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.Materials and 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

Effect of tenoxicam on biochemical serum parameters of rats

Tenoxicam is a nonsteroidal analgesic of the oxicam group, which possesses both antipyretic and anti-inflammatory characteristics. The use of tenoxicam has recently increased and it is reported in the literature that treatments lasting between a few weeks to three months caused increases in serum alanine transferase (ALT), aspartate transferase (AST), gamma glutamyl transferase (GGT) and bilirubin in humans. Toxic dose treatments to rats caused alterations in renal parameters. To verify these observations, various biochemical parameters were examined following administration of nontoxic doses of tenoxicam to rats. Rats were divided into three groups. One group received tenoxicam 0.6 mg/kg/day; the second group received 1.2 mg/kg/day i.p. The control group received normal saline i.p. At the end of 15 days, blood samples from the animals' hearts were taken for routine biochemical tests. No statistically significant changes were observed in serum urea, uric acid, creatinine, electrolytes, ALT, AST, total protein, bilirubin or glucose levels between the treatment groups and control groups. Increases in GGT levels were found to be statistically significant in both of the treatment groups compared with the control group

The effects of acute hyperglycemia on oxidative stress and nitric oxide bioavailability in platelets

Objective: Platelet activation and thrombus formation tendency increase in diabetes mellitus. Nitric oxide (NO) synthesized by platelets plays important role in platelet functions and limits the recruitment of new platelets to the platelet-rich thrombus. However, NO may also be deleterious by elevating oxidative/nitrosative stress in cells. The aim of the present study was to examine effects of acute high glucose in both resting and collagen-stimulated platelets on platelet aggregation, oxidative stress and NO bioavailability. In addition, we investigated the role of superoxide production on NO bioavailability using quercetin as scavengers of superoxide anion. Methods: Washed platelets were incubated with 5mM D-glucose (physiological concentration, n:7) or 25mM D-glucose (pathophysiological concentration, n:7) or quercetin (10 mu M) plus 25 mM D-glucose (n:7) for 1 h. Superoxide production, lipid peroxidation (LPO), NO, nitrotyrosine (NT) levels and advanced glycation end products (AGE) in platelets were measured after incubation. Platelet aggregation was also investigated. Results: Incubation of resting and collagen-activated platelets with high glucose resulted in significant elevations in LPO, NT and AGE levels when compared to 5mM glucose concentration (p<0.05). High glucose significantly increased platelet aggregation, superoxide formation and NO levels in collagen-activated platelets (p<0.05). Quercetin markedly increased production and bioavailability of NO and suppressed oxidative stress in high glucose incubated platelets (p<0.05). Conclusion: High glucose increases oxidative stress and reduces bioavailability of NO in resting and especially collagen activated platelets. Hyperglycemia mediated superoxide production could be taken part on these effects. These findings would be suggested that there might be beneficial effects of quercetin in the prevention of platelet-related complications in diabetes mellitus

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

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/10(9) platelets and 35.67 +/- 3.01 mU/10(9) 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/10(9) platelets to 41.66 mU/10(9) platelets for gamma glutamyl-p-nitroanilide and from 45.45 mU/10(9) platelets to 38.46 mU/10(9) platelets for glycylglycine. Optimum pH of GGT activity was 8.2, and optimum temperature was 37 degrees C. It had thermal stability with a 64% relative activity at 56 degrees C for 30 min. Serine against berate 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/10(9) platelets to 31.80 +/- 12.21 mU/10(9) platelets in 2 hs. Cholesterol, high-density lipoprotein cholesterol in the membrane fractions, and platelet glutathione levels were unaltered. The lipid peroxidation (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

The apoptotic actions of platelets in acute ischemic stroke

Stroke is a disease that affects the blood vessels that supply blood to the brain. Although platelets are implicated in the pathophysiology of stroke the mechanism is still not clear and there antiplatelet agents available for the prevention and treatment of stroke. We herein examined the relationship between the potential cytokine, TNF-alpha platelet activation and apoptosis in acute ischemic stroke patients. We selected 60 patients (mean age 57.9 +/- A 10.2 years) who had not taken any antiplatelet drugs for 14 days. A group of 45 participants (mean age 51.05 +/- A 9.07 years) were selected as the control group. For both the patients and for the control group, P-selectin (CD62p) and Annexin-V binding, cytochrome-c levels, caspase-3 gene expression and caspase-3 releasing and plasma TNF-alpha levels were measured in platelets. The results showed significant increase in plasma TNF-alpha and platelet Annexin-V, CD62p, cytochrome-c and caspase-3 gene expression in stroke patients compared to the control group. The data of this work suggests that inflammation may have a role in platelet apoptosis in stroke which may suggest a new aspect of the role of inflammation in the development of acute ischemic stroke