Search

Background—Platelet-leukocyte interaction is an early important event for thrombogenesis, and this process is mainly mediated by P-selectin on platelets. Although α-tocopherol has been shown to inhibit thrombotic disorders, the effect of α-tocopherol on platelet P-selectin expression and platelet-leukocyte interaction is little known.Methods and Results—We examined whether α-tocopherol inhibited human platelet P-selectin expression and platelet-leukocyte interaction. α-Tocopherol (50 to 500 μg/mL) inhibited thrombin-induced or phorbol 12-myristate 13-acetate (PMA)-induced P-selectin expression on platelets. α-Tocopherol suppressed platelet–mononuclear cell (MNC) interaction, platelet aggregation, and platelet protein kinase C (PKC) activity stimulated with either PMA (100 nmol/L) or thrombin. Inhibitory actions of α-tocopherol against the platelet functions were mimicked by staurosporine, a selective PKC inhibitor. After oral supplementation of α-tocopherol (300 mg/d for 3 weeks) in healthy subjects, thrombin-mediated or PMA-mediated P-selectin expression, platelet-MNC interaction, and platelet aggregation ex vivo were suppressed.Conclusions—α-Tocopherol inhibited P-selectin expression on human platelets and thereby attenuated platelet-MNC interactions, which were mediated at least in part by the inhibition of intraplatelet PKC activity. These actions of α-tocopherol on platelet functions provide new insights into the antithromboatherogenic properties of α-tocopherol.

Lysophosphatidylcholine (LPC), a lysolipid contained in oxidized low-density lipoprotein, is an atherogenic molecule that induces endothelial dysfunction and platelet activation and inhibits angiogenesis. Although studies showed that vitamin E has antiatherogenic properties, the effects of vitamin E on LPC-induced endothelial dysfunction and platelet activation are little known. We examined whether vitamin E has protecting actions against LPC-induced alterations of endothelial and platelet functions. Incubation of cultured bovine aortic endothelial cells (BAECs) with LPC (10 microM) significantly inhibited bradykinin (1 microM)-stimulated nitric oxide release, which was prevented by cotreatment with vitamin E (50, 100, and 500 microg/ml) in a concentration-dependent manner. In isolated human platelets, LPC stimulated P-selectin expression and induced leukocyte-platelet interaction, which functionally depends on P-selectin expressed on the platelet surface. Vitamin E treatment significantly prevented the LPC-induced platelet P-selectin expression and leukocyte-platelet interaction. As LPC-induced endothelial dysfunction and platelet activation have been shown to involve the protein kinase C (PKC)-dependent signal transduction pathway, we examined the effects of vitamin E on LPC-induced PKC activation in human platelets and BAECs. Vitamin E significantly inhibited LPC (10 microM)-stimulated PKC activation in a concentration-dependent manner. It is concluded that (a) Vitamin E prevented LPC-induced endothelial dysfunction and preserved endothelial nitric oxide release, (b) vitamin E inhibited LPC-induced platelet activation (P-selectin expression) and leukocyte-platelet interaction, and (c) these mechanisms appeared to be at least partly mediated by suppression of the PKC in endothelial cells and platelets. The present findings may provide new insights into antiatherogenic mechanisms of vitamin E.

Impairment of endothelium-derived nitric oxide (EDNO) has been demonstrated in patients with coronary risk factors in some studies, as well as impaired platelet-derived nitric oxide (PDNO) in other studies. However, no study has examined whether these impairments coexist. In 24 patients with coronary risk factors, femoral vascular endothelial function was assessed with acetylcholine (ACh: 50, 100, 200 and 400 μg/min) and endothelium-independent vascular function with nitroglycerin (NTG; 50, 100, 200 μg/min) using a Doppler flow-wire technique, as well as ADP (5 μmol/L)-induced PDNO release with an NO-specific electrode. The ACh-mediated percent change in femoral vascular resistance index (% change of FVRI) and PDNO release had a significant correlation with the number of risk factors. The ACh-mediated % change of FVRI, but not that with NTG, significantly correlated with the PDNO release. Both EDNO and PDNO bioactivities are impaired in patients with coronary risk factors and there is a common mechanism. (Circ J 2002; 66: 837 - 840)

OBJECTIVES We investigated whether impaired platelet-derived nitric oxide (PDNO) bioactivity and augmented platelet aggregability in chronic smokers are related to the imbalance of the intraplatelet redox state through increased oxidative stress. BACKGROUND Chronic smoking impairs PDNO release and augments platelet aggregability. However, their mechanisms are unknown. METHODS Collagen-induced PDNO release, platelet aggregation, plasma and intraplatelet vitamin C and reduced glutathione (GSH), intraplatelet cyclic guanosine 3',5'-monophosphate (cGMP) and intraplatelet nitrotyrosine production, which is a marker of the peroxynitrite formation, were measured in 11 chronic smokers and 10 age-matched nonsmokers. RESULTS Release of PDNO and levels of intraplatelet cGMP were lower, and platelet aggregation was greater, in smokers than in nonsmokers. Intraplatelet vitamin C and GSH levels were lower in smokers than in nonsmokers. Intraplatelet nitrotyrosine production was greater in smokers than in nonsmokers. Next, we investigated the effects of oral vitamin C administration (2 g). After vitamin C administration, intraplatelet vitamin C levels were increased and not different at 2 h between the two groups. Then, PDNO release, intraplatelet cGMP levels and platelet aggregation in smokers were restored to the levels of nonsmokers. In smokers, PDNO release and consumption of GSH during platelet aggregation were inversely correlated, and consumption was much less after vitamin C administration. Vitamin C administration decreased intraplatelet nitrotyrosine production in smokers. CONCLUSIONS Impaired PDNO bioactivity and augmented platelet aggregability may be caused by an imbalance of the intraplatelet redox state through increased oxidative stress in smokers.

Abstract —Platelet aggregation is inhibited through a negative feedback mechanism by the l -arginine/nitric oxide (NO) pathway found in platelets themselves. We have shown that long-term smoking impairs the bioactivity of platelet-derived NO (PDNO), resulting in an increased platelet aggregability. However, little is known about the relation between other coronary risk factors and PDNO release. Accordingly, this study was undertaken to examine whether other coronary risk factors are related to the impairment of PDNO bioactivity. We measured collagen-induced PDNO release with an NO-selective electrode in 61 subjects (mean age 47 years, range 24 to 74 years) who underwent complete physical and laboratory examinations. There was a significant inverse correlation between PDNO release and the number of coronary risk factors ( r =−0.61, P r =−0.33, P r =−0.40, P r =−0.31, P r =−0.33, P P r 2 =0.51), with smoking ( F =37.8), age ( F =7.1), and mean arterial pressure ( F =5.1). Thus, we demonstrated that coronary risk factors are associated with an impairment of PDNO release by human platelets. Our findings may contribute to the understanding of the pathophysiological link between coronary risk factors and atherothrombotic disease.

Advanced glycation end products (AGEs) are elevated in diabetes. We have demonstrated that AGEs trigger thrombogenic responses in cultured cells. We investigated here whether serum AGE levels were positively correlated with thrombogenic markers in humans. Data for fasting serum AGE levels of 186 nondiabetic subjects were obtained from a general population in Japan. We measured body mass index, blood pressure, total cholesterol, low-density lipoprotein and high-density lipoprotein cholesterol, triglycerides, fasting plasma glucose, glycosylated hemoglobin A(1c), insulin, creatinine, uric acid, high-sensitive C-reactive protein, plasminogen activator inhibitor 1 (PAI-1), and fibrinogen. Uni- and multivariate analyses were applied for the determinants of serum AGE levels. The average AGE levels were 4.11 +/- 0.74 U/mL in males and 4.10 +/- 0.93 U/mL in females. In the univariate analysis, PAI-1 (P < .05) and fibrinogen (P < .05) were significantly associated with AGE levels. After performing multivariate analyses, PAI-1 (P < .05) and fibrinogen (P < .05) still remained significant independently. In conclusion, the present study is the first demonstration that PAI-1 and fibrinogen levels were positively associated with serum AGE levels. Advanced glycation end products may be associated with thrombogenesis in humans.

We investigated whether platelet responsiveness to nitroglycerin (NTG) is maintained in long-term smokers and if not, the mechanism. In the absence or presence of NTG, intraplatelet reduced glutathione (GSH) levels and ADP-induced platelet aggregation and intraplatelet cGMP levels were measured in 10 long-term smokers and 10 age-matched nonsmokers. The intraplatelet GSH level was significantly lower in smokers than in nonsmokers ( P N -acetylcysteine (1 mmol/L), an exogenous thiol agent, significantly potentiated NTG-induced platelet inhibition in nonsmokers but not in smokers. The ADP-induced intraplatelet cGMP level was significantly greater in the presence of NTG in nonsmokers but not so in smokers. Because the effects of long-term smoking are multifactorial, a rabbit model was made by chronic administration of buthionine sulfoximine (BSO, n=6) to decrease intraplatelet GSH. The intraplatelet GSH level was significantly lower in BSO-treated rabbits than in saline-treated rabbits ( P N -acetylcysteine–induced potentiation was not observed in BSO rabbits, whereas significant potentiation was found in saline rabbits. These findings were similar to those of long-term smokers. In contrast, the intraplatelet GSH-to–oxidized glutathione ratio, which represents the redox state of glutathione, was significantly lower in smokers than in nonsmokers, whereas no difference was found between saline rabbits and BSO rabbits. In conclusion, long-term smoking causes NTG resistance to aggregation in platelets, possibly through the depletion of intraplatelet GSH.

Abstract —Cell adhesion molecules may play an important role in the disease process of acute coronary syndromes. We have shown a neutralizing anti-P-selectin monoclonal antibody and a sialyl Lewis x -containing oligosaccharide (SLe x -OS), an analogue of selectin ligand on leukocytes, reduce cyclic flow variations (CFVs) in a canine model of recurrent coronary arterial thrombosis, suggesting the important interaction between P-selectin and SLe x for the pathophysiology of these syndromes. However, the functional role of these adhesion molecules in the thrombotic process remains unclear. Therefore, we investigated effects of SLe x -OS on CFVs, platelet P-selectin expression, and morphology of the stenotic site in the same model. Anesthetized open-chest dogs (n=34) were randomly divided into 4 groups after developing CFVs. Dogs intravenously received saline or graded doses of SLe x -OS (5, 20, or 40 mg/kg bolus) infusion followed by a continuous infusion (5 mg · kg −1 · h −1 ) for 60 minutes. By flow cytometric analysis, P-selectin expression on platelets after CFVs was significantly upregulated during CFVs. Immunohistochemical analysis revealed the incorporation of platelets with upregulated P-selectin within thrombi at the stenotic site. Microscopic observations revealed the presence of numerous platelets adhered to leukocytes at the stenotic site on the damaged endothelium. SLe x -OS significantly reduced CFVs, inhibited the P-selectin expression on platelets, and prevented the adherence of platelets and leukocytes. These findings further support the notion that the adhesive interaction between P-selectin on platelets and SLe x on leukocytes plays an important role in platelet-mediated thrombus formation in this model.

Background P-selectin in platelets and endothelial cells mediates adhesive interaction with leukocytes to form thrombi. The purpose of the present study was to investigate the plasma levels of P-selectin in patients with unstable angina and in those with stable effort angina of different pathophysiologies. Methods and Results Plasma P-selectin levels were determined by a monoclonal antibody–based enzyme immunoassay on plasma samples taken from 12 patients with unstable angina, 11 patients with stable effort angina, and 15 healthy volunteers. Patients with unstable angina had angina at rest associated with ECG changes. In patients with unstable angina, plasma P-selectin levels within 1 hour (361±90 ng/mL) and at 3 hours (282±56 ng/mL) after angina were significantly ( P Conclusions Plasma P-selectin levels after angina increased significantly in patients with unstable angina but did not in patients with stable effort angina. These findings may contribute to understanding of the pathophysiology of the acute coronary syndrome of unstable angina.