Turay Yardimci

Apo A-I Binding to Platelets Detected by Flow Cytometry

Lipoprotein-platelet interactions are very important in atherosclerosis and thrombosis. Several studies have been carried out on specific binding of various lipoproteins to platelets. But there is considerable disagreement about the details of these binding sites. Although low-density lipoprotein (LDL) receptors of several cells have been studied extensively, there is little datum about high-density lipoprotein (HDL) receptors. Apolipoprotein (apo) A-I may play a major role in the determination of the specificity of HDL receptors. In this study, binding of apo A-I to platelets was investigated by using a flow cytometric method. Citrated blood samples were obtained from five healthy and seven hypercholesterolemic subjects. Apo A-I antibody was incubated with the citrated whole blood before and after activation with ADP or thrombin receptor agonist peptide (TRAP). Then fluorescein isothiocyanate (FITC)-labeled secondary antibodies were added and analyzed on a Becton-Dickinson FACSort flow cytometer. In the hypercholesterolemic group, apo A-I binding to platelets was found to be significantly decreased after activation with TRAP (P<.05), but not after activation with ADP. In the control group, after platelet activation with ADP or TRAP, the apo A-I MFI values were not found to be significantly different from the values of resting platelets (P>.05). In this study, we demonstrated that apo A-I can bind to platelets, and this supports the hypothesis that apo A-I may play a major role in HDL binding to platelets.

Flow cytometric assay of platelet glycoprotein receptor numbers in hypercholesterolemia

In this study, platelet glycoprotein (Gp) receptor numbers were measured by a flow cytometric assay using Cytoquant Gp in seven hypercholesterolemic and five normal subjects. Thrombin receptor agonist peptide (TRAP) was used to activate platelets. In hypercholesterolemia the Gp receptor numbers per resting platelet were found to be: 38 629 - 8538 (GpIIb/IIIa), 22 269 - 5628 (GpIb), 37 037 - 9810 (GpIIIa), 224 - 504 (CD62-P). After activation, receptor numbers were determined to be: 56 399 - 9003 (GpIIb/IIIa), 10 970 - 5319 (GpIb), 50 715 - 7904 (GpIIIa), 1222 - 687 (P-selectin). In the normal group before the activation, receptor numbers were: 43 828 - 8862 (GpIIb/IIIa), 22 166 - 3847 (GpIb), 42 351 - 1049 (GpIIIa), 62 - 139 (CD62-P), After activation, receptor numbers were determined to be: 60 573 - 4294 (GpIIb/IIIa), 13 003 - 4118 (GpIb), 52 067 - 1039 (GpIIIa), 3608 - 1508 (CD62-P). In hypercholesterolemic subjects, GpIIb/IIIa and GpIIIa receptor numbers on activated platelets increased significantly, whereas P-selectin numbers remained unchanged. However, the GpIb levels decreased significantly. In the control group, after activation, GpIIb/IIIa and P-selectin receptors increased significantly, GpIIIa receptor numbers did not change significantly, whereas GpIb receptor numbers decreased significantly. When the GpIIb/IIIa, GpIb, GpIIIa receptor numbers of the control group and hypercholesterolemic group were compared before and after activation, no significant changes were observed ( P > 0.05). But P-selectin receptor numbers were significantly decreased in hypercholesterolemic patients compared to normals following TRAP activation ( P < 0.05). In this study, the effect of hypercholesterolemia on platelet function was observed. The striking observation about present study was the marked decrease in P-selectin expression after activation in the hypercholesterolemics compared to normals. This finding suggests some sort of platelet dysfunction in these individuals.

Postoperative Statin Therapy Attenuates the Intensity of Systemic Inflammation and Increases Fibrinolysis After Coronary Artery Bypass Grafting

A total of 25 patients undergoing coronary artery bypass grafting (CABG) were included in the study. Patients received statin (20 mg daily) postoperatively for 2 weeks. All analyses were performed at 2 different time points: preoperatively (group 1) and 2 weeks after operation (group 2). Interleukin (IL)-6, IL-8, plasminogen activator inhibitor 1 (PAI-1), tumor necrosis factor α (TNF-α), tissue plasminogen activator (t-PA) levels, and tissue factor pathway inhibitor (TFPI) were evaluated. Statin treatment caused a significant reduction in the plasma level of PAI-1 (preop: 15.04 ± 0.13 ng/mL vs postop: 13.89 ± 2.14 ng/mL; P < .05) and increased t-PA levels (preop: 109.74 ± 0.13 vs postop: 231.40 ± 1.22 ng/mL; P < .001). Plasma TNF-α and IL-6 levels did not change with treatment. Statin treatment caused a significant reduction in plasma IL-8 level (279.70 ± 3.42 ng/mL vs postop: 207.18 ± 3.63 ng/mL, P < .05), and TFPI (4.87 ± 2.05 ng/mL vs postop: 6.27 ± 1.25 ng/mL; P < .05). The results demonstrate that atorvastatin attenuates systemic inflammatory reaction after cardiac surgery.

Oxidative Modification of Fibrinogen Affects Its Binding Activity to Glycoprotein (GP) IIb/IIIa

Aim: Proteins are sensitive biomarkers of human diease condition associated with oxidative stress. Alteration of protein structures by oxidants may result in partial or complete loss of protein functions. We have investigated the effect of structural modifications induced by metal ion catalyzed oxidation of fibrinogen on its binding capacity to glycoprotein IIb/IIIa (GpIIb/IIIa) and human platelets. Methods: We identified and quantified of binding capacity of native and oxidized fibrinogen to its receptor in vitro by flow cytometer. Dityrosine formation on oxidized fibrinogen were detected spectrophotometrically. Elevated degradation products of fibrinogen after oxidation were revealed in the HPLC analysis. The native and oxidized fibrinogen were analyzed on mass spectrum upon digestion with tyripsin. Results: Oxidatively modified fibrinogen showed less binding activity than native fibrinogen to GpIIb/IIIa coated micro beads and human platelets whereas slightly higher binding capaticity to ADP induced stimulated platelets. Formation of dityrosines in the amino acid side chains of fibrinogen were observed upon oxidation. Decreased binding capacity of oxidized fibrinogen correlated with intensities of dityrosine formation. Oxidized fibrinogen had more ion-mass intensities at higher than native fibrinogen. Clinical implications: Important point is decreased of binding capacity of the oxidized fibrinogen to own receptor. The decreased rate of binding, leading to effect in the diseases of clot formation may acount for the association between oxidation of fibrinogen and the incidence of effect in human diseases.

Effect of Oxidized Fibrinogen on Hemostatic System: In Vitro Study

Standard coagulation assays were performed with control and oxidized fibrinogen (Fg), using prothrombin time (PT; 12.5 ± 0.4 vs 25 ± 0.8 seconds, P < .001) and activated partial thromboplastin time (aPTT; 33 ± 2.5 vs 63 ± 4.7 seconds, P < .001). Fibrin clot (MA), clot formation initiation (r), and rate of clot lysis (LY30) were measured, a reflection exposure of Fg to Fe3+/ ascorbate oxidative system by thrombelastograph (TEG) analysis (0, 6, 12, 24, and 48 hours, 6.2 ± 1.3 vs 5.5 ± 1.2, 4.3 ± 1.0 [P < .01], 3.9 ± 1.6, 3.2 ± 0.8, [P < .001]). Maximum amplitude level was found to be lower than control (69.1 ± 7.2 vs 67.9 ± 12.4, 64.0 ± 11.4, 60.2 ± 21.2, 42.2 ± 15.2, P < .001). The lysis rate was changed according to oxidation time between Fg exposed to Fe3+/ascorbate and control exposed to Fe 3+/ascorbate for the same treatment time (1.9 ± 0.71 vs 7 ± 0.5, 1.6 ± 0.1, 1.2 ± 0.5, 0.9 ± 1.3, P < .001). We revealed dysregulation of hemostatic system with contribution of oxidized Fg, which was in direct proportion to the intensity of Fg oxidation.

Determination of the N-Terminal Amino Acid Sequence of the Purified Prothrombin from a Patient with Liver Cirrhosis

The reasons for the decreased functional activity of prothrombin in liver diseases are still speculative. When a highly purified preparation of prothrombin from a patient with liver cirrhosis is available, the cause of prothrombin abnormalities may be researched on a molecular basis. In this study, prothrombin (6.7 mg) was purified from the ascites fluid (1130 mL) of a patient with liver cirrhosis by barium citrate adsorption, ammonium sulfate elution, DEAE Sephacel and Heparin Sepharose CL-6B column chromatography steps. The molecular weight of this prothrombin was the same as that of normal prothrombin purified from a normal plasma pool. The specific activities were found to be 3.36 U/mg in the one stage clotting assay and 28.9 U/mg in the staphylocoagulase/chromogenic substrate assay, while the normal prothrombin specific activities were 3.92 U/mg and 30.1 U/mg respectively. When N-terminal amino acid sequence analysis was carried out, it was seen that the first 20 residues were identical to the normal human prothrombin excepting the Gla at position #14.

Clopidogrel Provides Significantly Greater Inhibition of Platelet Activity Than Aspirin When Combined With Atorvastatin After Coronary Artery Bypass Grafting: A Prospective Randomized Study:

Objective: We aimed to compare the effects of 2 different antiplatelet agents on platelet activity in patients receiv- ing atorvastatin after coronary artery bypass grafting (CABG). Methods: We prospectively randomized 50 patients undergoing CABG into 2 groups; group 1 started to receive atorvastatin (10 mg) plus clopidogrel (75 mg; C + A, n = 25) and group 2 atorvastatin (10 mg) and acetylsalicylic acid (ASA; 300 mg, ASA + A, n = 25) daily on postoperative day 1 and continued for 6 months after operation. Adenosine diphosphate (ADP)–induced pla- telet aggregation and the expressions of glycoprotein (Gp) IIb, GpIIIa, P-selectin, and fibrinogen (Fg) and low-density lipoprotein (LDL) binding to platelets were assessed preoperatively and at postoperative days 7, 90, and 180. Results: The mean age of the patients was 59.6 ± 7.6 years, and 82% of the patients were males. The combination of C + A markedly inhibited ADP-induced platelet aggregation compared with ASA + A at postoperative days 90 and 180 (52% ± 6.0% vs 56% ± 7.25% and 19.6% ± 3.2% vs 37% ± 4.1%, P = .039 and P = .0001, respectively). The therapy of C + A significantly suppressed the expressions of GpIIIa at postoperative days 7, 90, and 180 (P = .0001, P = .0001, and P = .0001, respectively) and P-selectin at postoperative days 90 and 180 (P = .035 and P = .002, respectively) when compared to ASA + A. The expression of GpIIb was also significantly depressed at postoperative day 180 in group 1 when compared to group 2 (P = .0001). Low-density lipoprotein binding was significantly increased at day 180 postoperatively in both the groups (basal: 42.9% ± 5.6% vs 45.3% ± 4.4% and day 180: 60.3% ± 4.6% vs 61.8% ± 5.7%, P = .0001). Conclusions: Our results demonstrate that the combination of C + A is more effective than that of ASA + A in inhibiting ADP-mediated platelet aggregation and expression of major platelet receptors after CABG.

Effect of Fluconazole on Human Polymorphonuclear Leucocyte Functions ex vivo against Candida albicans

Polymorphonuclear leucocytes (PMNs) are important components of host defence against fungi. We investigated the ex vivo effect of fluconazole on chemotaxis, adherence, superoxide anion (O–2) generation and intracellular killing of Candida albicans blastoconidia after the administration of fluconazole (300 mg per os) to healthy volunteers. With regard to chemotaxis in response to zymosan-activated serum (ZAS), as measured using an agarose gel technique, fluconazole neither increased, nor decreased the chemotaxis of PMNs. The adherence was significantly enhanced after exposure of PMNs to fluconazole under ex vivo conditions, whereas, O–2 production after stimulation of PMNs with ZAS was not affected by fluconazole. The effect of fluconazole on intracellular killing of C. albicans blastoconidia by PMNs was determined by viable colony count, after release of yeast cells from disturbed neutrophils. Fluconazole under in vitro conditions, at a therapeutic concentration, significantly increased the intracellular killing of C. albicans by PMNs at 30 min when compared with the results obtained in ex vivo experiments (p < 0.001). During 90 min of exposure, no significant difference was found between in vitro and ex vivo conditions (p > 0.05).

Characterization of Platelet Gamma Glutamyltransferase and Its Alteration in Cases of Atherosclerosis

Among the various functional and biochemical alterations in the platelets of cases of atherosclerosis, the membrane alterations occupy an important place. The platelet intrinsic membrane protein gamma glutamyltransferase (GGT), which is involved in glutathione metabolism, has shown decreased activity in cases of atherosclerosis. To add new insights into the pathogenesis of atherosclerosis, GGT is characterized and correlated with other alterations. Triton X-100 solubilized membrane fractions of frozen and thawed platelets of atherosclerotic and normal subjects had 18.66 ± 2.86 mU/109 platelets and 35.67 ± 3.01 mU/109 platelets, respectively. The K m values were the same, 2.08 mmol/L for gamma glutamyl- p-nitroanilide and 5.87 mmol/L for glycylglycine. The V max values were reduced from 100 mU/109 platelets to 41.66 mU/109 platelets for gamma glutamyl-p-nitroanilide and from 45.45 mU/109 platelets to 38.46 mU/109 platelets for glycylglycine. Optimum pH of GGT activity was 8.2, and optimum temperature was 37°C. It had thermal stability with a 64% relative activity at 36°C for 30 min. Serine against borate was detected as the competitive inhibitor and bromcresol green as the noncompetitive inhibitor. In vivo administration of the antithrombotic drug defibrotide increased the platelet GGT levels to those of normals, from 14.72 ± 7.27 mU/109 platelets to 31.80 ± 12.21 mU/109 platelets in 2 hs. Cholesterol, high-density lipoprotein cholesterol in the membrane fractions, and platelet glutathione levels were unaltered. The lipid per-oxidation (membrane malondialdehyde) level was increased, and glucose and histidine active transport systems were impaired in atherosclerotics. All of these changes are discussed in relation to GGT. Key Words: Human platelets—Atherosclerosis—Gamma glutamyltransferase—Gamma glutamyl transpeptidase— Defibrotide—Biological membranes.

New in vitro effects of clopidogrel on platelets in hyperlipidemic and healthy subjects

OBJECTIVE We aimed to detect novel in vitro effects of clopidogrel on platelets by assessment of the following parameters: malondialdehyde, glutathione, nitrite, aggregation response, and expressions of P-selectin, fibrinogen, apolipoprotein A1, apolipoprotein B, and phosphatidylserine. METHODS Platelets were obtained from healthy (n: 9) and hyperlipidemic (n: 9) volunteers. Expressions of P-selectin, fibrinogen, apolipoproteins A1/B and phosphatidylserine with and without clopidogrel were assayed by flow cytometry. Malondialdehyde, glutathione, aggregation and nitrite levels were also assayed. RESULTS Without clopidogrel, the baseline values of platelet aggregation, malondialdehyde, and expressions of P-selectin, fibrinogen and phosphatidylserine were significantly higher, whereas nitrite and expression of apolipoproteins A1/B were significantly lower in hyperlipidemics than in the healthy group. In both groups, clopidogrel significantly reduced aggregation and expression of fibrinogen, but it elevated nitrite levels. Clopidogrel significantly decreased P-selectin and phosphatidylserine expression and malondialdehyde but increased expressions of apolipoproteins A1/B only in hyperlipidemics. CONCLUSION It seems that clopidogrel has some new in vitro antiplatelet effects. The present study is a basic in vitro study to suggest new insights into the effects of clopidogrel on platelet functions.