Thrombomodulin Concentrations Research Articles

Markery komórek śródbłonka w chorobie niedokrwiennej serca

Background: Endothelial injury, activation and dysfunction play an important role in atherosclerosis progression and development of cardiovascular disease. Aim: To evaluate biochemical markers assessing the vascular endothelium state in patients with coronary artery disease. Material and methods: We examined 57 patients with coronary artery disease (CAD): 27 patients with acute myocardial infarction (AMI) and 30 patients with stable angina pectoris (SA). The control group comprised 23 patients without symptoms of CAD. The concentration of von Willebrand factor (vWF), thrombomodulin (sTM), endothelin-1 (ET-1), and adhesion molecules (soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1)) was analysed in plasma or serum. Results: A significant increase in vWF, sICAM-1, sVCAM-1 and ET-1 concentrations was found in AMI compared to the control group. Increased vWF and sICAM-1 concentrations were found in SA compared to the control group. The AMI group was characterized by significantly higher vWF concentration than the SA group. Thrombomodulin concentration did not differ significantly between any patient groups and the control group. There was a positive correlation between vWF concentration and sVCAM-1 and sTM concentrations, and an inverse correlation between ET-1 and sICAM-1 concentrations in AMI. A sICAM-1 correlated positively with sVCAM-1 concentration in SA. Conclusions: von Willebrand factor is more useful than sTM, endothelin-1, and cell adhesion molecules sICAM-1 and sVCAM-1 to assess endothelium state in patients with CAD. The increase in plasma vWF concentration confirms endothelial injury and/or activation in CAD and indicates a greater severity of these disorders in AMI than in SA.

The effect of erythropoietin on platelet and endothelial activation markers: A prospective trial in healthy volunteers

Erythropoietin (EPO) enhances formation of red blood cells and also affects thrombopoiesis and platelet function. We hypothesized that the effect of EPO may be reflected by changes in thromboxane B2 (TXB2) and endothelial cell function. Six male and six female subjects received recombinant human epoetin alpha (Erypo®) intravenously (300 U/kg). Biomarker levels were assessed at baseline and 4, 24, 48 and 72 hours after infusion. Epoetin alpha increased TXB2 levels by 140%, which reached significance at 48 hours (6.6 ± 5 ng/ml vs. 15 ± 9 ng/ml; p = 0.044) and remained at that level at 72 hours. In line, epoetin alpha increased E-selectin levels by 25% already at 24 hours (39 ± 21 ng/ml vs. 49 ± 26 ng/ml; p < 0.001) which stayed at this level until 72 hours (p < 0.001). The raise in platelet activation markers corresponded to an 88% increase in reticulocyte count (43 ± 10 × 109/l vs. 81 ± 17 × 109/l; p < 0.001) and a 9% increase in platelet count at 72 hours (224 ± 45 × 109/l vs. 244 ± 52 × 109/l; p = 0.005). Thrombomodulin and von Willebrand factor concentrations were not significantly altered by epoetin alpha. Interestingly, gender differences in the baseline levels of E-selectin and thrombomodulin were observed. E-selectin and thrombomodulin levels were doubled in men compared to women (51 ± 24 and 28 ± 10 ng/ml; p = 0.025 and 30 ± 5 ng/ml vs. 16 ± 5 ng/ml; p = 0.002, respectively). EPO increases TXB2 serum levels and soluble E-selectin. Further studies are needed to investigate whether these markers might be useful for estimation of thromboembolic risk during EPO-therapy and whether inhibition of thromboxane formation may lower thrombotic complications during EPO treatment: NCT01392612.

The estimation of selected endogenous anticoagulation system parameters in patients with subclinical Cushing's syndrome

An increased tendency towards thromboembolic events is observed in patients with Cushing's syndrome. There are much fewer publications available about thromboembolic complications in patients with subclinical Cushing's syndrome (SCS). Therefore, a question arises whether hemostatic disturbances appear in this particular disease phase. Estimation of protein C (PC), free protein S (FPS), antithrombin (AT) activity, thrombomodulin (TM) concentration and activated PC resistance (APCR) in patients with SCS. We studied 35 patients with SCS. The control group consisted of 33 healthy volunteers. The activity of PC, AT, FPS, APCR and the concentration of TM was estimated in all representatives. The comparison of the examined coagulation parameters between the patients with SCS and the healthy individuals revealed significantly higher mean PC activity and mean FPS activity in the SCS group. Mean TM concentration was significantly lower in patients with SCS compared with the control group. The differences in APCR and AT activity were not significant. We did not prove any statistically significant correlations between the examined coagulation parameters and hormonal parameters. We did not find any correlation between the concentration of cortisol and basic coagulation parameters such as international normalized ratio, activated partial thromboplastin time or fibrinogen in the group with SCS either. The patients with SCS present disturbances in endogenous anticoagulation system defined as PC, FPS activity and TM concentration. This finding suggests an impact of mild autonomic cortisol overproduction on coagulation system.

The Resistance Training with Blood Flow Reduction and Vascular Endothelial Function in Healthy Old Volunteers

Not only aerobics but also resistance training is considered to improve the vascular endothelial function. Recently, it was reported that the training with blood flow reduction (BFR) performed with compressing the stem of upper or lower extremities induced muscle hypertrophy even at lower intensity workload, and increased the muscle strength of extremities at shorter training period. Although the hypoxic condition in tissue is known to increase the NO production from vascular endothelium, it is unclear whether or not the training with BFR increases the vascular endothelial function. PURPOSE:The purpose of the present study was to investigate whether long-term training with BFR improved the vascular endothelial function. METHODS:Seventeen healthy old male volunteers, 72±3 years old, performed the resistance training for the extremities with or without BFR by cuff compression: 9 subjects in BFR group and 8 in control group, respectively. The resistance training composed of 4 manners: leg extension, leg press, rowing and chest press was carried out 3 sets a day and 3 times a week for 4 weeks, while each manner was repeated 20 times by the 20%of 1 repetition maximum (1RM). The vascular endothelial function and muscle strength were assessed twice before and after the 4-week intervention of the resistance training. We measured reactive hyperaemic index (RHI) using Endo-PAT and blood concentrations of von willebrand factor (vWF) and thrombomodulin (TM) as indices of the vascular endothelial function. We also measured 1RM of leg extension, leg press, rowing and chest press as indices of muscle strength. RESULTS:The RHI, and 1RM of leg extension and leg press increased significantly from 1.7 ± 0.4, 45.1 ± 12.8kg and 130 ± 38.6kg before the intervention to 1.9 ± 0.3 (P <0.05), 59.4 ± 19.7kg (P <0.01) and 149 ± 39.5kg (P <0.05) after the intervention, respectively, in the BRF group. The 1RM of rowing increased significantly from 41.4 ± 7.5kg to 49.0 ± 5.8 in the BRF group (P <0.05) and from 44.0 ± 5.7kg to 49.3 ± 5.8kg in the control group (P <0.05). There were no significant differences between the vWF, TM or 1RM of chest press measured before and after the intervention in the two groups. CONCLUSIONS:We concluded that the low intensity resistance training with BFR elevated not only muscle strength but also the vascular endothelial function.

Increased thrombin generation measured in the presence of thrombomodulin in women with early pregnancy loss

Compared with 537 parous controls with no history of pregnancy loss, a lower thrombomodulin-related inhibition of the endogenous thrombin potential was measured in 264 cases with previous unexplained pregnancy loss, especially when losses occurred between 9 and 12 weeks of gestation. Adjusting age, protein S, factor VIII, factor V Leiden, and prothrombin G20210A did not change the results.

Levels of circulating endothelial cells in normotensive and severe preeclamptic pregnancies

Preeclampsia is a disease hypothesized to originate from widespread endothelial dysfunction or damage. This study investigated whether circulating endothelial cells (CEC) can serve as a surrogate marker for disease severity in patients with preeclampsia, and if their number correlates to serum endothelial biomarkers for activation, dysfunction, or damage of those cells. Blood was drawn consecutively from 30 patients admitted with a diagnosis of severe preeclampsia. Thirty healthy, normotensive, patients matched for age, body mass index, and gestational age served as a control group. We determined the number of CEC and serum concentrations of biomarkers indicative of endothelial damage (thrombomodulin) and activation (E-selectin), and the antiangiogenic protein (endoglin), which reflects endothelial dysfunction. Median CEC counts did not differ significantly between preeclamptic patients and the control group (median 5.3 vs. 3.5 CEC/mL, respectively) and were mostly within the normal range (i.e., <20 CEC/mL). However, serum concentrations of thrombomodulin (median 3.6 vs. 5.2 ng/mL; P = 0.006), E-selectin (median 32.0 vs. 42.9 ng/mL; P = 0.02), and especially endoglin (median 5.0 vs. 76.2 ng/mL; P < 0.0001) were significantly increased in severe preeclamptic patients. CEC counts did not correlate with any of the clinical parameters or routinely determined laboratory indices. Preeclampsia is characterized by endothelial dysfunction and activation rather than actual endothelial damage as characterized by increased CEC counts.

EXPRESSION OF THROMBOMODULIN ON MONOCYTES IS ASSOCIATED WITH EARLY OUTCOMES IN PATIENTS WITH CORONARY ARTERY BYPASS GRAFT SURGERY

Thrombomodulin (TM) mediates blood coagulation and inflammation and is expressed constitutively on resting monocytes. This expression might be a key regulator of monocyte-related inflammation. Conventional cardiopulmonary bypass (CPB), beating-heart CPB, and off-pump techniques have been used widely in cardiac surgery. Although beating-heart CPB and off-pump techniques have reduced postoperative inflammation significantly, the underlying mechanisms remain unclear. Whether CPB affects the expression of TM and changes the actual immune capacity of monocytes is also unknown. In this study, we analyzed TM expression on monocytes and in plasma among patients undergoing elective coronary artery bypass graft surgery. The days spent in an intensive care unit (ICU) and incidence of fever in the ICU were significantly lower in the beating-heart CPB and off-pump groups than in the conventional CPB group. Enzyme-linked immunosorbent assay showed a significant increase in TM at 30 min after the commencement of CPB and at the end of surgery in the conventional CPB group, whereas the level increased less markedly in the beating-heart CPB group. Flow cytometry showed that conventional CPB markedly reduced the expression of TM on monocytes. Based on monocyte chemotaxis analysis and an actin polymerization assay, we propose that TM expression on monocytes is associated with systemic inflammation. We conclude that the beating-heart CPB and off-pump techniques have a lower impact on patients than conventional CPB. The reduced incidence of fever and shorter ICU stay seem to be associated predominantly with the lower concentration of TM in plasma and with a higher expression of TM on monocytes.

Dabigatran enhances clot susceptibility to fibrinolysis by mechanisms dependent on and independent of thrombin-activatable fibrinolysis inhibitor.

Anticoagulants are expected to promote fibrinolysis by counteracting the antifibrinolytic effects of thrombin, which include thrombin-activatable fibrinolysis inhibitor (TAFI) activation and clot structure enhancement. However, the efficiency of anticoagulants may vary remarkably, and the ability of direct thrombin inhibitors to facilitate clot lysis remains controversial. To evaluate the profibrinolytic effect of dabigatran, a new, direct thrombin inhibitor, using different in vitro models. The resistance of tissue factor-induced plasma clots to fibrinolysis by exogenous tissue-type plasminogen activator (t-PA) (turbidimetric method) was reduced by dabigatran in a concentration-dependent manner, with > or = 50% shortening of lysis time at clinically relevant concentrations (1-2 microm). A similar effect was observed in the presence of low (0.1 and 1 nm) but not high (10 nm) concentrations of thrombomodulin. Acceleration of clot lysis by dabigatran was associated with a reduction in TAFI activation and thrombin generation, and was largely, although not completely, negated by an inhibitor of activated TAFI, potato tuber carboxypeptidase inhibitor. The assessment of the viscoelastic properties of clots showed that those generated in the presence of dabigatran were more permeable, were less rigid, and consisted of thicker fibers. The impact of these physical changes on fibrinolysis was investigated using a model under flow conditions, which demonstrated that dabigatran made the clots markedly more susceptible to flowing t-PA, by a mechanism that was largely TAFI-independent. Dabigatran, at clinically relevant concentrations, enhances the susceptibility of plasma clots to t-PA-induced lysis by reducing TAFI activation and by altering the clot structure. These mechanisms might contribute to the antithrombotic activity of the drug.

Fibrinogen Promotes Early Atherosclerotic Changes of the Carotid Artery in Young, Healthy Adults

To determine whether high plasma levels or activities of different hemostatic proteins contribute to the development of early atherosclerotic vessel wall changes. Elevated levels of various hemostatic proteins and markers of inflammation have been linked to an increased risk of ischemic cardiovascular events; however, the mechanisms by which these molecules might contribute to this increased risk is not clear. The intima-media thickness of the common carotid arteries (CCA-IMT) of 125 healthy young volunteers without known cardiovascular risk factors was measured by high-resolution ultrasound. Plasma concentrations of fibrinogen, thrombomodulin, protein Z and CRP were quantified, and the plasma activities of protein C, plasminogen and factor VIII were measured. Other established risk factors, such as body mass index (BMI) and plasma levels of cholesterol, triglycerides and homocysteine, were also determined. Furthermore, the carotid arteries were examined for the presence of plaques and stenoses. Univariate analysis showed a significant negative correlation between CCA-IMT and HDL cholesterol, and positive correlations with age, BMI, LDL cholesterol, triglycerides, homocysteine, fibrinogen and thrombomodulin, but not with total cholesterol, lipoprotein(a) and hsCRP. CCA-IMT was also statistically independent of the activities of protein C, factor VIII and plasminogen. Multivariate analysis revealed a significant correlation of CCA-IMT with age, BMI and fibrinogen. Our data suggest that fibrinogen levels correlate with early atherosclerotic changes of the carotid artery in a population with very low cardiovascular risk.

Pericytes in the macrovascular intima: possible physiological and pathogenetic impact

The frequently observed de-endothelialization of venous coronary bypass grafts prepared using standard methods exposes subendothelial prothrombotic cells to blood components, thus endangering patients by inducing acute thromboembolic infarction or long-term proliferative stenosis. Our aim was to gain deeper histological and physiological insight into these relations. An intricate network of subendothelial cells, characterized by histological features specific for true pericytes, was detected even in healthy vessels and forms, coupled to the luminal endothelium, a second leaflet of the macrovascular intima. These cells, and particularly those in the venous intima, express enormous concentrations of tissue factor and can recruit additional amounts of up to the 25-fold concentration within 1 h during preincubation with serum (intimal pericytes of venous origin activate 30.71 +/- 4.07 pmol coagulation factor x.min(-1).10(-6) cells; n = 15). Moreover, decoupled from the endothelium, they proliferate rapidly (generation time, 15 +/- 2.1 h, n = 8). Central regions of atherosclerotic plaques, as well as of those of restenosed areas of coronary vein grafts, consist almost completely of these cells. In stark contrast with the prothrombogenicity of the intimal pericytes, intact luminal endothelium recruits high concentrations of thrombomodulin (CD 141) specifically within its intercellular junctions, activates Protein C rapidly (42 +/- 5.1 pmol/min.10(6) venous endothelial cells at thrombin saturation; n = 15), can thus actively prevent coagulatory processes, and never expresses histologically detectable and functionally active tissue factor. Given this strongly prothrombotic potential of the intimal pericytes and their overshooting growth behavior in endothelium-denuded vascular regions, they may play important roles in the development of atherosclerosis, thrombosis, and saphenous vein graft disease.

Modulation of Substrate Specificity of the Thrombin-Thrombomodulin Complex for Protein C Versus TAFI Activation by Platelet Factor 4 and Non-Anticoagulant Heparin-Derivatives.

Abstract 3181 Poster Board III-108 The endothelial cell receptor thrombomodulin (TM) is an essential cofactor for thrombin-mediated activation of protein C and the generation of activated protein C (APC), an enzyme with anticoagulant and multiple cytoprotective activities. TM also stimulates thrombin-mediated activation of Thrombin Activatable Fibrinolysis Inhibitor (TAFI), resulting in anti-fibrinolytic and anti-inflammatory effects. Based on these properties of TM, several potential strategies for therapeutic intervention have been proposed. In hemophilia patients with inhibitory antibodies, stimulation of TM-dependent TAFI activation might diminish bleeding complications by protection of clots against premature lysis. Alternatively, stimulation of endogenous APC generation in patients with severe sepsis might prove to augment the beneficial effects of APC, as pharmacologic applications of APC in these patients increased survival. Platelet Factor 4 (PF4), a platelet alpha-granule protein, acts as a soluble cofactor for protein C activation by the thrombin-TM complex and stimulated endogenous generation of APC in vivo. Here we show that in contrast to stimulation of protein C activation, PF4 dose-dependently inhibited activation of TAFI by thrombin-TM in a purified system, in normal pooled plasma and on TM-expressing cells. Functional consequences of inhibition of TAFI activation by PF4 were determined in hemophilia A plasma by analysis of clot resistance to tPA-induced fibrinolysis. In hemophilia A plasma, activation of TAFI was dependent on TM due to the ineffective amplification of thrombin generation via the tenase complex. PF4 diminished TM-dependent prolongation of clot lysis times but did not affect clot lysis in the absence of TM. Inhibition of TM-dependent prolongation of clot lysis time by PF4 was dependent on both the TM and tPA concentration used, consistent with the proposed threshold mechanism for inhibition of fibrinolysis by TAFIa. To determine the effect of PF4 on TM-dependent TAFI activation in normal plasma, the inactivation of bradykinin (BK) in plasma by TAFIa during tissue factor induced coagulation was analyzed. Addition of TM to plasma accelerated BK cleavage, i.e., generation of des-Arg9-BK, consistent with TM-mediated stimulation of TAFI activation and subsequent BK cleavage by TAFIa. PF4 neutralized the TM-enhanced BK inactivation and reversed BK and des-Arg9-BK levels to those seen in the absence of TM. Thus, PF4 alters the substrate specificity of the thrombin-TM complex by selectively enhancing protein C activation while inhibiting TAFI activation, such that PF4 prevents generation of TAFIa's anti-fibrinolytic and anti-inflammatory activities. To find an antidote for PF4's inhibitory effects on TAFI activation, heparin-derivatives were screened for high affinity PF4 binding but greatly reduced anticoagulant activity. N-acetylated heparin (NAc-Hep), but not N-desulfated without N-acetylation or N-acetylated and O-desulfated heparins, retained high affinity binding to PF4 and effectively blocked PF4 binding to immobilized TM. Consistent with high affinity binding of NAc-Hep to PF4, sulfylamine group oxygens were relatively minor determinants of the interactions between heparin and PF4 based on the available structural model. NAc-Hep did not show detectable anticoagulant activity in APTT clotting assays but restored BK conversion to des-Arg9-BK by TAFIa in the presence of PF4, similar to that seen in the absence of PF4. In addition, NAc-Hep reversed PF4-mediated inhibition of TAFI activation and TAFIa's antifibrinolytic functions in clot lysis assays on TM-expressing cells using hemophilia A plasma. Thus, NAc-Hep reversed PF4-mediated inhibition of TAFI activation. In summary, PF4 modulates the substrate-specificity of the thrombin-TM complex by selectively enhancing protein C activation while inhibiting TAFI activation. NAc-Hep, a non-anticoagulant heparin derivative with high affinity for PF4, effectively reversed PF4-mediated inhibition of TM-dependent TAFI activation and restored TAFIa's antifibrinolytic and anti-inflammatory functions. Accordingly, NAc-Hep or similar non-anticoagulant heparin derivatives might provide therapeutic benefits by diminishing bleeding complications in hemophilia A via restoration of TAFIa-mediated protection of clots against premature lysis. Disclosures No relevant conflicts of interest to declare.

Relationship between disseminated intravascular coagulation and levels of plasma thrombinogen segment 1+2, D-dimer, and thrombomodulin in patients with multiple injuries

To explore the relationship between disseminated intravascular coagulation (DIC) and levels of plasma thrombinogen segment 1+2 (F1+2), D-dimer (D-D), and thrombomodulin (TM) in patients with severe multiple injuries. In this study, 66 patients (49 males and 17 females, aged 15-74 years, mean=38.4 years) with multiple injuries, who were admitted to our hospital within 24 hours after injury with no personal or family history of hematopathy or coagulopathy, were divided into a minor injury group (ISS<16, n=21) and a major injury group (ISS>or=16, n=45) according to the injury severity. The patients in the major injury group were divided into a subgroup complicated with DIC (DIC subgroup, n=12) and a subgroup complicated with no DIC (non-DIC subgroup, n=33). Ten healthy people (7 males and 3 females, aged 22-61 years, mean=36.5 years+/-9.0 years), who received somatoscopy and diagnosed as healthy, served as the control group. Venous blood samples were collected once in the control group and 1, 3 and 7 days after trauma in the injury groups. The F1+2 and TM concentrations were determined by enzyme linked immunosorbent assay (ELISA), and D-D concentrations were measured by automated latex enhanced immunoassay. F1+2, D-D and TM levels were higher in the minor and major injury groups than in the control group. They were markedly higher in the major injury group than in the minor injury group. In the non-DIC subgroup, F1+2 levels declined gradually while D-D and TM levels declined continuously. In the DIC subgroup, F1+2 and D-D levels remained elevated while TM levels exhibited an early rise and subsequent decrease. Plasma F1+2, D-D and TM levels were higher in the DIC patients than in the non-DIC patients. Injury-induced increases in F1+2, D-D and TM plasma levels had significant positive correlation with each other at each time point. Besides being related to trauma severity, F1+2, D-D and TM levels correlate closely with the occurrence of posttraumatic DIC. Therefore, changes in plasma F1+2, D-D and TM levels may predict the occurrence of DIC.

Thrombomodulin as a Marker for Bleeding Complications During Warfarin Treatment

The major adverse effect of warfarin treatment is hemorrhage. Several risk factors for bleeding complications are also risk factors for thromboembolic events, making the clinical decision to initiate or withhold anticoagulant treatment difficult. Specific markers that solely identify patients at high risk of bleeding would have great clinical impact. This study aimed to test if thrombomodulin (TM) concentrations were associated with bleeding complications, cardiovascular events, or mortality in long-term anticoagulant-treated patients. In a longitudinal cohort study we followed up 719 patients receiving warfarin treatment for a mean duration of 4.2 years. All bleeding complications causing hospitalization were registered and classified. Soluble TM antigen (sTM) concentration in plasma was measured with an enzyme-linked immunosorbent assay method. During the follow-up time, 113 clinically relevant bleeding events and 73 major bleeding events occurred. Increased concentration of sTM was associated with both clinically relevant bleeding and major bleeding events after adjustment for age. In the multivariable models, hazard ratios for the highest tertiles compared with the lowest were 2.29 (95% confidence interval, 1.35-3.89) and 2.33 (95% confidence interval, 1.21-4.48), respectively. No association between sTM concentration and nonfatal ischemic cardiovascular events or all-cause mortality was found. Increased levels of sTM are associated with bleeding complications during warfarin treatment but not with cardiovascular events or all-cause mortality. Soluble TM antigen concentration has potential as a new specific marker to identify patients at high risk of bleeding during warfarin treatment.

In Vitro Plasma-Induced Endothelial Oxidative Stress and Circulating Markers of Endothelial Dysfunction in Preeclampsia: An Observational Study

Objective: To investigate plasma-induced endothelial reactive oxygen species (ROS) production in vitro and its relation to endothelial dysfunction in preeclampsia (PE). Methods: Plasma was drawn from 17 PE patients, 17 matched healthy pregnant (HP) women, 17 matched non-pregnant healthy volunteers (NP), and 10 septic shock (SC) patients. In vitro plasma-induced ROS production was assessed in cultured human endothelial cells. In vivo endothelial activation and injury were assessed through measurements of plasma von Willebrand factor (vWF) and soluble thrombomodulin (sTM) concentrations, respectively. Results: Endothelial ROS production was not induced by PE, HP, and NP plasmas. However, it was significantly increased in SC compared to other groups (p < .005). Pregnancy (PE and HP) was associated with higher vWF compared to NP. Among pregnancies, vWF was higher in PE compared to HP women (p < .05). sTM was unchanged between PE, HP and NP. In SC, vWF and sTM were significantly increased compared to other groups (p < .01). Simultaneously, endothelial ROS production and sTM concentration were correlated (p = .673; p < .05). Conclusion. Plasma does not induce in vitro endothelial ROS production in PE women for which endothelial dysfunction is limited to activation but not injury. By contrast, SC patients demonstrate both endothelial activation and injury, closely related to plasma-induced endothelial oxidative stress.

The Role of the Coagulation and Fibrinolytic Pathway in Acute Lung Injury

Acute lung injury (ALI) is a common, life-threatening cause of acute respiratory failure, which is ultimately caused by a variety of local and systemic insults. Alterations in the coagulation and fibrinolysis profiles are present in almost all the patients suffering with ALI. The classic histologic findings in ALI patients include alveolar fibrin formation and microthrombi in the pulmonary vasculature. Decreased circulating levels of protein C and increased concentrations of thrombomodulin are present in patients with septic and nonseptic ALI. The circulating and pulmonary concentrations of plasminogen activator inhibitor-1 (PAI-1) are increased in the setting of ALI, and the degree of elevation in the PAI-1 level directly correlates with mortality. The need for new specific therapies has led a number of investigators to examine the role of altered coagulation and fibrinolysis in the pathogenesis of ALI. This review summarizes the current understanding of coagulation and fibrinolysis in ALI with an emphasis on the pathways that could be potential therapeutic targets, including the tissue factor pathway, the protein C pathway and the modulation of fibrinolysis via plasminogen activator inhibitor-1.

Low Plasma Activated Protein C-Protein C Inhibitor Complex Concentration Is Associated with Vascular Access Failure in Hemodialysis Patients

Background: Vascular access failure is a common cause of morbidity in patients with end-stage renal failure on hemodialysis (HD). Activation of the coagulation system and formation of a thrombus play important roles in recurrent arteriovenous fistula/graft (AVFG) failure. Thrombin in complex with thrombomodulin (TM) activates the anticoagulant protein C and creates activated protein C (APC), which is subsequently inactivated by the protein C inhibitor (PCI). The plasma concentration of the complex between APC and PCI (P-APC-PCI complex) is increased in hypercoagulable states and is therefore a sensitive indicator of the degree of activation of blood coagulation. Methods: Thirty-five HD patients dialyzed through a functioning AVFG were studied. The period of patency of AVFGs was recorded. Blood was drawn before and after the HD session for the analysis of the APC-PCI complex, soluble TM concentration and activity, von Willebrand factor antigen and homocysteine. Results: Patients with frequent AVFG failures (n = 8) had a significantly lower level of P-APC-PCI complex (median 0.09 µg/l) than those with less frequent AVFG failures (median 0.18 µg/l; n = 27; p = 0.04). No other significant differences were found between the groups. Conclusion: Thus, a low level of P-APC-PCI complex may be associated with an increased risk of AVFG failure in HD patients. Further prospective studies are needed to confirm these results and to evaluate the possibility of prophylactic measures.

Proinflammatory and atherogenic activity of monocytes in Type 2 diabetes

Cytokines secreted by the monocyte–macrophage system play a key role in the progression of atherosclerotic lesions in Type 2 diabetes. The objectives of this study were to assess the influence of cytokine gene expression in monocytes from patients with Type 2 diabetes on direct markers of endothelial injury with regard to clinically manifest atherosclerosis. Methods Monocytes from 58 patients with Type 2 diabetes and from 22 age-matched healthy volunteers of a control group were isolated in order to assess expression of tumor necrosis factor α (TNFα), interleukin (IL)-6, IL-8 and IL-10 cytokines (RTPCR, Applied Biosystems). Thrombomodulin concentration was determined using a Diagnostica Stago Immunoenzymatic assay, and circulating endothelial cell numbers were assayed using immunofluorescence studies with CLB-HEC19 antibodies. Results In 28 patients, TNFα expression in monocytes was observed. In these patients, as compared to those with undetectable levels of this cytokine's expression, higher hemoglobin A 1c ( P=.012) and thrombomodulin ( P=.005) concentrations were found. IL-8 expression was determined in 36 patients. Higher expression of TNFα ( P=.048) and IL-8 ( P=.049) was detected in patients with peripheral arterial disease in contrast to those free from this complication. Conclusion TNFα and IL-8 play a significant role in the proatherogenic activity of monocytes in Type 2 diabetes. The TNFα-connected activity of monocytes may directly determine endothelial dysfunction and injury. The location of atherosclerosis should be taken into account in the assessment of the proinflammatory activity of peripheral blood monocytes.

N-Glycans and the N Terminus of Protein C Inhibitor Affect the Cofactor-enhanced Rates of Thrombin Inhibition

Protein C inhibitor (PCI) is a serine protease inhibitor, displaying broad protease specificity, found in blood and other tissues. In blood, it is capable of inhibiting both procoagulant and anticoagulant proteases. Mechanisms that provide specificity to PCI remain largely unrevealed. In this study we have for the first time provided a full explanation for the marked size heterogeneity of blood-derived PCI and identified functional differences between naturally occurring PCI variants. The heterogeneity was caused by differences in N-glycan structures, N-glycosylation occupancy, and the presence of a Delta6-N-cleaved form. Bi-, tri-, and tetra-antennary complex N-glycans were identified. Fucose residues were identified both on the core GlcNAc and as parts of sialyl-Le(a/x) epitopes. Moreover, a glycan with a composition that implied a di-sialyl antenna was observed. PCI was N-glycosylated at all three potential N-glycosylation sites, Asn-230, Asn-243, and Asn-319, but a small fraction of PCI lacked the N-glycan at Asn-243. The overall removal of N-glycans affected the maximal heparin- and thrombomodulin-enhanced rates of thrombin inhibition differently in different solution conditions. In contrast, the Delta6-N-region increased both the heparin- and the thrombomodulin-enhanced rates of thrombin inhibition at all conditions examined. These results thus demonstrate that the N-linked glycans and the N-terminal region of blood-derived PCI in different ways affect the cofactor-enhanced rates of thrombin inhibition and provide information on the mechanisms by which this may be achieved. The findings are medically important, in view of the documented association of PCI with atherosclerotic plaques and the promising effect of PCI on reducing hypercoagulability states.

Protein C System in Patients with Idiopathic Pulmonary Fibrosis—Preliminary Report

The natural anticoagulant-activated protein C system plays an important role in the pathogenesis of idiopathic pulmonary fibrosis. The purpose of this study was to evaluate the concentration of protein C (PC), protein S (PS), thrombomodulin (TM), selectin E (sSelE), and thrombin-antithrombin complex (TAT) in patients with idiopathic pulmonary fibrosis (IPF). Study group consisted of 11 patients aged 51.5 +/- 8.62 years with idiopathic pulmonary fibrosis and 20 healthy adults as control. Concentration of PC, PS TM, sSelE and TAT in plasma with ELISA method was assessed. We observed significantly lower plasma concentration of PC (98.24 +/- 16.17% vs. 130.59 +/- 19.03%), PS (71.31 +/- +/- 12.95% vs. 93.47 +/- 18.63%), TM (2.67 +/- 0.40 ng/ml vs. 3.99 +/- 1.16 ng/ml) and significantly higher level of TAT complex (Me = 4.00 mg/ml vs. 2.20 mg/ml) and sSelE (Me = 36.40 ng/ml vs. 22.84 ng/ml) in patients with idiopathic pulmonary fibrosis as compared to controls. In presented pilot study we observed decreased activity of protein C system and increased thrombin generation in peripheral blood of patients with idiopathic pulmonary fibrosis.

Clinical and Virological Characteristics of 15 Patients with Chronic Active Epstein‐Barr Virus Infection Treated with Hematopoietic Stem Cell Transplantation

Chronic active Epstein-Barr virus (EBV) infection is characterized by recurrent infectious mononucleosis-like symptoms, and infected patients have high viral loads in their peripheral blood. Standard therapy for the disease has not yet been established. Recently, hematopoietic stem cell transplantation (HSCT) has been introduced and has the potential to become a standard treatment, although guidelines for HSCT to treat chronic active EBV infection have not yet been proposed. Fifteen patients were retrospectively analyzed, both clinically and virologically, to investigate the factors associated with prognosis of chronic active EBV infection treated with HSCT. After HSCT, 7 patients died after survival periods that ranged from 1 to 16 months (mean duration of survival after HSCT, 5 months). Three patients were considered to have died of transplantation-related complications. The duration between infection onset and diagnosis was significantly longer in patients who died than in those who survived. Five of the 7 patients who died experienced > or =3 life-threatening complications. The plasma concentrations of interferon-gamma, interleukin-10, thrombomodulin, and soluble E-selectin did not differ significantly between the groups of patients. With regard to sequence variations in the EBV latent membrane protein 1 gene, no specific patterns were found in the patients who died. Importantly, the plasma EBV load at diagnosis was significantly higher in patients who died than in living patients. Moreover, plasma viral load was shown to be an important factor to monitor during follow-up for patients after HSCT. The number of life-threatening complications and plasma viral load are indicative of the stage of disease progression and may be useful factors for predicting the outcome of HSCT.