of 1.1-1.3 for another 4 weeks. Blood samples were taken at the end of each of these periods and 4 weeks after the end of warfarin treatment. The vitamin K-dependent coagulation factors VII, IX, and X, as well as the inhibitor protein C and its cofactor protein S, all showed a highly significant correlation with treatment intensity. This was to some extent also true for the coagulation activation markers, prothrombin fragment 1+2 and thrombin-antithrombin complex. Ratios of pro- and anticoagulant factors in some instances showed a decrease at therapeutical (International Normalised Ratio) levels, and also sometimes with reduced warfarin treatment intensity. Taken together, our results encourage further research addressing issues of varying treatment intensity with warfarin and alternative methods for monitoring of anti-vitamin K treatment.

Evaluation of a new screening assay ProC Global for identification of defects in the protein C/protein S anticoagulant pathway.

In the present study a new assay, ProC Global, globally estimating the activity of the main plasma components of anticoagulant protein C/protein S pathway, was evaluated with respect to test characteristics and its sensitivity in the detection of deficiency states of protein C and protein S and of increased aPCR. In the ProC Global assay procedure protein C is activated in patient's plasma by an activator reagent (venom from agkistrodon contortrix). The extent of the prolongation of a sample's aPTT, caused by the activation of protein C, is taken as a measure for its anticoagulant capacity. Ninety-eight patients with one of the above mentioned defects were investigated. Decreased plasma protein C activity and increased aPCR were detected with a sensitivity of 1.0, while only 11 of 14 patients with decreased levels of free protein S antigen showed abnormal results in the ProC Global assay (sensitivity = 0.79). The test can be used in heparinized samples up to 1.0 anti Xa U/ml heparin (UFH and LMWH). When samples from patients on oral anticoagulant treatment are prediluted with factor V deficient plasma the test is sensitive for increased aPCR.

Effects of long-term treatment with recombinant human erythropoietin on physiologic inhibitors of coagulation.

Recombinant human erythropoietin (rHu-EPO) in the treatment of renal anemia might predispose to an increased risk of thrombotic complications. In an attempt to comprehend the involvement of the physiologic inhibitors of coagulation in this process, we studied 2 groups of hemodialysis patients. Group I included 21 patients receiving a starting dose of 90 IU/kg/week s.c., and group II included 17 patients without rHu-EPO. The following coagulation tests were performed before rHu-EPO treatment, and after 1, 6 and 12 months: prothrombin time; activated partial fistula thromboplastin time; fibrinogen; plasminogen activity; antithrombin III activity; protein C activity; total and free protein S antigens, and C4b binding protein. Only the latter three parameters were changed in group 1, while high baseline levels of protein S antigens were found in both groups. A decrease in total and free protein S was observed within 1 month of treatment. At the 6th month total protein S returned to near pretreatment values, whereas a significant fall in free protein S (p = 0.007) was observed. All three parameters returned to near baseline values by 12 months. These results suggest that protein S activity can be altered at the beginning of EPO therapy, a change that under favoring circumstances might contribute to the thrombotic events reported during the early phase of rHu-EPO treatment.

[Activity of endogenous anticoagulants (antithrombin III, protein C and protein S) in women after menopause treated with hormone replacement therapy (transdermal 17 beta-OH estradiol and oral medroxyprogesterone acetate)]. / Aktywnosc endogennych antykoagulantów (antytrombiny III, bialka C i bialka S) u kobiet po menopauzie leczonych hormonalna terapia zastepcza (17 beta-OH estradiol przezskórnie i octan medroksyprogesteronu doustnie).

The influence of hormone replacement therapy (HRT) on coagulation system has not been completely explained yet. We measured plasma activities of antithrombin III, protein C and protein S before and after 6 and 12 months of HRT in 48 postmenopausal women. HRT consisted of transdermal estradiol and orally medroxyprogesterone acetate. We observed no statistically significant differences in plasma levels of the above endogenous anticoagulants before and during treatment.

Influence of long-term continuous intravenous administration of pentoxifylline on endothelial-related coagulation in critically ill patients.

OBJECTIVE: To determine the influence of pentoxifylline on endothelial-associated coagulation. DESIGN: Prospective, randomized, placebo-controlled study. SETTING: A surgical intensive care unit of a university hospital. PATIENTS: Consecutive patients (n = 60) with trauma or sepsis secondary to major (nontrauma) surgery. All patients received controlled mechanical ventilation. INTERVENTIONS: According to a randomized sequence, the patients either received pentoxifylline continuously over 5 days (1.5 mg/kg/hr iv) (trauma-pentoxifylline group [n = 15], sepsis-pentoxifylline group [n=15] or saline solution as placebo (trauma-control group [n = 15], sepsis-control group [n = 15]. MEASUREMENTS AND MAIN RESULTS: In addition to the standard coagulation screen, thrombomodulin, protein C, (free) protein S, and thrombin-antithrombin plasma concentrations were measured by enzyme-linked immunosorbent assays. Intensive care therapy, hemodynamics, and changes of Acute Physiology and Chronic Health Evaluation II score were comparable for pentoxifylline-treated and nontreated patients. An average dose of 2.5 g/day of pentoxifylline (range 2.2 to 2.9) was infused into the pentoxifylline-treated patients. At baseline, plasma thrombomodulin concentrations were higher in the septic patients than in the trauma patients. Thrombomodulin plasma concentrations increased significantly more in the control patients (trauma: from 38.9 +/- 10.5 to 59.9 +/- 10.1 ng/mL; sepsis: from 49.7 +/- 12.1 to 72.3 +/- 11.2 ng/mL) than in the pentoxifylline-treated patients (trauma: from 37.9 +/- 11.9 to 50.2 +/- 9.2 ng/mL; sepsis from 51.9 +/- 10.1 to 63.3 +/- 10.2). Starting from similar baseline values, protein C concentration increased significantly more in the sepsis-pentoxifylline patients (from 52.0 +/- 11.1% to 69.1 +/- 11.1%) than in the untreated septic patients (from 50.1 +/- 10.0% to 52.5 +/- 9.5%). There were no significant differences between the pentoxifylline-treated and nontreated groups for protein S and thrombin-antithrombin concentrations. Standard coagulation parameters (fibrinogen, activated partial thromboplastin time, antithrombin III) did not differ between these groups either. CONCLUSIONS: Continuous intravenous administration of pentoxifylline for 5 days beneficially influenced the thrombomodulin/protein C/protein S system in both the trauma and septic patients.

TNF-alpha suppresses IL-6 upregulation of protein S in HepG-2 hepatoma cells.

The pathogenesis of disseminated intravascular coagulation (DIC) has, in part, been attributed to the impairment of the natural anticoagulant protein C/protein S pathway. DIC, which frequently occurs during sepsis, has been linked to cytokines that can induce or modulate procoagulant activity. Three of these cytokines, IL-1 alpha, IL-6, and TNF-alpha have been reported to be increased in the early stages of sepsis. In the present study, we have stimulated HepG-2 hepatoma cell cultures with recombinant human IL-1 alpha, IL-6, TNF-alpha, and oncostatin M (OSM). The results demonstrated that TNF-alpha, and to a lesser degree, IL-1 alpha, could significantly suppress IL-6 upregulation of protein S, whereas the effects of OSM was only suppressed by the combination of IL-1 alpha and TNF-alpha. The combination of IL-1 alpha and TNF-alpha also suppressed protein S production below that of control or basal levels. These results indicate that IL-1 alpha and TNF-alpha may play important regulatory roles in coagulation.