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(Circulation. 1997;95:1554-1559.)
© 1997 American Heart Association, Inc.

Articles

Effects of Monoclonal Antibody to P-Selectin and Analogue of Sialyl Lewis X on Cyclic Flow Variations in Stenosed and Endothelium-Injured Canine Coronary Arteries

Takahisa Ueyama, MD; Hisao Ikeda, MD, PhD; Nobuya Haramaki, MD, PhD; Kazunori Kuwano, MD, PhD; Tsutomu Imaizumi, MD, PhD

From the Third Department of Internal Medicine and the Institute of Cardiovascular Diseases, Kurume (Japan) University School of Medicine.

Correspondence to Hisao Ikeda, MD, PhD, The Third Department of Internal Medicine, Kurume University School of Medicine, 67 Asahi-machi, Kurume, 830 Japan.

	Abstract

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Background A fundamental role of cell adhesion molecules is implicated in the disease processes of acute coronary syndromes. We have shown an increase in the soluble form of P-selectin in these syndromes, suggesting the important interaction between P-selectin and sialyl Lewis X (SLe^X) for the pathophysiology of these syndromes. To further test this, we examined the effects of a monoclonal antibody against P-selectin (PB1.3) and a carbohydrate analogue of SLe^X (SLe^X-OS) on cyclic flow variations (CFVs) in stenosed and endothelium-injured canine coronary arteries.

Methods and Results Anesthetized, open-chest dogs (n=48) were divided into six groups after CFVs were established. Dogs received intravenous normal saline, PB1.3 (1 mg/kg bolus), a low dose (5 mg/kg bolus) or a high dose (40 mg/kg bolus) of SLe^X-OS followed by an infusion (5 mg·kg^-1·h^-1) for 60 minutes, a combination of PB1.3 and SLe^X-OS (low dose), or a combination of a nonblocking antibody against P-selectin (PNB1.6, 1 mg/kg) and SLe^X-OS (low dose). Although saline, PB1.3, SLe^X-OS (low dose), and the combination of PNB1.6 and SLe^X-OS (low dose) did not affect CFVs, the high dose of SLe^X-OS and the combination of PB1.3 and SLe^X-OS (low dose) significantly reduced CFVs.

Conclusions These findings indicate that the high dose of SLe^X-OS and the combination of PB1.3 and the low dose of SLe^X-OS provide protection against CFVs. Thus, the adhesive interaction between P-selectin and SLe^X may play an important role in mediating CFVs in this model.

Key Words: selectins • platelets • sialyl Lewis X • leukocytes • thrombosis

	Introduction

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Platelet aggregation and thrombus formation secondary to plaque disruption at the sites of coronary atheroma play a prominent role in the pathophysiology of acute coronary syndromes, including unstable angina and myocardial infarction.^{1 2 3} In clinical studies, coronary angiographic⁴ and angioscopic⁵ observations have clearly demonstrated the presence of thrombus formation in the early course of these syndromes. Thus, it is likely that acute vascular injury at the site of an atherosclerotic coronary artery activates thrombus formation through a complex cascade of cellular interactions.

Adhesion molecules such as integrins, selectins, and immunoglobulin superfamilies have been implicated in the disease processes of acute coronary syndromes^{6 7} and may play an important role in the first step of the thrombus formation at the culprit lesion of the coronary artery. Indeed, blockade of platelet IIb/IIIa integrin by the monoclonal antibody 7E3 has been shown to inhibit platelet aggregation in experimental⁸ and clinical studies,⁹ indicating the importance of cell-cell adhesive interactions on the thrombotic process. However, whether the in vivo action of other adhesion molecules contributes to platelet-mediated thrombosis is largely unknown. Among adhesion molecules, the glycoprotein P-selectin is a member of the selectin family and is located in both -granules of platelets¹⁰ and Weibel-Palade bodies of endothelial cells.^{11 12} After cellular activation by agonists such as thrombin¹³ and oxygen free radicals,¹⁴ P-selectin is rapidly translocated onto the cell surface and binds a sialylated carbohydrate structure, SLe^X, expressed on leukocytes through a calcium-dependent lectinlike mechanism.^{15 16} Furthermore, analysis of the complementary DNA demonstrated the possible presence of a soluble form of P-selectin in blood that possesses a deleted transmembrane segment derived from alternative splicing of mRNA.^{17 18} Recently, we showed that the soluble form of P-selectin markedly increased in acute coronary syndromes of unstable angina¹⁹ and myocardial infarction.²⁰ These findings may suggest that the pathophysiology of the acute coronary syndromes is closely related to coronary arterial thrombi through the cellular interactions among platelets, leukocytes, and endothelial cells associated with P-selectin and SLe^X. In the present study, to further elucidate the pathophysiology of acute coronary syndromes, we used an experimental canine model to test the hypothesis that adhesive interactions of P-selectin and SLe^X are involved in platelet-mediated thrombus formation in vivo. Three different agents, PB1.3, PNB1.6, and SLe^X-OS, were used to test this hypothesis in dogs with CFVs in stenosed and endothelium-injured coronary arteries.

	Methods

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Surgical Preparation
All experiments were conducted in accordance with the guidelines for animal experimentation of the Animal Research Committee of the Kurume University School of Medicine. Healthy mongrel dogs (15 to 20 kg) anesthetized with sodium pentobarbital (30 mg/kg) were ventilated with a ventilator. A thoracotomy was performed in the fifth left intercostal space, and the heart was suspended in a pericardial cradle. Polyethylene catheters were placed in the right atrium for drug infusion. A segment of the left anterior descending coronary artery was gently dissected free from the surrounding tissue, and a pulsed Doppler flow probe (Hartley Instruments) was placed proximal to the constricting cylinder. CBF velocity was measured with a pulsed Doppler flow system (model VF-1, Crystal Biotech). Arterial blood gasses and body temperature were maintained within normal physiological ranges. Aortic pressure was monitored with a micromanometer (Millar Instruments, Inc) placed in the femoral artery. Before the coronary arterial constriction, dogs were allowed to stabilize for 30 minutes, then control hemodynamics, including heart rate, systolic and diastolic aortic blood pressures, and phasic and mean CBF velocities, were recorded on an eight-channel recorder (Recti-Horiz-8K, San-ei). After control measurements were obtained, the endothelium of the exposed left anterior descending coronary artery was injured by gentle squeezing of the artery with cushioned forceps. Then a cylindrical constrictor was placed around the injured coronary artery distal to the flow probe to reduce the phasic CBF velocity to 40% of the control level, eliminating reactive hyperemia after 15 seconds of temporary coronary occlusion. Subsequently, CFVs developed in 48 of 56 dogs.

Experimental Protocol
After 60 minutes of stabilization of CFVs, drugs were administered intravenously according to the following protocols. Dogs (n=48) were divided into six treatment groups. Group 1 (n=10) received a bolus of saline followed by a continuous infusion of saline (1 mL/h). Group 2 (n=7) received a bolus of PB1.3 (1 mg/kg). Group 3 (n=8) received a bolus of low-dose SLe^X-OS (5 mg/kg) followed by a continuous infusion of 5 mg·kg^-1·h^-1 for 60 minutes. Group 4 (n=6) received a bolus of high-dose SLe^X-OS (40 mg/kg) followed by a continuous infusion of 5 mg·kg^-1·h^-1 for 60 minutes. Group 5 (n=11) received a combination of PB1.3 and low-dose SLe^X-OS at the same doses and in the same manners as used for groups 2 and 3, respectively. Group 6 (n=6) received a combination of PNB1.6 (1 mg/kg bolus) and low-dose SLe^X-OS at the same dose and in the same manner as used in group 5. To assess effects of treatments, the severity of CFVs was evaluated by monitoring mean CBF (mL/min), phasic and mean coronary nadir flow velocities (% control), and the frequency (cycles/h) for 60 minutes before and after treatments. For determination of CBF, flow velocity near the center of the vessel was recorded by use of the pulsed Doppler principle, and flow velocity was calculated by a digital planimeter. The cross-sectional area of the vessel was approximated to an inside diameter of the Doppler flow probe, ranging from 2.0 to 2.5 mm. Then mean CBF was derived by multiplication of mean flow velocity by the cross-sectional area.²¹ The peak and nadir flow velocities in both phasic and mean CBF were expressed as a percentage of unconstricted CBF velocity (control). The nadir flow velocity was calculated by averaging the three lowest flow velocities recorded before and after treatments, as done by Ashton et al.^{22 23 24} In dogs that exhibited only two flow restorations after the treatment, nadir flow velocity was calculated by averaging the two.

Statistical Analysis
Values are presented as mean±SEM. The effects of treatments on CFVs at different time periods were compared by repeated-measures ANOVA with a post hoc Scheffé's test. Differences were considered statistically significant at P<.05.

	Results

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The hemodynamic data from the six treatment groups of dogs are shown in the Table and Figures.

View this table: [in this window] [in a new window]   Table 1. Hemodynamic Variables Before and After Treatments

Before Development of CFVs (Stenosis in Table)
Endothelial injury and coronary constriction decreased the averaged peak phasic CBF velocity to 38% to 42% of baseline (control) and mean CBF velocity to 47% to 51% of baseline (control). Heart rate, aortic pressure, and peak phasic and mean flow velocities were similar among 6 groups.

After Development of CFVs and Before Treatment (60-Minute CFVs in Table)
No significant changes were observed in heart rates and in aortic systolic and diastolic blood pressures after the development of CFVs. The peak phasic and mean CBF velocities were similarly decreased among the six groups. The phasic coronary nadir flow velocity was decreased to 7% of control and mean coronary nadir flow velocity to 10% to 13% of control (P=NS among the groups). The frequency and the mean CBF of CFVs was 8.2 to 8.9 cycles/h and 6.5 to 6.8 mL/min, respectively (Fig 2). These values were also similar among the groups. Thus, the severity of CFVs was similar among them. These values were similar to those of other reports.^{22 23 24}

View larger version (43K): [in this window] [in a new window]   Figure 2. Effects of saline (group 1), PB1.3 alone (group 2), low-dose SLeX-OS alone (group 3), high-dose SLeX-OS alone (group 4), combination of PB1.3 and low-dose SLeX-OS (group 5), and combination of PNB1.6 and low-dose SLeX-OS (group 6) on frequency (top) and mean CBF (bottom) of CFVs. Data during 60 minutes of stabilization before treatment (open bars), between 0 and 60 minutes after treatment (solid bars), and between 30 and 60 minutes after treatment (hatched bars) are shown. High-dose SLeX-OS alone and the combination of PB1.3 and low-dose SLeX-OS significantly reduced CFVs. *P<.05 compared with before treatment.

Effects of Treatments on CFVs (After Treatment in Table)
The effects of saline, PB1.3 alone, low-dose SLe^X-OS alone, high-dose SLe^X-OS alone, a combination of PB1.3 and low-dose SLe^X-OS, and a combination of PNB1.6 and low-dose SLe^X-OS are shown in the Table and Figs 1 and 2. There were no significant effects of treatments on heart rate and aortic pressure in the six groups. Treatment with saline (group 1), PB1.3 alone (group 2), or low-dose SLe^X-OS alone (group 3) did not cause a significant change in the coronary nadir flow velocity or the frequency or the mean CBF of CFVs. In group 4, high-dose SLe^X-OS alone significantly increased the coronary nadir flow velocity (P<.05) and the mean CBF (P<.05) and significantly decreased the frequency of CFVs (P<.05). In group 5, a combination of PB1.3 and low-dose SLe^X-OS significantly increased the coronary nadir flow velocity (P<.05) and the mean CBF (P<.05) and significantly decreased the frequency of CFVs (P<.05). During the period between 30 and 60 minutes after treatment in groups 4 and 5, the increases in the coronary nadir flow velocity and mean CBF and the decreases in the frequency of CFVs were more apparent. In group 6, the combination of PNB1.6 and low-dose SLe^X-OS did not affect the coronary nadir flow velocity or the frequency or the mean CBF of CFVs.

View larger version (88K): [in this window] [in a new window]   Figure 1. Representative recordings showing effects of administration of saline (group 1), PB1.3 alone (group 2), low-dose SLeX-OS alone (group 3), high-dose SLeX-OS alone (group 4), combination of PB1.3 and low-dose SLeX-OS (group 5), and combination of PNB1.6 and low-dose SLeX-OS (group 6) on phasic left anterior descending coronary artery (LAD) flow pattern during 60 minutes after treatment. Note that treatments in groups 4 and 5 were effective in reducing CFVs. Open arrows indicate representative points at which nadir flow velocity was measured. Solid arrows indicate points at which drugs were administered.

	Discussion

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The episodes of CFVs, as originally described by Folts et al,²⁵ are caused by recurrent platelet aggregation and subsequent dislodgment at the coronary stenotic site with endothelial injury. Furthermore, we have previously shown that conscious dogs with CFVs manifest the pathophysiology of acute coronary syndromes in humans, including unstable angina, myocardial infarction, and ischemic sudden death.²⁶ In the clinical setting, CFVs have also been observed during coronary angioplasty in some patients with unstable angina.²⁷ In the present study, the most important findings were that high-dose SLe^X-OS or the combination of PB1.3 and low-dose SLe^X-OS significantly reduced CFVs. These findings may suggest that the adhesive interaction of P-selectin and SLe^X is involved in mediating CFVs in stenosed and endothelium-injured coronary arteries.

In the present study, to examine the role of the adhesion molecules in the thrombotic process, we used a canine model of coronary artery thrombosis that is characterized by CFVs. This model has many characteristics similar to those of acute coronary syndromes in humans, such as ST-segment elevation, depending on the fluctuation of CBF during the episode of CFVs.²⁶ Previous histological studies demonstrated in the animal model with CFVs that numerous platelets and leukocytes were present on the stenotic site adjacent to the thrombi,²² suggesting that cellular interactions between platelets and leukocytes may be involved in the thrombotic process of CFVs. Thus, interactions between P-selectin expressed on platelets and SLe^X expressed on leukocytes may play a role in CFVs in this model. To test this possibility, we examined the effects of PB1.3 and SLe^X-OS on CFVs.

In this study, PB1.3 was used for investigating the potential role of P-selectin in the thrombotic process of CFVs. This monoclonal antibody is of the IgG1 isotype and reacts with P-selectin on the activated platelet surface in not only humans but also other mammals.^{28 29 30} Furthermore, the action of PB1.3 is specific to P-selectin, because it does not cross-react with other selectin families, including E- and L-selectins.²⁸ A previous study showed that the intravenous administration of PB1.3 provides a dose-dependent protection against leukocyte-mediated lung injury in rats and that the maximum and plateau protections are observed with a dose of 1 mg/kg.²⁸ In other studies, it was reported that doses of 1 to 2 mg/kg of PB1.3 were effective for ameliorating myocardial reperfusion injury.^{29 30 31} Therefore, 1 mg/kg of PB1.3 was chosen in the present study. Sialic acid and fucose are known to be crucial components of carbohydrate ligands for selectin-mediated adhesion. Oligosaccharide-containing structures of both sialic acid and fucose, such as SLe^X-OS, could function as ligands to selectins. It was reported that SLe^X-OS competes with native SLe^X expressed on leukocytes, leading to the inhibition of platelet-leukocyte adhesion.³² The intravenous administration of SLe^X analogues protected against leukocyte-mediated rat lung injury in a dose-dependent manner from 0.25 to 2.5 mg/kg.³³ In another study using cats, administration of 10 mg/kg IV resulted in significant cardioprotective effects against ischemia-reperfusion injury, but 3 mg/kg did not.³⁴ Lefer et al³² demonstrated significant cardioprotection against canine ischemia-reperfusion injury with injection of 5 mg/kg IV. In addition, it has been observed that a continuous infusion of SLe^X-OS (5 mg·kg^-1·h^-1) combined with a bolus injection (5 mg/kg) is as effective in blocking the interaction between SLe^X and P-selectin as 1 mg/kg bolus administration of PB1.3 (S. Tojo and coworkers, unpublished work). Accordingly, we chose a dose of 5 mg/kg as a bolus injection along with 5 mg·kg^-1·h^-1 continuous infusion in group 3 (low-dose group). In this study, we did not use a nonfucosylated analogue of SLe^X-OS (sialyl lactosamine) missing a key fucose residue required for recognition by P-selectin. Thus, it was not known whether the effect of SLe^X-OS on CFVs was specific. However, previous studies clearly demonstrated that a nonfucosylated analogue of SLe^X-OS did not protect against ischemia-reperfusion injury in various experimental animals,^{32 35 36} P-selectin–mediated rat lung injury,³³ traumatic shock–induced rat tissue injury,³⁷ and thrombin- or histamine-mediated leukocyte rolling in rat mesenteric venules.^{38 39} Thus, it is likely that the SLe^X-OS used in this study provided beneficial effects as a specific selectin blocker.

Because P-selectin and SLe^X could act together on cellular adhesions, we anticipated that the single blockade of P-selectin or SLe^X should theoretically reduce CFVs. However, we observed no effect of either PB1.3 or low-dose SLe^X-OS alone on CFVs. There are several possibilities to account for this. First, the interaction between P-selectin and SLe^X may not play a role in this dog model. Second, the doses of PB1.3 and SLe^X-OS may have been too small. Third, it may be necessary to block both P-selectin and SLe^X. To examine these possibilities, a higher dose of SLe^X-OS (40 mg/kg bolus injection with 5 mg·kg^-1·h^-1 continuous infusion) was administered, which significantly reduced CFVs. The combination of PB1.3 and low-dose SLe^X-OS significantly reduced CFVs. A nonblocking antibody against P-selectin, PNB1.6, combined with low-dose SLe^X-OS did not affect CFVs. These results indicate that blocking the interaction between P-selectin on platelets and SLe^X on leukocytes and blocking either site were effective in reducing CFVs. These findings suggest that the adhesive interaction between P-selectin on platelets and SLe^X on leukocytes plays an important role in mediating CFVs in stenosed and endothelium-injured canine coronary arteries.

Our results suggest the activation of P-selectin and SLe^X in the present model. Although we cannot elucidate mechanisms of the activation of these adhesion molecules from this study, several possibilities may be considered. First, it is possible that thrombin may have upregulated the surface expression of P-selectin,¹³ because the surface expression of P-selectin occurs immediately on exposure to thrombin. Moreover, because it has been shown that thrombin is an important mediator of CFVs,⁴⁰ thrombin levels appear to be quite high in this model. Second, it is possible that oxygen free radicals may have upregulated the activation of adhesion molecules. We^{41 42} and others⁴³ have shown that oxygen free radicals, such as superoxide anion and hydrogen peroxide, are other important mediators of CFVs. Recently, oxygen free radicals were shown to induce the prolonged expression of P-selectin on the endothelial cell surface, which results in enhanced leukocyte adherence.¹⁴ Thus, it is possible that P-selectin on endothelial cells may be upregulated during the episode of CFVs. However, since the coronary vascular wall at the stenotic site is mechanically injured in the present model and the endothelium may no longer be present, the possibility that P-selectin expressed on endothelial cells plays a role in mediating CFVs may be small.

The present study demonstrated for the first time, to the best of our knowledge, that the inhibition of an adhesive interaction between P-selectin and SLe^X could reduce CFVs in stenosed and endothelium-injured canine coronary arteries. Our results may suggest that the adhesive interaction between P-selectin and SLe^X contributes importantly to the pathophysiology of acute coronary syndromes in humans. The inhibition of the adhesive interaction between P-selectin and SLe^X could become an attractive therapeutic modality of acute coronary syndromes in humans.

	Selected Abbreviations and Acronyms

 

CBF = coronary blood flow CFV = cyclic flow variation PB1.3 = specific murine monoclonal antibody against P-selectin PNB1.6 = nonblocking monoclonal antibody against P-selectin SLeX = sialyl Lewis X SLeX-OS = SLeX-containing oligosaccharide, unique carbohydrate analogue of SLeX

	Acknowledgments

 
We gratefully acknowledge the generous supply of PB1.3, PNB1.6, and SLe^X-OS from Shinichiro Tojo, PhD, of the Sumitomo Pharmaceutical Co (Osaka, Japan). The authors are also very grateful to Kimiko Kimura for her technical assistance.

Received August 14, 1996; revision received October 24, 1996; accepted November 4, 1996.

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