PLATELET CD62p EXPRESSION AND MICROPARTICLE FORMATION IN MURINE ACQUIRED IMMUNE DEFICIENCY SYNDROME AND CHRONIC ETHANOL CONSUMPTION

Yinhong Chen1,2, Grace Davis-Gorman2, Ronald R. Watson1,* and Paul F. McDonagh2

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


    ABSTRACT
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Aims: Abnormal platelet counts have been noticed in acquired immune deficiency syndrome (AIDS) patients. However, the actual state of platelets in AIDS is unclear. We hypothesize that platelets are activated and platelet-derived microparticles increase in murine AIDS. Methods: To elucidate the ethanol effects on platelets in murine AIDS, we studied four groups: control, murine AIDS, ethanol, and ethanol plus murine AIDS. Platelet CD62p as a platelet activation marker and CD61+ microparticles as platelet microparticles (PMPs) were measured by flow cytometry. Results: Platelets were significantly activated in mice with murine AIDS and chronic ethanol consumption. Increased platelet CD62p expression and increased PMPs were most pronounced in advanced stages of murine AIDS. Chronic ethanol consumption persistently enhanced platelet activation and PMP formation. Conclusions: Elevated platelet CD62p and PMPs may represent a pro-thrombotic status that have important pathological consequences.


    INTRODUCTION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Immune-related thrombocytopenia has been described extensively in acquired immune deficiency syndrome (AIDS) patients (Godwin and Kripas, 1996Go; Sood et al., 1996Go; Ehmann et al., 1997Go). The reduction in platelet counts may be due to antibodies against platelets and megakaryocytes (Sato et al., 2000Go). However, platelet function, specifically adhesion molecule expression and platelet-derived factors in murine AIDS, have not been investigated. Platelets play a major role in haemostasis and wound healing. The increase in myocardial infarction (Rickerts et al., 2000Go) and high incidence of thrombosis (Sullivan et al., 2000Go; Witz et al., 2000Go) in AIDS patients may be related to chronically activated platelets. Recently, platelets have been identified as inflammatory and immunological mediators. Activated platelets express adhesion molecules to facilitate direct interaction with leucocytes. Platelet– leucocyte and endothelial cell conjugation may plug coronary capillaries, mechanically blocking blood flow (Xiao et al., 1999Go). Inflammatory mediators, such as interleukin (IL)-1 (Hawrylowicz et al., 1989Go), IL-8 (Su et al., 1996Go), RANTES (regulated on activation and normally T-cell-expressed and presumably secreted), macrophage inflammatory protein (MIP)-1{alpha} (Klinger et al., 1995Go) and platelet-activating factor (PAF) released by activated platelets amplify the inflammatory response. Thus overall, progressive cardiovascular manifestations in AIDS may be related to mechanical or chemical injuries by activated platelets.

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., 1991Go); (3) PAF is released by retrovirus-infected cells (Westmoreland et al., 1996Go; Sei et al., 1997Go; Serradji et al., 2000Go). 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)-{alpha}-programmed apoptosis, neutrophil chemotaxis, degranulation, and the oxidative burst (O’Flaherty et al., 1984Go; Westmoreland et al., 1996Go; Huang et al., 1998Go; Falk et al., 1999Go; Clavijo et al., 2001Go). 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., 1997Go), but the plasma level of PAF is unknown. Iwamoto et al. (1997)Go 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, 2000Go). Some (Redmond et al., 2000Go; Serebruany et al., 2000Go) 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.


    MATERIALS AND METHODS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Animals
Female C57BL/6N mice (National Cancer Institute, Bethesda, MD, USA) at 8–12 weeks of age and weighing ~20–22.5 g were randomly assigned to four different groups: control, murine AIDS, ethanol, and murine AIDS plus ethanol. Mice were housed in transparent plastic cages with a stainless wire lid in a room at 20–22°C with constant humidity and a 12-h light:12-h dark cycle. Murine AIDS was induced by LP-BM5 murine leukaemia retrovirus infection. The LP-BM5 viruses were administered intraperitoneally with 0.1 ml of an LP-BM5 inoculum with a titre of 4.5 log10 plaque forming units/ml. Infection leads to the rapid induction of clinical symptomatology that is similar to human AIDS. In the first week, 10% (v/v) of ethanol in autoclaved tap water was made available to the chronic ethanol group mice in a 300 ml plastic bottle with a stopper. The ethanol concentration was increased to 20% (v/v) in the second week and kept at 20% (v/v) for the rest of the treatment periods. The average of 20% ethanol intake per mouse was 2.8 ml/day. Mice were active at night; therefore, we took blood samples at night (after lights turned off for 31/2h) to estimate blood-ethanol concentration (Sigma Diagnostics Alcohol Kit). The average blood-ethanol concentration was 66.9 mg/dl (0.0669%). The non-ethanol-fed mice were given the same diet, except that their bottles contained pure water only. No difference in weight was found at the end of the experiment between the non-ethanol and ethanol-fed mice.

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. 1Go), 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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Fig. 1. Representative FACScans for platelet CD62p and platelet microparticle (PMP) CD61 expression. Dot plots (A) given representative side scatter (SSC) vs forward scatter (FSC). Regions in the ellipse and rectangle were platelet and microparticle populations, respectively. Background fluorescence for platelets was given (B) by gating the platelet region and was designated by marker 2 (M2). Platelets that were CD62p+ exhibited a shift in fluorescence intensity into the marker 1 (M1) region (C). Background fluorescence for microparticles was given (D) by gating the microparticle region and was designated by marker 2 (M2). CD61+ microparticles exhibited a shift in fluorescence intensity into the M1 region (E). Microparticle-expressed CD61 was defined as PMPs. Data were expressed as the percentages of positives.

 
Analysis of platelet CD62p and PMP
Collected data from flow cytometry were analysed using WinMDI 2.8. Dotplots (Fig. 1AGo), represented as SSC vs FSC were used to sort platelet and microparticle populations. Platelet (ellipse) and microparticle (rectangle) regions were created using a tool bar. The same file was reopened as a histogram that represented total events. From this original histogram, individually gating ellipse and rectangle regions subsequently made two histograms, one represented as the platelet population (Fig. 1BGo) and the other as the microparticle events (Fig. 1DGo). Background fluorescence for platelets (Fig. 1BGo) and microparticles (Fig. 1DGo) was measured from a sample containing blood only and was designated by marker 2 (M2). Platelets that were CD62p+, and microparticle CD61+ exhibited a shift in fluorescence intensity into the marker 1 (M1) region (Fig. 1C, EGo). Data were analysed by built-in statistics and reported as the percentage of gated cells and mean fluorescence of gate in M1. We used the percentage of gated cells for further statistical analysis. The reasons were: (1) as soon as platelets were activated, platelet CD62p was expressed, and then shed into plasma. The percentage of CD62p+ was more indicative of CD62p expression than the mean fluorescence of gate; (2) PMP identification was dependent on CD61 expression only. Any microparticles carried on CD61 were platelet-derived microparticles.

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 Newman–Keuls 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.


    RESULTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Platelet CD62 expression
Platelet activation was studied on the basis of platelet CD62 expression. Figure 2Go presents the results of platelet CD62p expression in all groups and over a 3-month period. The percentage of positive platelet CD62p in the control mice (25.34 ± 2.62) was considered as the basal level of CD62p expression. In murine AIDS, platelet CD62p expression significantly increased after 3 months of LP-BM5 infection, with up to 77.98 ± 2.9% of platelets expressing CD62p (P < 0.001). There was no significant difference between uninfected control and 1 or 2 months of murine retrovirus infection groups. However, 2 months of LP-BM5 infection significantly decreased platelet CD62p expression compared to 1 month of infection (P < 0.05). In ethanol-consuming mice, platelet CD62p expression consistently increased and continued to increase as mice chronically consumed ethanol over 3-month periods. After 3 months of ethanol consumption, more than half of their platelets (57.47 ± 7.4%, P < 0.01) were activated. Activation increased to 76.65 ± 10.15% after 3 months of ethanol consumption (P < 0.001). In retrovirus-infected mice subjected to chronic ethanol consumption, the pattern of platelet CD62 expression over the 3-month period was unchanged but higher than controls (P < 0.01). Around 52–62% of the platelets expressed CD62p. Overall, platelet CD62p expression increased in murine AIDS, murine AIDS plus ethanol, and ethanol-consuming mice.



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Fig. 2. Effect of murine AIDS and ethanol on platelet CD62p expression over a 3-month period. Values were means ± SEM in eight mice for each group. Flow cytometry counted 5000 total platelet events. Platelet CD62p was expressed as the percentage of positive events. CD62p in the control mice (month zero) was considered as the basal level of CD62p expression. P < 0.001, control vs 3-month murine AIDS, control vs 2-month and 3-month ethanol consumption; P < 0.01, control vs 1-month ethanol consumption, control vs murine AIDS plus ethanol consumption over 3-month periods; P < 0.05, 1-month murine AIDS vs 2-month murine AIDS.

 
Measurement of PMPs
We identified CD61+ microparticles as PMPs. Figure 3Go presents the percentage of PMPs in total microparticles in the different groups over the 3-month period. The fraction of PMPs in control mice (15.48 ± 2.47%) was regarded as a basal level of PMPs. In murine AIDS the fraction of PMPs increased after 2 months of LP-BM5 infection (24.11 ± 1.61%, P < 0.05) and then jumped to 54.35 ± 1.03% after 3 months (P < 0.001). In chronic ethanol-consuming mice, PMPs were elevated after 1 month of ethanol intake (P < 0.05), compared to the basal levels. Thereafter, PMPs continually increased to 58.37 ± 3.2% (P < 0.001) and reached a plateau at 2 and 3 months of ethanol consumption. In murine AIDS with chronic ethanol-consumption, PMPs remained consistently elevated over the 3-month period (P < 0.001). Thus, the fraction of PMPs in the microparticle region was elevated in murine AIDS mice in the presence or absence of chronic ethanol consumption as well as in the non-murine AIDS ethanol-consuming mice. PMPs reached maximal levels at 3 months of intervention in all three groups.



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Fig. 3. Effect of murine AIDS and ethanol on platelet microparticle (PMP) formation over a 3-month period. Values were means ± SEM in eight mice for each group. CD61+ microparticles in the microparticle region were PMPs. PMPs were expressed as the percentage of CD61+ in total microparticles. PMPs in the control mice (month zero) were considered as the basal level of CD61 expression. P < 0.001, control vs 3-month murine AIDS, control vs 2- and 3-month ethanol consumption, control vs murine AIDS plus ethanol consumption over 3-month periods. P < 0.05, control vs 2-month murine AIDS, control vs 1-month ethanol consumption.

 
Platelet counts
Table 1Go summarizes the platelet counts. Platelets in all groups were in the normal ranges with no significant differences among the groups.


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Table 1. Blood platelet counts in different groups at 3-month intervention
 

    DISCUSSION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Our present study demonstrated platelet activation with normal platelet counts in murine AIDS and chronic ethanol consumption. An increased platelet CD62p expression and elevated number of PMPs, reflecting enhanced platelet activation, were most pronounced in the advanced stages of murine AIDS with or without chronic ethanol consumption. In addition, chronic ethanol consumption alone caused chronic platelet activation.

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 platelet–neutrophil 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., 1992Go). The brain level of PAF is increased in AIDS (Westmoreland et al., 1996Go; Sei et al., 1997Go), 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., 1997Go). 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., 1988Go, 1989Go; Gilbert et al., 1991Go; Thiagarajan et al., 1991; Dahlback et al., 1992Go). The surface of PMPs is enriched by GPIIb/IIIa, which can bind to neutrophils, vascular endothelial cells and platelets through a fibrinogen bridge. PMP–cell 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., 1996Go; Chi et al., 2000Go). 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., 1989Go; Larsen et al., 1989Go; Abrams and Shattil, 1991Go). Antibodies could also bind to the platelet Fc receptor (Fc gamma RII) through their Fc portion and activate platelets (Asch et al., 1987Go; McGregor et al., 1989Go). The signal transduction pathway induced by antibodies may ultimately lead to platelet CD62 expression. Bettaieb et al. (1996)Go 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., 2000Go) along with the HIV co-receptor CXCR4 (Kowalska et al., 1999Go) 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., 1989Go; Klinger et al., 1995Go; Su et al., 1996Go). 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., 1992Go). Besides their stimulatory effects, CD62p expressed on activated platelets serves as a ligand to facilitate platelet–leucocyte and platelet–endothelial 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., 2000Go; 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., 1990aGo,bGo; Renaud and Ruf, 1996Go). Ethanol inhibits collagen-induced platelet aggregation, secretion, arachidonate mobilization, and thromboxane A2 formation (Nguyen et al., 1999Go) but does not inhibit platelet adhesion to de-endothelialized rabbit aortae (Rand et al., 1987Go). Platelet CD62p is an adhesion molecule associated with platelet–platelet, platelet–endothelial cell and platelet–neutrophil interaction. The effects of ethanol on platelet adhesion have been reported in vitro (Rand et al., 1988Go; McKenzie et al., 2002Go) and ex vivo (Rand et al., 1987Go). 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)Go 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.


    ACKNOWLEDGEMENTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
This study was supported by NIH HL 63667 and 59794.


    FOOTNOTES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
* Author to whom correspondence should be addressed at: Division of Health Promotion Science, 1501 N. Campbell, College of Public Health, University of Arizona, P. O. Box 245155, Tucson, AZ 85724, USA. Back


    REFERENCES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
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