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Article
Nature Medicine  9, 61 - 67 (2002)
Published online: 16 December 2002; | doi:10.1038/nm810

Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein E

Yuqing Huo1, Andreas Schober2, S. Bradley Forlow1, David F. Smith1, Matthew Craig Hyman1, Steffen Jung3, Dan R. Littman4, Christian Weber2 & Klaus Ley1

1 Department of Biomedical Engineering and Cardiovascular Research Center, University of Virginia, Health Science Center, Charlottesville, Virginia, USA

2 Department of Molecular Cardiovascular Research, University Hospital Aachen, Aachen, Germany

3 Skirball Institute of Biomolecular Medicine, New York, New York, USA

4 Howard Hughes Medical Institute, New York, New York, USA

Correspondence should be addressed to Klaus Ley klausley@virginia.edu
We studied whether circulating activated platelets and platelet−leukocyte aggregates cause the development of atherosclerotic lesions in apolipoprotein-E−deficient (Apoe-/-) mice. Circulating activated platelets bound to leukocytes, preferentially monocytes, to form platelet−monocyte/leukocyte aggregates. Activated platelets and platelet−leukocyte aggregates interacted with atherosclerotic lesions. The interactions of activated platelets with monocytes and atherosclerotic arteries led to delivery of the platelet-derived chemokines CCL5 (regulated on activation, normal T cell expressed and secreted, RANTES) and CXCL4 (platelet factor 4) to the monocyte surface and endothelium of atherosclerotic arteries. The presence of activated platelets promoted leukocyte binding of vascular cell adhesion molecule-1 (VCAM-1) and increased their adhesiveness to inflamed or atherosclerotic endothelium. Injection of activated wild-type, but not P-selectin−deficient, platelets increased monocyte arrest on the surface of atherosclerotic lesions and the size of atherosclerotic lesions in Apoe-/- mice. Our results indicate that circulating activated platelets and platelet−leukocyte/monocyte aggregates promote formation of atherosclerotic lesions. This role of activated platelets in atherosclerosis is attributed to platelet P-selectin−mediated delivery of platelet-derived proinflammatory factors to monocytes/leukocytes and the vessel wall.
Atherosclerosis is a multi-factorial vascular disease involving endothelial cells, vascular smooth muscle cells, mononuclear cells, platelets, growth factors and cytokines1. The response-to-injury hypothesis of atherogenesis2 has been modified dramatically over the past three decades. The original version of this hypothesis proposed that the first step in atherosclerosis was endothelial denudation and the key events in the development of atherosclerosis were the release of growth factors from deposited platelets and consequent smooth-muscle proliferation2, 3. In addition, platelets interacting with monocyte-like cells have also been shown to contribute to foam cell formation4.

Atherogenesis is a chronic inflammatory process in which monocytes and T cells interact with structurally intact but dysfunctional endothelium of arteries1. It was further demonstrated that interventions to reduce mononuclear cell recruitment to vessels, a key step in the initiation of atherosclerosis, were able to protect animals from atherosclerosis5, 6, 7, 8. This inflammation hypothesis of atherosclerosis, emphasizing the role of mononuclear cells in the development of atherosclerosis, made questionable the involvement of platelets in the development of spontaneous atherosclerotic lesions.

Activated platelets are present in the circulating blood of atherosclerotic individuals. The presence of circulating activated platelets was found in the circulating blood of patients with unstable atherosclerosis9, 10, 11, 12, stable coronary disease13 and hypercholesterolemia14, 15. Activated platelets in blood are prone to bind leukocytes, preferentially monocytes, to form platelet−leukocyte aggregates13, 16, 17. Therefore, platelet activation is one of the major characteristics present throughout the atherosclerotic process.

Circulating activated platelets might affect endothelial inflammation and leukocyte−endothelial interactions, which are crucial events in atherosclerosis. P-selectin expressed on activated platelets increases monocytoid cell adhesion to endothelial cells in an in vitro assay18. Activated platelets bind to circulating lymphocytes and may support lymphocyte homing to lymph nodes19. Activated platelets also release proinflammatory cytokines (for example, CD40L20 and interleukin (IL)-1beta21), resulting in endothelial activation. We have previously found that the CC chemokine CCL5 (regulated on activation, normal T cell expressed and secreted, RANTES) secreted by stimulated platelets is immobilized on microvascular or aortic endothelium and triggers monocyte arrest22. Although these observations are suggestive, the role of circulating activated platelets and platelet−leukocyte aggregates in the formation of atherosclerotic lesions in vivo has not been tested so far.

Here, we investigated the interactions of circulating activated platelets with monocytes/leukocytes and atherosclerotic carotid arteries of Apoe-/- mice in vivo. Lesion formation in Apoe-/- mice was studied following repeated injections of activated platelets.

Activated platelets interact with leukocytes and endothelium
Activated, fluorescently labeled platelets infused into C57BL/6 mice through a jugular vein were able to bind to leukocytes immediately. Monocytes and neutrophils (Gr-1 and Mac-1 positive cells), but not CD3+ lymphocytes, bound activated but not resting or P-selectin−deficient platelets (data not shown). In normal C57BL/6 mice, circulating platelet−leukocyte aggregates were no longer detectable at 3 to 4 hours after a single injection of activated platelets. Perfusion of activated platelets caused leukocytes and preferentially monocytes to be removed from the circulation. Following infusion of activated platelets into C57BL/6 mice, the monocyte population disappeared from the circulation almost immediately and returned at 3 to 4 hours, when most of the platelet−leukocyte aggregates were disengaged (Fig. 1). Most neutrophils also disappeared, but returned back to the circulation much sooner than monocytes, at 30 to 80 minutes. Infusion of GFP-expressing neutrophils demonstrated that the recovery of neutrophils was due to leukocytes returning to the circulation rather than release from bone marrow. Leukocytes returning to the circulation were no longer decorated with platelets (data not shown).

Figure 1. Interactions of activated platelets with leukocytes in vivo.
Figure 1 thumbnail

Injection of activated wild-type platelets into wild-type mice caused sequestration of neutrophils for 80 min and monocytes for 180 min. Top right corner, minutes after injection of platelets. R1, granulocytes; R2, monocytes; R3, lymphocytes.



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In an in vitro parallel plate flow chamber assay, activated, but not resting, wild-type platelets were able to interact with IL-1beta−activated human aortic endothelial cells (HAECs). The interactions were mainly characterized by transient tethering and rolling, whereas firm adhesion only rarely occurred. Activated platelets without P-selectin (Selp-/-) showed attenuated interactions with inflamed endothelium (Fig. 2a). Similar to these in vitro data, fluorescently labeled, activated wild-type platelets, but not Selp-/- platelets, interacted with atherosclerotic carotid arteries of Apoe-/- mice, but not carotid arteries of age-matched C57BL/6 mice (Fig. 2b). Fluorescently labeled platelets not only tethered and rolled but also arrested on atherosclerotic endothelium (Fig. 2c). However, activated platelets adherent on atherosclerotic arteries often detached to re-enter the flowing blood within a short time. These interactions mainly occurred on the early atherosclerotic lesions and on the shoulders, but not in the central regions, of established atherosclerotic lesions.

Figure 2. Interactions of activated platelets with atherosclerotic arteries.
Figure 2 thumbnail

a, Activated wild-type () but not Selp-/- (P-/-) platelets () or resting platelets (shaded square) interacted with IL-1beta−treated aortic endothelial cells under physiological shear stress in vitro. Data are expressed as interactions per mm2 per minute. n = 4. *P < 0.01 compared with Selp-/- platelets or resting platelets. b, Labeled platelets interacted with atherosclerotic mouse carotid arteries in vivo following perfusion of activated wild-type but not Selp-/- platelets or resting platelets. n = 6. *P < 0.01 compared with Selp-/- platelets or resting platelets perfusion groups. c, Interactions of activated platelets with an atherosclerotic artery in vivo. A calcein-labeled platelet (black circle) tethers, rolls and arrests on endothelium of atherosclerotic carotid artery, and detaches back to blood flow. The lesion (L) is outlined by broken lines.



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Deposition of platelet-derived chemokines
To determine whether deposition of platelet-derived proinflammatory factors on endothelium was associated with platelet−endothelial interactions, we perfused activated platelets on IL-1beta−treated aortic endothelial cells under shear flow. A substantial granular deposition of RANTES (Fig. 3a) and immobilization of platelet factor-4 (PF-4) in a linear pattern (data not shown) were detected on the surface of inflamed HAEC following perfusion of activated human or mouse wild-type platelets. In contrast, the perfusion of activated Selp-/- platelets resulted in few interactions with endothelium and a reduced immobilization of RANTES (Fig. 3a). Feeding Apoe-/- mice with a western diet for 6 weeks induced an inflammatory, atherosclerotic phenotype of the endothelium of carotid arteries without visible atherosclerotic lesions23. Injected activated wild-type platelets, but not Selp-/- platelets, showed robust interactions with these carotid arteries. Consistent with this observation, en face immunostaining showed that much higher levels of RANTES (Fig. 3b) and PF-4 (data not shown) were present on the carotid arterial endothelium of Apoe-/- mice receiving activated wild-type platelets than those perfused with activated Selp-/- platelets.

Figure 3. Deposition of platelet-derived proinflammatory factors on atherosclerotic endothelium.
Figure 3 thumbnail

a, Perfusion of activated wild-type but not Selp-/- (P-/-) platelets over activated aortic endothelial cells deposited RANTES. b, En face preparation of carotid arteries of Apoe-/- mice fed with a western diet for 6 weeks. Injection of activated wild-type but not Selp-/- platelets deposited RANTES on the atherosclerotic endothelium of mouse carotid arteries. c, Mean fluorescence intensity determined by flow cytometry of activated aortic endothelial cells stained for RANTES. d, RANTES deposition on en face preparation of carotid arteries from Apoe-/- mice measured by image processing. n=4, p<0.01.



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Activated platelets were also able to deposit proinflammatory factors on monocytes. Using confocal microscopy, we found that platelets adherent on the monocyte surface stained positively for RANTES and PF-4. We found that RANTES was distributed diffusely on the monocyte membrane areas where platelets were bound (Fig. 4a). No staining of RANTES or PF4 was found on monocytes not bound with platelets.

Figure 4. Deposition of platelet-derived proinflammatory mediators on the surface of monocytes.
Figure 4 thumbnail

a, Activated platelets (labeled with FITC-conjugated anti-P-selectin) released RANTES (labeled with PE-conjugated anti-RANTES) to the monocyte (MM6 cell) surface. Release can be complete (middle platelet) or incomplete. b, Whole blood flow cytometry shows that monocytes (MM6 cells) incubated with activated platelets bind more VCAM-1−IgG. VCAM-1−IgG binding was significantly inhibited on monocytes pretreated with pertussis toxin or in the presence of P-selectin blocking antibody (G1). Pretreatment of MM6 cells with HP1/2, a VLA-4 blocking antibody, completely abrogated VCAM-1−IgG binding. n = 4. *P < 0.01, compared with no treatment.



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The very late antigen-4 (VLA-4) and vascular cell adhesion molecule-1 (VCAM-1) pathway is known to be crucial for monocyte adhesion to the vessel wall to initiate atherosclerosis6. To test whether activated platelets could activate monocytes and increase the affinity of their VLA-4 integrins for ligand binding, we measured VCAM-1−IgG binding to Mono Mac 6 (MM6) cells, a monocytic cell line expressing phenotypic and functional features of mature monocytes24. Using flow cytometry, we show that VLA-4 of MM6 cells incubated with activated platelets was able to bind more VCAM-1−IgG (Fig. 4b). This result is consistent with a change in VLA-4 affinity. Pretreatment of monocytes with pertussis toxin (PTX) or platelets with a P-selectin blocking antibody inhibits binding of VCAM-1−IgG with VLA-4 on monocyte−platelet aggregates (Fig. 4b). This finding indicates that VLA-4 activation requires adhesive contact between platelets and monocytes and proceeds through PTX-sensitive G-protein coupled receptors. The RANTES receptors CCR1, 3 and 5 are known to couple through PTX-sensitive G proteins.

Activated platelets promote leukocyte adhesion
Under the epifluoresence intravital microscope, interactions of leukocytes in vivo labeled with rhodamine 6G with atherosclerotic carotid arteries were rare, consistent with the chronic nature of atherosclerosis. Following perfusion of activated but not resting wild-type platelets or the supernatant of activated wild-type platelets, substantial interactions of rhodamine 6G labeled leukocytes with atherosclerotic carotid arteries occurred immediately and persisted throughout the experiment (1−2 hours; Fig. 5a). To investigate whether activated platelets affect monocyte arrest on atherosclerotic arteries, EGFP-expressing monocytes isolated from CX3CR1−EGFP mice25 were injected into Apoe-/- mice via tail veins. Few GFP-expressing monocytes interacted with atherosclerotic carotid arteries. However, an increase in monocyte arrest on atherosclerotic endothelium appeared after perfusion of activated wild-type but not Selp-/- platelets. Similar to the behavior of activated platelets, monocytes also mainly interacted with the early atherosclerotic lesions and the edges of advanced lesions (Fig. 5b). To investigate the associations between platelets and leukocytes interacting with atherosclerotic lesions in vivo, we used scanning electron microscopy. Consistent with the intravital study, we found many more leukocytes adherent on atherosclerotic carotid arteries of Apoe-/- mice treated with activated wild-type platelets, but not with resting platelets or activated Selp-/- platelets. Platelets were present on atherosclerotic carotid arteries as platelet−leukocyte aggregates, but rarely as individual platelets directly adherent on atherosclerotic endothelium (Fig. 5c). This indicates that activated platelets bound to leukocytes rapidly promote leukocyte arrest.

Figure 5. Monocyte/leukocyte−endothelial interactions in the presence of activated platelets.
Figure 5 thumbnail

a, Rhodamine 6G labeled leukocyte tethering, rolling and adhesion on atherosclerotic carotid arteries of Apoe-/- mice in vivo were increased following perfusion of activated wild-type platelets but not resting platelets or the supernatant of activated wild-type platelets. n = 9. *P < 0.01. b, EGFP-expressing mouse monocytes isolated from CX3CR1-EGFP mice substantially interacted with the edge of mouse carotid atherosclerotic lesions 20 min following injection of activated wild-type but not Selp-/- or resting wild-type platelets. The lesions (L) are indicated by broken lines. c, Leukocyte accumulation on an atherosclerotic lesion at 30 min after activated platelet perfusion as seen by scanning EM at low (top) and high (bottom, bar, 10 mum) magnification. Platelets (arrows) associated with leukocytes adherent on atherosclerotic lesions of Apoe-/- mice following perfusion of activated wild-type platelets (left) but not activated platelets lacking P-selectin (right). d, Arrest of MM6 cells on IL-1 stimulated aortic endothelial cells was increased when activated wild-type, but not Selp-/-, platelets or the supernatant of activated wild-type platelets were pre-perfused over the endothelial monolayer. n = 4. *P < 0.01. e, Blocking mouse endothelial P-selectin with RB40.34 only partially inhibited leukocyte adhesion on atherosclerotic endothelium in vivo in the presence of activated human platelets. n = 4. *P < 0.01.



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To investigate the role of endothelium pre-perfused with activated platelets in the subsequent arrest of monocytes, we used an in vitro parallel plate flow system. Pre-perfusion of activated wild-type platelets, but not resting platelets, activated Selp-/- platelets or the supernatant of activated wild-type platelets through a cultured inflamed endothelial monolayer increased the arrest of Mono Mac 6 cells (Fig. 5d).

Perfusion of activated human platelets into Apoe-/- mice also caused an increase in leukocyte interactions with atherosclerotic carotid arteries. Pretreatment of Apoe-/- mice with monoclonal antibody RB40.34, an antibody against mouse P-selectin, blocked the mouse endothelial P-selectin function, but not P-selectin on perfused human activated platelets. In contrast to the crucial role of endothelial P-selectin in monocyte arrest in the absence of platelets in an ex vivo model23, endothelial P-selectin blockade only partially inhibited increased leukocyte adhesion (by 45 plusminus 5%; Fig. 5e) due to the presence of activated platelets, indicating a significant contribution of platelet P-selectin.

Activated platelets accelerate atherosclerosis
To investigate whether circulating activated platelets eventually contribute to the formation of atherosclerotic lesions, we injected activated wild type or Selp-/- platelet suspensions into the tail veins of Apoe-/- mice. Each mouse received an injection of activated mouse platelets at 3 times 107 per 20-g mouse weight every 5 days for 12 weeks. The number of injected activated platelets corresponds to 5−7% of total platelets in the mouse, which would increase by two- to three-fold the number of activated circulating platelets in Apoe-/- mice (data not shown). This number of activated wild-type platelets caused 10−15% of mouse leukocytes to be decorated with platelets as determined by flow cytometry (data not shown). Perfusion of activated platelets did not cause a difference in the number of leukocytes and profiles of blood cholesterol at the time when mice were sacrificed. However, atherosclerotic lesions in Apoe-/- mice injected with activated wild-type platelets were 39 plusminus 6% larger than those of Apoe-/- mice treated with activated Selp-/- platelets. Injection of the supernatant of activated wild-type platelets in a similar way did not increase the size of atherosclerotic lesions of Apoe-/- mice (Fig. 6).

Figure 6. Repeated injections of wild-type but not Selp-/- activated platelets or the supernatant of activated wild-type platelets exacerbate atherosclerosis in Apoe-/- mice.
Figure 6 thumbnail

The 8-week-old male Apoe-/- mice fed with a western diet were injected with activated wild-type platelets, Selp-/- activated platelets or the supernatant of activated platelets via the tail vein every 5 d for 12 weeks. En face analysis showed that lesion sizes of mice treated with activated wild-type platelets were increased by 39 plusminus 6% compared with those treated with activated Selp-/- platelets or supernatant of activated wild-type platelets. a, Representative oil red O-stained aortas from Apoe-/- mice with different treatment. b, Quantitative data on lesion size from the whole aorta; each data point represents a value from a single mouse. n = 10. *P < 0.01.



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Discussion
We have shown that circulating activated platelets promote monocyte recruitment to atherosclerotic arteries and accelerate the formation of atherosclerotic lesions in Apoe-/- mice. Activated platelets interact with monocytes and the endothelium of the vessel wall, depositing chemokines on the cell surface. These processes are likely to occur in patients with atherosclerosis, in whom activated platelets are commonly observed9, 10, 11, 12, 13, 14, 15. We infused activated platelets to illustrate the disease process in a compressed time frame.

A variety of pathways are involved in the interaction of platelets with endothelium and leukocytes. Endothelial P-selectin26, von Willebrand factor27, 28, platelet glycoproteins (GP) Ib29, 30 and IIb/IIIa30, 31 have important roles in platelet−endothelial interactions in different models. Platelet P-selectin is required for platelet interaction with leukocytes16, 32. Here, we show that platelet P-selectin is indispensable for interactions of activated platelets with atherosclerotic arteries and leukocytes/monocytes in vivo. The interactions of activated platelets with vessel walls occur in a transient way, resulting in little platelet accumulation on the endothelial surface of atherosclerotic lesions. This may be one of the reasons why the involvement of platelets in the formation of atherosclerotic lesions was not appreciated in histological studies. P-selectin on activated platelets, required to initiate platelet−leukocyte interactions, is also crucial to maintain the aggregates between leukocytes and activated platelets33. These aggregates likely cause monocytes and neutrophils to disappear from the circulation. Neutrophils return to the circulation within 60−80 minutes following a single injection of activated platelets, whereas monocyte numbers do not recover until 180−240 minutes. During the time when monocytes disappear from the circulation, increased monocyte adhesion was observed on atherosclerotic lesions in carotid arteries. This indicates that platelet−monocyte aggregation is one of the ways in which circulating activated platelets may participate in the formation of atherosclerotic lesions.

Our study shows that activated platelets can deliver the chemokines RANTES and PF4 to endothelium and leukocytes/monocytes. This is consistent with the observation that platelets present proinflammatory mediators on their membrane surface upon degranulation34. When direct interactions of platelets with endothelium and leukocytes are abrogated, chemokine delivery is abolished, because proinflammatory factors secreted by activated platelets may be rapidly diluted in the bloodstream. It is also possible that P-selectin engagement is required for chemokine release. Alternatively, or in addition, platelet-derived substances may be actively cleared from circulating blood. For example, Duffy antigen/receptor, a promiscuous chemokine receptor expressed on the surface of erythrocytes35, binds CXC and CC chemokines, including RANTES36.

Platelet contact-mediated deposition may also be relevant for the deposition of other platelet-derived mediators contributing to atherosclerosis. Epidermal growth factor, platelet-derived growth factor, beta-thromboglobulin and products of the lipoxygenase pathway, both mitogenic and chemotactic37, are candidates that may induce monocyte recruitment and/or smooth muscle cell proliferation. Consistent with this idea, abrogation of platelet interactions with endothelium and monocytes/leukocytes by removal of platelet P-selectin is particularly effective at delaying the onset of atherosclerotic disease38 and reducing neo-intima formation after vascular injury (D.R. Manka, manuscript submitted) in Apoe-/- mice.

Deposited platelet-derived mediators activate monocytes and cause monocyte recruitment. Consistent with previous in vitro studies22, 39, platelet-derived mediators deposited on the endothelium cause increased monocyte arrest, a process mediated by monocyte integrin activation induced by endothelium-associated proinflammatory mediators. In this study, we show that activation of monocyte integrins can also be triggered by platelet-derived mediators deposited on the monocyte surface. An upregulation of VCAM-1−IgG binding to monocytes was demonstrated, indicating increased affinity of VLA-4 integrin for ligand40, 41, 42. In addition, platelet interaction with monocytes may also increase monocyte−integrin avidity as a result of clustering, which was not measured in our study. Other integrins43 may also participate in this platelet-mediated increased monocyte arrest.

Our data provide the first direct evidence for an active contribution of circulating activated platelets in the formation of atherosclerotic lesions. Platelet P-selectin−mediated interactions lead to deposition of platelet-derived proinflammatory factors to the vessel wall and monocytes, resulting in activation of monocyte integrins, increased monocyte recruitment and exacerbation of atherosclerosis. Prevention of platelet activation and/or abrogation of platelet interactions with leukocytes/monocytes and the vessel wall, and neutralization of platelet-derived pro-inflammatory factors may become interesting means for therapeutic or preventive interventions in atherosclerosis.

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Methods
Mice.
Male Apoe-/- mice and Selptm1Bay/tm1Bay mice were obtained either from The Jackson Laboratory (Bar Harbor, Maine) or as a gift from A. Beaudet (Baylor University, Houston, Texas). Wild-type C57BL/6 mice were from Hilltop Farms (Scottsdale, Pennsylvania). CX3CR1-EGFP mice were a gift from D.R. Littman (Howard Hughes Medical Institute) and maintained as a heterozygous breeding colony at the University of Virginia.

Antibodies and reagents.
The monoclonal antibodies G1 (blocking) and S12 (non-blocking) against human P-selectin were provided by R. McEver (University of Oklahoma, Oklahoma). Thrombin-receptor activating peptide (TRAP, SFLLRNP) was obtained from Peninsula Laboratories Inc. (San Carlos, California), recombinant IL-1beta from PeproTech, Inc. (Rocky Hill, New Jersey), and human thrombin and hirudin from Sigma Chemical Chemical (St. Louis, Missouri). Rat antibodies against mouse CD11b (clone M1/70), mouse P-selectin (IgG1; RB40.34) and control rat IgG1 were purchased from PharMingen (San Diego, California), polyclonal rabbit antibodies against human RANTES and human PF-4 from Santa Cruz Biotechnology (Santa Cruz, California), and antibodies against human IgG conjugated PE and rabbit antibodies conjugated to Texas red from Vector Laboratories (Burlingame, California). Human VCAM-1−IgG was from R&D Systems Inc. (Minneapolis, Minnesota).

Cell culture, platelet isolation and activation.
Human aortic endothelial cells (HAECs) (Clonetics, San Diego, California) and human monocytic Mono Mac 6 cells, provided by P.C. Weber (Munich, Germany) were cultured as described22. Human and mouse platelets were isolated by gel-filtration44. Platelet activation was achieved by treating human platelets with TRAP for 10 minutes at 2 muM and mouse platelets with thrombin for 15 min at 0.0 5U/ml, followed by neutralization with equimolar dose of hirudin.

Interactions of platelets and Mono Mac 6 cells with cultured endothelial cells in parallel plate flow chamber assays.
Laminar flow assays were carried out as described22. Confluent HAECs grown in petri dishes (for monocyte arrest) or on glass coverslips (for immunostaining) were activated with IL-1beta (10 ng/ml) for 12 h and assembled as the lower wall of a flow chamber. Activated platelets (108 platelets/ml) were perfused at a wall shear stress of 1.5 dyne/cm2 for 20 min at 37 °C. Mono Mac 6 cells (106 cells/ml) were perfused for 5 min. The interactions of calcein-labeled platelets and Mono Mac 6 cells with endothelial cells were quantified in multiple fields. Immunostaining was carried out on fixed HAECs treated with an antibody against RANTES or a rabbit polyclonal PF-4 antibody and TRITC- or Texas red-conjugated secondary antibody. Images were recorded with a fluorescence microscope (times100 oil immersion objective). Endothelial cells were detached mechanically and RANTES deposition was measured by flow cytometry.

Flow cytometry.
Whole blood drawn from mouse carotid arteries was heparinized and fixed with PFA at 1% for 60 min. Fixation was stopped and red blood cells were lysed by Tris solution and Tyrode's buffer. Samples were incubated with monoclonal antibodies against Mac-1, Gr-1 or CD3 conjugated with PE for 30 min and analyzed by flow cytometry on a FAC-Scan (Becton Dickinson; Palo Alto, California).

Intravital microscopy of Apoe-/- mouse carotid arteries.
Apoe-/- mice were anesthetized, followed by cannulation of the trachea and right jugular vein. The peri-adventitial tissues around the left carotid arteries were carefully separated from the vessel. Most of the common carotid artery, external bifurcation and external branch were exposed and left intact. Following perfusion of calcein AM labeled platelets, the interactions of platelets and platelet−leukocyte aggregates with atherosclerotic carotid arteries were observed by intravital microscopy (Axioskop FS; Carl Zeiss, Thornwood, New York) with a saline immersion objective (SW 20, 0.5 numerical aperture) and stroboscopic epifluoresence illumination (60 s-1; Strobex, Chadwick-Helmuth, Mountain View, California). Rhodamine 6G (1 mg/mL, Molecular Probes, Inc., Eugene, Oregon) was injected I.V. to label leukocytes in vivo. Interactions between leukocytes or platelets and endothelium lasting less than 1 s were defined as tethering, more than 1 s as rolling. Leukocytes or platelets not moving for more than 30 s were defined as adhered.

VCAM-1−IgG binding assay.
We suspended 106 MM6 cells with or without pertussis toxin (250 ng/ml for 3 h at 37 °C) or monoclonal antibody HP1/2 (10 mug/ml for 20 min at 37 °C) treatment in 1 ml of whole blood (buffy coat removed) containing 20 mug VCAM-1-IgG and placed them in 24-well plates rotated at a rate of 60 rpm. A volume of 50 mul (5 times 106) activated platelet suspension was added to each well for 3 min and fixed by adding 0.5 ml of 4% paraformaldehyde at 22 °C. Red blood cells were lysed with Tris:glycine solution (250 mM Tris, 500 mM glycine). Binding of VCAM-1−IgG was detected with PE-conjugated goat anti-human IgG by flow cytometry.

Deposition of platelet-derived proinflammatory factors on atherosclerotic endothelium.
Aortas of Apoe-/- mice were harvested and fixed with 4% PFA/PBS. Immunostaining was carried out using primary antibodies against RANTES, PF4 and secondary goat anti-rabbit conjugated with Texas red. Sytox green (Molecular Probes, Eugene, Oregon) was used to label nuclei of endothelial cells. Images of the endothelial cell monolayer were obtained by using a Bio-Rad MRC-1024ES confocal microscope equipped with a krypton/argon laser and a times60 1.4-numerical aperture objective (Nikon).

Measurement of atherosclerotic lesion size of Apoe-/- mice.
The aortas of Apoe-/- mice were collected and stained with oil red O as described45. Images were scanned into a Macintosh computer and the percent surface areas occupied by oil red O−stained lesions were determined using image analysis software (NIH Image).

All animal experiments and care were approved by the University of Virginia Animal Care & Use Committee, in accordance with AAALAC guidelines.

Statistical analysis.
Data are represented as the mean plusminus s.e.m. of at least 4 independent experiments and were compared using a two-tailed Student's t-test. The null hypothesis was rejected at P < 0.05.

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Received 12 July 2002; Accepted 22 November 2002; Published online: 16 December 2002.

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