BACKGROUND

Thrombin promotes angiogenesis and cell proliferation in cancer. Whether thrombin turnover influences cancer incidence is unknown.

OBJECTIVE

To explore the relation between the status of the coagulant pathway and cancer incidence by population survey.

METHODS

Of 4,009 middle-aged men clinically free of malignancy, 305<em>2</em> (76.<em>1</em>%) were recruited. Measurements of hemostatic status were made annually for 4 years, and follow-up for morbidity and mortality was maintained thereafter. Persistent activation of the coagulant pathway was diagnosed when <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> and fibrinopeptide A concentrations exceeded the upper quartiles of the population distribution in two consecutive annual examinations. Cancer incidence rates in men developing persistent activation (taking the time of onset of activation as baseline) were compared with those in men remaining free of this condition.

RESULTS

Persistent activation of the hemostatic pathway was a distinct entity found in <em>1</em><em>1</em><em>1</em> men [43 expected by chance alone (P <0.00<em>1</em>)], and associated with activation throughout the coagulation pathway. Total mortality (/<em>1</em>000 person-years) was higher in those with persistent activation than in others (<em>1</em>7.<em>1</em> and 9.7, respectively, P=0.0<em>1</em>5), owing to a higher mortality from all cancers (<em>1</em><em>1</em>.3 and 5.<em>1</em>, respectively, P=0.0<em>1</em>), due in turn largely to a higher mortality from cancers of the digestive tract (6.3 and <em>1</em>.9, respectively, P=0.004). Trends were similar for non-fatal cancers.

CONCLUSIONS

Persistent activation of the coagulant pathway plays a role in the preclinical phase of cancer and is associated with an increased incidence of clinical malignancy, especially of the digestive tract.

BACKGROUND

Previous studies have suggested that statins exert beneficial effects beyond their favorable lipid lowering effect. Particularly, the modification of thrombus formation and degradation, alteration in inflammatory response, plaque stabilization and improved endothelial function are thought to be responsible for additional reduction of morbidity and mortality due to cardiovascular events. To date, however, it is still unclear whether these effects are elicited by all statins.

RESULTS

We set out to compare in a controlled, randomized, double-blind study design the effects of almost equieffective cholesterol lowering doses of three chemically and pharmacokinetically different statins (atorvastatin, simvastatin, pravastatin) on hemostatic and inflammatory markers in 99 hypercholesterolemic patients. At entry and 3 months after onset of statin therapy plasma cholesterol and von Willebrand factor antigen (vWf-Ag), fibrinogen, d-dimer, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>.<em>2</em>) and C-reactive protein (CRP) were measured. The effect on plasma values of F<em>1</em>.<em>2</em>, vWf-Ag, d-dimer and CRP was not significantly different between the three treatment groups. The effect of simvastatin on fibrinogen (p = 0.005) was more pronounced than the effects of atorvastatin (p = 0.48 n.s.) and pravastatin (p = 0.<em>1</em>5 n.s.). Plasma levels of F<em>1</em>.<em>2</em> and vWf-Ag (when data of all statins were pooled) were significantly reduced by 7% and <em>1</em>0% versus baseline, respectively. No significant reduction was observed for d-dimer (p = 0.<em>2</em>6) and CRP (p = 0.5). Total plasma cholesterol levels decreased significantly (p < 0.000<em>1</em> in all groups) between <em>2</em><em>2</em>% and <em>2</em>9% compared to baseline.

CONCLUSIONS

The present study shows similar short-term (3 months) effects of atorvastatin, simvastatin and pravastatin on selected hemostatic and inflammatory parameters in plasma in patients with hypercholesterolemia. Thus, chemical and pharmacological differences between statins appear to exert no major influence on these parameters.

The effects of chronic cigarette smoking on the coagulation system were examined in <em>2</em>964 men aged 50 to 6<em>1</em> years and clinically free of cardiovascular disease. Factor VII activity (VIIc), factor VII antigen (VIIag), <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>.<em>2</em>), fibrinopeptide A (FPA) and fibrinogen were measured in all participants, and activated factor VII (VIIa), factor IX activation peptide (IX pep) and factor X activation peptide (X pep) in a large sub-sample. The levels of all indices except FPA differed significantly between non-smokers, ex-smokers and current smokers. After adjustment for other conventional cardiovascular risk factors, mean VIIc was raised slightly by 3% in ex-smokers and current smokers as compared with non-smokers, owing to increases in VIIa and VIIag. Plasma IX pep, X pep, F<em>1</em>.<em>2</em> and fibrinogen concentration were highest in current smokers, intermediate in ex-smokers and lowest in non-smokers. These findings accord with the increased risk of arterial thrombosis in smokers.

BACKGROUND

Lipopolysaccharide (LPS) is a major trigger of sepsis-induced disseminated intravascular coagulation (DIC) via the tissue factor (TF)/factor VIIa-dependent pathway of coagulation. Experimental endotoxemia has been used repeatedly to explore this complex pathophysiology, but little is known about the effects of clinically used anticoagulants in this setting. Therefore, we compared with placebo the effects of unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) on LPS-induced coagulation.

RESULTS

In a randomized, double-blind, placebo-controlled trial, 30 healthy male volunteers received LPS <em>2</em> ng/kg IV followed by a bolus-primed continuous infusion of UFH, LMWH, or placebo. In the placebo group, activation of coagulation caused marked increases in plasma levels of <em>prothrombin</em> <em>fragment</em> F(<em>1</em>+<em>2</em>) (P<0.0<em>1</em>) and polymerized soluble fibrin, termed thrombus precursor protein (TpP; P<0.0<em>1</em>); TF-positive monocytes doubled in response to LPS, whereas levels of activated factor VII slightly decreased and levels of TF pathway inhibitor remained unchanged. UFH and LMWH markedly decreased activation of coagulation caused by LPS, as F(<em>1</em>+<em>2</em>) and TpP levels only slightly increased; TF expression on monocytes was also markedly reduced by UFH. TF pathway inhibitor values increased after either heparin infusion (P<0.0<em>1</em>). Concomitantly, factor VIIa levels dropped by >50% at 50 minutes after initiation of either heparin infusion (P<0.0<em>1</em>).

CONCLUSIONS

This experimental model proved the anticoagulatory potency of UFH and LMWH in the initial phase of experimental LPS-induced coagulation. Successful inhibition of thrombin generation also translates into blunted activation of coagulation factors upstream and downstream of thrombin.

This study was set up to examine whether an influenza vaccine or an influenza vaccine in combination with pneumococcal vaccine can be used as a model to study responses to mild stimulation of the inflammatory system. In this study, <em>1</em>9 subjects received the influenza vaccine, <em>2</em>0 subjects the combination of influenza and pneumococcal vaccine. CRP and <em>prothrombin</em> <em>fragment</em> <em>1</em> and <em>2</em> (F<em>1</em>+<em>2</em>) were measured at baseline, and two times after vaccination. Influenza vaccination increased CRP by 0.<em>2</em>0 mg/L, and influenza in combination with pneumococcal vaccine increased CRP by 0.60 mg/L. F<em>1</em>+<em>2</em> increased 0.<em>1</em>5 nmol/L after the combined vaccination; an increase in response to the influenza vaccination was not statistically significant. Our findings show that the influenza vaccine alone as well as the combination of the influenza and pneumococcal vaccine increases CRP-levels with a peak <em>2</em> days after vaccination.

Atrial fibrillation (AF) is associated with hemostatic abnormalities and increased risk of thrombotic cardiovascular events even during oral anticoagulant therapy (OAT). The aim of our study was to evaluate the predictive value of hemostatic markers for the risk of major cardiovascular events during OAT. The study group comprised <em>1</em><em>1</em>3 patients with chronic AF (70.<em>2</em> +/- 5.4 years old, 60% men), referred for OAT. Established clinical risk factors and levels of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), thrombin-antithrombin complexes (TAT), D-dimer, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor <em>1</em> (PAI-<em>1</em>) antigen and activity, before and during OAT (after 3.9 +/- 0.7 months; INR <em>2</em>.57 +/- 0.57) were determined. In all patients OAT significantly suppressed levels of F<em>1</em>+<em>2</em> by 67%,TAT by 30% and D-dimer by 48% (all p <0.00<em>1</em>). During an average follow-up of 44 months <em>2</em><em>2</em>/<em>1</em><em>1</em><em>1</em> (<em>2</em>0%) patients suffered a combined cardiovascular event (stroke, myocardial infarction, peripheral vascular occlusion or vascular death). Patients with cardiovascular events were significantly older, had more frequent heart failure/systolic dysfunction and had significantly increased levels of D-dimer at entry (63 vs 39 ng/mL, p = 0.005) and during OAT (33 vs <em>1</em>8 ng/mL, p = 0.00<em>2</em>), and of t-PA antigen at entry (<em>1</em>4.3 vs <em>1</em>0.9 ng/mL, p = 0.0<em>2</em>) and during OAT (<em>1</em>5.0 vs <em>1</em><em>1</em>.<em>2</em> ng/mL, p = 0.05) (all values are medians). In multivariate Cox proportional hazard models, heart failure/systolic dysfunction (hazard ratio <em>2</em>.9<em>1</em>; 95% CI <em>1</em>.<em>1</em>7-7.<em>2</em>6; p = 0.0<em>2</em>), high levels of D-dimer on OAT (top vs. lower two quartiles) (hazard ratio 4.78, 95% CI <em>1</em>.39-<em>1</em>6.4<em>1</em>; p = 0.0<em>1</em>) and t-PA antigen levels (continuous variable) (hazard ratio <em>1</em>.09; 95% CI <em>1</em>.0<em>1</em>-<em>1</em>.<em>1</em>7; p = 0.0<em>2</em>) were significantly associated with combined cardiovascular events. In conclusion, high levels of D-dimer and t-PA antigen during OAT are significant predictors of combined cardiovascular events in AF patients and, on this basis, could be useful additional markers of cardiovascular risk in such patients.

A previous study has shown that simvastatin reduces in vivo clotting activation and monocyte tissue factor (TF) expression. This effect, however, was only in part attributable to the reduction of serum cholesterol, suggesting that more than one mechanism may be involved. Furthermore, it was not investigated if the inhibition of clotting activation was dependent upon the reduced expression of monocyte TF. In order to assess if simvastatin directly affects clotting activation, we developed an in vitro method in which clotting system is activated by monocytes stimulated with LPS. Monocytes were prepared from blood taken from healthy volunteers or patients with hypercholesterolemia and incubated with heparinized plasma plus either simvastatin (0.0<em>1</em>-<em>1</em>0 microM) or medium as control. Samples were then stimulated with LPS (4 microg/ml) and after 6 h the rate of thrombin generation, assessed by <em>prothrombin</em> <em>fragment</em> (F) <em>1</em>+<em>2</em>, was measured. In separate experiments, we measured the expression of TF by monocytes which were incubated with simvastatin and then stimulated with LPS. The study showed that compared to control, LPS-stimulated monocytes induced abundant formation of F<em>1</em>+<em>2</em>, which was inhibited by simvastatin in a dose-dependent manner. Simvastatin also inhibited dose dependently the monocyte expression of TF. This study suggests that simvastatin inhibits the rate of thrombin generation by directly interfering with the monocyte expression of TF.

BACKGROUND

Activation markers of coagulation and fibrinolysis are increased in individuals at risk of coronary-artery disease and other thrombotic disorders--a condition defined as the prethrombotic state. We aimed to find out the extent to which the prethrombotic state is determined by genetic factors.

METHODS

We analysed concentrations of <em>prothrombin</em>, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, thrombin-antithrombin complex, crosslinked fibrin degradation product D-dimer, and thrombin-activatable fibrinolysis inhibitor by ELISA in <em>1</em><em>1</em>8 monozygotic and <em>1</em><em>1</em><em>2</em> dizygotic unselected female twins aged <em>2</em><em>1</em>-73 years from the St Thomas' UK Adult Twin Registry. We used quantitative genetic-model fitting to estimate heritability.

RESULTS

We found significant heritabilities in concentrations of the activation markers in plasma. Genetic factors contributed 45, 40, and 65% of the variation in concentrations of <em>fragment</em> <em>1</em>+<em>2</em>, thrombin-antithrombin complex, and D-dimer, respectively. Age was important only in <em>fragment</em> <em>1</em>+<em>2</em> concentrations, in which it accounted for <em>1</em><em>2</em>% of the variation. The remaining variation could be attributed to unique environmental factors. Variation in concentrations of precursor <em>prothrombin</em> in plasma was determined by 57% heritability, and that of zymogen thrombin-activatable fibrinolysis inhibitor showed a very strong genetic component (8<em>2</em>%).

CONCLUSIONS

The activation mechanisms of the coagulation and fibrinolytic systems, and therefore the prethrombotic state, are controlled to a substantial degree by genetic factors. Genes influencing activation of haemostasis are likely to be an important component of the overall thrombotic tendency in the general population.

OBJECTIVE

To establish the plasma evolution of <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em> (F <em>1</em>+<em>2</em>), thrombin-antithrombin III complexes (TAT), fibrin <em>fragment</em> D-Dimers (DD), von Willebrand factor antigen (vWf), Type <em>1</em> plasminogen activator inhibitor antigen (PAI) and blood platelet count during normal pregnancy and to compare these values with those obtained in hypertensive or pre-eclamptic pregnancies.

METHODS

Cross-sectional study.

METHODS

Forty-seven healthy pregnant women with gestational age ranging between 5 and 40 weeks, and fourteen women with gestational age ranging between <em>2</em>5 and 38 weeks presenting with either gestational hypertension (n = 4) or pre-eclampsia (n = <em>1</em>0). Numbers of nulliparous women in the control, hypertension and pre-eclampsia groups were <em>1</em>3/47 (<em>2</em>8%), <em>1</em>/4 (<em>2</em>5%) and 9/<em>1</em>0 (90%), respectively.

RESULTS

All six markers increased with gestational age in normal pregnant women (P < 0.0<em>1</em>). Using the upper limit of 95% prediction interval obtained from regression curves as normality threshold, TAT showed the best sensitivity (7<em>1</em>% vs < 30% for F<em>1</em>+<em>2</em>, DD, vWf, PAI and platelet count).

CONCLUSIONS

TAT appears to be an interesting marker for detecting haemostatic system alterations in pregnancies complicated by hypertension or pre-eclampsia. A large prospective study to determine its clinical usefulness for such complicated pregnancies is currently in progress.

The conversion of <em>prothrombin</em> to thrombin requires the cleavage of two peptide bonds and is catalyzed by the <em>prothrombin</em>ase complex composed of factors Xa and Va assembled on a membrane surface. Presteady-state kinetic studies of the effects of membranes on the proteolytic reaction were undertaken using model membranes composed of phosphatidylcholine and phosphatidylserine (PCPS). The concentration of PCPS was varied to alter the concentration of free phospholipid available for substrate binding without influencing the concentration of membrane-assembled <em>prothrombin</em>ase. In fluorescence stopped-flow measurements, increasing concentrations of PCPS resulted in an increase in the rate of product formation. Assessment of bond cleavage by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following rapid chemical quench using <em>1</em><em>2</em>5I-<em>prothrombin</em> revealed that the activation reaction proceeded through the ordered cleavage at Arg3<em>2</em>3-Ile3<em>2</em>4 followed by cleavage at Art<em>2</em>74-Thr<em>2</em>75 at all concentrations of PCPS. Increasing the PCPS concentration resulted in a large increase in the Arg3<em>2</em>3-Ile3<em>2</em>4 cleavage reaction with a much smaller effect on the subsequent cleavage at Arg<em>2</em>74-Thr<em>2</em>75, thereby leading to an increase in the extent of accumulation of the intermediate, meizothrombin. Fluorescence stopped-flow and rapid chemical quench measurements were also conducted using prethrombin <em>2</em> plus <em>fragment</em> <em>1</em>.<em>2</em> or meizothrombin as substrates to assess the influence of PCPS on the individual cleavage reactions. The rate of cleavage at Arg3<em>2</em>3-Ile3<em>2</em>4 by <em>prothrombin</em>ase was increased approximately 60-fold with increasing PCPS, whereas the cleavage at Arg<em>2</em>74-Thr<em>2</em>75 was increased by a factor of approximately 5. These differential effects of PCPS on the two cleavage reactions adequately explain changes in the extent of accumulation of meizothrombin during <em>prothrombin</em> activation. Proteolytic removal of the membrane binding <em>fragment</em> <em>1</em> domain of the substrates, meizothrombin and prethrombin <em>2</em>-<em>fragment</em> <em>1</em>.<em>2</em>, had no effect on the cleavage at Arg<em>2</em>74-Thr<em>2</em>75 at saturating PCPS but completely eliminated the membrane-dependent rate enhancement for cleavage at Arg3<em>2</em>3-Ile3<em>2</em>4. Thus, membrane binding by the substrate is essential for the first cleavage reaction at Arg3<em>2</em>3-Ile3<em>2</em>4, which leads to the conversion of <em>prothrombin</em> to meizothrombin. In contrast, the substrate-membrane interaction mediated by the <em>fragment</em> <em>1</em> domain has no detectable effect on the second cleavage reaction at Arg<em>2</em>74-Thr<em>2</em>75 which is required for the conversion of meizothrombin to thrombin.

Staphylocoagulase (SC) is a potent nonproteolytic <em>prothrombin</em> (ProT) activator and the prototype of a newly established zymogen activator and adhesion protein family. The staphylocoagulase <em>fragment</em> containing residues <em>1</em>-3<em>2</em>5 (SC-(<em>1</em>-3<em>2</em>5)) represents a new type of nonproteolytic activator with a unique fold consisting of two three-helix bundle domains. The N-terminal, domain <em>1</em> of SC (D<em>1</em>, residues <em>1</em>-<em>1</em>46) interacts with the <em>1</em>48 loop of thrombin and prethrombin <em>2</em> and the south rim of the catalytic site, whereas domain <em>2</em> of SC (D<em>2</em>, residues <em>1</em>47-3<em>2</em>5) occupies (pro)exosite I, the fibrinogen (Fbg) recognition exosite. Reversible conformational activation of ProT by SC-(<em>1</em>-3<em>2</em>5) was used to create novel analogs of ProT covalently labeled at the catalytic site with fluorescence probes. Analogs selected from screening <em>1</em>0 such derivatives were used to characterize quantitatively equilibrium binding of SC-(<em>1</em>-3<em>2</em>5) to ProT, competitive binding with native ProT, and SC domain interactions. The results support the conclusion that SC-(<em>1</em>-3<em>2</em>5) binds to a single site on fluorescein-labeled and native ProT with indistinguishable dissociation constants of <em>1</em>7-7<em>2</em> pM. The results obtained for isolated SC domains indicate that D<em>2</em> binds ProT with approximately <em>1</em>30-fold greater affinity than D<em>1</em>, yet D<em>1</em> binding accounts for the majority of the fluorescence enhancement that accompanies SC-(<em>1</em>-3<em>2</em>5) binding. The SC-(<em>1</em>-3<em>2</em>5).(pro)thrombin complexes and free thrombin showed little difference in substrate specificity for tripeptide substrates or with their natural substrate, Fbg. Lack of a significant effect of blockage of (pro)exosite I of (pro)thrombin by SC-(<em>1</em>-3<em>2</em>5) on Fbg cleavage indicates that a new Fbg substrate recognition exosite is expressed on the SC-(<em>1</em>-3<em>2</em>5).(pro)thrombin complexes. Our results provide new insight into the mechanism that mediates zymogen activation by this prototypical bacterial activator.

BACKGROUND

Apixaban, a factor Xa (FXa) inhibitor, is a new oral anticoagulant for stroke prevention in atrial fibrillation (AF). However, little is known about its efficacy and safety as a periprocedural anticoagulant therapy for patients who had undergone catheter ablation (CA) for AF.

RESULTS

We evaluated 34<em>2</em> consecutive patients who underwent CA for AF between April <em>2</em>0<em>1</em>3 and March <em>2</em>0<em>1</em>4 and received apixaban (n = <em>1</em>05) and warfarin (n = <em>2</em>37) for uninterrupted periprocedural anticoagulation. We retrospectively investigated the occurrence of bleeding and thromboembolic complications during the procedural period and compared them between the apixaban group (AG) and warfarin group (WG). Thromboembolic complications occurred in one (0.4%) patient in the WG. Major and minor bleeding complications occurred in one (<em>1</em>%) and four (4%) patients in the AG, and three (<em>1</em>%) and <em>1</em><em>2</em> (5%) patients in the WG. No significant difference in complications was observed between the AG and WG. Of importance, adverse event rates did not differ between the two groups after adjusting by a propensity score analysis. In preoperative tests of blood coagulation, there were significant differences in the <em>prothrombin</em> time, activated partial thromboplastin time, FXa activity, and <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em>+<em>2</em>) levels between the AG and WG.

CONCLUSIONS

The use of apixaban during the periprocedural period of AF ablation seemed as efficacious and safe as warfarin.

OBJECTIVE

The purpose of this study was to examine the association between hemostatic activation and stroke severity, and to provide data on hemostatic variables in acute ischemic stroke.

METHODS

The patient material comprised 76 consecutive patients with acute ischemic stroke (median <em>1</em>6 h, interquartile range 3-48). Levels of hemostatic variables were determined in blood samples collected on the day of hospitalization. Stroke severity was assessed on admission by the Oxfordshire Community Stroke Project (OCSP) classification, and on discharge (median 9 days, interquartile range 6-<em>1</em>4) by Barthel Index (BI, scores 0-50, 55-90, or 95-<em>1</em>00) and modified Rankin Scale (mRS, scores 0-<em>1</em> or <em>2</em>-6). Associations were assessed by multiple linear regression analyses.

RESULTS

Levels of the fibrin degradation product D-Dimer and the activation peptide <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>) were linearly related to stroke severity, whether assessed on admission (P = .00<em>1</em> and.03, respectively, for the OCSP classification), or on discharge (P = .009 and.43, respectively, for BI; and.00<em>1</em> and.05, respectively, for mRS). High levels of D-Dimer and F(<em>1</em> + <em>2</em>), as well as low levels of antithrombin and protein C were also present in patients with a presumed embolic source, and low antithrombin or protein C was borderline significantly associated with atrial fibrillation (P = .07<em>2</em> and.058, respectively). Low levels of protein C or protein S, and the presence of antiphospholipid antibodies, including lupus anticoagulant (LA), was detected in <em>1</em>3/73 (<em>1</em>8%) and <em>1</em>5/70 (<em>2</em><em>1</em>%) of the patients, respectively.

CONCLUSIONS

Activation of the hemostatic system is independently related to acute stroke severity and short-term outcome. Low levels of coagulation inhibitors or presence of antiphospholipid antibodies is a relatively frequent finding in unselected patients with acute ischemic stroke, but a causative role cannot be inferred from our study.

OBJECTIVE

To investigate platelet and leukocyte activation and interaction in patients with rheumatoid arthritis (RA) and the effect of methotrexate (MTX) or anti-tumor necrosis factor-a (TNF-a) treatment on these variables.

METHODS

Four-color flow cytometry analysis was performed for quantitative measurement of platelet (P-selectin, PAC-<em>1</em>) and leukocyte (CD<em>1</em><em>1</em>b, CD64) activation markers and estimation of percentage of leukocyte-platelet complexes in whole blood in <em>2</em>0 patients with RA before and after 6 weeks of therapy and in <em>2</em>0 controls. In addition, measures of soluble P-selectin (sP-selectin), beta-thromboglobulin, fibrinogen, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, D-dimer, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), interleukin 6 (IL-6), and TNF-a and tender and swollen joint counts were carried out.

RESULTS

Before therapy, PAC-<em>1</em> binding, expression of CD<em>1</em><em>1</em>b and CD64 on monocytes and neutrophils, circulating levels of monocyte (CD<em>1</em><em>1</em>b+ or CD64+)-platelet complexes, monocyte-PAC-<em>1</em>+ platelet complexes, CRP, ESR, IL-6, TNF-a, fibrinogen, D-dimer and sP-selectin were significantly higher in RA patients compared to controls. The anti-TNF-a therapy significantly reduced levels of monocyte-PAC-<em>1</em>+ platelet complexes, sP-selectin, CRP, ESR, IL-6, TNF-a, fibrinogen, and D-dimer and tender and swollen joint counts. CD64 expression on monocytes was significantly decreased by MTX therapy. PAC-<em>1</em> binding was not inhibited by MTX or anti-TNF-a.

CONCLUSIONS

Increased platelet and leukocyte activation and increased formation of leukocyte-platelet complexes in patients with RA suggest a status of simultaneous activation of the immune and hemostatic systems.

The effects of steroids on the outcome of sepsis are dose dependent. Low doses appear to be beneficial, but high doses do not improve outcome for reasons that are insufficiently understood. The effects of steroids on systemic inflammation as a function of dose have not previously been studied in humans. To determine the effects of increasing doses of prednisolone on inflammation and coagulation in humans exposed to LPS, 3<em>2</em> healthy males received prednisolone orally at doses of 0, 3, <em>1</em>0, or 30 mg (n = 8 per group) at <em>2</em> h before i.v. injection of Escherichia coli LPS (4 ng/kg). Prednisolone dose-dependently inhibited the LPS-induced release of cytokines (TNF-alpha and IL-6) and chemokines (IL-8 and MCP-<em>1</em>), while enhancing the release of the anti-inflammatory cytokine IL-<em>1</em>0. Prednisolone attenuated neutrophil activation (plasma elastase levels) and endothelial cell activation (von Willebrand factor). Most remarkably, prednisolone did not inhibit LPS-induced coagulation activation, measured by plasma concentrations of thrombin-antithrombin complexes, <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em>, and soluble tissue factor. In addition, activation of the fibrinolytic pathway (tissue-type plasminogen activator and plasmin-alpha(<em>2</em>)-antiplasmin complexes) was dose-dependently enhanced by prednisolone. These data indicate that prednisolone dose-dependently and differentially influences the systemic activation of different host response pathways during human endotoxemia.

BACKGROUND

Recent investigations suggest that activation of coagulation and fibrinolysis occurs in patients with ulcerative colitis (UC). However, the role of the hypercoagulable state in UC has not been determined. On the other hand, there are no reports dealing with coagulation in ischemic colitis (IC), in which acute bowel inflammation and reversible vascular occlusion affect the colon. Thus, our aim was to evaluate the hyper states of coagulation and fibrinolysis in UC by comparing activations of coagulation and fibrinolysis in patients with active UC and in those with IC.

METHODS

Twenty-four patients with active UC and <em>1</em><em>2</em> patients with IC were studied, with <em>1</em>8 patients with inactive UC serving as controls. We investigated the activation of the coagulation system, including platelet counts, activated partial thromboplastin time (APTT), thrombin time (TT), <em>prothrombin</em> time (PT), serum concentrations of von Willebrand factor (vWF), activated factors XII, XI, X, IX, VIII, VII, V, II, fibrinogen, <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), thrombin-antithrombin complexes (TAT), protein S, protein C, plasminogen, alpha-<em>2</em> plasminogen inhibitor (alpha-<em>2</em>PI) and D-dimer (D-D).

RESULTS

Median serum vWF concentrations, F<em>1</em>+<em>2</em>, TAT, fibrinogen, activated factor XI, IX, VIII and V were significantly elevated in patients with active UC and IC compared to those in patients with inactive UC. There was no significant difference between active UC and IC patients in the mean values of any of the factors that were measured.

CONCLUSIONS

The results of the present study indicate that the coagulation-fibrinolysis system is activated in patients with active bowel inflammation such as active UC and IC, and that the hyper states of coagulation and fibrinolysis in patients with active UC are secondary to bowel inflammation.

The effects of dietary n-3 fatty acids (n-3FAs) on the frequency of pain episodes and ex vivo blood tests of thrombosis have been evaluated in patients with sickle cell disease (SCD) utilizing a double-blind, olive oil-controlled clinical trial. Dietary n-3FA therapy (0.<em>1</em> g/kg/d) was provided as menhaden fish oil (0.<em>2</em>5 g/kg/d) containing <em>1</em><em>2</em>% eicosapentaenoic acid (EPA), and <em>1</em>8% docosahexaenoic acid (DHA). Within <em>1</em> month dietary n-3FAs exchanged with n-6FAs in plasma and erythrocyte membrane phospholipids (p <0.0<em>1</em> in all cases). Treatment with dietary n-3FAs for <em>1</em> year reduced the frequency of pain episodes requiring presentation to the hospital from 7.8 events during the preceding year to 3.8 events/year (p <0.0<em>1</em>; n = 5). By contrast, subjects receiving control dietary olive oil (n = 5) experienced 7.<em>1</em> pain events/year, compared to 7.6 during the previous year (p >0.4). The reduction in episodes in n-3FA-treated subjects was also significant when compared to control subjects (p <0.0<em>1</em>). Dietary n-3FA therapy was not associated with hemorrhagic, gastrointestinal or other adverse effects. Compared to <em>1</em>0 asymptomatic African-American controls, sickle cell subjects demonstrated significantly increased pretreatment: <em>1</em>) flow cytometric expression of platelet membrane P-selectin (CD6<em>2</em>p; p <0.0<em>1</em>) and annexin V binding sites (p = 0.0<em>2</em>); <em>2</em>) plasma levels of platelet-specific secretory proteins platelet factor 4 (PF4) and beta-thromboglobulin (betaTG) (p <0.0<em>1</em> in both cases); 3) plasma products of thrombin generation, <em>prothrombin</em> <em>fragment</em> <em>1</em>.<em>2</em> (F<em>1</em>.<em>2</em>) and thrombin:antithrombin (TAT) complex (p <0.0<em>1</em> in both cases); and 4) plasma levels of thrombolytic products, D-dimer and plasmin:antiplasmin (PAP) complex (p <0.0<em>1</em> in both cases). Treatment with dietary n-3FAs concurrently decreased plasma levels of F<em>1</em>.<em>2</em>, D-dimer, and PAP (p <0.05, compared to olive oil controls), implying that the reduction in pain events was related to n-3FA-dependent inhibition of thrombosis. We conclude that dietary n-3FAs reduce the frequency of pain episodes perhaps by reducing prothrombotic activity in sickle cell disease.

Stress-induced activation of haemostasis may be involved in the triggering of acute coronary syndromes. We compared the effects of mental stress, dynamic exercise and adrenaline infusion on platelet sensitivity to thrombin using flow-cytometric analysis of platelet fibrinogen binding in whole blood, and platelet aggregability using filtragometry ex vivo, in healthy volunteers. Furthermore, we assessed thrombin generation [<em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) and thrombin-antithrombin complexes in plasma] and thrombin activity (fibrinopeptide A in plasma). Exercise (bicycle ergometry) enhanced thrombin-induced platelet fibrinogen binding (P<0.05) and platelet aggregability (P<0.0<em>1</em>), and elevated F<em>1</em>+<em>2</em>, thrombin-antithrombin complexes and fibrinopeptide A (P<0.05 for all three). Adrenaline infusion enhanced thrombin-induced platelet fibrinogen binding and platelet aggregability (P<0.05), and elevated thrombin-antithrombin complexes (P<0.05), whereas F<em>1</em>+<em>2</em> and fibrinopeptide A levels were not significantly affected. Mental stress increased platelet sensitivity to high concentrations of thrombin only, and produced small increases in levels of thrombin-antithrombin complexes. Time control experiments showed no important changes with repeated measurements during rest. Platelet responses to exercise and adrenaline were reversible, with recovery 60 min later. Thus, heavy exercise and high levels of adrenaline reversibly increased platelet aggregability and platelet sensitivity to thrombin, and enhanced thrombin formation; the effects were most pronounced during exercise. Mental stress only weakly affected these parameters.

BACKGROUND

Intravascular fibrin deposition and platelet sequestration occur with porcine xenograft rejection by baboons. Disseminated intravascular coagulopathy may arise either as a direct consequence of the failure to fully deplete xenoreactive natural antibodies and block complement, or because of putative cross-species molecular incompatibilities in this discordant species combination.

METHODS

Three baboons were conditioned with retrovirally transduced autologous bone marrow to induce tolerance to swine antigens. Xenoreactive natural antibodies and complement were depleted by plasmapheresis and the use of Gal alpha<em>1</em>-3Gal column adsorptions; baboons were then splenectomized and underwent renal xenografting from inbred, miniature pigs. Soluble complement receptor type-<em>1</em> with protocol immunosuppression (mycophenolate mofetil, <em>1</em>5-deoxyspergualin, steroids, and cyclosporine) was administered.

RESULTS

A bleeding diathesis was clinically evident from days 5 to <em>1</em>2 after transplantation in two baboons. Low levels of circulating C3a, C3d, and iC3b were measured despite the absence of functional circulating complement components. Profound thrombocytopenia with abnormalities in keeping with disseminated intravascular coagulopathy were observed. Prolongation of prothrombin and partial thromboplastin times was accompanied by evidence for tissue factor-mediated coagulation pathways, high levels of thrombin generation (prothrombin fragment F(<em>1</em>+2) production and thrombin-antithrombin complex formation), fibrinogen depletion, and production of high levels of the fibrin degradation product D-dimer. Importantly, these disturbances resolved rapidly after the excision of the rejected xenografts in two surviving animals. Histopathological examination of the rejected xenografts confirmed vascular injury, fibrin deposition, platelet deposition, and localized complement activation.

CONCLUSIONS

Systemic coagulation disturbances are associated with delayed xenograft rejection.

The haemostatic system was examined in <em>2</em>95<em>1</em> men aged 50 to 6<em>1</em> years, clinically free of cardiovascular disease, who were ranked according to a risk score for fatal coronary heart disease (CHD). Risk was judged from their serum cholesterol concentration, systolic blood pressure, body mass index and smoking habit. The status of the factor VII-tissue factor pathway was estimated from the plasma levels of factor VII coagulant activity, factor VII antigen and activated factor VII. Activation of factor IX was assessed from the plasma concentration of factor IX activation peptide. Activity within the common pathway was measured as the plasma concentrations of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> and fibrinopeptide A. All 6 markers of haemostatic status were positively and statistically significantly associated with risk, providing further evidence for a hypercoagulable state in men at high risk for fatal CHD. Plasma fibrinogen and serum triglyceride concentrations were also graded positively with risk.

OBJECTIVE

To study expression of tissue factor (TF) in pancreatic cancer and its role in the development of thromboembolism.

METHODS

TF expression was studied in eight human pancreatic carcinoma cell lines by Northern blot and indirect immunofluorescence. Expression of alternatively spliced TF (asTF) was assessed by RT-PCR. In addition, TF expression was determined by immunofluorescence in pancreatic tissues of <em>1</em>9 patients with pancreatic adenocarcinoma (PCa), 9 patients with chronic pancreatitis (CP) and <em>2</em>0 normal controls. Plasma samples (30 PCa-patients, <em>1</em>3 CP-patients and <em>2</em>0 controls) were investigated for soluble TF levels and coagulation activation markers [thrombin-antithrombin III complex (TAT), <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>)].

RESULTS

All pancreatic carcinoma cell lines expressed TF (8/8) and most of them expressed asTF (6/8). TF expression at the protein level did not correlate with the differentiation of the carcinoma cell line. All but two pancreatic cancer tissue samples stained positive for TF (<em>1</em>7/<em>1</em>9). In all samples of CP weak staining was restricted to pancreatic duct cells, whereas only a few subendothelial cells were positive in 9/<em>2</em>0 of normal controls. TF and TAT levels in PCa patients were significantly elevated compared to controls whereas elevated F<em>1</em> + <em>2</em> levels did not reach statistical significance compared to controls. In CP patients TAT and F<em>1</em> + <em>2</em> levels proved to be significantly elevated compared to controls, although TAT elevation was less pronounced than in PCa patients.

CONCLUSIONS

We conclude that in addition to the upregulated expression of TF on the cell membrane, soluble TF might contribute to activation of the coagulation system in pancreatic cancer.

BACKGROUND

Orthopedic surgery, especially total knee and total hip arthroplasty, is considered a risk factor for peri-operative venous thromboembolism.

OBJECTIVE

This study evaluates how accelerated inflammatogenic cellular interactions and the subsequent production of tissue factor and CD40 ligand play an important role in the pathogenesis of venous thromboembolism.

METHODS

Twenty-four patients undergoing total knee arthroplasty were randomly assigned to groups with (Ti; n = <em>1</em><em>2</em>) and without (Tn; n = <em>1</em><em>2</em>) pneumatic tourniquet inflation.

RESULTS

Numbers of leukocyte-platelet aggregates, especially those comprising monocytes-platelets in central venous blood from the Ti group, were increased during the peri-operative period (P < 0.0<em>1</em>), and returned to the baseline level at <em>2</em>4 h after starting surgery. Levels of PAC-<em>1</em>, P-selectin, CD40 ligand, tissue factor, Mac-<em>1</em> expression on monocytes including monocyte-platelet aggregates, and the number of microparticles including those of endothelial cell origin were noticeably increased in central venous blood from the Ti group (P < 0.0<em>1</em>). Whole blood coagulability was also obviously increased in central venous blood from the Ti group (P < 0.0<em>1</em>). Furthermore, the concentrations of venous plasma tissue factor antigen, CD40 ligand, platelet factor 4, beta-thromboglobulin, the soluble fibrin monomer complex and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> were also increased (P < 0.05).

CONCLUSIONS

This study showed that platelet, leukocyte and endothelium activities as well as their interactions are enhanced during the peri-operative period of total knee arthroplasty, particularly in venous blood from the lower half of the body, which consequently augments blood coagulability. Further, tourniquet inflation during surgery exaggerates these responses.

BACKGROUND

It has been claimed that regional citrate anticoagulation (RCA) improves unfavorable calcium and magnesium dependent cellular and humoral events due to blood/dialyzer membrane interactions during hemodialysis (HD). This study aimed to verify whether the favorable effect of RCA on biocompatibility is independent from coagulation pathway modulation.

METHODS

A randomized controlled cross-over single blind trial comparing the activity of the coagulation pathway (thrombinantithrombin complexes (TAT), fibrinopeptide A (FPA), <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em> (F <em>1</em>+<em>2</em>) and D-dimer (DD)), complement activation (C3a) and interleukin-<em>1</em> beta secretion (IL-<em>1</em>beta) in nine chronic HD patients treated with RCA or heparin. Blood samples were obtained from the arterial (C3a, IL-<em>1</em>beta, TAT, F <em>1</em>+<em>2</em>, FPA and DD) and venous (TAT, F <em>1</em>+<em>2</em>, FPA) lines <em>2</em> min after starting treatment and repeatedly during the procedure after <em>1</em>5 min (C3a and IL-<em>1</em>beta), 30 min (C3a), 45 (C3a) and <em>1</em>80 min (TAT, F <em>1</em>+<em>2</em>, FPA and DD).

RESULTS

In both treatment protocols significant enhancement was observed in the coagulation activity during the dialysis session, documented by an increase in TAT (p<0.00<em>1</em>), F <em>1</em>+<em>2</em> (p<0.00<em>1</em>) and FPA (p=0.00<em>1</em>). Comparing the two anticoagulation modalities, no differences were noticed in the activity of the coagulation pathway, but a significantly higher complement activity (C3a=886 (83<em>2</em>-908) vs. 770 (645-857) ng/mL, p<0.05) and lower IL-<em>1</em>beta secretion (<em>2</em>35 (<em>2</em>06-<em>2</em>85) vs. 538 (346-974) pg/mL, p<0.05) was observed in RCA.

CONCLUSIONS

Due to an RCA protocol guaranteeing the same extent of anticoagulation activation as standard heparin, we demonstrated that the significantly lower IL-<em>1</em>beta secretion obtained with RCA is independent from the anticoagulation modulation and dissociated from the complement activity.

Patients with functional deficiency of C<em>1</em>-inhibitor (C<em>1</em>-INH) suffer from recurrent acute attacks (AA) of localized oedema associated with activation of the contact system, complement and fibrinolysis. To unravel further the role of coagulation and fibrinolysis in the pathophysiology of C<em>1</em>-INH deficiency, we performed simultaneous thrombin and plasmin generation measurements in plasma from patients with hereditary angioedema (HAE) due to C<em>1</em>-INH deficiency during AA (n = <em>2</em>3), in remission (R) (n = <em>2</em>0) and in controls (n = <em>2</em>0). During AA thrombin generation after in-vitro activation of plasma was higher than in controls, as demonstrated by shorter thrombin peak-time (P < 0·05), higher thrombin peak-height (P < 0·00<em>1</em>) and increased area under the curve (AUC) (P < 0·05). Additionally, elevated levels of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (P < 0·000<em>1</em>) were observed in non-activated plasma from the same patients. In contrast, in activated plasma from patients during AA plasmin generation estimated as plasmin peak-height (P < 0·05) and plasmin potential (P < 0·05) was reduced, but non-activated plasma of the same patients showed elevated plasmin-anti-plasmin (PAP) complexes (P < 0·00<em>1</em>). This apparent discrepancy can be reconciled by elevated soluble thrombomodulin (sTM) (P < 0·0<em>1</em>) and thrombin activatable fibrinolysis inhibitor (TAFI) in patients during AA providing possible evidence for a regulatory effect on fibrinolysis. Plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>) was reduced in patients during AA indicating, together with the observed reduction of plasmin generation, the consumption of fibrinolytic factors. In conclusion, our results support the involvement of coagulation and fibrinolysis in the pathophysiology of HAE and show the possible application of simultaneous measurement of thrombin and plasmin generation to evaluate different clinical conditions in HAE patients.