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(The FASEB Journal. 1998;12:79-90.)
© 1998 FASEB

RESEARCH COMMUNICATION

Enhanced activation of platelets with abnormal release of RANTES in human immunodeficiency virus type 1 infection

Pål André Holmea, Fredrik Müllera,b, Nils Olav Soluma, Frank Brosstada, Stig S. Frølanda,b, and Pål Aukrusta,b,1

a Research Institute for Internal Medicine, Medical Department A, The National Hospital, University of Oslo, Norway
b Section of Clinical Immunology and Infectious Diseases, Medical Department A, The National Hospital, University of Oslo, Norway


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Besides their role in hemostasis, platelets are involved in inflammatory and immunological processes, and we hypothezise that platelet activation may play an immunopathogenetic role in HIV-1 infection. Blood was drawn from 15 controls and 20 HIV-1-infected patients with normal platelet counts, classified into groups of non-AIDS and AIDS. Platelet activation was detected using flow cytometry with mAbs against the release markers P-selectin and CD63, mAb against GPIb, and the probe annexin V detecting surface exposure of aminophospholipids. The amount of microvesicles was measured using mAb against GPIIIa. Compared to controls, blood samples from HIV-1-infected patients showed significantly enhanced levels of microvesicles and activated platelets as detected by their exposure of P-selectin, CD63, and aminophospholipids, as well as reduction in GPIb expression. Increased expression of P-selectin and amounts of microvesicles were most pronounced in advanced clinical and immunological disease. When studying the effect of HIV-1 protease inhibitor therapy (indinavir) on platelet activation, we found that concomitant with a profound decrease in plasma viral load, there was a near normalization of several of the parameters reflecting enhanced platelet activation. Finally, we demonstrated that platelets may be an important source of the chemokine RANTES in HIV-1-infected patients. Although both unstimulated and SFLLRN-stimulated platelets from asymptomatic patients had enhanced release of RANTES, platelets from AIDS patients were characterized by markedly enhanced spontaneous, but decreased SFLLRN-stimulated release of this chemokine. Taken together, these results, which demonstrate for the first time increased platelet activation in HIV-1-infected patients with normal platelet counts, may represent a previously unrecognized immunopathogenic factor in HIV-1 infection.—Holme, P. A., Müller, F., Solum, N. O., Brosstad, F., Frøland, S. S., Aukrust, P Enhanced activation of platelets with abnormal release of RANTES in human immunodeficiency virus type 1 infection. FASEB J. 12, 79–90 (1998)


Key Words: HIV • RANTES • platelets • antiretroviral therapy • P-selection


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THROMBOCYTOPENIA IS AN IMPORTANT complication in patients infected with human immunodeficiency virus type 1 (HIV-1)2 (1). However, studies of platelets in HIV-1 infection may be of pathophysiological interest not only in patients with thrombocytopenia. It appears that blood platelets, in addition to playing a major role in hemostasis and wound healing, may also participate in inflammatory and immunological processes (2, 3). Thus, platelets have been shown to modulate the activity of neutrophils and monocytes, and this platelet–leukocyte interaction is thought to include soluble factors (4–7) as well as contact-dependent events (6, 8). For example, several cytokines—interleukin (IL)-1 (9), transforming growth factor-ß (10), and the chemokines RANTES, IL-8, ENA-78, and macrophage inflammatory protein (MIP)-1{alpha} (11–14)—are released from or expressed on the surface of activated platelets. In vitro, platelets, or platelet-derived products have been shown to modulate production of reactive oxygen species (ROS), phagocytosis, adhesion to endothelial cells, and degranulation of granulocytes (4, 5, 8, 15), as well as the production of chemokines, ROS generation, and activation of the transcriptional factor NF{kappa}B in monocytes (6, 8). Such effects may also be of clinical importance. Platelets adhere to leukocytes in numerous inflammatory and thrombotic disorders, and abnormal platelet–leukocyte interaction may be a contributory factor in several inflammatory diseases such as acute respiratory distress syndrome, certain forms of glomerulonephritis, and occlusion-reperfusion myocardial injury (2, 3, 5).

Despite several studies of platelet function in HIV-1-infected patients with thrombocytopenia, platelets in HIV-1-infected patients with normal platelet counts have, to our knowledge, not been investigated. We hypothesized that abnormal platelet activation may also be a characteristic of HIV-1 infection in patients without thrombocytopenia. In the present study we used different experimental approaches to explore this hypothesis by analyzing platelets from HIV-1-infected patients in different clinical and immunological stages of disease, always with platelet counts within normal limits.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and controls
Blood samples were obtained from 20 HIV-1-infected patients, clinically classified according to the revised criteria from Centers for Disease Control and Prevention ( Table 1) (16). Twelve of the patients were infected through homosexual, and 8 through heterosexual sex. None of the patients were infected through intravenous drug abuse, and none of the patients were abusing drugs or alcohol at the time of the study. Patients with ongoing acute infections or exacerbation of chronic infections at the time of blood collection (1 month before to 2 wk after) were not included in the study.


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  		Table 1. Clinical and immunological characteristics of the study groupa

At the time of the study, 12 patients were receiving antiretroviral therapy: 2 didanosine, 3 zidovudine alone, 5 zidovudine + lamivudine, 1 zidovudine + lamivudine + indinavir and 1 zalcitabine + lamivudine + ritonavir. Twelve patients received Pneumocystis carinii prophylaxis (2 aerosolized pentamidine, 7 dapsone and 3 trimetoprim-sulfametoxazole). None of the patients had received analgetics, acetylsalicylic acid, or any other nonsteroid anti-inflammatory drug during the last month prior to blood testing. All patients had platelet counts within the normal range and equal to controls ( Table 1). Hemophiliacs, patients with known history of thromboembolic or hemostatic disorder, and patients with a family history of coronary heart or any other atherosclerotic disease were not included in the study.

Controls were 15 healthy, volunteer, HIV-1-seronegative health care workers ( Table 1). The percentage of smokers was equal in controls and in HIV-1-infected patients. Informed consent was obtained from all patients and controls.

Blood sampling protocol
To study platelet activation by flow cytometry, blood was drawn and anticoagulated on sterile vacuum tubes (Becton Dickinson, San Jose, Calif.) containing 1/10th volume of 0.129 M tri-sodiumcitrate. To study platelet exposure of aminophospholipid with annexin V, blood was drawn into tubes containing the irreversible thrombin inhibitor PPACK (5x10-5 M). The first collected tube was disregarded for flow cytometry analyses. The blood was immediately centrifuged at 240 g and room temperature for 7 min to provide platelet-rich plasma (PRP), and subsequently processed for flow cytometry. Plasma for HIV-polymerase chain reaction (PCR) and tumor necrosis factor {alpha} (TNF-{alpha}) analyses was drawn on sterile vacuum blood collection EDTA tubes (Becton Dickinson), immediately immersed in melting ice, and centrifuged within 15 min at 1200 g and 4°C for 10 min. Plasma was stored at -80°C until analyzed, and samples were frozen and thawed only once.

Preparation of platelet samples for flow cytometry
Generally, 1 x 106 platelets were added to polystyrene tubes containing filtered phosphate-buffered saline (pH 7.4) or Tris-buffered saline (TS) containing 2.5 mM CaCl2 for experiments studying annexin V binding, at a final volume of 100 ml after addition of the fluorescent probes. The various fluorescein isothiocyanate (FITC) -labeled probes were added in final concentrations of 5 µg/ml (FITC-Y2/51 against GPIIIa; Dako A/S, Glostrup, Denmark), 5 µg/ml (FITC-AN51 against GPIb; Dako A/S), 4 µg/ml (FITC-CLB-gran 12 against CD63; Immunotech, Marseille, France), 5 µg/ml [FITC-AK4 against P-selectin (CD62p); Pharmingen, San Diego, Calif.], 21.4 µg/ml (FITC-PAC-1, which recognizes an epitope on activated GPIIb-IIIa; The Cell Center, University of Pennsylvania), and 33.3 µg/ml (FITC-annexin V; Bender Wien, Vienna, Austria). The mixtures were incubated in the dark at room temperature for 20 min, diluted with 1 ml of the same incubation buffer, and processed for flow cytometry.

Flow cytometry
Platelets labeled with the FITC-conjugated probes were analyzed in a FACScan flow cytometer (Becton Dickinson) as previously described (17). Briefly, the light scatter and fluorescence channels were set at logarithmic gain, and platelets were gated based on forward and side scatter properties. A fluorescence threshold was set at the upper limit of native control platelets, and cells with a fluorescence intensity exceeding this threshold level were considered positive. The relative amounts of GPIIb-IIIa and GPIb on the platelet surface were measured as mean fluorescence intensity from the bound fluorochrome labeled monoclonal antibodies (mAbs) Y2/51 and AN51, respectively.

To study the amounts of platelet microparticle formation and to resolve platelet-derived microparticles from background light scatter, acquisition was gated so as to include only positive events for antibody bound to GPIIIa (Y2/51). Microparticles and platelets were separated analytically on the basis of their characteristics in forward and side scatter. To quantitate and discriminate between platelets and microparticles, the lower limit of the platelet gate was set at the left-hand border of the forward scatter profile of unactivated platelets. The number of microparticles present was expressed as the number of particles below this limit in percent of the total number of fluorescent particles counted (i.e., platelets plus microparticles). Altogether, 10,000 positive events were analyzed each time; the Cellquest program (Becton Dickinson) were used for data processing.

Stimulation of platelets in PRP
For preparation of PRP, blood was drawn into sterile vacuum blood collection tubes containing citrate as anticoagulant (Becton Dickinson) and centrifuged at 240 g and room temperature for 10 min immediately after blood collection. A volume of 475 µl PRP containing less than 0.02 x 109/l leukocytes for all patients and controls was incubated by gentle tilting for 30 min at room temperature after addition of 25 µl of the thrombin receptor agonist peptide SFLLRN (stimulated sample) or TS only (unstimulated sample). The final concentration of SFLLRN [synthesized at the Biotechnology Center of Oslo, Norway (17)] was 100 µM, and the TS buffer consisted of 148 mM NaCl and 20 mM Tris-HCl (pH 7.4). At baseline and after 30 min, equal volumes of PRP were centrifuged at 11,000 g and 4°C for 10 min, and platelet free supernatant and platelet pellet with 500 µl TS were stored separately at -80°C until analyses. The platelet pellets were lysed by freezing and thawing three times, and the concentrations of RANTES were analyzed in the lysates. The increase in RANTES levels in supernatants from unstimulated and stimulated platelets is expressed as concentration in supernatant at the end of the experiment minus concentration in supernatant at baseline. The concentration of RANTES is expressed as ng/108 platelets in PRP. The release of RANTES is also given as the increase in RANTES levels in unstimulated or SFLLRN-stimulated supernatants divided on the RANTES levels in the platelet pellet before stimulation x 100 (percent release).

Enzyme immunoassay (EIA) for detection of RANTES and TNF-{alpha}
RANTES was measured by EIA (R&D System, Minneapolis, Minn.). Samples with high RANTES levels were diluted and reassayed. In our laboratory conditions, the intra- and interassay coefficients of variation were less than 9% and the recovery of exogenously added recombinant RANTES from PRP (citrate) was 96%. Plasma levels of TNF-{alpha} were quantified by EIA (Medgenix, Fleurus, Belgium), as previously described (18).

Quantification of HIV-1 RNA copy numbers in plasma
HIV-1 RNA levels were measured in EDTA plasma by quantitative reverse PCR (Amplicor HIV Monitor, Roche Diagnostic Systems, Branchburg, N.J.). The limit of detection was 200 copies/ml plasma.

Quantification of lymphocyte subsets in peripheral blood
The numbers of CD4+ and CD8+ lymphocytes in peripheral blood were determined by immunomagnetic quantification (18).

Statistical analysis
For comparison of two groups of individuals, the Mann-Whitney U test (two-tailed) was used. For comparison of parameters be~fore and during antiviral therapy within the same group of individuals, the Wilcoxon signed rank test for paired data (two-tailed) was used. Coefficients of correlation (r) were calculated by the Spearman's rank test. Results are given as medians and 25th to 75th percentiles if not otherwise stated. P values are two-sided and considered significant when < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Platelet activation detected by flow cytometry
Platelet activation was studied in blood from 20 HIV-1-infected patients and 15 normal controls by flow cytometry using a panel of mAbs. First, platelet secretion was detected by using mAbs against the {alpha}-granule membrane protein P-selectin (also known as CD62p, GMP-140, and PADGEM protein) and the lysosomal integral membrane protein CD63, as these appear on the platelet surface when the granule membranes fuse with the platelet membrane during secretion. A significant increase in P-selectin expression was found both in the non-AIDS [asymptomatic (n=8) + symptomatic non-AIDS patients (n=4)] and in the AIDS group (n=8), compared to controls (~220% increase, Fig. 1 and Fig. 2). The highest single levels were found within the AIDS group, although the median value was somewhat higher for the non-AIDS patients ( Fig. 1). In fact, all four patients with more than 5% of the platelets expressing P-selectin on the surface were in the AIDS group. Also, significantly increased CD63 expression was demonstrated in both clinical groups of HIV-1-infected patients compared to controls (~320% increase in both groups, Fig. 1). In contrast, no statistically significant increase in activation of GPIIb-IIIa, detected as binding of the monoclonal antibody PAC-1 (19), could be observed on the platelets among the HIV-1-infected patients (data not shown).



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  		Figure 1. Platelet activation as determined by flow cytometry in two clinical groups of HIV-1-infected patients [12 non-AIDS patients (8 asymptomatic + 4 symptomatic non-AIDS patients) and 8 AIDS patients] and in 15 healthy controls. The following parameters were studied: the amount of microvesicles (MV) per 100 GPIIIa stained particles in peripheral blood, the percentage of platelets expressing P-selectin (P-sel) and CD63 on the surface, the percentage of platelets binding annexin V (Ann V) indicating exposure of phosphatidylserine on the platelet surface, and the mean fluorescence intensity of GPIb on the platelet surface. Data are given as medians and 25th–75th percentiles. *P < 0.03 and **P < 0.005 vs. controls.



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  		Figure 2. Platelet-derived microvesicles (A, B) and expression of P-selectin (C, B) from one representative AIDS patient (left panels) and one healthy control (right panel). In panels A and B, acquisition was gated so that only particles stained by anti-GPIIIa were included. Microvesicles and platelets were separated on the basis of their characteristics in foward and side scatter. In panels C and D, staining with anti-P-selectin FITC (FL1-height) is plotted against side scatter. The percentage of microvesicles (microvesicles per 100 GPIIIa stained particles, panels A and 4B) and the percentage of platelets expressing P-selectin on their surface (C, D) are shown on the plots.

Platelet activation resulting in a procoagulant surface with exposure of phosphatidylserine was detected by annexin V, which binds with high affinity to aminophospholipids in the presence of Ca2+ (20). Significantly increased annexin V binding was detected both in the non-AIDS group and the AIDS group compared to healthy controls, with the most marked increase among AIDS patients (~120% and 160% increase, respectively; Fig. 1).

Platelets generate procoagulant microvesicles upon activation with various stimulants such as thrombin and collagen (17, 21). Platelet-derived microvesicles have been detected in several clinical conditions such as activated coagulation and fibrinolysis (22) and idiopathic thrombocytopenic purpura (23), but their potential significance under physiological and pathophysiological conditions remains unclear. Nevertheless, they represent a parameter for platelet activation, and by using a platelet specific mAb against GPIIIa, we found a significant increase in the amounts of platelet-derived microvesicles among the HIV-1-infected patients in both the non-AIDS and AIDS group compared to controls, with the highest levels among the AIDS patients (~30% and 80% increase, respectively; Figs. 1 and 2).

During platelet activation, the number of available GPIIb-IIIa complexes on the platelet surface will increase due to fusion of the GPIIb-IIIa containing {alpha}-granule membrane with the surface membrane, whereas GPIb expression may decrease due to redistribution to the open canalicular system or to proteolytic cleavage (24). Thus, as another indication of increased platelet activation, the HIV-1-infected patients showed a significant decrease in surface expression of GPIb both in the non-AIDS and AIDS patients compared to normals (~20% decrease in both groups, Fig. 1). However, no significant differences in the amounts of (nonactivated) GPIIb-IIIa could be observed between patients and controls (data not shown).

Twelve of the patients were receiving antiretroviral therapy or P. carinii prophylaxis (see Materials and Methods), and these medications may possibly influence the degree of platelet activation. However, a similar pattern of platelet activation was also found when these 12 patients were excluded from the study (data not shown).

Thus, platelets from HIV-1-infected patients without thrombocytopenia demonstrate significantly elevated expression of P-selectin and CD63, increased annexin V binding, increased amount of platelet-derived microvesicles, and decreased GPIb expression, with the most pronounced abnormalities in the AIDS group. These findings suggest enhanced platelet activation during HIV-1 infection in patients with normal platelet counts.

Relationships between platelet activation markers and immunological parameters
Among HIV-1-infected patients, a significant negative correlation was observed between the P-selectin expression on the platelet surface and the CD4+ lymphocyte counts in peripheral blood (r=-0.49, P<0.03; Fig. 3). Also, the amounts of microvesicles, but no other parameters of platelet activation, tended to be negatively correlated with CD4+ lymphocyte counts (r=-0.39, P=0.08).



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  		Figure 3. Correlation between percentage of platelets expressing P-selectin on the surface and CD4+ lymphocyte counts in peripheral blood in 20 HIV-1-infected patients.

Persistent immune activation seems to play an important pathogenic role in HIV-1 infection (25), and sustained TNF-{alpha} activation is a distinctive feature of this inappropriate immune activation (18, 25). Among HIV-1-infected patients, there was a significant positive correlation between plasma levels of TNF-{alpha} and both P-selectin expression on the platelet surface (r=0.50, P<0.03; Fig. 4) and the amounts of microvesicles (r=0.45, P<0.05; Fig. 4), but not with any of the other parameters of enhanced platelet activation (data not shown). Thus, patients with the most advanced immunodeficiency and most marked immune activation in vivo (reflected as high plasma levels of TNF-{alpha}) seem to have the highest P-selectin expression on the platelet surface and the highest amount of platelet-derived microvesicles in peripheral blood.



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  		Figure 4. Correlation between percentage of platelets expressing P-selectin on the surface (A), the amount of microvesicles per 100 GPIIIa stained particles in peripheral blood (B), and plasma levels of TNF-{alpha} in 20 HIV-1-infected patients.

Correlation between platelet activation and viral load in plasma and the effect of antiretroviral therapy
To better understand the role of platelet activation in HIV-1 infection, we examined the relationship between parameters of platelet activation and HIV-1 RNA copies in plasma. When the patients were divided into two groups according to the percentage of platelets expressing P-selectin on the surface, we found that the patients with the highest levels had significantly higher viral load in plasma than the other HIV-1-infected patients ( Fig. 5). Furthermore, the expression of P-selectin on the platelet surface tended to be positively correlated with numbers of HIV-1 RNA copies in plasma (r=0.41, P=0.07). None of the other parameters of platelet activation were correlated with viral load in plasma (data not shown).



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  		Figure 5. HIV-1 RNA copy number in plasma in two subgroup of HIV-1-infected patients classified according to the percentage of platelets expressing P-selectin on the surface (>2% P-selectin+ platelets, n=10; <2% P-selectin+ platelets, n=10). *P < 0.03.

Recently, inhibitors of HIV-1 protease have been found to have a potent down-regulatory effect on HIV-1 replication in vivo (26). To further elucidate the relationship between HIV-1 replication and enhanced platelet activation, we therefore analyzed the degree of platelet activation before and during therapy with the HIV-1 protease inhibitor indinavir (dosage: 800 mgx3 per day) combined with zidovudine (250 mgx2) and lamivudine (150 mgx2) in five AIDS patients. As also described by others (26), there was a marked fall in HIV-1 RNA copies in plasma after initiating therapy (median reduction in HIV-1 RNA copies/ml: 2.94 log10, range 0.69–3.28 log10; Fig. 6). Concomitant with the decrease in HIV-1 RNA copies, there was a near normalization of P-selectin and CD63 expression on the platelet surface in all patients without any significant changes in platelet counts, most probably reflecting a profound down-regulation of platelet secretion and thus platelet activation during potent antiviral therapy (~90% and 70% reduction, P-selectin and CD63, respectively; Fig. 6). Binding of annexin V decreased during indinavir therapy, although the difference did not reach statistical significance (~50% reduction, Fig. 6). In contrast, microvesicles persisted elevated during therapy despite the profound fall in viral load ( Fig. 6).



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  		Figure 6. The effect of potent antiretroviral therapy (indinavir in combination with zidovudine and lamivudine) in five AIDS patients on the HIV-1 RNA copy number in plasma (A), percentage of platelets expressing P-selectin (B) and CD63 (C) on the surface, percentage of platelets expressing phophatidylserine on the platelet surface (D) as detected by annexin V binding, and the number of microvesicles (MV) per 100 GPIIIa stained particles in peripheral blood (E). The line corresponding to 200 HIV-RNA copies/ml plasma in panel A indicates the limit of detection. The shaded areas in panels B–E indicate the range of the control levels. The P values in panels B and C represent the difference between baseline (0) levels and levels at the first time point in number of weeks after therapy was initiated.

Release of RANTES in PRP
RANTES belongs to a family of immunoregulatory cytokines called chemokines (27, 28). RANTES, in combination with two other members of the C-C chemokine subfamily (MIP-1{alpha} and MIP-1ß), has recently been demonstrated to suppress HIV-1 replication in vitro (29). RANTES was orginally found to be released from CD8+ lymphocytes (30); other cell types, including platelets, have also been implicated as important cellular sources for this chemokine (6, 11, 12, 27). To elucidate a possible immunopathogenic role for enhanced platelet activation in HIV-1 infection, we therefore analyzed both the platelet content of RANTES and the release of this chemokine in unstimulated and SFLLRN-stimulated platelets in PRP in five asymptomatic HIV-1-infected patients with moderately enhanced platelet activation, five AIDS patients with markedly enhanced platelet activation, and six healthy controls.

At baseline, platelets in pellets from AIDS patients had markedly lower levels of RANTES than pellets from healthy controls, as calculated per platelet (~50% decrease, Fig. 7). In contrast, platelets in pellets from asymptomatic HIV-1-infected patients had slightly higher RANTES levels than controls (~10% increase, Fig. 7).





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  		Figure 7. Levels of RANTES in platelet pellets and unstimulated and SFLLRN-stimulated PRP in five asymptomatic HIV-1-infected patients, five AIDS patients, and six healthy controls. RANTES levels in platelets from pellets before stimulation are given as ng/108 platelets (A). The increase in RANTES levels in unstimulated (B) and SFLLRN-stimulated (C) platelet-free supernatant are given as ng/108 platelets (left panels, bars represent median values), and the increase in RANTES levels in platelet free supernatant divided on the RANTES levels in platelet pellet before stimulation x 100, i.e., percent release (right panels, data are given as median and ranges).

As shown in Fig. 7, both clinical groups of HIV-1-infected patients showed significantly enhanced liberation of RANTES as measured in unstimulated PRP supernatant after 30 min as compared to controls, with particularly high levels in the AIDS group (~200% and 800% increase, asymptomatic and AIDS patients, respectively). Indeed, whereas only ~0.5% of the amount of RANTES in platelets from healthy controls was released in unstimulated PRP, ~2% and 11% were released from platelets in asymptomatic HIV-1-infected patients and AIDS patients, respectively ( Fig. 7).

In contrast, whereas asymptomatic HIV-1-infected patients had significantly enhanced SFL-LRN-stimulated release of RANTES (~20% increase compared with controls, Fig. 7), AIDS patients released significantly lower levels of RANTES into PRP supernatant after stimulation by SFLLRN than did healthy controls (~50% reduction, Fig. 7). Platelets from AIDS patients released a lower percentage of their content of RANTES after SFLLRN stimulation, whereas platelets from asymptomatic HIV-1-infected patients released a higher percentage compared with platelets from controls; however, these differences did not reach statistical significance ( Fig. 7). Thus, whereas AIDS patients are characterized by markedly enhanced spontaneous and decreased SFLLRN-stimulated release of RANTES in PRP, asymptomatic HIV-1-infected pa~tients have enhanced release of RANTES compared with healthy controls in both unstimulated and SFLLRN-stimulated PRP.

The effect of antiretroviral therapy on the release of RANTES in PRP
We also examined the release of RANTES from platelets in PRP of all five AIDS patients after 12 wk of indinavir therapy in combination with zidovudine and lamivudine, with the same dosage as described above. Concomitant with a profound fall in viral load (data not shown), there was a marked increase in RANTES levels both in platelet pellets and in SFLLRN-stimulated release from platelets ( Fig. 8). In contrast, there was a fall in spontaneous release of RANTES, although the levels were still significantly above levels in healthy controls ( Fig. 8). In three of the patients, we also obtained flow cytometry data on the changes in platelet activation during antiretoviral therapy (see above); simultaneously with the changes in release of RANTES, there was a significant decrease in P-selectin and CD63 expression on the platelet surface, suggesting that these changes in RANTES release reflect a decrease in platelet activation.





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  		Figure 8. The effect of potent antiretroviral therapy (indinavir in combination with zidovudine and lamivudine) in five AIDS patients on RANTES levels in platelet pellets and unstimulated and SFLLRN-stimulated PRP. The RANTES levels in platelets from pellets before stimulation (A) and the increase in RANTES levels in unstimulated (B) and SFLLRN-stimulated (C) platelet-free supernatants are given as ng/108 platelets. The shaded areas indicate the range of the control levels. The P values represent the difference between baseline (0) levels and levels after 12 wk of therapy.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates for the first time enhanced platelet activation in HIV-1-infected patients with normal platelet counts in peripheral blood. The increased expression of P-selectin on the platelet surface and the high amount of platelet-derived microvesicles reflecting enhanced platelet activation were most pronounced in advanced clinical and immunological disease. Furthermore, after a profound decrease in plasma viral load induced by potent antiretroviral therapy, near normalization of several of the parameters reflecting platelet activation occurred. Finally, platelet activation in HIV-1-infected patients was associated with dysregulated release of RANTES from platelets in these patients. These findings suggest that enhanced platelet activation may be involved in immunopathogenetic processes in HIV-1 infection.

Although the absolute increase in the parameters for platelet activation in HIV-1-infected patients may seem low, this represents a marked enhancement, possibly of biological and clinical significance. In fact, the percentage of activated platelets in HIV-1-infected patients are in accordance with other studies of enhanced platelet activation in vivo under different clinical conditions (31–34). Also, a recent study from our laboratory showed the range of 4–15% for activated platelets after perfusion through an ex vivo flow chamber at an unphysiological shear rate of 10,500 s-1 at an eccentric stenosis (35). Thus, the platelet activation detected in this study is in the range of what could be expected for clinically observable platelet activation with the techniques used.

In contrast to the other markers of platelet activation, there was no increase in PAC-1 binding. This may have several explanations. Fibrinogen already bound to activated GPIIb-IIa on activated platelets remaining in the circulation may block the access of PAC-1 to activated GPIIb-IIIa (36). Furthermore, "platelet activation" is a collective term used to describe quite different phenomena that may be regulated differently and not even occur simultaneously or in concert. For example, binding of the mAb PAC-1 and the mAb against P-selectin of the platelet surface represents two totally different phenomena: a conformational change in the GPIIb-IIIa complex vs. secretion from the intracellular located {alpha}-granules. Thus, results of assays of platelet activation may differ somewhat between clinical disorders involving different activation processes (37).

The mechanisms leading to enhanced platelet activation in HIV-1-infected patients are unknown, but several factors may be involved. The profound effect of antiviral therapy suggests that the degree of HIV-1 replication is associated with the activation of platelets, and one possibility is that HIV-1 replication per se induces this activation: HIV-1 infection of megakaryocytes and effects of HIV-1-derived proteins on platelets have been reported (38, 39). However, platelet activation may also be related to other manifestations of immunological hyperactivity characteristic of HIV-1 infection. Monocytes and neutrophils, which are persistently activated during HIV-1 infection (25, 40), and several inflammatory cytokines (e.g., IL-1 and TNF-{alpha}) known to be raised during HIV-1 infection (18, 25) appear to promote platelet activation (5, 41). Indeed, in the present study there was a significant positive correlation between plasma levels of TNF-{alpha} and both the expression of P-selectin on the platelet surface and the amount of microvesicles. Finally, increased thrombin activation during HIV-1 infection has been suggested (42, 43), and this may also induce platelet activation in vivo in these patients.

For whatever reasons, enhanced platelet activation may have clinically and immunologically relevant consequences in HIV-1-infected patients. Activated platelets may release soluble factors (chemokines and other cytokines) that may modulate several monocyte and neutrophil functions, including ROS production, adhesion to endothelial cells, phagocytosis, and cytokine production (4–6, 8, 15). It appears, however, that in addition to soluble factors, contact-dependent mechanisms that involve the platelet adhesion molecule P-selectin are of major importance for platelet–leukocyte interaction (6, 8). Thus, besides its own stimulatory effects (44, 45), P-selectin may ligate activated platelets to leukocytes and thereby facilitate the action of soluble mediators released from platelets (e.g., RANTES) (6). Furthermore, it seems that although only a small proportion of lymphocytes may bind to P-selectin on activated platelets under normal physiological conditions, chronically stimulated CD4+ lymphocytes, as found in HIV-1-infected patients (25), may bind P-selectin, modulating the release of proinflammatory cytokines from these cells (46). Thus, activated platelets with up-regulation of P-selectin significantly correlated with enhanced TNF-{alpha} activity, as found in HIV-1-infected patients in the present study, may possibly induce an inappropriate activation of monocytes and neutrophils as well as lymphocytes. Products released from these cells (e.g., inflammatory cytokines such as TNF-{alpha}) may, in turn, further enhance platelet activation (5, 41). It is tempting to hypothesize that abnormally activated platelets and leukocytes in HIV-1 infection may represent a vicious circle contributing to the persistent immune activation characteristics of HIV-1 infection (18, 25).

A major finding in the present study, suggesting a possible immunopathogenic role for enhanced platelet activation in HIV-1 infection, was the demonstration of abnormal release of RANTES from platelets in HIV-1-infected patients. RANTES in combination with MIP-1{alpha} and MIP-1ß has recently been found to suppress HIV-1 replication in vitro (29), and these chemokines have been claimed to be the major HIV-1 suppressive factors produced by CD8+ lymphocytes (29). However, these chemokines, and in particular RANTES, may also be produced by a variety of other cell types (6, 11, 12, 27), and the present study demonstrates for the first time that platelets are an important cellular source of RANTES in HIV-1-infected patients. Several studies have failed to demonstrate any differences in the secretion of RANTES from peripheral blood mononuclear cells (47) and CD8+ lymphocytes (48) by HIV-1-infected patients vs. controls. It may well be that the altered release of RANTES from platelets in AIDS patients may be a contributor to the decrease in circulating RANTES levels in the advanced clinical stages of disease (P. Aukrust, F. Müller, and S. S. Frøland, unpublished results).

We found that whereas platelets from asymptomatic HIV-1-infected patients showed enhanced release of RANTES both spontaneously and after SFLLRN stimulation, platelets from AIDS patients showed markedly enhanced spontaneous, but depressed stimulated release of RANTES. This might suggest that although inflammatory cytokines or other soluble factors present in HIV-1 infection (18, 25) may have priming effects on SFLLRN-stimulated release from platelets in asymptomatic patients with moderately enhanced platelet activation (5, 41), the persistently activated platelets in AIDS patients may be refractory to further stimulation, reflecting a state of "platelet exhaustion." It is possible that the decreased stimulated release of RANTES in platelets from AIDS patients may contribute to enhanced HIV-1 replication in these patients. On the other hand, whereas enhanced levels of RANTES may be beneficial as a suppressive factor for HIV-1 replication (29), RANTES and other chemokines may also augment inflammatory and nonbeneficial processes in disorders characterized by persistent T lymphocyte activation, such as HIV-1 infection (49–51). Thus, the immunological and virological consequences of dysregulated release of RANTES in platelets from HIV-1-infected patients is unclear and warrant further studies.

In the present study, we found that potent antiretroviral therapy with the HIV-1 protease inhibitor indinavir induced a marked decrease in platelet activation and altered release of RANTES from platelets, with an increase in stimulated and a decrease in spontaneous release. Whereas all other platelet activation markers were down-regulated during indinavir therapy, the levels of microvesicles were unchanged. This may have several explanations. First, it is possible that microvesicles are at least partly derived from cellular sources other than platelets (52). However, in the present study the microvesicles were detected by using a platelet-specific mAb against GPIIIa. Furthermore, the different parameters used to describe "platelet activation" represent quite different biological phenomena that may be regulated differently and are not necessarily coordinated (37). Thus, we do not find it peculiar that a tendency toward normalization may be more clear-cut when studied with some parameters rather than others. Moreover, the clearance of microvesicles may be different from that of the activated platelets. Finally, our data suggest that some degree of platelet activation also persists during potent antiretroviral therapy, possibly reflected in persistently increased amount of microvesicles. More long-term studies will be needed to fully elucidate the effects of potent antiretroviral therapy on platelet activation in HIV-1-infected patients. Nevertheless, our findings may represent a previous unrecognized effect of such therapy.

The present study is the first demonstration of enhanced platelet activation in HIV-1-infected patients with platelet counts within normal limit. This activation, probably closely related to the persistent activation of leukocytes during HIV-1 infection, may possibly have important pathophysiological consequences, and may represent a previously unrecognized immunopathogenic factor in HIV-1 infection.


   ACKNOWLEDGMENTS
 
We thank Lisbeth Wikeby and Bodil Lunden for excellent technical assistance. This work was supported by the Norwegian Cancer Society, the Research Council of Norway, Anders Jahre's Foundation, Medinnova Foundation, the Norwegian Council on Cardiovascular Disease, Professor Paul A. Owren's Fund, and Odd Kãre Rabben's Fund for AIDS Research.


   FOOTNOTES
 
1 Correspondence: Section of Clinical Immunology and Infectious Diseases, Medical Department A, Rikshospitalet, N-0027 Oslo, Norway. E-mail: pal.aukrust{at}klinmed.uio.no

2 Abbreviations: mAb, monoclonal antibody; TS, Tris-buffered saline; HIV-1, human immunodeficiency virus type 1; MIP, macrophage inflammatory protein; IL, interleukin; ROS, reactive oxygen species; PRP, platelet-rich plasma; PCR, polymerase chain reaction; TNF, tumor necrosis factor; EIA, enzyme immunoassay; FITC, fluorescein isothiocyanate.

Received for publication September 3, 1997. Accepted for publication October 1, 1997.


   REFERENCES
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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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