Azize Sener

In vitro effects of nitric oxide donors on apoptosis and oxidative/nitrative protein modifications in ADP-activated platelets

Nitric oxide (NO) is an important physiological signaling molecule. However, when produced in excessive amounts, NO can also have toxic effects. The aim of this study is to investigate the effects of exogenous- and endogenous-derived NO on oxidative modifications of proteins and apoptosis in activated platelets. Washed platelets were incubated with l-arginine or nitroso-glutathione (GSNO) in the presence of adenosine diphosphate (ADP). After incubation, caspase-3 activity, phosphatidylserine (PS) externalization and the potential of mitochondrial membrane as markers of apoptosis were measured. In addition, the alterations in protein carbonylation (PCO) and nitrotyrosine (NT) formation as markers of protein oxidation were examined. Platelet activation with ADP (20 µM) significantly increased PCO and NT levels and apoptotic events. After incubation with l-arginine, platelet NO production increased significantly. This l-arginine-induced increase caused decreases in formerly increased PCO and NT levels associated with ADP-induced platelet activation. Stimulation of NO production with l-arginine protected platelets from apoptosis. GSNO caused an increase in protein NT levels. Despite this change, GSNO was effective in inhibition of P-selectin expression, platelet aggregation, protein carbonylation and apoptosis. The results suggest that l-arginine and GSNO-mediated NO leads to the inhibition of key apoptotic processes including caspase-3 activation, PS exposure and low mitochondrial membrane potential in washed platelets. The inhibitory effect of platelet clearance of l-arginine and GSNO may be a novel useful therapeutic property in clinical application.

Exogenous l-Arginine and HDL Can Alter LDL and ox-LDL-Mediated Platelet Activation Using Platelet P-Selectin Receptor Numbers

The aim of this study is to investigate the effects of exogenous l-arginine and HDL on LDL and oxidized LDL (ox-LDL)-mediated platelet activation. Adenosine diphosphate (ADP)-activated platelets have been incubated with lipoproteins with or without l-arginine. P-selectin receptor numbers per platelet have been measured by flow cytometry. After incubation with only l-arginine (without lipoproteins), platelet nitric oxide (NO) levels and P-selectin receptor numbers significantly increased compared to the controls (P < .05). After incubation with LDL or ox-LDL, receptor numbers of P-selectin significantly increased (P < .001). However, P-selectin receptor numbers in platelets treated with l-arginine + LDL or l-arginine + ox-LDL decreased compared to the levels in platelets treated with only LDL or ox-LDL (P < .01, P < .001, respectively). Addition of HDL to l-arginine + ox-LDL caused significant reduction in P-selectin receptor numbers as in the control values (P < .001).We have concluded that l-arginine causes enhanced platelet NO levels and blocks the effects of LDL or ox-LDL on platelet P-selectin receptor numbers and HDL also strengthens this effect of l-arginine.

Platelets Proteomic Profiles of Acute Ischemic Stroke Patients

Platelets play a crucial role in the pathogenesis of stroke and antiplatelet agents exist for its treatment and prevention. Through the use of LC-MS based protein expression profiling, platelets from stroke patients were analyzed and then correlated with the proteomic analyses results in the context of this disease. This study was based on patients who post ischemic stroke were admitted to hospital and had venous blood drawn within 24 hrs of the incidence. Label-free protein expression analyses of the platelets’ tryptic digest was performed in triplicate on a UPLC-ESI-qTOF-MS/MS system and ProteinLynx Global Server (v2.5, Waters) was used for tandem mass data extraction. The peptide sequences were searched against the reviewed homo sapiens database (www.uniprot.org) and the quantitation of protein variation was achieved through Progenesis LC-MS software (V4.0, Nonlinear Dynamics). These Label-free differential proteomics analysis of platelets ensured that 500 proteins were identified and 83 of these proteins were found to be statistically significant. The differentially expressed proteins are involved in various processes such as inflammatory response, cellular movement, immune cell trafficking, cell-to-cell signaling and interaction, hematological system development and function and nucleic acid metabolism. The expressions of myeloperoxidase, arachidonate 12-Lipoxygenase and histidine-rich glycoprotein are involved in cellular metabolic processes, crk-like protein and ras homolog gene family member A involved in cell signaling with vitronectin, thrombospondin 1, Integrin alpha 2b, and integrin beta 3 involved in cell adhesion. Apolipoprotein H, immunoglobulin heavy constant gamma 1 and immunoglobulin heavy constant gamma 3 are involved in structural, apolipoprotein A-I, and alpha-1-microglobulin/bikunin precursor is involved in transport, complement component 3 and clusterin is involved in immunity proteins as has been discussed. Our data provides an insight into the proteins that are involved in the platelets’ activation response during ischemic stroke. It could be argued that this study lays the foundation for future mechanistic studies.

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.

The effect of competition on gamma-glutamyl transferase, creatinine and protein levels of taekwondo players.

Post-exercise proteinuria and increased urinary gamma-glutamyl transferase (GGT) levels can be predictive of exercise induced renal damage. In the literature, numerous studies exist on exercise induced proteinuria, but studies investigating the effects of exercise on urinary GGT levels are quite few in martial arts. The purpose of this study was to determine that changes in serum and urinary GGT activity and creatinine levels, and also urinary protein levels in order to assess any potential exercise induced tubular damage on taekwondo players. The study was performed on 18 female and 17 male taekwondo players who were participants in Istanbul Taekwondo Championships. Blood and urine samples collected pre- and 1 h post competition were analyzed for serum GGT and creatinine, urinary GGT, creatinine and protein levels. The post competition serum creatinine level (p = 0.002), urinary GGT (p = 0.010) and protein levels (p = 0.000) were higher than the pre- competition levels in female players. The post competition serum creatinine level (p = 0.006), urinary GGT (p = 0.005) and protein levels (p = 0.000) were higher than the pre-competition levels in male players. We suggest that high-intensity short duration exercise does not lead to increase in serum GGT levels, but lead to increase in serum creatinine levels, as well as induced excretion urinary GGT and protein levels.