Ewa Stępień

EVpedia: a community web portal for extracellular vesicles research

MOTIVATION Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. RESULTS We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research. AVAILABILITY AND IMPLEMENTATION The web site was implemented in PHP, Java, MySQL and Apache, and is freely available at http://evpedia.info.

Hyperglycemia Is Associated With Enhanced Thrombin Formation, Platelet Activation, and Fibrin Clot Resistance to Lysis in Patients With Acute Coronary Syndrome

OBJECTIVE—Acute hyperglycemia on admission for acute coronary syndrome worsens the prognosis in patients with and without known diabetes. Postulated mechanisms of this observation include prothrombotic effects. The aim of this study was to evaluate the effect of elevated glucose levels on blood clotting in acute coronary syndrome patients. RESEARCH DESIGN AND METHODS—We studied 60 acute coronary syndrome patients within the first 12 h after pain onset, including 20 subjects with type 2 diabetes, 20 subjects with no diagnosed diabetes but with glucose levels >7.0 mmol/l, and 20 subjects with glucose levels <7.0 mmol/l. We determined generation of thrombin-antithrombin complexes (TATs) and soluble CD40 ligand (sCD40L), a platelet activation marker, at the site of microvascular injury, together with ex vivo plasma fibrin clot permeability and lysis time. RESULTS—The acute coronary syndrome patients with no prior diabetes but elevated glucose levels had increased maximum rates of formation and total production of TATs (by 42.9%, P < 0.0001, and by 25%, P < 0.0001, respectively) as well as sCD40L release (by 16.2%, P = 0.0011, and by 16.3%, P < 0.0001, respectively) compared with those with normoglycemia, whereas diabetic patients had the highest values of TATs and sCD40L variables (P < 0.0001 for all comparisons). Patients with hyperglycemia, with no previously diagnosed diabetes, had longer clot lysis time (by ∼18%, P < 0.0001) similar to that in diabetic subjects, but not lower clot permeability compared with that in normoglycemic subjects. CONCLUSIONS—Hyperglycemia in acute coronary syndrome is associated with enhanced local thrombin generation and platelet activation, as well as unfavorably altered clot features in patients with and without a previous history of diabetes.

Altered Fibrin Clot Structure/Function in Patients With Cryptogenic Ischemic Stroke

Background and Purpose— We tested the hypothesis that fibrin structure/function is unfavorably altered in patients with cryptogenic ischemic stroke. Methods— Ex vivo plasma fibrin clot permeability, turbidimetry, and efficiency of fibrinolysis were determined in 89 patients with patent foramen ovale (PFO) and a history of first-ever stroke, 58 patients with first-ever stroke and no PFO, and 120 healthy controls. Results— Stroke patients, evaluated 3 to 19 months after the event, and controls did not differ with regard to age, sex, smoking, and fibrinogen. Stroke patients with or without PFO had lower clot permeability (P<0.0001), faster fibrin polymerization (P<0.0001), prolonged clot lysis time (P<0.0001), higher maximum D-dimer levels released from clots (P<0.0001), and maximum rate of D-dimer release (P=0.02) than controls. Time from stroke occurrence showed no association with any clot variables. Scanning electron microscopy of fibrin clots showed increased fiber diameter and density in stroke patients. Clots from stroke patients with PFO were more permeable and showed shorter lysis time compared to those without PFO, and this was related to lower proportion of smokers in the former group. Conclusions— Altered fibrin clot structure and resistance to fibrinolysis are associated with cryptogenic stroke.

Fibrin clot properties are altered in patients with chronic obstructive pulmonary disease. Beneficial effects of simvastatin treatment.

Increased risk of thrombotic events occurs in chronic obstructive pulmonary disease (COPD). Elevated fibrinogen and C-reactive protein (CRP), being common in COPD, are associated with formation of dense fibrin clots resistant to lysis. Statins have been found to display anti-inflammatory and antithrombotic effects. We investigated fibrin clot properties in COPD patients prior to and following statin therapy. Ex vivo plasma fibrin clot permeability, compaction, and fibrinolysis were assessed in 56 patients with stable COPD, aged 64.9 +/- 9.2 years (mean FEV(1), 54.7 +/- 15.9% predicted), versus 56 controls matched for age, sex and cardiovascular risk factors. Patients were then randomly assigned to receive simvastatin 40 mg/day (n = 28) or to remain without statins for three months (n = 28). Patients with COPD had lower clot permeability (6.1+/- 1.07 versus 9.2 +/- 0.9 10(-9) cm(2), p < 0.0001), decreased compaction (44.9 +/- 4.5 versus 63.9 +/- 6.1%, p < 0.0001), higher maximum D-dimer levels released from clots (4.23 +/- 0.55 versus 3.53 +/- 0.31 mg/l, p < 0.0001) with a decreased rate of this release (75.0 +/- 8.3 versus 80.9 +/- 8.0 microg/l/min, p = 0.03) and prolonged lysis time (9.84 +/- 1.33 versus 8.02 +/- 0.84 min, p < 0.0001) compared with controls. Scanning electron microscopy confirmed denser clot structure in COPD. Multiple linear regression analysis after adjustment for age and fibrinogen showed that in the COPD patients, CRP was the only independent predictor of permeability (R(2) = 0.47, p < 0.001) and lysis time (R(2) = 0.43, p < 0.001). Simvastatin improved clot properties (p < 0.05) despite unaltered CRP and irrespective of cholesterol reduction. Our study shows that fibrin clots in COPD patients are composed of much denser networks that are more resistant to lysis, and these properties can be improved by statin administration.

Number of Microparticles Generated During Acute Myocardial Infarction and Stable Angina Correlates with Platelet Activation

BACKGROUND AND AIMS Elevated levels of circulating microparticles (MPs) have been reported in patients with acute myocardial infarction (AMI) and coronary artery disease. Platelet activation and inflammation have been recognized during AMI and stable angina (SA). We hypothesize that the origin and count of MPs in AMI and SA patients are related to markers of inflammation and platelet activation. METHODS Platelet, monocytes and endothelial MPs and surface P-selectin were determined in 12 AMI patients, 10 SA patients and 9 controls by flow cytometry. Plasma P-selectin, CD40 ligand (sCD40L) and interleukin 6 (IL-6) levels were evaluated by ELISA methods. RESULTS The total MP count was compared in control subjects, AMI, and SA patients: 12,765 (8465) vs. 38,750 (11,931) vs. 29,715 (12,072) counts/μl (p = 0.01), respectively. Patients with AMI displayed higher levels of total and platelet origin- tissue factor-positive (CD42/CD142) MPs than patients with SA: 72.8 (6.2) vs. 56.2 (6.4) %, p = 0.001. Levels of soluble P-selectin were significantly elevated in patients with AMI as compared to SA patients: 146 (6.5) vs. 107 (2.7) ng/mL, p = 0.005; significant correlation between total MP count and relative number of CD34, CD51, CD42-positive MPs, and the P-selectin expression was observed in patients with AMI. CONCLUSIONS Platelet activation in AMI is associated with increased generation of MPs not only from platelets, but also monocytes and endothelial cells. It suggests that interactions between platelets, monocytes and endothelial cells play an important role in the pathogenesis of myocardial ischemia.

Lipoprotein(a) as a modifier of fibrin clot permeability and susceptibility to lysis.

Lipoprotein(a) [Lp(a)] is the unique lipoprotein particle with apolipoprotein(a) [apo(a)] bound to apoB by a disulfide linkage [1]. It has been demonstrated that elevated plasma Lp(a) is associated with cardiovascular disease [2]. Kringle domains of apo(a) are homologous with the kringle domains IV and V in the plasminogen molecule [1]. A varying number of type 2 kringles IV determines a heterogenous size of apo(a), with the smaller isoforms being the most pathogenic [3]. There is an inverse correlation between the number of KIV repeats and Lp(a) concentrations [1]. Apo(a) competes with plasminogen for fibrin(ogen) binding sites, resulting in the inhibition of clot lysis [4]. Very recently, in apo(a) transgenic mice, Sha et al. [5] have reported that apo(a) produces a marked prothrombotic effect by a mechanism independent of plasminogen. However, it is not known whether elevated Lp(a) levels and apo(a) isoforms can alter clot properties in human subjects. We studied two groups: (i) 52 apparently healthy men, aged 38–63 years (mean 50 years); and (ii) 24 male survivors of myocardial infarction (MI), aged 42–65 years (mean 51 years). Exclusion criteria were: diabetes; renal insufficiency; acute vascular event within the preceding 6 months; severe concomitant diseases. Healthy individuals and patients with MI (despite clear indications) did not take any medication at least 6 weeks prior to the enrolment. Lipid profile was measured by routine laboratory methods, and fibrinogen and C-reactive protein (CRP) by nephelometry (Dade Behring, Marburg, Germany). Lp(a) was determined using a commercial assay (American Diagnostica, Greenwich, CT, USA). To better evaluate the relationship between the fibrin clot properties and Lp(a), we evaluated two features of the apo(a) gene locus that appear to determine Lp(a) levels. The number of KIV repeats that affects Lp(a) particle size was determined by immunoblotting [6]. Genotyping of the apo(a) pentanucleotide polymorphism at position )1373 was performed as described previously [7]. Study groups were stratified by a dominant allele of the KIV polymorphism (£22 or >22 KIV repeats) to evaluate the effect of the apo(a) pentanucleotide polymorphism according to the study by Holmer et al. [8]. Fibrin clot permeation was determined in a pressure-driven tube system and expressed as a permeation coefficient (Ks), which indicates the pore size [9]. The kinetics of fibrin formation were evaluated by turbidimetry and a slope of the polymerization curve, along with maximum absorbancy, were recorded [9]. Fibrinolysis was evaluated bymeasuring the fibrin assembly kinetics by spectrophotometry upon addition of recombinant tissue plasminogen activator (rt-PA, Boerhinger Ingelheim, Ingelheim, Germany), along with thrombin [10] and the time required for a 50%decrease in clot turbidity (t50%) was a measure of the clot susceptibility to fibrinolysis. All measurements were performed in duplicate by an investigator blinded to the origin of samples. The intraindividual variabilities of results were 6–8%. Data are given as medians (95% confidence interval; CI) or otherwise stated. Inter-group comparisons were performed using the Mann–Whitney U-test and correlations were assessed by the Spearman test. Multiple linear regression analysis (the backward stepwise method) was used to determine predictors of Ks and t50%. A value of P < 0.05 was considered significant. Patients with MI had higher cholesterol, fibrinogen, and CRP levels than healthy subjects while other variables were similar (Table 1). Fibrin clot permeability was reduced and clot lysis, expressed by t50%, was slower inMI patients as compared with healthy individuals (Table 1). In MI patients, we also found lower maximum absorbancy of the clots than in healthy controls (Table 1). The median Lp(a) concentration was higher in patients than in healthy subjects (28.8 vs. 13.2 mg dL; P 1⁄4 0.002). In healthy individuals with a cutoff of Lp(a) Correspondence: Anetta Undas, Institute of Cardiology, Jagiellonian University School of Medicine, 80 Pradnicka Str., 31-022 Cracow, Poland. Tel.: +48 12 6143145; fax: +48 12 4233900; e-mail: mmundas@ cyf-kr.edu.pl

Folic acid administration and antibodies against homocysteinylated proteins in subjects with hyperhomocysteinemia

Growing evidence indicates that elevated total homocysteine (tHcy) levels can elicit autoimmune response in vivo. Antibodies against Nepsilon-Hcy-proteins have been shown to be associated with stroke and premature myocardial infarction. The aim of the current study was to investigate the effect of treatment with folic acid on anti-Nepsilon-Hcy-albumin and -hemoglobin antibodies. We recruited 20 apparently healthy men and 12 male patients with documented coronary artery disease (CAD). All participants had plasma fasting tHcy levels >15 microM. At baseline, and after three and six months of treatment with folic acid 1 mg daily, we determined tHcy, serum folate and vitamin B12 levels, along with serum anti-Nepsilon-Hcy-albumin and -hemoglobin IgG antibodies using the home-made immunoenzymatic assays. Both groups did not differ with regard to age, tHcy, folate, lipid profile, and CRP. The only significant difference between healthy subjects and CAD patients was levels of antibodies against Nepsilon-Hcy-albumin. As expected, folic acid administration led to significant decreases in tHcy and increases in folate levels in both groups. Levels of both anti-Nepsilon-Hcy-albumin and -hemoglobin antibodies fell markedly following a three-month folic acid administration in healthy subjects, but not in CAD patients, without any changes at six months in either group. Folic acid administration resulted in a loss of significant correlations between tHcy and antibodies both following three and six months of the therapy in healthy subjects, in contrast to CAD patients. Carriers of the methylenetetrahydrofolate reductase (MTHFR) 677T allele with CAD had significantly higher levels of anti-Nepsilon-Hcy-albumin before and during folic acid administration as compared to healthy subjects. In conclusion, our findings suggest that Hcy-related autoimmune response is resistant to folic acid administration in CAD patients, while in healthy subjects reduced tHcy levels are associated with suppressed production of antibodies against Nepsilon-Hcyproteins. These observations might explain at least in part the failure of vitamin therapy to reduce the risk of cardiovascular events as recently reported.

Increased levels of bone remodeling biomarkers (osteoprotegerin and osteopontin) in hypertensive individuals

OBJECTIVES Osteoprotegerin (OPG) and osteopontin (OPN) are bone metabolism biomarkers which are involved in the regulation of vascular calcification processes and prediction of future adverse cardiac events. DESIGN AND METHODS OPG, OPN levels and classic risk factors were determined in 130 asymptomatic and hypertensive subjects. Receiver operator characteristic (ROC) analysis was performed and the area under the curve (AUC) was calculated. RESULTS The hypertensive subjects had elevated OPG, OPN, fibrinogen, CRP and fasting glucose levels in comparison to the normotensive ones. There were significant correlations between age, CRP and OPG. Multiple regression analysis showed that as well as inflammation (CRP), age and hypertension were predictors of increased OPG levels. OPN increase was correlated with CRP and glucose levels. The AUCs were similar for OPG and OPG biomarkers. CONCLUSIONS Plasma OPG and OPN levels were significantly associated with inflammation and arterial hypertension. They might be useful as additional biomarkers for monitoring endothelial dysfunction and prognosis of cardiovascular diseases.

No-Reflow Phenomenon After Acute Myocardial Infarction Is Associated With Reduced Clot Permeability and Susceptibility to Lysis

Objective— We assessed the relationship between fibrin clot properties and the no-reflow phenomenon after primary coronary intervention (PCI). Methods and Results— Epicardial blood flow was assessed by TIMI scale and corrected TIMI frame count (cTFC), and perfusion by TIMI Myocardial Perfusion Grade (TMPG) after PCI during ST-segment elevation myocardial infarction (STEMI). Fibrin clot permeability (Ks) and susceptibility to lysis in assays using exogenous thrombin (t50%) and without thrombin (tTF) were determined in 30 no-reflow patients (TIMI ≤2) and in 31 controls (TIMI-3) after uneventful 6 to 14 months from PCI. Patients with TIMI ≤2 had lower Ks by 18% (P<0.0001) and prolonged fibrinolysis by 33% for t50% (P<0.0001) and by 45% for tTF (P<0.0001). cTFC was correlated with Ks (r=−0.56, P<0.0001), t50% (r=0.49, P<0.001), and tTF (r=0.54, P<0.001). Ks increased in a stepwise fashion with TIMI flow (P<0.0001) and TMPG (P<0.0001), whereas both fibrinolysis times decreased with TIMI flow (P<0.0001 for both) and TMPG (P<0.01 for both). Multiple regression models showed that only Ks and fibrinogen were independent predictors of cTFC (P<0.05 for both), TIMI ≤2 flow (P<0.05 for both) and TMPG-0/1 (P<0.05 for both). Conclusions— Survivors of myocardial infarction with a history of the no-reflow after PCI are characterized with more compact fibrin network and its resistance to lysis.

Effect of aortic valve stenosis on haemostasis is independent from vascular atherosclerotic burden

OBJECTIVE The aim of study was to assess whether activation of blood coagulation and platelets is enhanced in aortic stenosis (AS) and if so, to determine factors that might modulate these processes. PATIENTS/METHODS Seventy-five patients with AS (48 males, 27 females, aged 65+/-10 years) were enrolled in the study. A control group comprised 75 age- and sex-matched subjects. We determined markers of thrombin generation (thrombin-antithrombin complex [TAT], prothrombin fragment 1+2 [F1+2]), platelet activation (soluble CD40 ligand [sCD40L], beta-thromboglobulin [beta-TG], P-selectin) in peripheral blood plasma. The extent of atherosclerosis in the carotid and coronary arteries was assessed as a potential confounding factor. RESULTS Mean concentrations of thrombin and platelet markers were higher approximately two-fold in the AS group than in controls (p<0.005 for all comparisons). Maximal gradient was positively associated with TAT (r=0.61, p<0.001), F1+2 (r=0.60, p<0.001), sCD40L (r=0.52, p<0.01) and beta-TG (r=0.70, p<0.001). Aortic valve area (AVA) negatively associated only with one platelet marker, beta-TG (r=-0.30, p<0.05). The presence of concomitant atherosclerotic plaque in the carotid (in 65% of patients) or coronary arteries (in 43% of patients) did not influence thrombin generation and platelet activation in patients with AS. CONCLUSIONS AS predisposes to prothrombotic state. Maximal gradient as an index of turbulent flow associated with activation of coagulation and platelets. In contrast, the small aortic valve area was not closely related to these parameters.