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Stepien E.,Jagiellonian University | Stepien E.,Laboratory for Molecular Biology and Research | Gruszczynski K.,Holycross Cancer Center | Kapusta P.,Laboratory for Molecular Biology and Research | And 2 more authors.
Biochemia Medica | Year: 2015

Introduction: Centrifugation is an essential step for plasma preparation to remove residual elements in plasma, especially platelets and plateletderived microparticles (PMPs). Our working hypothesis was that centrifugation as a preanalytical step may influence some coagulation parameters. Materials and methods: Healthy young men were recruited (N = 17). For centrifugation, two protocols were applied: (A) the first centrifugation at 2500 x g for 15 min and (B) at 2500 x g for 20 min at room temperature with a light brake. In protocol (A), the second centrifugation was carried out at 2500 x g for 15 min, whereas in protocol (B), the second centrifugation involved a 10 min spin at 13,000 x g. Thrombin-antithrombin (TAT) and plasmin-antiplasmin (PAP) complexes concentrations were determined by enzyme-linked immunosorbent assays. PMPs were stained with CD41 antibody and annexin V, and analyzed by flow cytometry method. Procoagulant activity was assayed by the Calibrated Automated Thrombogram method as a slope of thrombin formation (CAT velocity). Results: Median TAT and PAP concentrations did not differ between the centrifugation protocols. The high speed centrifugation reduced the median (IQR) PMP count in plasma from 1291 (841-1975) to 573 (391-1010) PMP/µL (P = 0.001), and CAT velocity from 2.01 (1.31-2.88) to 0.97 (0.82-1.73) nM/min (P = 0.049). Spearman’s rank correlation analysis showed correlation between TAT and PMPs in the protocol A plasma which was (rho = 0.52, P < 0.050) and between PMPs and CAT for protocol A (rho = 0.74, P < 0.050) and protocol B (rho = 0.78, P < 0.050). Conclusion: Centrifugation protocols do not influence the markers of plasminogen (PAP) and thrombin (TAT) generation but they do affect the PMP count and procoagulant activity. © 2015, Croatian Society of Medical Biochemistry and Laboratory Medicine. Source

Stepien E.,Laboratory for Molecular Biology and Research | Stepien E.,Jagiellonian University | Miszalski-Jamka T.,Center for Diagnosis Prevention and Telemedicine | Kapusta P.,Laboratory for Molecular Biology and Research | And 2 more authors.
Journal of Thrombosis and Thrombolysis | Year: 2011

To examine the associations between cigarette smoking and preferable clot properties. Plasma fibrin clots from 21 randomly selected current smokers (n = 7), former smokers (n = 7) and non-smokers (n = 7) were analyzed, using scanning electron microscopy (SEM). With the use of the turbidimetric clotting and lysis assay in plasma, the maximum absorbance (MaxAbsC, MaxAbs L) was measured and lysis time (Lys50%) was calculated. Smoking cessation significantly influenced fibrin fiber branching and density. Median fiber diameter was not changed. Lys50%) was the highest in current smokers and was reduced in former smokers to the non-smoker level (2120 ± 385 versus 1771 ± 122 and 1724 ± 272 s; P = 0.04). Smoking cessation improves fibrin clot architecture which results in the lesser resistance to lysis. © Springer Science+Business Media, LLC 2011. Source

Stepien E.,Laboratory for Molecular Biology and Research | Stepien E.,Jagiellonian University | Wypasek E.,Laboratory for Molecular Biology and Research | Wypasek E.,Jagiellonian University | And 4 more authors.
Clinical Biochemistry | Year: 2011

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. © 2011. Source

Sekula M.,Jagiellonian University | Janawa G.,Jagiellonian University | Stankiewicz E.,Laboratory for Molecular Biology and Research | Stepien E.,Jagiellonian University | Stepien E.,Laboratory for Molecular Biology and Research
Cellular and Molecular Biology Letters | Year: 2011

Microparticles (MPs) are small membrane vesicles released by stimulated or apoptotic cells, including the endothelium. Hyperhomocysteinemia (HHcy) is a blood disorder characterized by an increase in the plasma concentrations of total homocysteine (Hcy). The plasma Hcy level is determined by environmental factors (dietary habits, i.e. the intake of folic acid, FA) and genetic factors (N5,N10-methylenetetrahydro-folate reductase, MTHFR, polymorphism 677C>T). To evaluate whether moderate Hcy concentrations induce endothelial MP formation, the role of FA supplementation and the influence of MTHFR polymorphism were analysed. Human umbilical vein endothelial cells (HUVEC) were treated in vitro with 50 μM of Hcy and methionine (Met). The MP number and apoptotic phenotype were analyzed using flow cytometry. Increasing doses of FA (5, 15 and 50 μM) were used to reduce the HHcy effect. The MTHFR 677C>T polymorphism was determined. HUVEC stimulated by Hcy produced significantly more MPs than HUVEC under the control conditions: 3,551 ± 620 vs 2,270 ± 657 kMP (p = 0.02). Supplementation with FA at concentrations of 5, 15 and 50 μM reduced the MP count in the cell culture supernatant to 345 ± 332, 873 ± 329, and 688 ± 453 kMP, respectively (p = 0.03). MTHFR 677C>T heterozygosity was associated with a significant increase in MP formation after stimulation with Hcy compared to the control conditions: 3,617 ± 152 vs 1,518 ± 343 kMP (p = 0.02). Furthermore, the MTHFR genotype altered MP formation after Met loading. On average, 24% of the entire MP population was apoptotic (annexin V-positive). Endothelial function impairment due to HHcy is related to MP shedding, which may involve platelets and other blood and vascular cells. MP shedding is a physiological response to moderate HHcy. © 2010 by the University of Wroclaw, Poland. Source

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