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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1185-1191.)
© 1997 American Heart Association, Inc.

Articles

Importance of Platelets in Neutrophil Adhesion and Vasoconstriction After Deep Carotid Arterial Injury by Angioplasty in Pigs

Yahye Merhi; Patrick Provost; Robert Guidoin; ; Jean-Gilles Latour

From the Laboratory of Experimental Pathology, Montreal Heart Institute (Y.M., P.P., J.-G.L.); the University of Montreal; and the Department of Surgery, Laval University (R.G.), Quebec, Canada.

Correspondence to Yahye Merhi, PhD, Experimental Pathology, Montreal Heart Institute, 5000 Belanger St East, Montreal, Quebec H1T 1C8, Canada.

	Abstract

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Abstract In previous studies we have shown that platelets can support neutrophil adhesion to the injured vessel wall in vitro and that neutrophils contribute to vascular tone regulation after arterial injury in vivo. In this study, we investigated the implication of platelets in neutrophil adhesion and the vasomotor response to arterial injury in vivo. ¹¹¹In-labeled neutrophil adhesion and angiographic vasoconstriction were quantified after deep carotid arterial injury by balloon angioplasty in normal (n=8), thrombocytopenic (n=7), and aspirin-treated (2 mg/kg IV, n=7) pigs. Thrombocytopenia was produced by a polyclonal antiplatelet serum that depleted circulating platelet count by 84% without influencing neutrophil count. In the control animals, neutrophil adhesion (x10⁴/cm²) at the site of deep arterial injury averaged 26.8±4.0 and decreased significantly to 11.5±2.3 and 11.2±2.4 in the thrombocytopenic and aspirin groups, respectively. The degree of vasoconstriction was also reduced significantly, from 55.5±3.8% in the control group to 31.4±6.2% after platelet depletion and to 23.6±4.5% in the aspirin-treated group. Neutrophil adhesion to intact noninjured adjacent arterial segments was low in all groups and was not affected by the antiplatelet serum or by aspirin. In in vitro superfusion flow chambers, neutrophil adhesion to damaged arterial segments increased in the presence of platelets in a concentration-dependent manner and was not influenced by the anti-platelet serum. This study demonstrates that platelets can modulate neutrophil adhesion to the deeply injured arterial wall and that both elements may influence the degree of postangioplasty vasoconstriction in vivo.

Key Words: platelet • neutrophil • arterial injury • vasoconstriction

	Introduction

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Platelet deposition after arterial wall injury promotes mural thrombus formation, vasoconstriction, and restenosis.^{1 2 3 4} Platelets can influence vascular tone³ and the activation of other blood cells^{5 6 7} through the release of a variety of bioactive substances, such as thromboxane A₂ (TxA₂), serotonin, ADP, and platelet-activating factor (PAF).^{8 9 10} Although platelet inhibitors and antithrombins can significantly reduce platelet deposition and vasoconstriction induced by arterial injury,¹¹ they do not completely prevent these responses, suggesting that either residual platelets or other cellular elements or mediators are involved in the pathophysiological response to arterial injury. Recently we have demonstrated that in addition to platelets, neutrophils are involved in the acute response to arterial injury in vivo.^{12 13 14}

Neutrophils, which have been shown to be activated after balloon angioplasty,¹⁵ are capable of influencing vascular tone^{16 17 18} as well as activating platelets¹⁹ through the release of biologically active products such as cathepsin G, elastase, oxygen-free radicals, PAF, and leukotrienes (LTs).^{20 21 22 23 24 25 26} In an experimental model of arterial injury by angioplasty, we have shown that neutrophil adhesion and the vasoconstrictive response are influenced by the severity of arterial injury.¹² In addition, we have reported, in the same animal model, that postangioplasty vasoconstriction is influenced by neutrophils, since neutropenia decreases it, independently of any effect on platelet function.¹³ In these neutropenic animals, administration of aspirin was associated with inhibition of platelet aggregation and deposition and with a further decrease in vasoconstriction. These results suggest that in addition to platelets, neutrophils are implicated in these phenomena. Neutrophils are able to interact with the injured vessel wall, an interaction that can be enhanced by platelets through a heterotypic interaction involving adhesion molecules on the surface of each cell type.²⁷ In fact, we have demonstrated in ex vivo bioassay experiments that similar to platelets, neutrophil adhesion to damaged porcine arteries with the media exposed is time and shear rate dependent and that platelet depletion inhibits this shear rate–dependent neutrophil adhesion.²⁸

Therefore, in the present study we examined the importance of platelets in neutrophil adhesion and the implication of these interactions in the vasomotor response after carotid arterial wall injury by balloon angioplasty in normal, thrombocytopenic, and aspirin-treated pigs. Since abolition of platelet deposition could not be achieved by conventional platelet inhibitors or anticoagulants, thrombocytopenia^{28 29} was developed in an experimental porcine model to study neutrophil adhesion and the vascular response to injury in the absence of platelets on the arterial wall.

	Methods

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Animal Preparation
Experiments were performed on 22 normal cross-breed Yorkshire pigs (mean weight, 15.4±0.4 kg) prepared according to the Canadian Council on Animal Care regulation. Animals were sedated by an IM injection of 200 mg ketamine (Rogarsetic, Rogar/STB Inc) and 100 mg azaperone (Stresnil, Janssen Pharmaceuticals). The pigs were intubated, mechanically ventilated with ambient air, and anesthetized with 0.5% halothane (Fluothane, Ayerst) as previously described.^{12 13 14}

Isolation and Labeling of Neutrophils
On the day of the experiment, 50 mL of autologous blood collected in acid-citrate dextrose was used for isolation and radiolabeling of neutrophils.^{12 14 28 30} After sedimentation with 4% dextran 250, the leukocyte-rich plasma was washed with HBSS-HEPES buffer and then layered on Ficoll and Monopoly resolving medium gradients (ICN Biomedicals Inc) and centrifuged at 300g for 30 minutes. The neutrophil layer was collected, washed, and incubated with 500 µCi ¹¹¹In-tropolone (Merck Frosst Canada Inc) for 30 minutes. The suspension was centrifuged to remove unbound ¹¹¹In, and the labeled neutrophil preparation was resuspended and reinjected into the animal. Neutrophil viability, as determined by trypan blue exclusion, exceeded 91%. The labeling efficiency with ¹¹¹In exceeded 90%. To determine whether the isolation and the labeling procedures affected neutrophil function, a nitroblue tetrazolium reduction function test was carried out on the labeled neutrophils, and the activated neutrophils were <5% of the total injected. The free ¹¹¹In in the circulating blood was <10% and remained unchanged during the experiment.

To assess the influence of the antiplatelet serum and of platelet concentration on neutrophil adhesion, in additional experiments neutrophils were isolated, radiolabeled as described above, and resuspended in HBSS-HEPES buffer containing 1.3 mmol/L Ca²⁺ and 0.8 mmol/L Mg²⁺, at 5x10⁶ neutrophils per milliliter for use in an in vitro neutrophil adhesion assay. Platelets were also isolated as detailed previously by differential centrifugation.^{12 13 30} Platelet suspensions, ranging from 50 to 500x10⁶ platelets per milliliter, were prepared in HBSS-HEPES buffer with Ca²⁺ and Mg²⁺ and used to determine the influence of platelets on neutrophil adhesion in vitro.

Induction of Thrombocytopenia
Thrombocytopenia was induced, as described previously,²⁸ with an intravenous injection of 8 to 10 mL of a polyclonal rabbit antiplatelet serum (1 mL every 15 minutes). The antiplatelet serum was prepared as detailed previously.^{28 29} Briefly, pure porcine platelets were homogenized in Freund's complete adjuvant and injected subcutaneously in rabbits. The second immunization was performed 10 days later by IM injection of platelets in Freund's incomplete adjuvant. Ten days after the second immunization, the rabbits were bled and the antiserum was inactivated at 56°C for 40 minutes and stored at -70°C. Nonimmune serum was obtained from nonimmunized rabbits and served in control animals. The angioplasty procedure was begun immediately after thrombocytopenia was achieved. Blood sampling for platelet and neutrophil counts was obtained in each animal before the injection of the antiserum or its control and before the angioplasty procedure.

Experimental Groups and Carotid Arterial Injury
Carotid arterial injury was produced by angioplasty in eight control, seven thrombocytopenic, and seven animals receiving aspirin (2 mg/kg IV, aspégic, Synthélabo) 60 minutes before the dilation procedure. Angioplasty was performed using a 7F polyethylene balloon dilation catheter (size 8 mmx3 cm, Meditech Inc). After a single bolus of heparin (100 USP U/kg), the catheter was advanced under fluoroscopic control through the right femoral artery into the left and right common carotid arterial segments between the fourth and fifth cervical vertebrae. Five inflations were performed at 6 atm pressure, each for 30 seconds, with 60-second intervals between inflations.^{12 13 14} The vasoconstrictive response localized at the site of the distal tapering end of the balloon, where endothelial injury occurs without any balloon stretching of the arterial wall, was quantified by using angiograms obtained before and immediately after the dilation. The diameter of each artery was measured and the vasoconstrictive response defined as the lumen diameter after dilation, expressed as percentage of the lumen diameter before dilation, as detailed previously.^{12 13 14} The angiograms obtained before and during balloon dilation were used to determine the balloon-to-artery ratio for each artery.

Quantification of Neutrophil Adhesion
Immediately after the angioplasty procedure, the pigs were euthanatized and the carotid arteries fixed in situ, with a buffered solution of 2% glutaraldehyde and 1% paraformaldehyde perfused anterogradely. The fixed carotid arteries were then removed and cleaned of all adventitia. Each dilated artery was examined macroscopically and the injured portion divided into two segments. The internal diameter and length of each segment were measured with an electronic caliper to determine the surface area (in square centimeters) of each segment. A distal denuded segment at the site of vasoconstriction and an uninjured segment with intact endothelium were also measured. After surface measurements, the segments were stored in 10% formalin for surface analysis by scanning electron microscopy.³¹ The radioactivity of each segment, as well as that of reference blood samples, was counted for 5 minutes in a gamma counter (Minaxi 5000, Packard Instruments). Knowing blood neutrophil counts of each animal, the radioactivity of ¹¹¹In in the blood and on the arterial segments, and the surface area of each segment, neutrophil adhesion per square centimeter was calculated as detailed previously.²⁸

Histological Analysis
After isolation, the arteries were examined macroscopically and the dilated region was delineated and divided as described in the previous section. After radioactivity counting, representative sections were processed for histological analyses to determine the degree of arterial injury after Movat pentachrome staining, which produces intense staining of the internal elastic lamina. The presence of deep arterial wall injury was detected by the presence of tears through the internal elastic lamina resulting in exposure of the arterial media.^{12 13 14} In addition, deep injury is characterized angiographically by a balloon-to-artery ratio higher than 1.1¹² and by the absence of vasoconstriction in the dilated area of the arteries, which can be related to severe injury to the smooth muscle cells in the media.³² According to these analyses, deep arterial injury was found in 12 of 16 dilated arteries in the control, 8 of 14 in the thrombocytopenic, and 9 of 14 in the aspirin-treated groups. Equal surface areas of injured arteries were obtained in the control (3.08±0.38 cm2), thrombocytopenic (3.13±0.37 cm2), and aspirin-treated (3.40±0.13 cm2) animals.

Neutrophil Adhesion Assay
To determine the influence of the antiplatelet serum and of platelet concentration on neutrophil adhesion, we performed an in vitro adhesion assay. In these experiments we have used Plexiglas acrylic plastic superfusion flow chambers that mimic the tubelike and cylindrical shape of blood vessels.^{28 30 33} Each chamber contains a window (2.0 mm inner diameter), allowing direct exposure of damaged arterial segments to flowing platelet or neutrophil suspensions. These damaged arterial segments were prepared from porcine aortas, which were dissected free of surrounding tissues, cut into rings, and longitudinally opened. Damaged arterial segments were then prepared by lifting and peeling off the intima, together with a thin portion of the subjacent media, and cutting to fit within the superfusion flow chambers, as previously described.^{28 30} The flow within the chambers was adjusted to 10 mL/min with a peristaltic pump. The chambers were placed in parallel in a thermostatically controlled water bath at 37°C, permitting simultaneous parallel pairwise superfusion over arterial tissues of control or treated neutrophil suspensions.

In the first set of experiments, we exposed the arterial segments for 5 minutes (in the flow chambers) to ¹¹¹In-labeled neutrophils (5x10⁶/mL) pretreated for 5 minutes with 10 µL/mL of either the antiplatelet serum or its nonimmune control serum.

In the second set of experiments, the arterial tissues were exposed to buffer control on one side of the system and to increasing concentrations of platelets (50, 100, 250, and 500x10⁶/mL) on the parallel side. After 5 minutes of perfusion, the buffer and platelet suspensions were replaced by two suspensions of ¹¹¹In-labeled neutrophils (5x10⁶/mL), one suspension on each side of the system, which were allowed to circulate for 5 minutes over the arterial tissues that had been preexposed to buffer or platelets. The arterial tissues were then removed and the radioactivity was counted in a gamma counter. The level of neutrophil adhesion, expressed as neutrophilsx10³/cm², on the exposed arterial tissues was calculated as detailed previously.²⁸ The percentage change in neutrophil adhesion, relative to parallel control, was then calculated.

Statistics
Results are expressed as mean±SEM. Comparisons were evaluated by Student's unpaired t test and, when applicable, Student's paired t test. One-way ANOVA followed by Dunnett's test was used for comparisons with the control group. A value of P<.05 was accepted as being significant.

	Results

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Animal Characteristics
As shown in Table 1, the weight of the pigs and their leukocyte and neutrophil counts were similar between groups. However, the platelet count was significantly reduced to 126.7±23.3x10⁶/mL in the thrombocytopenic group, which represents almost 85% reduction (P<.05) relative to platelet counts in the control and aspirin-treated groups.

View this table: [in this window] [in a new window]   Table 1. Characteristics of the Experimental Groups

Neutrophil Adhesion In Vivo
Platelet depletion caused a significant reduction in neutrophil adhesion to the site of arterial injury. Fig 1 shows that neutrophil adhesion to the dilated arterial segments with deep injury was reduced from 26.8±4.0x10⁴/cm² in the control to 11.5±2.3x10⁴/cm² (P<.05) in the thrombocytopenic animals. Aspirin treatment was also associated with a similar decrease in neutrophil adhesion to the deeply injured arteries (Fig 1). In the control group, neutrophil adhesion to the dilated segments with deep arterial injury was significantly higher than to mildly injured segments with endothelial denudation or to intact uninjured segments. Despite a decrease in the aspirin group, there was no statistical difference between groups in neutrophil adhesion to arterial segments denuded of endothelium or to arterial segments with intact endothelium. Surface analyses by SEM (Fig 2) showed a thromboresistant endothelium on the intact uninjured arterial segments, whereas in the control pigs, the dilated arterial segments with deep injury were covered with a heavy thrombotic matrix composed mainly of platelets and leukocytes. In the thrombocytopenic group, the presence of platelets and blood cells on the injured arterial surface was negligible.

View larger version (15K): [in this window] [in a new window]   Figure 1. Neutrophil adhesion to the deeply injured dilated arterial segments and the denuded and uninjured arterial segments with intact endothelium in the control, thrombocytopenic, and aspirin-treated groups. *P<.05 vs control; P<.05 vs denuded and uninjured.

View larger version (175K): [in this window] [in a new window]   Figure 2. Surface analysis by scanning electron microscopy of injured (2 left panels) dilated segments and normal (2 right panels) segments with intact endothelium from control (2 upper panels) and thrombocytopenic (2 lower panels) pigs.

In Vivo Vasoconstriction
The vasoconstrictive response at the site of selective endothelial injury produced by the distal tapering end of the balloon was determined angiographically. As shown in Table 2, postangioplasty vasoconstriction averaged 55.5±3.8% in the control pigs and was significantly reduced, to 31.4±6.2%, after platelet depletion in the thrombocytopenic pigs and 23.6±4.5% in the aspirin-treated group. Although the diameter of the arteries before dilation and the balloon-to-artery ratio were similar in all groups, the lumen diameter of the dilated arteries at the site of endothelial injury was significantly higher in the thrombocytopenic and aspirin-treated groups than in controls.

View this table: [in this window] [in a new window]   Table 2. The Balloon-to-Artery Ratio, the Diameter of the Carotid Arteries Before and After Balloon Dilation, and the Degree of Vasoconstriction in the Control, Thrombocytopenic, and Aspirin-Treated Pigs

Neutrophil Adhesion In Vitro
To assess whether the antiplatelet serum interfered directly with the adhesive function of neutrophils, we performed in vitro adhesion experiments, in which ¹¹¹In-labeled neutrophils were pretreated with either the antiplatelet serum or its nonimmune control serum and then exposed to damaged arterial surfaces under flowing conditions. The level of neutrophil adhesion, which averaged 15.1±6.6x10³/cm² in control experiments, remained unchanged at 16.6±6.2x10³/cm² (P=not significant) after treatment with the antiplatelet serum.

In additional in vitro experiments, the influence of platelet concentration on neutrophil adhesion to the injured arterial wall was investigated and is illustrated in Fig 3. Neutrophil adhesion, presented as the ratio over control experiments, was not influenced by preexposure of the damaged arterial segments to 50x10⁶ platelets per milliliter but increased significantly by 19%, 30%, and 56% when these damaged arterial surfaces were preexposed to 100, 250, and 500x10⁶ platelets per milliliter, respectively.

View larger version (15K): [in this window] [in a new window]   Figure 3. Neutrophil adhesion to damaged arterial segments in in vitro superfusion flow chambers in the presence of increasing concentration of platelets. The results are presented as a ratio over control experiments without platelets. *P<.05 vs control.

	Discussion

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Increasing experimental evidence supports the concept that the interactions of platelets and leukocytes with the arterial wall modulate and enhance the pathophysiological response associated with arterial injury. In fact, we²⁸ and others³⁴ have shown that platelets can support neutrophil adhesion to the injured arterial wall in vitro. In the present study, the implication of platelets in neutrophil adhesion to the damaged artery in vivo was investigated and related to the degree of arterial vasoconstriction at the site of endothelial injury. Our results show that inhibition of neutrophil interaction with the damaged arterial wall by platelet depletion or cyclooxygenase inhibition is associated with a decreased vasoconstrictive response in vivo.

The implication of platelets in thrombosis and vascular tone modulation after arterial injury is well documented. Platelet reactivity is influenced by the nature of the arterial surface exposed¹² ; regulated by platelet glycoprotein receptors^{1 2 27} and procoagulant proteins found in the plasma and exposed arterial matrix; and amplified by the secretion of potent vasoactive substances such as TxA₂, serotonin, and ADP.^{1 2 3} In this porcine arterial injury model, balloon dilation induces a rupture of the internal elastic lamina, exposing the highly thrombogenic medial components to the circulation. Such severe or deep arterial injury can be produced clinically by balloon angioplasty of atherosclerotic plaques, where platelets play an important role in acute thrombotic events, vasospasm, and restenosis. Experimentally, the vasoconstrictive response associated with deep arterial injury has been related to the extent of platelet deposition.^{1 35} TxA₂ is an important platelet mediator in these events, since the cyclooxygenase inhibitor aspirin decreased platelet aggregation and deposition and inhibited the arterial vasoconstrictive response.^{13 35} Similar results were also obtained with a TxA₂ receptor antagonist.³⁶ Although the present study confirms a role for platelets in the vasoconstrictive response at the site of endothelial injury in vivo, other cellular elements may also be involved. In fact, the vascular response to injury is now recognized as a multicellular process. In this regard, we have previously demonstrated the importance of neutrophils in the vasoconstrictive response after deep arterial injury in a neutropenic animal model.¹³ In this study, thrombocytopenia induced a significant decrease in neutrophil adhesion to the site of deep arterial injury. The effect of platelets on neutrophil adhesion was directly related to platelet concentration, confirming our previous observations in vitro.²⁸ In that study, platelet depletion was shown to inhibit shear rate–dependent neutrophil adhesion. Inhibition of platelet function with aspirin was also associated with decreased neutrophil adhesion, suggesting that the presence of activated platelets on damaged arterial surfaces may enhance neutrophil adhesion, a feature related to the concentration of platelets. Together, these findings indicate that platelets can modulate neutrophil adhesion to damaged arteries. This modulation may be regulated by the expression on the surface of activated and adherent platelets of a member of the selectin family of adhesion molecules, P-selectin (GMP-140 or CD62).³⁷ P-selectin has been shown to mediate the interaction between platelets and neutrophils in vitro^{34 38} and in a thrombosis model in vivo.³⁹ It has been shown that aspirin, at doses that block dense granule secretion of platelets, does not inhibit -granule secretion and P-selectin expression.⁴⁰ Therefore, the effect of aspirin on neutrophil adhesion may be related to its effect on platelet deposition, in which P-selectin appears not to be involved. Recently,⁴¹ aspirin has been shown to increase nitric oxide production by neutrophils, which can contribute to decreased neutrophil adhesion.

It is possible that the persistence of vasoconstriction after thrombocytopenia may be associated with the residual neutrophil adhesion in the platelet-poor environment. This hypothesis is supported by physiological studies showing the vasoconstrictive properties of neutrophils on isolated arteries in vitro^{17 18 42} and more recently in vivo using the same experimental model of arterial injury in neutropenic pigs.¹³ However, the contribution of other metabolic pathways or cellular elements besides platelets and neutrophils cannot be excluded, and these require further evaluation.

Antiplatelet serum produces a severe thrombocytopenia without affecting leukocyte and neutrophil count and function. This thrombocytopenia was accompanied by a complete inhibition of platelet aggregation to ADP.²⁸ The antiplatelet serum did not interfere directly with neutrophils, as neutrophil adhesion to the damaged arterial wall was not influenced by the antiplatelet serum. In addition, adhesive neutrophils are still present in the circulation after thrombocytopenia, because neutrophil adhesion to normal adjacent arterial segments with intact endothelium was similar between the control and thrombocytopenic groups. Neutrophil–endothelial cell adhesion has been shown to be controlled by L-selectin on resting neutrophils, by the CD11/CD18 glycoprotein complex or ß₂-integrin expressed on activated neutrophils,^{43 44} by the intercellular adhesion molecule, by the endothelial-leukocyte adhesion molecule (E-selectin), and by P-selectin expressed on activated endothelial cells.⁴⁵ Recently, Del Mashio et al⁴⁶ suggested that resting platelets can inhibit neutrophil adhesion to the endothelium in vitro. However, in that study, the inhibitory effect of platelets was observed only after activation of neutrophils. In our in vivo study, neutrophil adhesion to the arterial wall was assessed without prior activation. Under these conditions, neutrophil–endothelia cell interaction was minimal and was not significantly influenced by thrombocytopenia or aspirin. The decrease in neutrophil adhesion to the intact endothelium in the presence of aspirin, although nonsignificant, can be related to its effect on neutrophil L-selectin, which instead of CD11b expression has been shown to be decreased by aspirin.⁴⁷

The pathophysiological response to arterial injury may involve many cell types and metabolic pathways. In addition to platelets, neutrophils may influence vascular tone through the release of potent vasoactive substances such as cysteinyl LTs¹⁷ and superoxide anions²³ and by transcellular metabolism of arachidonic acid, through the cyclooxygenase and lipoxygenase pathways, between platelets and neutrophils. It has been shown that the production of TxA₂ by platelets is enhanced in the presence of neutrophils.⁴⁸ Also, the vasoconstrictive LTC₄ can be synthesized by platelets from neutrophil LTA₄ through a steal phenomenon.⁴² In the absence of platelets, the production of many of these vasoactive substances can be decreased or even completely inhibited. In addition, we have recently found that a dual cyclooxygenase/lipoxygenase inhibitor¹⁴ and a selective leukotriene biosynthesis inhibitor (unpublished data, 1996) were very effective in reducing neutrophil adhesion and the vasoconstrictive response associated with deep arterial injury by balloon angioplasty. In this thrombogenic environment, neutrophils may be involved in thrombogenesis by the secretion of PAF, proteolytic enzymes, oxygen-free radicals, and LTs. These substances, secreted locally at the site of injury produced by balloon stretching of the arterial wall, may influence the vasoconstrictive response distally to the injured areas. Neutrophil adhesion and activation at the site of dilation may be involved in subsequent vessel injury, recruitment of other platelets, and enhancement of the vasoconstrictive response to injury.

In summary, like platelets, neutrophils can interact with the damaged artery after balloon angioplasty. Neutrophil adhesion, which is amplified by platelets, may enhance the vasoconstrictive response to injury. These interactions may have important implications in the pathogenesis of thrombosis and vasoconstriction after arterial wall injury in vivo.

	Acknowledgments

 
Dr Merhi is supported by the Heart and Stroke Foundation of Canada and the Fonds de la Recherche en Santé du Québec (FRSQ). P. Provost is the recipient of a studentship from the Medical Research Council of Canada. We thank Johanne Doucet for excellent technical assistance.

Received July 6, 1996; accepted August 2, 1996.

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