Effects of n-3 PUFA on endothelial function in patients with peripheral arterial disease: a randomised, placebo-controlled, double-blind trial.

As only limited evidence is available for potential benefits of n-3 PUFA supplementation in patients with peripheral arterial disease (PAD), we studied the effects of 4 g n-3 PUFA on endothelial function and inflammatory markers. Seventy patients with stable PAD classified as Rutherford stage 2 or 3 and good control of cardiovascular factors were randomised to receive either 4 g n-3 PUFA or placebo daily for 3 months in a double-blind fashion. Primary endpoint was endothelial function assessed by flow-mediated vasodilation (FMD). In addition, ankle-brachial index, maximum and pain-free walking distances were determined. Lipid parameters including the omega-3 index reflecting n-3 PUFA intake as well as pro-inflammatory, endothelial and platelet activation markers were measured over the same time interval. After 3 months of treatment with 4 g n-3 PUFA daily, a significant improvement of FMD was observed compared with placebo (n-3 PUFA, median Δ 3·7 (interquartile range (IQR) -1·8, 7·1) % v. placebo, Δ -0·5 (IQR -6·5, 3·0) %, P = 0·01 between the groups). After a 3-month washout period, this benefit was not sustained (n-3 PUFA, median Δ 0·6 (IQR -2·2, 5·6) % v. placebo, Δ -0·9 (IQR -6·6, 6·7) %, P = 0·20). In response to n-3 PUFA, an improvement of lipid parameters with a pronounced increase in the omega-3 index was seen. No changes were found for other parameters. In conclusion, in patients with PAD, 4 g/d n-3 PUFA improved cardiovascular risk in PAD patients, which needs testing in large-scale trials.

Preserved thrombin-inducible platelet activation in thienopyridine-treated patients.

BACKGROUND: Abundant thrombin generation may be a major reason for subsequent thromboembolic events in patients with cardiovascular disease receiving dual antiplatelet therapy. We therefore investigated the susceptibility of thienopyridine responders and nonresponders to thrombin receptor-activating peptide (TRAP)-6- and adenosine diphosphate (ADP)-inducible platelet activation. MATERIALS AND METHODS: Response to clopidogrel or prasugrel was determined by the vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay and multiple electrode aggregometry (MEA) in 317 patients undergoing angioplasty and stenting for cardiovascular disease. Baseline, TRAP-6-, and ADP-inducible P-selectin expression, activated glycoprotein IIb/IIIa (GPIIb/IIIa) and monocyte-platelet aggregate (MPA) formation were measured as sensitive parameters of platelet activation. RESULTS: In patients with high on-treatment residual ADP-inducible platelet reactivity (HRPR), baseline P-selectin expression, GPIIb/IIIa and MPA formation were similar to those in patients without HRPR (all P > 0.05). After platelet activation with TRAP-6 or ADP, patients with HRPR by both assays exhibited significantly higher levels of P-selectin expression, GPIIb/IIIa and MPA formation than patients with an adequate thienopyridine-mediated platelet inhibition (all P ≤ 0.02). However, high levels of TRAP-6-inducible P-selectin, GPIIb/IIIa and MPA formation also occurred in 20.4%, 19.1% and 20.1% of the good responders by the VASP assay, and in 19.6%, 16.6% and 20.6% of the good responders by MEA, respectively. CONCLUSIONS: Thienopyridine nonresponders are more susceptible to thrombin- and ADP-inducible platelet activation than patients with good platelet inhibition. However, even patients with adequate thienopyridine-mediated platelet inhibition often show a preserved responsiveness to thrombin. These patients may benefit from additional thrombin receptor blockage or inhibition of thrombin generation.

Chronic kidney disease is associated with increased platelet activation and poor response to antiplatelet therapy.

BACKGROUND: Chronic kidney disease (CKD) is a common co-morbidity of patients with atherosclerotic vascular disease, and may influence the response to antiplatelet therapy. We, therefore, sought to investigate its effect on platelet activation and on-treatment residual platelet reactivity. METHODS: We assessed platelet activation and the response to clopidogrel and aspirin in 316 patients after percutaneous intervention with stent implantation. CKD was defined as a glomerular filtration rate <60 mL/min/1.73 m(2) according to the Modification of Diet in Renal Disease formula. Surface expression of activated glycoprotein IIb/IIIa without the addition of agonists was determined to assess baseline platelet activation. GPIIb/IIIa in response to adenosine diphosphate (ADP) and arachidonic acid (AA), as well as the VerifyNow assays and light transmission aggregometry (LTA) were used to measure residual platelet reactivity. RESULTS: Baseline platelet activation was significantly increased in CKD patients compared with patients without renal insufficiency [3.1 versus 2.7 mean fluorescence intensity (MFI), P = 0.001]. Moreover, patients with CKD exhibited a more pronounced expression of GPIIb/IIIa in response to ADP (13 versus 9.6 MFI) and AA (6 versus 5.1 MFI; both P≤ 0.02) than patients without CKD. In the VerifyNow assays, CKD patients showed significantly higher platelet reactivity than patients without CKD (P2Y12 assay: 239 versus 182 P2Y12 Reaction Units; aspirin assay: 415 versus 399 Aspirin Reaction Units; both P≤ 0.03). Further, patients with CKD had significantly higher platelet reactivity by LTA in response to ADP (49.9 versus 43.2%, P = 0.01). Finally, high on-treatment residual ADP-inducible platelet reactivity by the VerifyNow P2Y12 assay and by LTA occurred significantly more frequent in patients with CKD (VerifyNow: 52.2 versus 26.2%, P < 0.001; LTA: 23.3 versus 12.1%, P = 0.01). CONCLUSIONS: Patients with CKD exhibit increased platelet activation, and an attenuated response to dual antiplatelet therapy compared with patients without renal insufficiency.

Soluble p-selectin, D-dimer, and high-sensitivity C-reactive protein after acute deep vein thrombosis of the lower limb.

BACKGROUND: Soluble p-selectin (sP-selectin), D-dimer, and C-reactive protein (CRP) are elevated in deep vein thrombosis (DVT), and may play a role as risk predictors of recurrent venous thromboembolism. However, these parameters have only been assessed at manifestation or at single time points after DVT so far. We therefore investigated the course of sP-selectin, D-dimer, and high-sensitivity (hs)-CRP after acute unprovoked DVT of the lower limb. METHODS: In this prospective, longitudinal study, sP-selectin, D-dimer, and hs-CRP were determined by enzyme-linked immunosorbent assay, quantitative latex assay, and particle enhanced immunonephelometry, respectively, in 44 patients with sonographically confirmed acute DVT at the time of diagnosis, and 1, 3, 6, and 12 months later. sP-selectin and hs-CRP were also measured in 88 age- and gender-matched healthy controls. Further, color duplex sonography was performed in all patients at each time point. RESULTS: At DVT diagnosis, sP-selectin and hs-CRP were significantly higher in patients compared with healthy controls. From baseline to 1 month, both parameters decreased significantly. In patients with oral anticoagulation (OAC) for 6 months (n = 35), levels of sP-selectin increased significantly after cessation of anticoagulant therapy (P = .002), while sP-selectin was similar to healthy controls in patients with ongoing OAC (n = 9) at 12 months (P = .49). In contrast, hs-CRP in both subgroups remained constantly low at levels seen in healthy controls. The course of D-dimer was similar to sP-selectin. Color duplex sonography showed no ongoing thrombus formation in any patient. Thirty-four (77.3%), 29 (65.9%), 26 (59.1%), and 25 (56.8%) patients had residual thrombosis 1, 3, 6, and 12 months after the acute event, respectively. D-dimer was significantly higher in patients with residual thrombosis compared with patients without residual thrombosis 1 month after DVT (0.58 µg/mL [range, 0.2-9.67 µg/mL] vs 0.25 µg/mL [range, 0.2-0.62 µg/mL]; P = .02). At all other time points, the levels of D-dimer and sP-selectin did not differ significantly between patients without and with residual thrombosis (all P > .05). CONCLUSIONS: Concentrations of sP-selectin and D-dimer after acute DVT seem to be strongly influenced by treatment with vitamin K antagonists. After withdrawal of oral anticoagulation, they rise again and could therefore reflect a prothrombotic state, which is susceptible to pharmacologic therapy.

Comparison of methods to evaluate aspirin-mediated platelet inhibition after percutaneous intervention with stent implantation.

Urinary 11-dehydro thromboxane B2 (d-TXB2) concentrations in the highest quartile have been associated with an increased risk of adverse outcomes in patients at high risk for atherothrombotic events. However, the determination of d-TXB2 is time consuming and not suitable for daily clinical routine. We therefore sought to determine the test correlating best with d-TXB2 to estimate aspirin-mediated platelet inhibition in 225 patients on dual antiplatelet therapy after percutaneous intervention with stent implantation. We selected light transmission aggregometry, the VerifyNow aspirin assay, the Platelet Function Analyzer-100, multiple electrode platelet aggregometry (MEA) and Impact-R for comparisons with d-TXB2. Cut-off values for high on-treatment residual arachidonic acid-inducible platelet reactivity (HRPR) were defined according to quartiles of each assay. Sensitivities and specificities of the different platelet function tests were based on d-TXB2 results. The results from all assays correlated poorly with d-TXB2. Only MEA showed a weak, but significant correlation with d-TXB2 (r = 0.14, p = 0.042). Further, the five platelet function tests showed a poor agreement with d-TXB2 regarding the determination of HRPR. Sensitivities and specificities for HRPR ranged from 17.5 to 44.6%, and from 70.8 to 77.9%, respectively. In conclusion, the evaluated assays did not mirror d-TXB2 results, suggesting that thromboxane inhibition can be circumvented in assays that determine platelet function. Therefore, large trials with clinical outcome data are needed to determine the diagnostic value of the various test systems and to define the gold standard method for assessing residual AA-inducible platelet reactivity.

Platelet-monocyte cross talk and tissue factor expression in stable angina vs. unstable angina/non ST-elevation myocardial infarction.

Tissue factor (TF), the major procoagulant in vivo, is usually absent from blood cells. However, since both monocyte TF (MoTF) expression and platelet activation are present in acute coronary syndrome we hypothesized that MoTF expression may in part depend on monocyte platelet aggregate (MPA) formation in coronary artery disease (CAD). Patients with unstable angina/non-ST-elevation myocardial infarction (UA/NSTEMI, n = 20) had significantly higher levels of MoTF (17.4 ± 3.1MFI) and MPAs (CD42b:273 ± 183MFI; CD62P:256.3 ± 48.5MFI) than patients with stable angina (SA, n = 40; MoTF:13.2 ± 2.2MFI, p = 0.001; CD42b:160 ± 113MFI, p = 0.025; CD62P:118.7 ± 24.5MFI, p = 0.018) as measured by whole blood flow cytometry on CD14+-cells. TF-activity of isolated mononuclear cells (MNC) was elevated in UA/NSTEMI (75 ± 27 pg/mL) in comparison to SA (47 ± 17 pg/mL, p = 0.001) as determined by chromogenic assay, and TF mRNA expression in isolated MNC was more frequent in UA/NSTEMI than in SA (50% vs. 18.2%; p = 0.017). MoTF expression significantly correlated with the constitutive platelet marker CD42b (r = 0.69, p < 0.001) and the platelet activation marker CD62P (r = 0.47, p = 0.001) on CD14+-cells suggesting its association with MPAs in UA/NSTEMI. In addition, MoTF expression correlated with MoTF activity of isolated MNC (r = 0.41, p = 0.01) and plasma levels of the F1.2 prothrombin fragment (r = 0.35, p = 0.02). In conclusion, MoTF and MPAs are elevated in UA/NSTEMI compared with SA. MoTF expression correlates with platelet mass and activity attached to monocytes.

The influence of proton pump inhibitors on the antiplatelet potency of clopidogrel evaluated by 5 different platelet function tests.

Proton pump inhibitors (PPIs) are metabolized by the cytochrome P450 enzyme system to a variable degree and may therefore interact with the metabolism of clopidogrel to its active form. The conflicting data on this potential interaction may be due to the use of different PPIs and platelet function tests in recent studies. We therefore evaluated the influence of different PPIs on the antiplatelet effect of clopidogrel by 5 platelet function tests in the same population. Adenosine diphosphate (ADP)-inducible platelet reactivity was assessed in 230 patients on dual antiplatelet therapy after angioplasty and stenting for cardiovascular disease by the light transmission aggregometry, VerifyNow P2Y12 assay, vasodilator-stimulated phosphoprotein phosphorylation assay, multiple electrode aggregometry, and Impact-R. ADP-inducible platelet reactivity showed no significant differences between patients without (n = 95, 41.3%) and with PPI therapy (n = 135, 58.7%) in each test system (all P > 0.05). Furthermore, we observed no significant differences of ADP-inducible platelet reactivity between patients without PPIs and patients with pantoprazole (n = 95, 70.4%), esomeprazole (n = 22, 16.3%), omeprazole (n = 9, 6.65%), or lansoprazole (n = 9, 6.65%) (all P > 0.3). High on-treatment residual ADP-inducible platelet reactivity occurred to a similar extent in patients without and with PPI therapy in each assay (all P > 0.05). In conclusion, concomitant treatment with PPIs did not attenuate the antiplatelet effect of clopidogrel in patients on dual antiplatelet therapy irrespective of the type of PPI and the used test system.

Is TRAP-6 suitable as a positive control for platelet reactivity when assessing response to clopidogrel?

Adenosine 5'-diphosphate (ADP) inducible aggregation is used to assess platelet response to thienopyridines. Thrombin receptor-activating peptide-6 (TRAP-6) inducible aggregation may serve as a positive control because it acts via the thrombin receptor protease-activating receptor-1, which is not blocked by thienopyridines. We therefore investigated if TRAP-6 is suitable as a positive control when assessing residual platelet reactivity to ADP. Platelet response to clopidogrel was assessed in 200 patients on dual antiplatelet therapy using ADP inducible platelet aggregation by light transmission aggregometry (LTA), multiple electrode aggregometry (MEA), and the shear-dependent Impact-R. Test specificities were monitored by TRAP-6 inducible platelet aggregation. The aggregation-independent vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay served for comparisons. ADP inducible aggregation was correlated to that by TRAP-6 (r = 0.33 to 0.72; p < 0.001 for all assays). A linear correlation was seen within MEA (r = 0.72). LTA TRAP-6 correlated weakly with the VASP assay (r = 0.19; p = 0.01), while there were no correlations of TRAP-6 responses by MEA or the Impact-R with the VASP assay (r = 0.03 and −0.09; p > 0.05). In all three assays, differences between ADP and TRAP-6 inducible aggregation varied considerably. Within MEA, TRAP-6 inducible aggregation was almost always stronger than ADP inducible aggregation, while within LTA and the Impact-R, weak responses to ADP were associated with both, weak and strong responses to TRAP-6. In conclusion, the application of TRAP-6 as a positive control for platelet reactivity has major limitations and results need to be cautiously interpreted on an individual basis.

Comparison of methods to evaluate clopidogrel-mediated platelet inhibition after percutaneous intervention with stent implantation.

A high on-treatment residual ADP-inducible platelet reactivity in light transmission aggregometry (LTA) has been associated with an increased risk of adverse outcomes after percutaneous coronary intervention (PCI). However, LTA is weakly standardized, and results obtained in one laboratory may not be comparable to those obtained in another one. We therefore sought to determine the test correlating best with LTA to estimate clopidogrel-mediated platelet inhibition in 80 patients on dual antiplatelet therapy after elective percutaneous intervention with stent implantation. We selected the VerifyNow P2Y12 assay, the vasodilator-stimulated phosphoprotein phosphorylation assay, multiple electrode platelet aggregometry and the Impact-R for comparisons with LTA. Cut-off values for residual ADP-inducible platelet reactivity were defined according to quartiles of each assay. Sensitivities and specificities of the different platelet function tests were based on the results from LTA. The results from all four assays correlated significantly with those from LTA. The VerifyNow P2Y12 assay revealed the strongest correlation (r = 0.61, p < 0.001). Sensitivities and specificities ranged from 35% to 55%, and from 78.3% to 85%, respectively. In conclusion, although all assays correlated significantly with LTA, they need to be improved to become clinically used diagnostic tests. Further, it may be too early to define the gold standard method for assessing residual ADP-inducible platelet reactivity and generally acceptable cut-off values.

Disaggregation Following Agonist-Induced Platelet Activation in Patients on Dual Antiplatelet Therapy.

Disaggregation as the difference between maximal and final platelet aggregation by light transmission aggregometry indicates the stability of platelet aggregates. We evaluated the extent of disaggregation after platelet stimulation with adenosine diphosphate (ADP), arachidonic acid (AA), collagen, epinephrine, and thrombin receptor-activating peptide (TRAP)-6 in 323 patients on dual antiplatelet therapy with daily aspirin and clopidogrel (group 1), prasugrel (group 2), or ticagrelor (group 3) therapy. All patients in group 1 underwent elective angioplasty and stenting, whereas all patients included in groups 2 and 3 suffered from acute coronary syndromes (STEMI or NSTEMI) and underwent urgent PCI. Significant differences between maximal and final platelet aggregation were observed with all agonists throughout the groups (all p<0.001). Disaggregation was highest using AA (clopidogrel 36.5%; prasugrel/ticagrelor 100%) and ADP (clopidogrel 21.7%; prasugrel/ticagrelor 100%). In contrast, low disaggregation was observed after platelet stimulation with collagen and TRAP-6 in clopidogrel-treated patients, and after platelet stimulation with collagen and epinephrine in prasugrel- and ticagrelor-treated patients. In conclusion, pathways of platelet activation that are not inhibited by standard antiplatelet therapy allow persisting platelet aggregation and may at least in part be responsible for adverse ischemic events.

Simvastatin blunts endotoxin-induced tissue factor in vivo.

BACKGROUND: Beyond lipid lowering, various antiinflammatory properties have been ascribed to statins. Moreover, in vitro studies have suggested the presence of anticoagulant effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, as lipopolysaccharide (LPS)-induced monocyte tissue factor (TF) was suppressed. In this study, we examined the role of statins in experimental endotoxemia on inflammatory and procoagulant responses in vivo. METHODS AND RESULTS: In this double-blind, placebo-controlled, parallel-group study, 20 healthy, male subjects were randomized to receive either simvastatin (80 mg/d) or placebo for 4 days before intravenous administration of LPS (20 IU/kg IV). Plasma high-sensitive C-reactive protein (hsCRP), monocyte chemoattractant protein (MCP-1), sCD40L, sCD40, and prothrombin fragment F1+2 (F1.2) were determined by ELISAs at baseline and at 4 and 8 hours after LPS administration. Monocyte TF expression and monocyte-platelet aggregates were measured by whole-blood flow cytometry over the same time course. The increases in hsCRP and MCP-1, both known inducers of TF, were significantly suppressed by statin treatment after LPS challenge. Statin premedication blunted the increase of monocyte TF expression in response to LPS. In parallel, endotoxin-induced formation of F1.2 was significantly reduced by simvastatin after 4 and 8 hours. LPS infusion affected neither the formation and activation of monocyte-platelet aggregates nor plasma levels of sCD40 and sCD40L. CONCLUSIONS: Simvastatin suppresses the inflammatory response to endotoxin and blunts monocyte TF expression but does not affect platelet activation.

Depletion of endothelial progenitor cells in the peripheral blood of patients with rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is characterized by increased cardiovascular morbidity and mortality that cannot be explained solely by traditional cardiovascular risk factors. Cardiovascular morbidity is related to disease activity and can be normalized by effective therapy. Because the quantity of endothelial progenitor cells (EPCs) in the peripheral blood is correlated inversely with cardiovascular risk, we studied whether such abnormalities could also be observed in patients with RA. METHODS AND RESULTS: EPCs were determined in 52 RA patients and in 16 healthy referents (HRs) by fluorescence-activated cell-sorting (FACS) analysis. Patients were divided into groups characterized by active disease (n=36) and low disease activity (n=16). Cells that were positive by flow cytometry for CD34/KDR/AC133 within the lymphocyte population were characterized as EPCs. Furthermore, in subgroups of patients, circulating EPCs were also quantified by a colony-forming unit (CFU) and a circulating angiogenic cell (CAC) assay. EPCs were significantly decreased in RA patients suffering from active disease compared with those from HRs, as measured by FACS analysis (0.026+/-0.002% versus 0.045+/-0.008%, respectively, P<0.05), CFU assay (mean of 5+/-2 versus 18+/-5 CFU/well in HRs, P<0.05), and CAC assay (mean of 7+/-2 versus 52+/-16 positive cells/high-power field, P<0.005). In contrast, the frequency of circulating EPCs from patients with low disease activity was comparable to that of healthy individuals (0.052+/-0.006% by FACS analysis), CFU assay (10+/-5 CFU/well), and CAC assay (mean of 25+/-5 positive cells). Moreover, EPC quantities in peripheral blood were correlated inversely with disease activity as assessed by the disease activity score (r=-0.38, P<0.01). CONCLUSIONS: Our observations indicate that active RA is associated with a depletion of circulating EPCs. This might be one of several factors contributing to the increased cardiovascular risk in RA.

Simvastatin suppresses endotoxin-induced upregulation of toll-like receptors 4 and 2 in vivo.

In addition to lipid lowering effects, statins appear to have pleiotropic immunomodulatory properties. As they particularly affect monocyte functions, we tested the influence of statin treatment on the monocyte activating toll-like receptors (TLR) 4 and 2 in response to lipopolysaccharides (LPS) in vivo. In this double-blind, placebo-controlled study, 20 healthy, male subjects were randomized to receive either simvastatin (80 mg/day) or placebo for 4 days before intravenous LPS administration (20 IU/kg). Simvastatin did not influence the increase in TLR transcripts after LPS administration measured in mRNA isolated from whole blood by quantitative RT-PCR. In contrast, the parallel upregulation of TLR4 and TLR2 on the surface of monocytes determined by flow cytometry was attenuated by more than half after LPS challenge (P<0.02). Suppressed TLR4 and TLR2 expression was associated with diminished circulating concentrations of tumor necrosis factor-alpha and monocyte chemoattractant protein-1. In conclusion, high-dose simvastatin pretreatment blunted TLR4 and TLR2 expression on monocytes in a human endotoxemia model on a posttranscriptional level. This suppressive effect of statins on key receptors of the innate immunity which was associated with a reduction of effector cytokines reveals a potential mechanism for their beneficial effects in sepsis and cardiovascular disease.

Endothelial progenitor cells in kidney transplant recipients.

BACKGROUND: Lower concentrations of endothelial progenitor cells (EPCs) may be associated with increased cardiovascular risk. EPC counts and their correlates have not yet been studied in kidney transplant recipients (KTR). METHODS: We cross-sectionally studied EPC counts in 105 middle-aged KTR (mean estimated glomerular filtration rate 45.2 ml/min/1.73 m; range: 5.4 to 117.5). Using univariate and multivariate linear regression assuming a gamma distribution of the outcome, we examined the associations between counts of cultured EPCs and traditional cardiovascular disease risk factors (hypertension, diabetes, hyperlipidemia, smoking), kidney function, and immunosuppressive agents, amongst others. RESULTS: The median count of cultured EPCs was 34 cells per high-power field (interquartile range: 19 to 64), comparable to healthy individuals. From multivariate analyses, we found independent inverse associations between counts of cultured EPCs and body mass index, mean arterial pressure, and history of cardiovascular disease. Statin use was associated with greater EPC counts, whereas patients receiving azathioprine or angiotensin II receptor treatment had lower EPC counts (all P<0.01). CONCLUSIONS: This study suggests negative associations in KTR between EPC counts and body mass index, and blood pressure, whereas statin use was associated with greater EPC counts. These findings raise the hypothesis whether EPCs are responsible, at least in part, for the well established associations between these factors and cardiovascular outcomes in KTR.

Endurance training increases the number of endothelial progenitor cells in patients with cardiovascular risk and coronary artery disease.

OBJECTIVE: As regular physical exercise improves endothelial dysfunction and promotes cardiovascular health, we investigated the effect of training on angiogenesis by measuring the number of circulating endothelial progenitor cells (EPC), the level of EPC-mobilizing growth factors and tested vascular function by flow-mediated dilation (FMD) in patients with coronary artery disease (CAD) and cardiovascular risk factors (CVRF). In addition, degradation products of the NO pathway (NOx) were determined. METHODS AND RESULTS: Twenty patients with documented CAD and/or CVRF joined a 12-week supervised running training. Circulating EPCs--defined by the surface markers CD34, KDR and CD133--were measured at baseline and after exercise training by flow cytometry. We found a significant increase in circulating EPCs (2.9+/-0.4-fold increase; P < .0001), which was positively correlated with both, the change of FMD (r = .81, P < .001) and the increase of NOx synthesis (r = .83, P < .001). Plasma VEGF and erythropoietin did not change in response to exercise. However, we observed a positive correlation between the number of EPCs and erythropoietin at baseline (r = .70, P < .01) and after training (r = .73, P < .01). CONCLUSIONS: Regular exercise training augments the number of circulating EPCs in patients with CVRF and CAD and is associated with improved vascular function and NO synthesis.

History of cardiovascular disease is associated with endothelial progenitor cells in peritoneal dialysis patients.

BACKGROUND: It is unknown whether traditional cardiovascular disease risk factors influence the number of endothelial progenitor cells (EPCs) and whether numbers of EPCs correlate with endothelial function in patients with end-stage renal disease. METHODS: In a cross-sectional study of 38 peritoneal dialysis patients, we examined numbers of circulating CD34+/KDR+/CD133+ cells, CD34+ hematopoietic stem cells, and EPCs cultured from peripheral blood. We also assessed conventional cardiovascular disease risk factors, such as history of vascular disease, diabetes, hypercholesterolemia, hypertension, and smoking. We determined endothelial function by measurement of endothelium-dependent and endothelium-independent reactivity of forearm resistance arteries by using strain-gauge plethysmography. RESULTS: Numbers of EPCs cultured from peripheral blood and forearm blood flow reactivity did not differ between erythropoietin-treated peritoneal dialysis patients and healthy individuals. A history of vascular disease was associated with number of cultured EPCs, but other cardiovascular disease risk factors showed no association. Furthermore, there was no association of endothelial-dependent and endothelial-independent forearm blood flow reactivity with EPCs in peritoneal dialysis patients. CONCLUSION: In this first study of EPCs in peritoneal dialysis patients, we found an association between history of vascular disease and EPCs, but no association of EPCs with endothelial function or other cardiovascular disease risk factors.

Atorvastatin reduces thrombin generation after percutaneous coronary intervention independent of soluble tissue factor.

INTRODUCTION: Statins were previously shown to suppress cellular tissue factor (TF) in vitro. Here, we investigated the effect of atorvastatin on the TF-pathway and thrombin generation after coronary angioplasty and stenting in vivo. MATERIALS AND METHODS: A cohort of 30 patients with coronary artery disease (CAD) was randomised to treatment with either none (n=10), 10 mg (n=10) or 80 mg (n=10) atorvastatin per day for the postinterventional period of 6 months starting the day before percutaneous coronary intervention (PCI). Fasting blood samples were collected on admission and after 6 weeks and 6 months of statin therapy to determine sTF, free tissue factor pathway inhibitor (TFPI) and prothrombin fragment F1.2 by immunoassay. RESULTS: Soluble TF (sTF) significantly correlated with thrombin generation as measured by prothrombin fragment F1.2 at baseline. This correlation was lost 6 weeks and 6 months after initiation of statin therapy. In vivo, F1.2 was significantly lowered after 6 months of statin therapy by both, low dose (0 vs. 10 mg: 1.3+/-0.3 vs. 0.7+/-0.2 ng/ml; P<0.05) and high dose (0 vs. 80 mg: 1.2+/-0.3 vs. 0.6+/-0.2 ng/ml; P=0.01) atorvastatin compared to control. However, sTF and free TFPI did not change significantly with atorvastatin therapy when compared to baseline or control. CONCLUSIONS: Our results demonstrate reduced in vivo generation of thrombin six months after percutaneous coronary intervention and statin therapy independent of sTF and free TFPI.

Effect of glycoprotein IIb/IIIa antagonist abciximab on monocyte-platelet aggregates and tissue factor expression.

OBJECTIVE: Activated platelets rapidly adhere to monocytes and upregulate the expression of tissue factor (TF), the major trigger of the coagulation cascade. In this study, we examined the effect of abciximab, a nonselective glycoprotein IIb/IIIa-receptor antagonist, on monocyte TF expression in thrombin receptor activator-stimulated whole blood in vitro. METHODS AND RESULTS: Abciximab (50 microg/mL) reduced the mass of platelets attached to monocytes, measured by the mean fluorescence intensity (MFI) of CD42b on CD14+ cells, 1 (CD42b, 471+/-197 versus 1073+/-217 MFI, mean+/-SD, P<0.05), 5, and 10 minutes after thrombin receptor activator stimulation of whole blood to the same extent as anti-P-selectin (50 microg/mL; 288+/-177 MFI, P<0.05) when determined by flow cytometry. In parallel, the expression of the platelet activation marker P-selectin colocalized with CD14+ monocytes was reduced up to 25% by abciximab at the same time points. Expression of monocyte TF antigen (CD14+/TF+, 39.9+/-8.7% versus 66.3+/-19.9%, P<0.05), chromogenic TF-activity (TF, 8.4+/-1.9 versus 13.2+/-2.8 U, arbitrary units, P<0.05), and TF mRNA was suppressed in the presence of abciximab as a consequence of reduced platelet mass attached to monocytes. CONCLUSIONS: Our data suggest that heterotypic monocyte-platelet aggregates are a target for abciximab, which suppresses monocyte TF because of a reduction of monocyte-platelet cross talk.

The role of intraluminal thrombus formation for expansion of abdominal aortic aneurysms.

BACKGROUND: Though the pathophysiology of initiation, formation, and expansion of abdominal aortic aneurysm (AAA) has been intensely researched, the distinct mechanisms driving these processes still remain unclear. In particular, human studies on predictors of AAA progression as a major determinant of rupture risk are scarce. METHODS: All consecutive abdominal aortic ultrasound sonographic examinations performed at the duplex laboratory of the Division of Angiology of the Medical University of Vienna between 1999 and 2012 were reviewed. Patients with repeated measurements of the infrarenal aortic diameter, who had no prior AAA repair were included. Detailed informations on AAA, including length, anterior-posterior and transversal measurements of diameter, and intraluminal thrombus formation/size were obtained from ultrasound examination; patients' comorbidities, cardiovascular risk factors, and medications were obtained from outpatient charts. The expansion rate of AAA in relation to intraluminal thrombus size, gender, age, comorbidities, cardiovascular risk factors, and pharmacotherapy was evaluated. Independent predictors of AAA growth were identified through mixed effects models. RESULTS: In total, 166 patients (123 men and 43 women, mean age 68 ± 9 years) were included. Patients were followed over a mean period of 1.4 ± 1.2 years with a mean number of follow-up investigations of 4.4 ± 2.7. Overall, mean maximum AAA diameter at baseline was 37.4 ± 8.2 mm. The average expansion rate of AAA diameter throughout the follow-up period was 2.0 mm per year (95 % confidence interval: 1.6-2.4). At initial investigation, intraluminal thrombus formation was present in 56.6 % of all patients. AAA diameter at baseline, time of follow-up as well as presence and size of intraluminal thrombus formation were identified as independent predictors of AAA expansion rate. Importantly, gender and presence of cardiovascular risk factors were not associated with AAA progression rate. CONCLUSIONS: Intraluminal thrombus formation seems to be a key determinant for progression of AAA diameter. Further prospective longitudinal studies are warranted to confirm the potential impact of thrombus formation on AAA development and its implication on monitoring and treatment decisions in patients with AAA.

Interleukin-6 and asymmetric dimethylarginine are associated with platelet activation after percutaneous angioplasty with stent implantation.

Data linking in vivo platelet activation with inflammation and cardiovascular risk factors are scarce. Moreover, the interrelation between endothelial dysfunction as early marker of atherosclerosis and platelet activation has not been studied, so far. We therefore sought to investigate the associations of inflammation, endothelial dysfunction and cardiovascular risk factors with platelet activation and monocyte-platelet aggregate (MPA) formation in 330 patients undergoing angioplasty with stent implantation for atherosclerotic cardiovascular disease. P-selectin expression, activation of glycoprotein IIb/IIIa and MPA formation were determined by flow cytometry. Interleukin (IL)-6, high sensitivity C-reactive protein and asymmetric dimethylarginine (ADMA) were measured by commercially available assays. IL-6 was the only parameter which was independently associated with platelet P-selectin expression and activated GPIIb/IIIa as well as with leukocyte-platelet interaction in multivariate regression analysis (all p<0.05). ADMA was independently associated with GPIIb/IIIa activation (p<0.05). Patients with high IL-6 exhibited a significantly higher expression of P-selectin than patients with low IL-6 (p=0.001), whereas patients with high ADMA levels showed a more pronounced activation of GPIIb/IIIa than patients with low ADMA (p=0.003). In conclusion, IL-6 and ADMA are associated with platelet activation after percutaneous angioplasty with stent implantation. It remains to be established whether they act prothrombotic and atherogenic themselves or are just surrogate markers for atherosclerosis with concomitant platelet activation.