1 Divison of Health Promotion Science, College of Public Health and
2 Cardiovascular and Thoracic Surgery, College of Medicine and The Sarver Heart Center, University of Arizona, Tucson, AZ 85724, USA
Received 23 November 2001; in revised form 14 July 2002; accepted 30 July 2002
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ABSTRACT |
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INTRODUCTION |
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Platelets may be chronically activated in murine AIDS. The reasons for activation are: (1) collagen may be exposed, because the integrity of the vascular endothelium is disrupted; (2) thromboxane is elevated by retrovirus infection (Ramis et al., 1991); (3) PAF is released by retrovirus-infected cells (Westmoreland et al., 1996
; Sei et al., 1997
; Serradji et al., 2000
). All the above are potent platelet-activating mediators. Upon platelet activation, CD62p, found in the alpha-granules of platelets, can be rapidly translocated to the plasma membrane, responding to a variety of stimulators. CD62p expressed on activated platelets interacting with neutrophils and vascular endothelial cells through the carbohydrate ligand, sialyl Lewis X, is a crucial step for blood aggregation, neutrophil activation and the inflammatory processes.
PAF has been implicated in the pathogenesis of human AIDS. PAF is a bioactive phospholipid-derived mediator. A variety of cell types, including platelets, basophils, neutrophils, monocytes/macrophages, and endothelial cells can release PAF in both secreted and cell-bound forms. In addition to platelet stimulation, PAF causes vasoconstriction, increases venular permeability, induces neutrophil adhesion molecule expression, enhances tumour necrosis factor (TNF)--programmed apoptosis, neutrophil chemotaxis, degranulation, and the oxidative burst (OFlaherty et al., 1984
; Westmoreland et al., 1996
; Huang et al., 1998
; Falk et al., 1999
; Clavijo et al., 2001
). Platelets and neutrophils tend to mutually enhance their activation by PAF. Thus, PAF can elicit most of the myocardial features of inflammation. It has been reported that the brain level of PAF is increased in AIDS (Sei et al., 1997
), but the plasma level of PAF is unknown. Iwamoto et al. (1997)
found that 80% of the PAF released from the platelets was recovered in the microparticle fraction. Thus, the number of platelet microparticles (PMP) could be related to the level of plasma PAF and the degree of platelet apoptosis.
AIDS patients experience significant behavioural changes, and 14% of HIV-infected patients misuse alcohol (Welch, 2000). Some (Redmond et al., 2000
; Serebruany et al., 2000
) have proposed that moderate ethanol consumption is associated with a reduction in thromboembolic complications of coronary artery disease, possibly partially attributable to modulation of platelet responses, but no direct evidence has verified that chronic ethanol consumption modulates platelet adhesion molecule expression or PMP formation. In the present study, we have investigated the effects of murine AIDS and chronic ethanol consumption on platelet activation as assessed by platelet CD62p expression and platelet-derived microparticle formation.
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MATERIALS AND METHODS |
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Flow cytometry for platelet CD62p and PMP
Platelet CD62p and PMPs were assessed in whole blood using flow cytometry. CD61 is a beta subunit of platelet GPIIb/IIIa. It is expressed consecutively and specifically on platelets. We found that >99% of platelets were CD61+ in control mice. Therefore, CD61+ microparticles were derived from platelets. We defined the CD61+ microparticles as PMP. In this procedure, 1.4:10 citrated whole blood was collected and 20 µl were added to 1 ml of filtered phosphate-buffered saline. A volume of 100 µl of the mixture was incubated with a saturating concentration of fluoroscein isothiocyanate (FITC)-conjugated anti-CD62p and anti-CD61 antibody for 10 min at room temperature. Samples were protected from light by wrapping polypropylene tubes in foil then fixed with 1% paraformaldehyde, until data acquisition in flow cytometry (Becton Dickinson, San José, CA; FACScan Clinical Flow Cytometry). During FACScanning, using side-scatter (SSC) and forward scatter (FSC) sorted the population of platelets (Fig. 1), and FL1 channel determined the fluorescence intensity of FITC. The data from the FACS processing was further analysed using WinMDI 2.8. Data were expressed as the percentage of positive events.
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Platelet counts
Platelet counts were determined by an automated cell counter (Model 9018 CP; Serono Diagnostics, Allentown, PA, USA) and then were verified manually on Wright-stained blood smears.
Statistical analysis
All statistics were calculated using Prism Statistical software (version 3.0). Comparisons among groups were made using analysis of variance with NewmanKeuls post hoc testing when significant differences were observed. Comparing the same blood samples in the absence and presence of stimulators was done using a paired t-test. P < 0.05 was considered statistically significant.
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RESULTS |
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DISCUSSION |
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Platelet CD62p is stored in the alpha-granules in the resting platelets. It can be rapidly translocated to the plasma membrane by a variety of stimulators. Platelet CD62p is associated with platelet adhesion, aggregation and plateletneutrophil interactions, and inflammatory responses may contribute to the severity of myocardial infarction in AIDS. The mechanism leading to enhanced platelet CD62p expression is not fully elucidated in murine AIDS, but several factors may be involved: (1) an increase in the plasma level of soluble agonists; (2) collagen-induced contact activation; and (3) pathological antigen and antibody-mediated platelet activation. All these factors may activate platelets through the combination of protein kinase C (PKC), PKA and arachidonic acid pathways.
PAF is a potent platelet agonist with procoagulant activity (Maier et al., 1992). The brain level of PAF is increased in AIDS (Westmoreland et al., 1996
; Sei et al., 1997
), whereas the plasma level of PAF has been not reported. In the present study, we measured PMPs, which are known to contain an abundance of soluble PAF (Iwamoto et al., 1997
). Our data clearly demonstrated chronically increased PMPs in murine AIDS and chronic ethanol-consuming mice. The concentrated PAF on the PMPs may further activate target cells, such as platelets and neutrophils. PAF binds with G protein-coupled receptors on platelets and activates the latter through both adenylyl cyclase and PLC pathways. PAF in PMPs may contribute to platelet CD62p expression in murine AIDS and/or chronic ethanol consumption. Thus, PMP formation and platelet activation may cause a chronic positive feedback loop. Platelet activation results in the formation of PMPs, which in turn cause platelet activation. In addition to containing PAF, PMPs are also highly pro-thrombogenic. The effective surface area of PMPs for binding coagulation factors is significant. PMPs are capable of binding clotting factors Va, VIII, Xa and protein S to exert profound pro-coagulant activity (Sims et al., 1988
, 1989
; Gilbert et al., 1991
; Thiagarajan et al., 1991; Dahlback et al., 1992
). The surface of PMPs is enriched by GPIIb/IIIa, which can bind to neutrophils, vascular endothelial cells and platelets through a fibrinogen bridge. PMPcell interactions may also involve pathological haemostatic and inflammatory events in murine AIDS and chronic ethanol consumption.
Collagen-induced contact platelet activation may occur in murine AIDS. Retrovirus infection directly or indirectly destroys the vascular endothelium (Zietz et al., 1996; Chi et al., 2000
). Collagen could be exposed due to endothelial injury. Collagen exposure to platelets results in several intracellular signalling events that lead to rapid platelet activation and expressed CD62p on platelet membranes.
In addition to activation by soluble agonists and collagen exposure, anti-platelet antibodies under pathological conditions could also activate platelets. Abnormal antiplatelet antibodies bind to platelet surface glycoproteins or phospholipids via the Fab portion of the IgG molecule (Johnson et al., 1989; Larsen et al., 1989
; Abrams and Shattil, 1991
). Antibodies could also bind to the platelet Fc receptor (Fc gamma RII) through their Fc portion and activate platelets (Asch et al., 1987
; McGregor et al., 1989
). The signal transduction pathway induced by antibodies may ultimately lead to platelet CD62 expression. Bettaieb et al. (1996)
found significant anti-platelet antibodies in AIDS patients. Cross-reactive antibodies between HIV-GP160/120 and platelet GPIIb/IIIa may lead to platelet activation. Platelets expressed CD4 (Sato et al., 2000
) along with the HIV co-receptor CXCR4 (Kowalska et al., 1999
) is the response to retrovirus infection. It may contribute to platelet activation.
No matter how platelet activation is induced, platelet CD62p expression and PMP formation may have pathologically relevant consequences in murine AIDS and chronic ethanol consumption. We found that almost 80% of platelets expressed CD62p in the late stage of murine AIDS in the presence or absence of ethanol consumption. Our findings suggest that a pro-thrombotic condition exists in AIDS. Activated platelets release soluble factors that may act as hormones, IL-1, IL-8, RANTES and PAF, which stimulate neutrophils and cause pathological inflammatory responses (Hawrylowicz et al., 1989; Klinger et al., 1995
; Su et al., 1996
). Activated platelets may undergo apoptosis to form PMPs that enrich PAF. PAF, like an autocrine, further activates platelets and exhibits pro-coagulant activity to accelerate the coagulation cascade (Maier et al., 1992
). Besides their stimulatory effects, CD62p expressed on activated platelets serves as a ligand to facilitate plateletleucocyte and plateletendothelial cell and platelet platelet interactions. Overall, platelet activation may contribute to vascular thrombosis formation and neutrophil activation.
Moderate ethanol consumption has cardioprotective effects (Redmond et al., 2000; Van Tol and Hendricks, 2001) by causing vascular relaxation, reducing fibrinogen level, and modulating platelet function. However, the effects of ethanol on platelets are controversial (Veenstra et al., 1990a
,b
; Renaud and Ruf, 1996
). Ethanol inhibits collagen-induced platelet aggregation, secretion, arachidonate mobilization, and thromboxane A2 formation (Nguyen et al., 1999
) but does not inhibit platelet adhesion to de-endothelialized rabbit aortae (Rand et al., 1987
). Platelet CD62p is an adhesion molecule associated with plateletplatelet, plateletendothelial cell and plateletneutrophil interaction. The effects of ethanol on platelet adhesion have been reported in vitro (Rand et al., 1988
; McKenzie et al., 2002
) and ex vivo (Rand et al., 1987
). However, few studies have been reported on the effects of ethanol on platelet CD62p expression in vivo. In addition, the mechanism by which ethanol affects platelets is not clarified. Serebruany et al. (2000)
reported that chronic ethanol consumption decreased platelet CD62p expression. However, in their study, chronic ethanol-consuming patients (n = 6) with acute myocardial infarction were defined as subjects reporting consumption of any alcohol beverage regularly (at least once weekly) during the preceding year. Our data do not agree with their reports. On the contrary, our study demonstrated that ethanol consumption persistently enhanced platelet CD62 expression and PMP formation over a 3-month period. An increased platelet CD62p expression was also observed in mice with chronic ethanol consumption plus murine AIDS. Therefore, further in vivo studies in a variety of subjects should be conducted to elucidate the role of ethanol in platelet adhesion molecule expression.
The mechanism of ethanol-induced platelet CD62 expression is distinct from that of retrovirus infection. It is possible that ethanol enhances platelet CD62 expression by an increase in reactive oxygen species (ROS) formation (unpublished data), directly increasing production of ROS by dehydrogenase, microsomal oxidation system and catalase. An elevated ROS may induce platelet apoptosis to form PMPs. The high concentration of PAF in PMP activates platelets to express CD62P. Activated platelets produce more PMPs. When this cycle is initiated by ethanol consumption, more platelets become activated and more PMPs are formed.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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