oxLDL – the molecule linking hypercoagulability with the presence of cardiovascular disease in hemodialyzed uraemic patients

Introduction

Patients with end-stage renal disease (ESRD) exhibit features of a hypercoagulable state, which may contribute to cardiovascular complications. Data from “in vitro” studies suggest that cell-free plasma lipids and lipoproteins may be capable to support thrombin generation. The aim of this study has been to establish the role of plasma oxidized LDL (oxLDL) in the coagulation activation in hemodialyzed (HD) patients with and without cardiovascular disease (CVD).

Materials and Methods

We examined relationship between a marker of coagulation activation – prothrombin fragments 1 + 2 (F_1 + 2), and plasma oxLDL levels in 60 HD patients with and without CVD and in 20 healthy controls.

Results

F_1 + 2 levels were significantly higher in HD patients than in controls, and they were higher in HD patients with CVD compared to those without CVD (p < 0.001). Conversely, oxLDL levels were similar in HD patients with CVD and healthy controls, whereas this parameter was lower in HD patients without CVD when compared to controls and patients with CVD (both p < 0.01). Close positive and independent association was between F_1 + 2 and oxLDL levels in HD patients. Nitrates treatment and the presence of pyelonephritis were associated with reduced oxLDL as well as hypercoagulability in HD patients with cardiovascular complications.

Conclusion

This study demonstrates the independent association between oxLDL and the marker of coagulation activation - F_1 + 2 in HD patients. This new observation may offer a better understanding of the complex mechanism leading to hypercoagulability, which is markedly intensified in these patients, particularly in those with CVD.     

Exploring soluble urokinase plasminogen activator receptor and its relationship with arterial stiffness in a bi-ethnic population: the SAfrEIC-study

Introduction

Elevated soluble urokinase-type plasminogen activator receptor (suPAR) indicates an inflammatory state caused by conditions such as HIV and cancer. Recently suPAR was identified as an indicator of cardiovascular disease (CVD). CVD is highly prevalent in black South Africans, but the potential role of suPAR is unknown. We investigated suPAR as a possible marker of arterial stiffness in Africans and Caucasians.

Methods

This study involved 207 Africans and 314 Caucasians (aged 20-70 yrs). C-reactive protein (CRP) and suPAR were determined in fasting blood samples. We measured blood pressure, pulse wave velocity (PWV) and Windkessel arterial compliance (Cwk).

Results

Africans displayed higher suPAR, CRP, PWV and lower Cwk (p < 0.001) compared to Caucasians. SuPAR was elevated in Africans irrespective of gender and smoking. We found strong relationships between PWV and suPAR (r = 0.27; p < 0.001) and Cwk and suPAR (r = − 0.39; p < 0.001) in the whole group, but found no independent relationship of any arterial stiffness measure and suPAR in Africans after adjustment for confounders. Caucasian men indicated a weak significant independent association between Cwk and suPAR (β = − 0.09; p = 0.028).

Conclusion

Africans had higher levels of suPAR and arterial stiffness than Caucasians (p < 0.001), but there was no independent relationship between arterial stiffness and suPAR in the Africans. It is speculated that due to the inflammatory role of suPAR, it will have stronger relationships with atherosclerosis, which has not yet manifested in this relatively young population group. SuPAR may therefore not be an ideal early marker of cardiovascular dysfunction, but may rather indicate established CVD.     

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.     

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.     

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.     

Hyperhomocysteinemia and the presence of cardiovascular disease are associated with kynurenic acid levels and carotid atherosclerosis in patients undergoing continuous ambulatory peritoneal dialysis

Introduction

Hyperhomocysteinemia and the activation of kynurenine (KYN) pathway have been reported as the factors participated in atherosclerosis in uraemic patients. The objective of this study was to verify whether hyperhomocysteinemia may be involved in atherosclerotic cardiovascular disease (CVD) in patients undergoing continuous ambulatory peritoneal dialysis (CAPD).

Materials and methods

We determined the plasma concentrations of KYN, KYNA, KYNA/KYN ratio, homocysteine (Hcy) and intima-media thickness (IMT) - an early reflection of the systemic atherosclerosis in CAPD patients both with and without CVD and healthy controls.

Results

KYNA concentrations and KYNA/KYN ratio were about 3 times higher in the patients with hyperhomocysteinemia (Hcy > 15 μM) compared to those with normal Hcy levels (< 15 μM), and they were significantly lower in CVD[+] than in CVD[−] patients in these studied groups. The presence of CVD was associated with higher Hcy levels only in the patients with Hcy > 15 μM. The positive association was between Hcy and KYNA, KYNA/KYN ratio in all CAPD patients and in CVD[+] patients with hyperhomocysteinemia. IMT was positively associated with Hcy levels in patients with hyperhomocysteinemia, whereas there was no relationship between IMT and KYNA concentrations in the studied groups.

Conclusions

These results showed the association between hyperhomocysteinemia and carotid atherosclerosis as well as the possible role of hyperhomocysteinemia in the activation of KYNA production in CAPD patients. The elevated KYNA levels observed in patients with hyperhomocysteinemia seem to be protective against Hcy-mediated atherosclerosis in these patients.     

Inhibition of factor XI reduces thrombus formation in rabbit jugular vein under endothelial denudation and/or blood stasis

Introduction

In addition to acquired and inherited risk factors, the growth of venous thrombus under static conditions and endothelial injury play important roles in the development of deep venous thrombosis (DVT), for which risk factors include increased plasma levels of coagulation factor XI (FXI). The aim of this study is to understand the role of FXI in venous thrombus formation under conditions of endothelial denudation and/or blood stasis.

Materials and methods

The contribution of FXI to venous thrombus formation was investigated in a rabbit model and a flow chamber system. Thrombi were induced in the rabbit jugular veins by (1) endothelial denudation, (2) vessel ligation (blood stasis) or (3) by combined endothelial denudation and vessel ligation. Blood samples were perfused on immobilized type III collagen at a wall shear rate of 70/s and then the surface area covered by platelets and fibrin was morphometrically evaluated. Prothrombin fragment 1 + 2 (F1 + 2) generation was also measured before and after perfusion.

Results

All thrombi induced in rabbit jugular veins were composed of platelets, fibrin and erythrocytes. Anti-FXI antibody significantly reduced ex vivo plasma thrombin generation initiated by ellagic acid but not by tissue factor, and in vivo thrombus formation under endothelial denudation and/or vessel ligation. The antibody significantly reduced surface areas covered by platelets and fibrin, as well as F1 + 2 generation at a wall shear rate of 70/s in flow chambers.

Conclusion

These results suggest that FXI contributes to venous thrombus growth under conditions of endothelial denudation and/or blood stasis, and that thrombin generation by FXI interaction promotes further platelet aggregation and fibrin formation at low shear rates.     

Activation of coagulation in amniotic fluid during normal human pregnancy

Introduction

Amniotic fluid (AF) is an important medium for fetal development which exhibits high procoagulant activities; however, the role of these procoagulants during pregnancy has not been elucidated and might be associated with pregnancy complications. The current study aimed to evaluate factor X (FX) activation and its association with tissue factor (TF), tissue factor pathway inhibitor (TFPI) and coagulation activation markers in AF during normal human pregnancy.

Methods

Activation of FX and concentration of TF, free TFPI, D-dimer and prothrombin fragments (F1 + 2) were evaluated in AF samples obtained for chromosome analysis from 91 women with normal pregnancy: 65 samples were taken from patients at 16-20 weeks of gestation, 9 samples were drawn at 21-30 weeks and 17 samples−after 30 weeks of gestation.

Results

Activation of FX in AF significantly increased during normal pregnancy (from 65 ± 41 to 205 ± 80 equivalent RVV ng/mg total protein, P < 0.0001). TF and TFPI levels in AF also rose with gestational age. In contrast, the AF concentration of D-dimer and F1 + 2, markers of coagulation activation significantly decreased when expressed per mg total protein. Levels of free TFPI correlated with TF (r = 0.5, P < 0.001), and were 8-fold higher than those of TF during pregnancy.

Conclusion

High levels of TFPI might be associated with the inhibition of procoagulant activity in amniotic fluid during normal pregnancy, which may account for the rarity of clinical amniotic fluid embolism.     

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.     

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.     

Inflammatory and thrombotic markers in patients with ST-elevation myocardial infarction treated with thrombolysis and early PCI: A NORDISTEMI substudy

Introduction

Both pharmacological and invasive treatment might influence the inflammatory and pro-thrombotic responses observed in acute ST-elevation myocardial infarction (STEMI). We aimed to study whether circulating levels of inflammatory and pro-thrombotic markers differ in STEMI patients treated with early angioplasty compared to standard therapy following thrombolysis. Furthermore, we wanted to study if levels of markers were related to infarct size.

Materials and Methods

This was a substudy of the NORwegian study on DIstrict treatment of ST-Elevation Myocardial Infarction (NORDISTEMI), in which STEMI patients treated with thrombolysis were randomized to early invasive or standard therapy. Fasting blood samples were collected in the morning 3 days and 3 months after onset of STEMI. Commercially available ELISA methods were used for determination of inflammatory and pro-thrombotic markers. Infarct size was assessed by SPECT after 3 months.

Results

246 patients were included in this substudy. At 3 days, levels of prothrombin fragment 1 + 2 and D-dimer were higher in the early invasive compared to the standard treatment group, whereas levels of soluble CD40 ligand were lower (p < 0.01 for all). No other differences between groups were found in any of the measured markers. Significant, although weak correlations were found between Day 3 levels of C-reactive protein, interleukin-6, prothrombin fragment 1 + 2 and D-dimer, and infarct size assessed by SPECT after 3 months.

Conclusions

An early invasive strategy following thrombolysis for STEMI was associated with higher subacute levels of D-dimer and prothrombin fragment 1 + 2, and lower levels of soluble CD40 ligand than standard treatment. Further studies are needed to establish the relation between these changes and clinical outcome.The NORDISTEMI was registered at www.clinicaltrials.gov, NCT00161005.     

Prothrombin fragment 1 + 2 in urine as a marker on coagulation activity in patients with suspected pulmonary embolism

Introduction

We have recently reported that increased levels of urine prothrombin fragment 1 + 2 reflected radiologically verified deep vein thrombosis. In this study we evaluated whether urine prothrombin fragment 1 + 2 was associated with pulmonary embolism in non-selected patients.

Materials and methods

Patients with clinical suspected pulmonary embolism were interviewed on comorbidities and medications. Urine was collected from each patient before radiological examination and snap frozen until analysed on urine prothrombin fragment 1 + 2 with an ELISA kit. Imaging of the pulmonary arteries were conducted with contrast enhanced computer tomography.

Results

Pulmonary embolism was diagnosed in 44/197 patients. Non-significantly higher urine prothrombin fragment 1 + 2 levels were found in non-selected patients with pulmonary embolism vs. those without (p = 0.324). Significantly higher urine prothrombin fragment 1 + 2 levels were found in the pulmonary embolism positive patients without comorbidities (n = 13) compared to the control group (n = 28) (p = 0.009). The calculated sensitivity, specificity and negative predictive value using the lowest detectable urine prothrombin fragment 1 + 2 level was 82%, 34% and 87%, respectively.

Conclusions

There was no significant urine prothrombin fragment 1 + 2 level difference in patients with and without pulmonary embolism. In non-comorbide pulmonary embolism positive patients the urine prothrombin fragment 1 + 2 levels were significantly higher compared to the control group. The negative predictive value found in this study indicates that uF1 + 2 has the potential to identify patients with a low risk of PE.     

Hypercoagulability in chronic kidney disease is associated with coagulation activation but not endothelial function

Introduction

Patients with chronic kidney disease exhibit features of a hypercoagulable state and have endothelial dysfunction, which may contribute to their increased cardiovascular risk. We examined the relationship between coagulation activation and vascular function in patients with chronic kidney disease.

Materials and Methods

We measured parameters of the tissue factor pathway of blood coagulation (tissue factor, factor VIIc and factor X); natural inhibitors (tissue factor pathway inhibitor, protein C, free and total protein S, antithrombin III) and markers of coagulation activation (thrombin-antithrombin complexes, prothrombin fragment 1 + 2) in 66 stage 4&5 chronic kidney disease patients and 36 healthy controls. Their relationship with markers of vascular function (flow mediated dilatation, soluble E-selectin and thrombomodulin) and a mediator of inflammation (interleukin-6) was determined.

Results

Up-regulation of the tissue factor pathway (increased tissue factor and factor VIIc), increased prothrombin fragment 1 + 2 and significant reductions in antithrombin III and the ratio of free protein S: total protein S were found in patients compared to healthy controls. Increased tissue factor antigen was significantly and independently correlated with creatinine and interleukin-6 (P < 0.001). Factor X and antithrombin III were both reduced in chronic kidney disease and correlated (r = 0.58; P < 0.001). Changes in coagulation and anti-coagulation were independent of all measures of endothelial function.

Conclusions

Significant activation of the TF pathway of coagulation and depletion or reduction of some natural anticoagulants in chronic kidney disease was correlated with the degree of renal dysfunction, but not correlated with the abnormalities of vascular function. These data are consistent with a hypercoagulable state in chronic kidney disease that may be independent of endothelial based regulation but associated with an inflammatory state.     

Effects of carvedilol or metoprolol on PAI-1, tPA-mass concentration or Von Willebrand factor in chronic heart failure - a COMET substudy

Introduction

In COMET (Carvedilol or Metoprolol European Trial), carvedilol reduced mortality compared with metoprolol in patients with chronic heart failure. We hypothesized that carvedilol might have greater effects on endothelial derived haemostatic factors than metoprolol. We aimed to study the effects of carvedilol or metoprolol on tissue plasminogen activator (tPA), its inhibitor PAI-1 and Von Willebrand factor (VWF) in patients with heart failure.

Material and Methods

We recruited 260 patients (134 on carvedilol, 126 on metoprolol), mean age 66 years and 84% of them men. Plasma mass concentrations of tPA and PAI-1and percent of VWF were measured at baseline and after one and two years of treatment.

Results

Plasma tPA, PAI-1 and VWF were similar between treatment groups at baseline and no significant differences between groups emerged after one or two years of treatment. In paired analyses in patients assigned to carvedilol, median PAI-1 level decreased from 37.2 to 32.1 µg/l at two years (p = 0.034) and of VWF decreased from baseline to one year (240 vs. 218%, p = 0.023) in patients assigned to carvedilol but were not reduced at any time in patients assigned to metoprolol. Plasma tPA increased over time in both treatment groups (p = 0.013 and 0.027 respectively).

Conclusion

We found no significant difference in the effects of carvedilol or metoprolol on tPA, PAI-1 and VWF. Comparison over time within treatment groups suggested that PAI-1 and VWF might have declined on carvedilol but not on metoprolol. Our hypothesis is not proved but this may reflect an inadequate sample size rather than lack of an effect.     

Comparison of a direct Factor Xa inhibitor, edoxaban, with dalteparin and ximelagatran: A randomised controlled trial in healthy elderly adults

Introduction

Edoxaban (the free base of DU-176b) is a new, oral direct Factor Xa inhibitor. This is the first study to compare the hemostatic response to edoxaban, ximelagatran, and dalteparin in healthy, elderly adults.

Materials and Methods

In this open-label, active-controlled clinical trial, 40 adults (65-75 years), were randomised to: oral edoxaban (60 mg, twice-daily, 7 doses), subcutaneous dalteparin (5000 IU, once-daily, 4 doses), oral ximelagatran (24 mg, twice-daily, 7 doses) or no drug. Blood samples were taken before, and 1.5, 4, 12, 24, 72, 84, 96, 108, 120, and 144 hours after, the first dose. The primary outcomes were changes in thrombin-antithrombin complex, prothrombin fragment 1 + 2 and D-dimer, and adverse events. Additional biomarkers of coagulation, and endothelial cell and platelet activation were compared (ANOVA).

Results

All subjects completed the study. Inhibition of thrombin generation lag time, peak, and constant velocity index were significantly greater, and extended for a longer period of time, following edoxaban administration, compared with dalteparin. We found that the traditional assay for anti-FXa activity was not appropriate for the new anticoagulants. Biomarker changes following edoxaban administration (including prolongation of prothrombin time) reflected inhibition of Factor Xa; there was no effect on platelet, tissue factor or endothelial activation. There were no clinically significant changes in primary outcomes. No serious adverse events were reported.

Conclusion

Oral administration of edoxaban resulted in effective Factor Xa and TG inhibition, and was well-tolerated. Studies are needed to confirm edoxaban (60 mg daily) use in clinical practice.

Sponsorship

Daiichi Sankyo Pharma Development.     

Different effects of enoxaparin and unfractionated heparin on some thrombogenesis markers during hemodialysis: a cross-over study

Background

Unfractionated heparin (UFH) and low molecular weight heparin constitute fundamental anticoagulants during hemodialysis (HD). We aimed to investigate the effect of UFH and enoxaparin on plasma levels of prothrombin fragment 1 + 2 (PF 1 + 2) and thrombin/antithrombin complex (TAT) as markers of intravascular thrombogenesis during HD.

Methods

We enrolled 22 chronic HD patients, who were randomly assigned to either iv enoxaparin (n = 11) or UFH (n = 11) anticoagulation, and followed prospectively for 12 weeks before crossing over to the alternate therapy for further 12 weeks. Plasma levels of PF 1 + 2 and TAT were measured by immunoassay at the start, at 10 and 180 min of HD session after each period of evaluation.

Results

The baseline PF 1 + 2 and TAT levels were comparable under enoxaparin and UFH treatment. PF 1 + 2 significantly decreased during both UFH (χ² ANOVA = 9.82, P = 0.007) and enoxaparin (χ² ANOVA = 29.40, P < 10^− 6) anticoagulated HD, while over-HD TAT levels changes differed depending on the type of heparin. The switch from enoxaparin to UFH treatment was connected with a significantly higher PF 1 + 2 after 10 and 180 min as well as higher TAT concentration after 180 min of HD. Only during enoxaparin anticoagulated HD 34% PF 1 + 2 decrease and TAT levels after 180 min of HD was closely associated with heparin dosage.

Conclusion

Single bolus of enoxaparin ensures efficient and convenient anti-thrombotic protection during HD procedure. Enoxaparin mean dose of 0.67 mg/kg, which is generally lower than manufacturer's instructions, can be recommended for over-dialytic regular use.     

Compartment-specific expression of plasminogen activator inhibitor-1 correlates with severity/outcome of murine polymicrobial sepsis

Introduction

Plasminogen activator inhibitor type 1 (PAI-1) co-induces septic coagulopathy. We aimed to characterize spatiotemporal PAI-1 gene/protein changes occurring in acute sepsis and tested whether PAI-1 fluctuations correlate with sepsis severity and early outcome.

Materials and Methods

Female mice underwent cecal ligation and puncture (CLP) in three experiments. I: mild (23G needle) CLP to compare circulating PAI-1 to its organ gene expression within 0-24 h. II: mild or severe (17G) CLP to asses differences in PAI-1 organ-specific expression and in coagulation/fibrinolysis. III: moderate (18G) CLP to characterize circulating PAI-1 in survivors (SUR), and to retrospectively compare it to dying (DIE) mice.

Results

In mild sepsis, the trajectory of circulating PAI-1 (1089 ng/ml peak at 24 h) was identical to PAI-1 gene expression in the left cranial vena cava (LCVC; 39-fold peak at 24 h). PAI-1 expression rise was immediate (60-fold at 6 h) and sustained in the liver, but marginal in the kidney, lungs and heart. Body temperature decrease correlated with the PAI-1 expression increase in the liver (rho = − 0.79), and blood (protein, rho = − 0.53). Regardless of severity, PAI-1 gene expression remained unaltered except the LCVC where it was > 3-fold higher in 17G (vs. 23G). Severe sepsis extended activated partial thromboplastin/pro-thrombin time and increased circulating PAI-1, while antithrombin and fibrinogen decreased at 6 and/or 24 h (vs. 23G). Within 24 h of death, circulating PAI-1 in DIE was > 3-fold higher versus SUR.

Conclusions

Polymicrobial sepsis caused a gradual circulating PAI-1 release and highly variable gene expression response pattern in organs. Only circulating PAI-1 and PAI-1 expression in the LCVC correlated with response severity and/or outcome.     

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.     

Tissue factor-induced Thrombin Generation in the Fasting and Postprandial State among Elderly Survivors of Myocardial Infarction

Introduction

Tissue factor (TF)-induced thrombin generation (TG) ex vivo has been suggested to be an important method to assess thrombotic risk. No studies have investigated the impact of postprandial lipemia on TF-induced TG. Since myocardial infarction (MI) is associated with elevated postprandial levels of triglycerides, we hypothesized a differential impact of postprandial lipemia on coagulation activation in MI-patients and healthy controls.

Material and Methods

Elderly survivors of acute MI (n = 44) and healthy age-and sex matched controls (n = 43) underwent a fat tolerance test (1 gram per kg body weight) to assess coagulation activation during postprandial lipemia.

Results

The incremental area under the curve (AUCi) for serum triglycerides was higher in MI-patients than in healthy age-and sex matched controls (5.64 ± 0.52 mmol/L?h and 3.94 ± 0.39 mmol/L?h, p = 0.012) during the postprandial phase. Subsequent endogenous activation of coagulation, assessed by FVIIa and thrombin generation (F1 + 2), was similar among groups and not related to levels of triglycerides during the postprandial phase. Healthy individuals had a gradual decline in TF-induced thrombin generation ex vivo, assessed by endogenous thrombin potential (ETP) (AUCi = - 542.4 ± 71.4 nM?min?h, p < 0.001), whereas MI-patients retained their ETP (AUCi = 127.4 ± 89.0 nM?min?h, p = 0.47) in plasma during the postprandial phase (p for group difference = 0.005).

Conclusions

MI-patients had elevated postprandial lipemia and retained their ability for TF-induced TG in plasma ex vivo in the postprandial phase, whereas the capacity gradually decreased in healthy individuals. Further studies are warranted to reveal underlying mechanism(s) and clinical implications.     

Microscopic clot fragment evidence of biochemo-mechanical degradation effects in thrombolysis

Introduction

Although fibrinolytic treatment has been used for decades, the interactions between the biochemical mechanisms and the mechanical forces of the streaming blood remain incompletely understood. Analysis of the blood clot surface in vitro was employed to study the concomitant effect of blood plasma flow and recombinant tissue plasminogen activator (rt-PA) on the degradation of retracted, non-occlusive blood clots. Our hypothesis was that a faster tangential plasma flow removed larger fragments and resulted in faster overall thrombolysis.

Materials and Methods

Retracted model blood clots were prepared in an optical microscopy chamber and connected to an artificial perfusion system with either no-flow, or plasma flow with a velocity of 3 cm/s or 30 cm/s with or without added rt-PA at 2 µg/ml. The clot surface was dynamically imaged by an optical microscope for 30 min with 15 s intervals.

Results

The clot fragments removed during rt-PA mediated thrombolysis ranged in size from that of a single red blood cell to large agglomerates composed of more than a thousand red blood cells bound together by partly degraded fibrin. The average and the largest discrete clot area change between images in adjacent time frames were significantly higher with the faster flow than with the slow flow (14,000 μm² and 160,000 μm² vs. 2200 μm² and 10,600 μm²).

Conclusions

On the micrometer scale, thrombolysis consists of sequential removal of clot fragments from the clot surface. With increasing tangential plasma flow velocity, the size of the clot fragments and the overall rate of thrombolysis increases.