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.     

Comparison of markers of coagulation activation and thrombin generation test in uncomplicated pregnancies

Introduction

Pregnancy is a well-established risk factor for venous thromboembolism, and is associated with a state of hypercoagulability or parameters of thrombin generation. Currently, there is a lack of consensual data on thrombin generation during pregnancy. This study aimed to find a sensitive and specific biological marker of coagulation activation and to identify parameters of thrombin generation.

Patients and methods

The population included 101 women with uncomplicated pregnancies. The objective of this study was to correlate thrombin generation test (measured at 5pM tissue factor, 4 μM lipids and without thrombomodulin), with fibrinogen and markers of blood coagulation activation: D-dimer, prothrombin fragments 1+2 (F1+2), thrombin-antithrombin complexes (TAT) and fibrin monomer complexes (FMC) in these women. Internal quality control was performed in each set of experiments.

Results

Fibrinogen, D-dimer, F1+2, and TAT concentrations increased significantly throughout pregnancy, and were correlated with term of pregnancy. In our study, thrombin generation seemed to increase early on, and then remained stable throughout normal pregnancy, in contrast with other markers of blood coagulation activation, excepting FMC. The latter are subject to large inter-individual variations, especially during second trimester. No correlation was demonstrated between thrombin generation parameters and other activation markers.

Conclusion

While markers of coagulation activation significantly increased during pregnancy, thrombin generation increased only early on and remains stable during pregnancy. Finding a sensitive and specific biological marker for vascular pregnancy complications, such as FMC and thrombin generation levels, requires further investigation.     

Coagulation activation and ultrasound characteristics in patients with carotid artery disease

Introduction

Elevated levels of markers for thrombin activation are associated with plaque echogenicity and degree of stenosis in patients with carotid artery stenosis. The Activated Protein C-Protein C Inhibitor (APC-PCI) complex reflects activation of the Protein C system and is a measure of thrombin generation. The aim of the present study was to examine APC-PCI complex in patients undergoing thrombendartherectomy for carotid artery stenosis, and to relate the findings to clinical characteristics and plaque morphology as determined by ultrasound.

Materials and Methods

Blood was obtained from 125 patients (39 female, median age 71 years) with carotid artery stenosis admitted from September 2005 to May 2007. The APC-PCI complex was measured using a sandwich immunofluorometric method and compared to an age- and sex-matched healthy control-group. Clinical and demographic characteristics, routine laboratory markers and ultrasound characteristics were analysed using univariate and multivariate analysis.

Results

APC-PCI complex concentration was significantly increased in patients with carotid artery stenosis (median 0.21 µg/L; 10th to 90th percentile 0.15-0.36) compared to a healthy control-group (0.19 µg/L; 0.11-0.31; P = .009). There was no significant difference in APC-PCI-values between asymptomatic (n = 48) and symptomatic (n = 77) patients with carotid artery stenosis (0.22 vs. 0.20 µg/L; p = 0.626). Patients with minor stroke (n = 31) had a higher median APC-PCI-concentration (0.27 µg/L; 0.15-0.63) than patients with amaurosis fugax (0.19 µg/L; 0.15-0.36) or transient ischemic attack (0.21 µg/L; 0.12-0.36) (p = 0.016). No association was found between APC-PCI-values and the degrees of carotid artery stenosis or the time from the latest neurological symptoms to blood sampling. Patients with echolucent plaques had significantly lower APC-PCI concentrations (0.20 µg/L; 0.14-0.35 vs. 0.24 µg/L; 0.15-0.60; p = 0.043), according to the Gray-Weale classification.

Conclusions

Patients with carotid artery disease exhibit increased concentrations of APC-PCI compared to a healthy control-group, particularly those patients with echogenic plaques, who have significantly higher APC-PCI levels than patients with echolucent plaques.     

The coagulation profile of preterm delivery

Introduction

Hypercoagulation was suggested to be involved in preterm birth etiology; however, the coagulation state of preterm parturients remains unelucidated. The study aim was to evaluate the haemostatic system of pregnant women with premature uterine contractions (PUC).

Materials and Methods

The cohort study population consisted of 76 healthy pregnant women admitted with regular PUC. The study group included 38 women who experienced preterm birth; 14 of them had preterm premature rupture of membranes (PPROM). The control group included 38 women who eventually had term delivery. Groups were matched for maternal age, number of births and gestational age at admission. Blood samples were tested for haemostatic parameters and coagulation activation markers.

Results

Significantly shorter PT and aPTT were documented in the study compared to control group (25.7 ± 2 vs. 27.4 ± 2.7 seconds, P = 0.003, and 9.96 ± 0.5 vs. 10.1 ± 0.4 seconds, P = 0.05, respectively), although differences in absolute values were small. There was no significant difference between the two groups in levels of: fibrinogen, D-dimer, protein C-global, free protein S antigen, factor VIII activity, Von Willebrand factor, plasminogen activator inhibitor-1, prothrombin fragments F1 + 2 (PT F1 + 2), tissue factor and tissue factor pathway inhibitor.Women with PPROM had significantly lower PT F1 + 2 levels compared to those who had preterm delivery with intact membranes (351 ± 99 vs. 561 ± 242 pmol/L, P = 0.003).

Conclusions

Shortened PT and aPTT, reflecting increased thrombotic activity in maternal plasma, could serve as a marker of real preterm labor in women with premature uterine contractions. Further prospective studies in a larger cohort are warranted to validate these findings.     

Increased activation of blood coagulation in pregnant women with the Factor V Leiden mutation

Background

The risk of venous thromboembolism is enhanced in pregnant carriers of the Factor V Leiden mutation. The primary aim of the study was to compare prothrombin fragments 1 + 2, soluble fibrin and D-dimer levels in pregnant Factor V Leiden mutation carriers with those in non-carriers. Secondary aims were to evaluate whether these biomarkers could predict placenta-mediated complications or venous thromboembolism, and to study blood coagulation after caesarean section with thromboprophylaxis and after vaginal delivery without thromboprophylaxis.

Material/Methods

Prothrombin fragments 1 + 2, soluble fibrin and D-dimer levels were studied longitudinally in 476 carriers with singleton pregnancies from gestational weeks 23–25 until 8–10 weeks postpartum.

Results

Prothrombin fragments 1 + 2 and D-dimer levels gradually increased during pregnancy. D-dimer levels were higher in carriers, both during pregnancy and puerperium, compared to non-carriers. D-dimer levels above 0.5 mg/l were found in about 30% and 20% of the heterozygous carriers at 4–5 and 8–10 weeks postpartum, respectively. Soluble fibrin levels were mainly unchanged during pregnancy, with no difference between carriers and non-carriers. Biomarker levels were similar in carriers with uncomplicated and complicated pregnancies.

Conclusion

Higher D-dimer levels indicate increased blood coagulation and fibrinolysis activity in carriers. The high proportion of carriers with D-dimer levels exceeding 0.5 mg/l postpartum must be considered when assessing the probability of venous thromboembolism. Large overlaps in biomarker levels in normal and complicated pregnancies suggest that these biomarkers cannot be used as predictors. Thromboprophylaxis following caesarean section may prevent increased activation of blood coagulation.     

Treatment with everolimus is associated with a procoagulant state

Introduction

Renal transplant recipients are at increased risk of venous thromboembolic events, which is in part caused by their treatment with maintenance immunosuppressive drugs. Because we observed an increased incidence of venous thromboembolic events in renal transplant recipients treated with the mTOR inhibitor (mTORi) everolimus, we aimed to identify prothrombotic mechanisms of this immunosuppressive drug.

Materials and Methods

In a single center study, nested in a multi-center randomized controlled trial, we measured parameters of coagulation, anti-coagulation and fibrinolysis in renal transplant recipients, receiving the mTORi everolimus (n = 16, mTOR group) and compared them to a similar patient group, receiving a calcineurin inhibitor and/or mycophenolate sodium (n = 20, non-mTOR group). All patients were at least 6 months following transplantation with a stable transplant function.

Results

The use of an mTORi was associated with significantly higher levels of von Willebrand factor, prothrombin fragment 1 + 2, thrombin-activatable fibrinolysis inhibitor and plasminogen activator inhibitor-1 as compared to a non-mTORi based immunosuppressive regimen.

Conclusions

Treatment with an mTORi leads to increased endothelial activation, thrombin formation and impaired fibrinolysis in renal transplant recipients. This suggests an increased risk of thrombotic events in renal transplant recipients treated with mTOR inhibitors. A prospective study to establish the precise risk of thrombotic events in these patients is urgently needed.     

Postprandial coagulation activation in overweight individuals after weight loss: Acute and long-term effects of a high-monounsaturated fat diet and a low-fat diet

Diet is important in the prevention of cardiovascular disease, and it has been suggested that a high-MUFA diet is more cardioprotective than a low-fat diet. We hypothesised that the postprandial thrombotic risk profile is improved most favourably by a high-MUFA diet compared with a low-fat diet. This was tested in a parallel intervention trial on overweight individuals (aged 28.4 (SD 4.7) years) randomly assigned to a MUFA-diet (35-45% of energy as fat; > 20% as MUFA, n = 21) or a low-fat (LF) diet (20-30% of energy as fat, n = 22) for 6 months after a weight loss of ~ 10%. All foods were provided free of charge from a purpose-built supermarket. Meal tests designed after the same principles were performed before and after the dietary intervention, and blood samples were collected at 8.00 h (fasting), 12.00 h, and 18.00 h and analysed for factor VII coagulant activity (FVII:C), activated FVII, fibrinogen, prothrombin fragment 1 + 2 (F1 + 2), D-dimer, plasminogen activator inhibitor (PAI:Ag), and thrombin activatable fibrinolysis inhibitor. There were significant postprandial increases in F1 + 2 and D-dimer before and after dietary intervention, with significantly lower values after 6 months. No significant differences were observed between the postprandial changes induced by the two diets. The postprandial decrease in FVII:C and PAI:Ag did not differ before and after intervention, irrespective of the diets. Our findings suggest postprandial coagulation activation in overweight subjects with more pronounced acute than long-term effects. We observed similar effects of the MUFA diet and the LF diet on the postprandial prothrombotic risk profile.     

Markers of endothelial dysfunction differ between subphenotypes in children with sickle cell disease

In adult patients with sickle cell disease two distinct subphenotypes have previously been defined: patients with the viscosity-vaso-occlusion subphenotype (VVO) suffer mainly from vaso-occlusive pain crises and have a relatively high hemoglobin concentration. Patients classified as the hemolysis-endothelial dysfunction subphenotype (HED) suffer from stroke and pulmonary hypertension and have an elevated concentration of lactate dehydrogenase. However, this classification is not possible in children due to low rates of complications. We used laboratory markers to classify children into the two subphenotypes, and measured vWF and vWF propeptide as markers of endothelial dysfunction. We included 106 children with sickle cell disease (mean age 8.7 years), 74 (70%) with HbSS/HbSβ° genotype and 32 (30%) with HbSC/HbSβ⁺ genotype. vWF and vWF propeptide were significantly elevated in patients with sickle cell disease; this was more pronounced in patients with the HbSS/HbSβ° genotype. Patients with the HED subphenotype had higher levels of vWF propeptide, and a trend towards higher levels of vWF compared to those with the VVO subphenotype. We demonstrated that even young children in a stable clinical condition show signs of persistent endothelial dysfunction. A prospective study should demonstrate whether elevated levels of vWF and its propeptide are associated with an increased risk of complications specific for the HED subphenotype.     

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.     

Stent implantation in the superficial femoral artery: Short thrombelastometry-derived coagulation times identify patients with late in-stent restenosis

Introduction

The mechanisms of restenosis, the recurrence of luminal narrowing, are complex and incompletely understood to date. Thrombin, the pivotal enzyme in haemostasis, presumably contributes to the formation of in-stent restenosis (ISR). It was therefore the aim of our study to investigate whether blood coagulation/thrombin generation plays a critical role in the formation of ISR in peripheral artery disease patients with stent angioplasty in the superficial femoral artery.

Materials and Methods

We aimed to examine in this retrospective study whether patients with high-degree restenosis (50-75% lumen diameter reduction, n = 20) are in a hypercoaguable state implying enhanced readiness to generate thrombin compared to patients with low-degree restenosis (< 50% lumen diameter reduction, n = 14).

Results

The coagulation tests calibrated automated thrombography, activated partial thromboplastin time, platelet aggregation, platelet adhesion, fibrinogen, and microparticles’ procoagulant activity did not indicate a different coagulation status in the two patient groups. However, the thrombelastometry-derived value Coagulation Time (CT) was significantly shorter in the high-degree restenosis group (p = 0.012), indicating a hypercoagulable state of patients with high-degree restenosis. Under our experimental conditions, CTs shorter than 444.5 s identify patients at high risk (sensitivity = 95%) for luminal narrowing.

Conclusions

Our study supports the assumption that blood coagulation/thrombin generation plays a critical role in the development of ISR in peripheral arteries after stent insertion and that the thrombelastometry-derived CT might be a suitable value to identify peripheral artery disease patients at risk for development of high-degree in-stent restenosis in the superficial femoral artery.     

Protein Z/protein Z-dependent protease inhibitor system in human non-small-cell lung cancer tissue

Introduction

NSCLC progression is often associated with VTE. Activation of factor X is an important step in blood coagulation activation in cancer patients. PZ)/ZPI contribute to direct factor Xa inhibition, and ZPI - attenuates factors IXa and XIa activity. The role of the PZ/ZPI in NSCLC is obscure. The aim of the study was to localize ZPI and PZ in NSCLC tissue in relation to factors X, IX and XI, as well as indicators of blood coagulation activation: prothrombin fragment F1 + 2 (F1 + 2) and fibrin.

Material & Methods

Immunohistochemical studies were performed on surgical NSCLC specimens employing antibodies against ZPI, PZ, coagulation factors X, IX, XI, as well as fibrinogen, F1 + 2 and fibrin. A semiquantitative analysis (acc. to immunoreactive score-IRS) was conducted.

Results

Medium expression of ZPI(IRS = 6.5), together with weak expression of PZ(IRS = 4), was observed in cancer cells. Strong or medium staining for factors IX, X, and XI(IRS = 8-9) was revealed in cancer cells. Fibrinogen(IRS = 10) and fibrin(IRS = 8) were demonstrated in tumor stroma and cancer cells. F1 + 2(IRS = 10) was localized in NSCLC cells. Endothelial cells (ECs) and tumor infiltrating macrophages (TAMs) were characterized by a positive staining for ZPI and PZ.

Conclusions

ZPI and PZ expression in NSCLC cells, ECs and TAMs may suggest a role for PZ/ZPI in the anticoagulant mechanisms at the tumor site. The presence of F1 + 2 and fibrin, along with a disproportional expression of ZPI and PZ, might point to impaired function of the coagulation inhibitory system in NSCLC tissue.     

Effect on perfusion chamber thrombus size in patients with atrial fibrillation during anticoagulant treatment with oral direct thrombin inhibitors, AZD0837 or ximelagatran, or with vitamin K antagonists

Introduction

AZD0837 and ximelagatran are oral direct thrombin inhibitors that are rapidly absorbed and bioconverted to their active forms, AR-H067637 and melagatran, respectively. This study investigated the antithrombotic effect of AZD0837, compared to ximelagatran and the vitamin K antagonist (VKA) phenprocoumon (Marcoumar^®), in a disease model of thrombosis in patients with non-valvular atrial fibrillation (NVAF).

Methods

Open, parallel-group studies were performed in NVAF patients treated with VKA, which was stopped aiming for an international normalized ratio (INR) of ≤ 2 before randomization. Study I: 38 patients randomized to AZD0837 (150, 250 or 350 mg) or ximelagatran 36 mg twice daily for 10-14 days. Study II: 27 patients randomized to AZD0837 250 mg twice daily or VKA titrated to an INR of 2-3 for 10-14 days. A control group of 20 healthy elderly subjects without NVAF or anticoagulant treatment was also studied. Size of thrombus formed on pig aorta strips was measured after a 5-minute perfusion at low shear rate with blood from the patient/control subject.

Results

Thrombus formation was inhibited by AZD0837 and ximelagatran. Relative to untreated patients, a 50% reduction of thrombus size was estimated at plasma concentrations of 0.6 and 0.2 μmol/L for AR-H067637 and melagatran, respectively. For patients receiving VKA treatment, the thrombus size was about 15% lower compared with healthy elderly controls.

Conclusions

Effects of AZD0837 and ximelagatran on thrombus formation were similar or greater than for VKA therapy and correlated with plasma concentrations of their active forms.     

Women with unexplained recurrent pregnancy loss do not have evidence of an underlying prothrombotic state: Experience with calibrated automated thrombography and rotational thromboelastometry

Introduction

Where unexplained recurrent pregnancy loss (RPL) is attributed to an underlying maternal prothrombotic state, empirical prophylactic anticoagulation may be recommended.

Materials and Methods

In the present study we used calibrated automated thrombography and rotational thromboelastometry to determine the procoagulant potential of these women as a rationale for anticoagulation. Fifty women with ≥ three consecutive unexplained losses prior to 14 weeks’ gestation or one loss after this time were compared with forty-one parous women with no miscarriages. Exclusion criteria included antiphospholipid syndrome, inherited thrombophilia and prior venous thromboembolism. Thrombin generation in platelet poor plasma and whole blood thromboelastometry was performed outside pregnancy to determine the presence or not of an underlying prothrombotic state.

Results

Peak thrombin and endogenous thrombin potential were not significantly increased in subjects relative to controls. The use of low tissue factor (1 pM) to better reflect physiological conditions and assay modification to better assess the protein C pathway (5 pM in the presence of thrombomodulin) provided no additional discrimination. Consistent results were shown with thromboelastometry; mean parameters were equivalent between subjects and controls.

Conclusions

These data demonstrate that global coagulation assays provide no evidence of an underlying hypercoagulable state in women with unexplained RPL; this is in keeping with the results of recent randomised controlled trials and strengthens the evidence base against use of anticoagulants in this setting.     

The mTOR signaling pathway in the prefrontal cortex is compromised in major depressive disorder

Recent studies demonstrate that rapid antidepressant response to ketamine is mediated by activation of the mammalian target of rapamycin (mTOR) signaling pathway, leading to increased synaptic proteins in the prefrontal cortex (PFC) of rats. Our postmortem studies indicate robust deficits in prominent postsynaptic proteins including N-methyl-d-aspartate (NMDA) receptor subunits (NR2A, NR2B), metabotropic glutamate receptor subtype 5 (mGluR5) and postsynaptic density protein 95 kDa (PSD-95) in the PFC in major depressive disorder (MDD). We hypothesize that deficits in the mTOR-dependent translation initiation pathway contribute to the molecular pathology seen in the PFC of MDD subjects, and that a rapid reversal of these abnormalities may underlie antidepressant activity. The majority of known translational regulation occurs at the level of initiation. mTOR regulates translation initiation via its downstream components: p70-kDa ribosomal protein S6 kinase (p70S6K), and eukaryotic initiation factors 4E and 4B (eIF4E and eIF4B). In this study, we examined the expression of mTOR and its core downstream signaling targets: p70S6K, eIF4E, and eIF4B in the PFC of 12 depressed subjects and 12 psychiatrically healthy controls using Western blot. Levels of eIF4E phosphorylated at serine 209 (p-eIF4E-Ser209) and eIF4B phosphorylated at serine 504 (p-eIF4B-Ser504) were also examined. Adjacent cortical tissue samples from both cohorts of subjects were used in our previous postmortem analyses. There was a significant reduction in mTOR, p70S6K, eIF4B and p-eIF4B protein expression in MDD subjects relative to controls. No group differences were observed in eIF4E, p-eIF4E or actin levels. Our findings show deficits in mTOR-dependent translation initiation in MDD particularly via the p70S6K/eIF4B pathway, and indicate a potential association between marked deficits in synaptic proteins and dysregulation of mTOR signaling in MDD.