Plasminogen activator inhibitor-1 (PAI-1) 4 G/5 G polymorphism and endometrial cancer. Influence of PAI-1 polymorphism on tissue PAI-1 antigen and mRNA expression and tumor severity

Plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism may have significance for PAI-1 expression. High levels of PAI-1 in endometrial cancer patients are associated with a poor prognosis. The objective of this study was to evaluate the PAI-1 4G/5G polymorphism in women with and without endometrial cancer and to analyze the influence of this polymorphism on PAI-1 expression in endometrial tissue.In 423 women (212 patients with endometrial cancer and 211 controls) PAI-1 4G/5G polymorphism was determined by PCR amplification using allele-specific primers. Quantitative real-time RT-PCR assay was used to quantify PAI-1 mRNA and PAI-1 protein levels were quantified by ELISA in tissue extracts from 33 patients with endometrial cancer and from 70 endometrial tissues from control women. The frequency of PAI-1 4G/4G genotype (P = 0.010) and the PAI-1 4G allele (P = 0.009) was significantly higher in patients than in controls. The frequency of PAI-1 4G allele was significantly higher in patients with stage IB than in those with stage IA (P = 0.03). Control women with the 4G/4G genotype had higher endometrial PAI-1 protein (P = 0.018) and mRNA (P = 0.004) levels than those with the 5G/5G genotype. A significant increase in PAI-1 protein and mRNA was observed in endometrial cancer tissue in comparison with the endometrial tissue from control women (P < 0.01). In conclusion, frequencies of the PAI-1 4G allele and 4G/4G genotype were found significantly more often in women with endometrial cancer than in controls. PAI-1 levels in endometrial tissue seem to be associated with PAI-1 4G/5G polymorphism. These findings suggest that the PAI-1 4G/4G genotype may be associated with the risk of endometrial cancer in a Caucasian population. Further studies with a larger number of patients are needed to clarify the influence of this PAI-1 polymorphism in endometrial cancer.     

Haemostatic system, biochemical profiles, kynurenines and the prevalence of cardiovascular disease in peritoneally dialyzed patients

Introduction

The haemostatic and biochemical abnormalities participate in the progression of cardiovascular disease (CVD) in peritoneally dialysed (PD) patients. Recently, the role of kynurenine (KYN) pathway of tryptophan (TRP) degradation in the development of CVD has been postulated.

Materials and methods

The present study was undertaken to investigate haemostatic parameters, biochemical profiles and kynurenines in PD patients both with and without CVD compared to age- and sex-matched healthy controls.

Results

The multiple biochemical abnormalities were present in PD patients, particularly in those with CVD. Tissue factor (TF), its inhibitor (TFPI), prothrombin fragment 1 + 2 (F_1 + 2)_, urokinase-type plasminogen activator (uPA), its soluble receptor (suPAR), plasmin/antiplasmin (PAP) complexes, KYN, kynurenic (KYNA) and quinolinic (QA) acids levels were significantly higher, whereas TRP was significantly lower in the PD patients than in the controls. Tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) were higher in the patients with CVD than in the patients without CVD and controls. PD patients with CVD had higher F_1 + 2, and they had lower suPAR and KYNA levels compared with PD patients without CVD. KYNA was positively associated with TFPI, whereas its was inversely associated with F_1 + 2 both in the whole PD group and in CVD patients. Logistic regression analysis showed that low KYNA, high glucose, low HDL-cholesterol levels and the duration of dialysis treatment were independently associated with the presence of CVD in PD patients.

Conclusions

The present study suggests a relationship between kynurenine pathway of tryptophan degradation, haemostatic and biochemical disturbances and CVD prevalence in peritoneally dialyzed patients.     

Fibrin clot properties in women heterozygous for factor V Leiden mutation: Effects of oral contraceptives

Introduction

Oral contraceptives (OC) in the presence of factor V Leiden mutation (FVL) markedly increase the risk of venous thromboembolism (VTE). Little is known about the OC and FVL-related alterations in fibrin clot properties.

Subjects and Methods

Plasma fibrin clot permeability (K_s) and efficiency of lysis, reflected by clot lysis time (CLT) and the rate of D-dimer release from clots (D-D_rate) induced by recombinant tissue plasminogen activator (tPA) were determined in 25 women with a family history of VTE who were heterozygous for FVL [FVL(+/−) - twice, on third-generation OC and after their discontinuation. Female non-carriers of FVL, matched for demographics, using OC and after their discontinuation served as controls (n = 25). All participants had no personal history of VTE.

Results

OC discontinuation in FVL(+/−) women resulted in shortened CLT (− 9%), and increased K_s (+ 4%) and D-D_rate (+ 1.4%; all p < 0.01). Alterations in fibrin clot properties were associated with decreased prothrombin fragments 1 + 2 (F1 + 2) (− 8%), plasminogen activator inhibitor-1 (PAI-1) antigen (− 11%), and thrombin activatable fibrinolysis inhibitor (TAFI) activity (− 20%; all p < 0.01). During OC use FVL(+/−) carriers compared with non-carriers had higher platelet count, activity of PAI-1, TAFI, and tPA, as well as prolonged CLT and higher D-D_max, along with lower D-D_rate and K_s. Multiple regression analysis adjusted for fibrinogen and age, showed that PAI-1 antigen and TAFI activity independently predicted CLT in FVL(+/−) women on OC.

Conclusion

FVL(+/−) is associated with hypofibrinolysis in apparently healthy women and third-generation OC administration unfavorably alters plasma clot characteristics in female FVL(+/−) carriers with a family history of thrombotic events.     

Atorvastatin has antithrombotic effects in patients with type 1 diabetes and dyslipidemia

Introduction

Diabetes is a prothrombotic state involving a more thrombogenic fibrin network. In the present study we investigated the effects of lipid-lowering therapy with atorvastatin on fibrin network structure and platelet-derived microparticles in patients with type 1 diabetes and dyslipidemia.

Materials and Methods

Twenty patients were treated with atorvastatin (80 mg daily) or placebo during 2 months in a randomized, double-blind, cross-over study. Fibrin network permeability, expression of glycoprotein IIIa, P-selectin and tissue factor on platelet-derived microparticles, plasma endogenous thrombin potential, plasminogen activator inhibitor-1 and tissue plasminogen activator antigen levels were assessed. Additionally, levels of plasma fibrinogen, high-sensitivity C-reactive protein and glycated haemoglobin were measured.

Results

During treatment with atorvastatin, fibrin network permeability increased (p = 0.01), while endogenous thrombin potential and expression of glycoprotein IIIa, P-selectin and tissue factor decreased (p < 0.01). In vitro experiments indicated that platelet-derived microparticles influence the fibrin network formation as fibrin network permeability decreased significantly when platelet-derived microparticles were added to normal plasma. Baseline levels of plasminogen activator inhibitor-1 and tissue plasminogen activator antigen as well as plasma fibrinogen and high-sensitivity C-reactive protein were within reference values and not significantly changed during atorvastatin treatment, while glycated haemoglobin increased 0.3% (p < 0.001).

Conclusions

Novel treatment effects were found in patients with type 1 diabetes and dyslipidemia during atorvastatin therapy, i.e. a more porous fibrin network, to which reduced expression of glycoprotein IIIa, P-selectin and tissue factor on platelet-derived microparticles may contribute. The observed impairment of glycemic control during long-term statin treatment deserves attention.     

Changes of plasma and placental tissue factor pathway inhibitor-2 in women with preeclampsia and normal pregnancy

Objectives

To investigate the maternal and fetal plasma changes of tissue factor pathway inibitor-2 (TFPI-2) and its placental expression in women with normal pregnancy and preeclampsia.

Material and methods

We assessed the plasma TFPI-2 level in non-pregnant, normal pregnant and postpartum women, detected fetal plasma level and expression in placenta, and compared the changes in women with preeclampsia. Time-resolved fluoroimmunoassay and immunohistochemistry were used for plasma and placenta tissue detection, respectively.

Results

Maternal plasma levels of TFPI-2 in normal pregnant women at 13 weeks of gestation increased 9.3 times as compared with healthy non-pregnant women (149.3 ± 17.1 versus 16.0 ± 3.6 ng/ml),reached a maximum level ( 282.6 ± 17.1 ng/ml) at 39 weeks of gestation, and dramatically decreased to nearly a non-pregnant level on the first day of postpartum (32.3 ± 7.1 ng/ml); similar change was found in the placental expression. Fetal plasma TFPI-2 was significantly lower than the maternal level at delivery. The maternal plasma TFPI-2 in preeclampsia was significantly lower compared with that in normal pregnancy, coupled with significantly higher placental expression.

Conclusions

Placenta may be the main site of the high level of TFPI-2 production in maternal circulation, and the dramatic changes in preeclampsia provide a clue to elucidate its pathogenesis.     

Redirection of the reaction between activated protein C and a serpin to the substrate pathway

BACKGROUND: Activated protein C (APC) reduces mortality in severe sepsis. Protecting APC in the circulatory system from inactivation by serine protease inhibitors (serpins) could improve its therapeutic efficiency. Significantly elevated levels of a serpin plasminogen activator inhibitor 1 (PAI-1) correlate with a lethal outcome in severe sepsis and disseminated intravascular coagulation. Intermolecular mechanisms were employed to redirect the reaction between APC and PAI-1 from the inhibitory to the substrate pathway, which results in the catalytic neutralization of the serpin. METHODS: The effects of anti-PAI-1 monoclonal antibodies (mAbs) and vitronectin, as well as their fragments, on the kinetics and stoichiometry of the reaction between PAI-1 and APC were studied using SDS PAGE and fluorescence spectroscopy. RESULTS: MAbs with epitopes at alpha-helix F redirected 70-80% of the reaction between PAI-1 and APC, to the substrate pathway. Vitronectin and its SMB domain did not affect the stoichiometry of acyl-enzyme formation, but enhanced the effect of mAbs. While vitronectin induced a more than two-fold increase in the rate of the reaction between PAI-1 and APC, neither mAbs (mAb fragments), nor SMB domain of vitronectin affected it. CONCLUSIONS: Ligands interacting with alpha-helix F of PAI-1 demonstrated a potential for the protection of APC from inactivation by PAI-1. Since the mechanism of proteinase/serpin interaction is universal, a similar design and approach could be employed for enhancing the inactivation of other serpins in order to preserve APC activity in the circulation. Rational pharmacological targeting of the inhibitors of APC could have therapeutic utility.     

Persimmon leaf flavonoid induces brain ischemic tolerance in mice

The persimmon leaf has been shown to improve cerebral ischemic outcomes; however, its mechanism of action remains unclear. In this study, mice were subjected to 10 minutes of ischemic preconditioning, and persimmon leaf flavonoid was orally administered for 5 days. Results showed that the persimmon leaf flavonoid significantly improved the content of tissue type plasminogen activator and 6-keto prostaglandin-F1 α in the cerebral cortex, decreased the content of thromboxane B2, and reduced the content of plasminogen activator inhibitor-1 in mice. Following optical microscopy, persimmon leaf flavonoid was also shown to reduce cell swelling and nuclear hyperchromatism in the cerebral cortex and hippocampus of mice. These results suggested that persimmon leaf flavonoid can effectively inhibit brain thrombosis, improve blood supply to the brain, and relieve ischemia-induced pathological damage, resulting in brain ischemic tolerance.     

Circulating platelet-derived microparticles and endothelium-derived microparticles may be a potential cause of microthrombosis in patients with osteonecrosis of the femoral head

Introduction

To test the hypothesis that the platelet microparticle (PMP) and endothelial microparticle (EMP) may contribute to the hypercoagulability associated with microvascular thrombosis in patients with nontraumatc osteonecrosis of the femoral head (ONFH).

Materials and methods

The study comprised 46 patients who had been diagnosed with ONFH and 20 control subjects. The plasma was ultracentrifuged, and then PMPs and EMPs were examined by the flow cytometry. The thrombotic and fibrinolytic disorders were investigated.

Results

The numbers of PMPs expressing P-selectin and CD42a and EMPs expressing E-selectin and CD31 in the ONFH patients were significantly higher than those in the controls (P < 0.001). The number of MPs was correlated with the level of the serum C-reactive protein (CRP) (r = 0.661, P < 0.001), but there was a poor correlation between the MPs counts and the risk factors for ONFH (P > 0.05). The mean levels PAI-1, F1 + 2, and TAT were higher in the patients with ONFH than in the controls (P < 0.05).

Conclusions

The elevated numbers of PMPs and EMPs may contribute to hypercoagulability in the ONFH patients. This may provide important pathophysiological insights into the hypercoagulability associated with nontraumatic ONFH and have implications for pharmacological prevention and treatment of ONFH.     

Parameters of the thrombogram are associated with serum 25-hydroxyvitamin D levels at baseline, but not affected during supplementation with vitamin D

Introduction

In vitro studies indicate an anticoagulant effect of 1,25-dihydroxyvitamin D, and sun exposure may lower the risk of thrombotic events. Accordingly, an effect on haemostatic parameters could be expected after supplementation with vitamin D.

Materials and Methods

158 obese or overweight subjects were included in a one year intervention study with supplementation with 40.000 IU vitamin D₃ per week or placebo. All subjects were given 500 mg calcium daily. Plasminogen activator inhibitor 1 (PAI-1), tissue plasminogen activator antigen (tPA Ag), and tissue factor-induced thrombin generation over time in plasma assessed by the calibrated automated thrombogram (CAT) method as a parameter of over all thrombotic activity, were measured before and at the end of the study.

Results

Mean baseline serum 25(OH)D level was 61.8 nmol/L and increased in the vitamin D group to 145.6 nmol/L at the end of the study. At baseline there was a significant decrease in the CAT variables lag time and time to peak of the thrombogram across increasing serum 25(OH)D quartiles, whereas no significant associations between serum 25(OH)D and PAI-1 or tPA Ag were found. After one year, no significant differences were found between the vitamin D and placebo groups regarding change in any of the haemostatic parameters.

Conclusions

The association between lag time and time to peak in the CAT assay and serum 25(OH)D levels could indicate a pro-thrombotic state in subjects with high serum 25(OH)D levels, whereas the lack of effect of high dose vitamin D supplementation questions the causality of this relation.     

In vitro fibrin clot formation and fibrinolysis using heterozygous plasma fibrinogen from gammaAsn319, Asp320 deletion dysfibrinogen, Otsu I

INTRODUCTION: We have reported a heterozygous dysfibrinogenemia, fibrinogen Otsu I, caused by the deletion of gammaAsn319 and gammaAsp320, which was originally identified in the dysfibrinogen Vlissingen/Frankfurt IV (V/FIV) associated with thrombosis. Unlike the V/FIV family, the Otsu propositus showed no thrombotic tendencies. To analyze the relationship between thrombosis and the heterozygous plasma variant fibrinogen, we used purified plasma fibrinogen from the Otsu patient and compared it with a normal control. MATERIALS AND METHODS: Thrombin-induced fibrin clot formation and clot structure were observed by fibrin polymerization and scanning electron microscopy, respectively. For in vitro observation of fibrinolysis, plasmin generation and clot lysis assays were performed by the addition of tissue type plasminogen activation (tPA) and plasminogen. RESULTS AND CONCLUSIONS: Polymerization of Otsu was markedly impaired, while fibrin fibers were much thicker and the density of the bundles of fibrin fibers was less and porous compared with normal. Lysis of the Otsu clot was not significantly different from normal when a tPA and plasminogen mixture was overlaid onto the clots. For Otsu, the penetration of the tPA/plasminogen mixture into the clot was much faster than normal and the protection against plasmin cleavage was impaired; however, tPA-induced plasmin activation of the Otsu fibrin was slower than that of normal fibrin, resulting in a clot lysis of Otsu similar to normal.     

Comparison of the effect of edoxaban,a direct factor Xa inhibitor,with a direct thrombin inhibitor,melagatran, and heparin on intracerebral hemorrhage induced by collagenase in rats

Introduction

Intracerebral hemorrhage (ICH) is a major clinical concern with anticoagulation therapy. The effect of a new oral direct FXa inhibitor, edoxaban, was determined in a rat model of ICH and compared with a direct thrombin inhibitor, melagatran, and heparin.

Methods

To induce ICH, 0.1 U collagenase type VII was injected into the striatum of male Wistar rats under anesthesia with thiopental or halothane. Immediately after ICH induction, edoxaban, melagatran, or heparin were infused intravenously. Five hours after ICH induction, the brain was removed and ICH size was measured. To estimate the margin of safety, antithrombotic effects were evaluated in a rat venous thrombosis model.

Results

Edoxaban at 6 mg/kg/h significantly increased ICH volume (1.8-fold) and prolonged prothrombin time (PT) 2.8-fold compared to the vehicle group. No deaths were observed with edoxaban. Melagatran at 1 mg/kg/h increased ICH volume at 1 mg/kg/h (2.8-fold) with 6.1-fold PT prolongation. At 3 mg/kg/h, all rats died due to severe ICH (3.9-fold). Heparin at both 100 and 500 U/kg/h significantly increased ICH. At 500 U/kg/h, 5 out of 8 rats died. The doses required for 50% inhibition of thrombosis of edoxaban, melagatran, and heparin were 0.045 mg/kg/h, 0.14 mg/kg/h, and 55 U/kg/h, respectively. The safety margins between antithrombotic and ICH exacerbation effects of these anticoagulants were 133, 7.1, and 1.8, respectively.

Conclusion

The safety margin of edoxaban was wider than that of melagatran or heparin. These results suggest that edoxaban may be preferable from the perspective of ICH exacerbation risk.     

Carbon monoxide (CO)-releasing molecule-derived CO regulates tissue factor and plasminogen activator inhibitor type 1 in human endothelial cells

Introduction

Heme oxygenase-1 (HO-1) is the rate limiting enzyme that catalyzes the conversion of heme into biliverdin, free iron, and carbon monoxide (CO). The first human case of HO-1 deficiency showed abnormalities in blood coagulation and the fibrinolytic system. Thus, HO-1 or HO-1 products, such as CO, might regulate coagulation and the fibrinolytic system. This study examined whether tricarbonyldichlororuthenium (II) dimer (CORM-2), which liberates CO, modulates the expression of tissue factor (TF) and plasminogen activator inhibitor type 1 (PAI-1) in human umbilical vein endothelial cells (HUVECs), and TF expression in peripheral blood mononuclear cells (PBMCs). Additionally, we examined the mechanism by which CO exerts its effects.

Materials and Methods

HUVECs were pretreated with 50 μM CORM-2 for 3 hours, and stimulated with tumor necrosis factor-α (TNF-α, 10 ng/ml) for an additional 0-5 hours. PBMCs were pretreated with 50-100 μM CORM-2 for 1hour followed by stimulating with lipopolysaccharid (LPS, 10 ng/ml) for additional 0-9 hours. The mRNA and protein levels were determined by RT-PCR and western blotting, respectively.

Results

Pretreatment with CORM-2 significantly inhibited TNF-α-induced TF and PAI-1 up-regulation in HUVECs, and LPS-induced TF expression in PBMCs. CORM-2 inhibited TNF-α-induced activation of p38 MAPK, ERK1/2, JNK, and NF-κB signaling pathways in HUVECs.

Conclusions

CORM-2 suppresses TNF-α-induced TF and PAI-1 up-regulation, and MAPKs and NF-κB signaling pathways activation by TNF-α in HUVECs. CORM-2 suppresses LPS-induced TF up-regulation in PBMCs. Therefore, we envision that the antithrombotic activity of CORM-2 might be used as a pharmaceutical agent for the treatment of various inflammatory conditions.     

Low affinity binding of the classical D1 antagonist SCH23390 in rodent brain: potential interaction with A2A and D2-like receptors

Whereas structurally dissimilar D(1) antagonists competing for [(3)H]-SCH23390 binding recognize primarily one site in striatum, two distinct affinity states are observed in both amygdala and hippocampus. The binding profile of SCH23390 is similar in both of these regions, with the high affinity site (K(D) approximately 0.4 nM) consistent with D(1)/D(5) receptors. The appearance of the low affinity site (K(D) approximately 300 nM) is dependent upon the absence of MgCl(2), but independent of D(1) expression (i.e., still present in D(1) knockout mice). Although the density of high affinity state receptor is lower in hippocampus or amygdala of D(1) knockout mice, some residual binding remains, consistent with the known expression of D(5) receptors in these regions. Remarkably, in hippocampus, the affinity of the low affinity site is shifted rightward in the presence of the D(2) antagonist domperidone and is largely absent in the hippocampus of D(2) knockout animals. Additionally, this site is also shifted rightward in the presence of the A(2A) ligands SCH58261, CSC, or NECA, or in the absence of A(2A) receptors. The affinity of SCH23390 for this low affinity site is greater than seen for SCH23390 binding to D(2) receptors in heterologous expression systems, consistent with the hypothesis that both D(2) and A(2A) receptors are involved in the low affinity binding site. Therefore, we suggest that the heteromerization of D(2) and A(2A) receptors reported previously in vitro also may occur in the brain of both rats and mice.     

Pharmacokinetic and pharmacodynamic profiles of the novel serotonin and norepinephrine reuptake inhibitor desvenlafaxine succinate in ovariectomized Sprague-Dawley rats

Desvenlafaxine succinate (DVS) is a novel serotonin (5-HT) and norepinephrine (NE) reuptake inhibitor (SNRI) that is currently in clinical development for the treatment of major depressive disorder and vasomotor symptoms associated with menopause. Previous studies have documented the pharmacokinetic and pharmacodynamic profiles of DVS in male rats. Similar studies, however, have not been performed in ovariectomized (OVX) rats, a model that mimics the loss of ovarian hormones that occurs at menopause. The goal of the present study, therefore, was to characterize the pharmacokinetic and pharmacodynamic properties of DVS in OVX rats. Desvenlafaxine levels peaked in plasma, brain (total brain minus hypothalamus) and hypothalamus at concentrations of 7.0, 10.8 and 9.5 microM (assuming 1 g = 1 ml), respectively, 30 min post-dosing DVS (30 mg/kg, oral). The apparent terminal half-lives of desvenlafaxine in plasma, brain and hypothalamus were 3.0, 2.1 and 2.5 h, respectively. Based on AUC(0-last), brain to plasma and hypothalamus to plasma ratios were 1.7 and 1.3, respectively. Microdialysis experiments in the medial preoptic area of the hypothalamus showed that DVS (30 mg/kg, s.c.), in the presence of WAY-100635 (5-HT(1A) antagonist), increased 5-HT levels 225% at 1 h post-dosing. Norepinephrine levels increased 44% at 3 h post-dosing while dopamine levels were unchanged. Thus, in OVX rats, DVS has good pharmacokinetic properties, rapid brain penetration, excellent brain penetrability and selectively increases 5-HT and NE levels in the hypothalamus. This work supports the notion that DVS could have utility for treating disorders in menopausal women in which changes in 5-HT and/or NE have been implicated.     

P-glycoprotein and cytochrome P450 3A4 involvement in risperidone transport using an in vitro Caco-2/TC7 model and an in vivo model

The possible involvement of P-glycoprotein (P-gp) and cytochrome P450 (CYP) 3A4 in risperidone transport was investigated using in vitro and in vivo models. Firstly, uptake studies were performed on a Caco-2/TC7 cell monolayer; the effects of 1 microg ml(-1) risperidone on apparent permeability were determined for secretory and absorptive directions, in the presence or absence of various P-gp and CYP3A4 inhibitors (verapamil, ketoconazole, erythromycin), and of an associated multidrug-resistant protein inhibitor (indomethacin). Secondly, on a conscious rat model, risperidone pharmacokinetic parameters, notably absorption parameters, were determined using compartmental and deconvolution methods. Three groups of seven rats received respectively an IV risperidone dose, an oral risperidone dose (PO group) and the same oral risperidone dose after verapamil administration (POV group). No formation of 9-hydroxyrisperidone was observed on Caco-2 cells after risperidone administration; there was no evidence that intestinal CYP3A4 is involved in risperidone metabolising. Risperidone secretory permeation was higher than absorptive permeation. Verapamil increased risperidone absorption permeation and decreased its secretory permeation. Indomethacin did not modify these permeation values. In rats, verapamil led to an increase in both risperidone and 9-hydroxyrisperidone plasmatic concentrations. The fraction absorbed in the verapamil group was 3.18 times higher than in the oral group (65.9% and 20.7% for POV group and PO group). The absorption rate constant was lower in the verapamil group. Our results indicate that P-gp decreases the intestinal absorption of risperidone and that intestinal CYP3A4 is not involved in risperidone metabolism.     

Degradation of cross-linked fibrin by leukocyte elastase as alternative pathway for plasmin-mediated fibrinolysis in sepsis-induced disseminated intravascular coagulation

An alternative pathway for fibrinolysis that comprises leukocyte elastase and its interaction with the plasminogen activator-plasmin system has been suggested. Plasma levels of cross-linked fibrin degradation product by leukocyte elastase (e-XDP) were significantly increased in patients with sepsis induced disseminated intravascular coagulation (DIC) compared with healthy subjects (18.6 ± 19.9 vs 0.58 ± 0.47 U/mL, p < 0.001). Twenty seven unique spots were identified from e-XDP dominant patients by immune-purification and two-dimensional difference gel electrophoresis, and they contained fibrinogen Bβ-chain derived fragments Bβ Asp-164, Ser-200, Gln-301, Ala-354, Ile-484 and γ-chain derivatives γ Val-274 at their amino-termini by acquired and processed tandem mass spectrometer. The Sequential Organ Failure Assessment Scores in patients with e-XDPs levels 3-10 U/mL were significantly lower than those with e-XDPs levels -3 U/mL, 10-30 U/mL, and 30- U/mL. The adjusted odds for 28-day mortality rate in patients with e-XDP levels less than 3 U/mL (hazard ratio, 4.432; 95% CI, 1.557-12.615 [p = 0.005]) were significantly higher than those in patients with e-XDP levels of 3-10 U/mL. These data suggest that leukocyte elastase might contribute to the degradation of cross-linked fibrin in sepsis-induced DIC.     

TAFI and PAI-1 levels in human sepsis

BACKGROUND: Plasminogen activator inhibitor type-1 (PAI-1) is considered to be the main inhibitor of fibrinolysis in sepsis. However, the contribution of TAFI to the inhibition of fibrinolysis in sepsis is currently unknown. METHODS: TAFI antigen and PAI-1 levels were measured in severe sepsis (n = 32) and septic shock (n = 8) patients. In addition, TAFI antigen levels had been determined in 151 controls. RESULTS: Septic patients had significantly (p < 0.0001) decreased TAFI levels (median: 78.9% [range: 32.4-172.6]) as compared to controls (108.1% [35.9-255.4]). TAFI levels were equal in septic shock and severe sepsis (68.9% [32.4-172.6] vs. 82.5% [32.7-144.9], p = 0.987) as well as in survivors and non-survivors (87.1% [32.7-172.6] vs. 65.8% [32.4-129.5], p = 0.166). PAI-1 levels were significantly (705.5 ng/ml [131-5788]) higher in septic shock as in severe sepsis patients (316.5 ng/ml [53-1311], p = 0.016) and were equal in survivors and non-survivors (342 ng/ml [53-1311] vs. 413 ng/ml [55-5788], p = 0.231). TAT/PAP ratio (R((TAT/PAP))) reflecting the dysbalance between coagulation and fibrinolysis was calculated. R((TAT/PAP)) significantly increased with fatality and was significantly dependent on PAI-1, but not on TAFI. PAI-1 levels (570.5 ng/ml [135-5788]) and R((TAT/PAP)) (1.6 [0.3-6.1]) were significantly (p = 0.008 and p = 0.047) higher in patients with overt DIC as compared to patients without overt DIC (310 ng/ml [53-1128] and 0.6 [0.1-4.3]), whereas no difference was found for TAFI levels (68.9% [32.7-133.2] vs. 86.4% [32.4-172.6], p = 0.325). CONCLUSIONS: Although inhibition in sepsis is mediated by both, PAI-1 might be involved early in the sepsis process, whereas TAFI might be responsible for ongoing fibrinolysis inhibition in later stages of sepsis.     

Postprandial changes in the phospholipid composition of circulating microparticles are not associated with coagulation activation

Introduction

Evidence is present that the phospholipid composition of circulating cell-derived microparticles (MP) affects coagulation in vivo, and that postprandial metabolic alterations may be associated with hypercoagulable state. Our objective was to investigate whether postprandial metabolic responses affect the phospholipid composition of MP, and whether such changes are associated with coagulation activation.

Materials and Methods

Twelve healthy males were studied twice and randomly received two consecutive meals or remained fasted. Blood was collected before and at 2, 4, 6 and 8 h following breakfast. Plasma concentrations of prothrombin-F_1 + 2 and thrombin-antithrombin-complexes were measured. Numbers and cellular origin of MP were determined by flowcytometry. The phospholipid composition of MP was determined by hpTLC. In vitro procoagulant activity of MP was studied by fibrin generation.

Results

During the meal visit, plasma glucose, triglyceride and insulin levels increased, compared to baseline and the fasting visit (all P < 0.05). Postprandially, the total numbers of MP increased in time compared to the fasting visit (P < 0.05). Erythrocyte-derived MP increased (6-fold) during the meal visit, but remained constant on the fasting day (P < 0.001). On the meal versus fasting day circulating MP contained increased phosphatidylcholine (P < 0.05) and decreased sphingomyelin (P < 0.05) amounts. The amount of phosphatidylserine did not change. Concentrations of plasma F_1 + 2 and thrombin-antithrombin were similar on both days, as was the ability of MP to generate fibrin in vitro.

Conclusion

Although numbers, cellular origin and phospholipid composition of MP alter during exposure to two consecutive meals in healthy subjects, this does not lead to changes in the coagulation activation in vivo.