Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932
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ABSTRACT |
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Platelets roll and adhere in venules exposed to ischemia-reperfusion (I/R). This platelet-endothelial adhesion may influence leukocyte trafficking because platelet depletion decreases I/R-induced leukocyte emigration. The objectives of this study were 1) to assess the time course of platelet adhesion in the small bowel after I/R and 2) to determine the roles of endothelial and/or platelet P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) in this adhesion. The adhesion of fluorescently labeled platelets was monitored by intravital microscopy in postcapillary venules exposed to 45 min of ischemia and up to 8 h of reperfusion. Peak platelet adhesion was observed at 4 h of reperfusion. To assess the contributions of platelet and endothelial cell P-selectin, platelets from P-selectin-deficient and wild-type mice were infused into wild-type and P-selectin-deficient mice, respectively. Platelets deficient in P-selectin exhibited low levels of adhesion comparable to that in sham-treated animals. In the absence of endothelial P-selectin, platelet adhesion was reduced by 65%. Treatment with a blocking antibody against PSGL-1 reduced adhesion by 57%. These results indicate that I/R induces a time-dependent platelet-endothelial adhesion response in postcapillary venules via a mechanism that involves PSGL-1 and both platelet and endothelial P-selectin, with platelet P-selectin playing a greater role.
postcapillary venules; cell adhesion molecules; endothelial cells; inflammation; P-selectin
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INTRODUCTION |
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ISCHEMIA-REPERFUSION (I/R) results in microvascular dysfunction that is related to endothelial cell activation and the creation of an inflammatory phenotype. The microvascular responses to I/R are characterized by endothelial cell adhesion molecule expression, neutrophil recruitment, oxidative stress, and albumin extravasation, all of which largely occur to postcapillary venules (1). Whereas numerous studies have implicated neutrophil-endothelial cell adhesion as a rate-determining step in the microvascular dysfunction elicited by I/R (1, 15), more recent studies have provided evidence implicating other blood cells in the pathogenesis of I/R injury. For example, mucosal addressin cell adhesion molecule-1 mediated T-cell recruitment has been demonstrated in postischemic venules (30) and these T-cells appear to contribute to neutrophil recruitment. There is also recent evidence implicating a role for platelets in the pathogenesis of I/R. Support of a role for platelets in this injury process is provided by studies demonstrating an accumulation of platelets in the postischemic intestine (22), retina (26), and liver (11). The pathophysiological consequences of platelet accumulation within venules of the ischemic and postischemic microvasculature have not been fully defined; however, recent studies in vitro have suggested that platelets, similar to lymphocytes, may be involved in the recruitment of neutrophils to venular endothelium (12, 13). These observations are supported by evidence in vivo, suggesting that platelets contribute to the increased leukocyte emigration observed in postischemic intestine (29) and kidney (31). Indeed, platelet depletion significantly decreases leukocyte emigration in the postischemic intestine of rats (29). Such a role for platelets may be mediated via P-selectin, because chimeric mice expressing only endothelial P-selectin demonstrate an attenuation of neutrophil-mediated postischemic renal failure (31).
Intravital microscopy has been used to monitor and quantify the
recruitment of platelets in postcapillary venules during acute inflammation, including endotoxemia (2), exposure to tumor necrosis factor- (6) and calcium ionophore
(5), and I/R (22, 23, 26). These studies have
revealed that some stimuli elicit a rapid platelet-adhesion response
(ionophore) while other stimuli require hours to initiate a significant
response (LPS). I/R has been shown to produce an intense
adhesion response that is seen within the first 30 min after
reperfusion (22). This early response was shown to be
mediated by endothelial P-selectin, binding to an undefined ligand on
the platelet, possibly P-selectin glycoprotein-1 (PSGL-1). The rapid
endothelial P-selectin binding of platelets is consistent with the time
course of P-selectin expression in the postischemic mouse
intestine where rapid upregulation of P-selectin likely reflects the
mobilization of the storage pool of this adhesion molecule. However,
the kinetics of P-selectin expression indicates that there is also a
slower more intense expression of P-selectin in the murine intestinal
vasculature that peaks at 5 h after reperfusion (3).
This raises the possibility that I/R may also elicit a time-dependent
increase in platelet adhesion that peaks several hours after
reperfusion and that is also P-selectin dependent. Because a different
compliment of inflammatory mediators and platelet activators may exist
in the postischemic bowel several hours (instead of minutes)
after reperfusion, it is also possible that the relative contribution
of endothelial versus platelet P-selectin may differ hours after I/R.
The objectives of this study were the following: 1) to assess the time-course of platelet adhesion in postcapillary venules after intestinal I/R, 2) to define the relative contributions of endothelial versus platelet P-selectin to the platelet adhesion observed several hours after reperfusion, and 3) to determine whether PSGL-1 also contributes to this later phase of I/R-induced platelet adhesion. Using intravital microscopy, we demonstrated that platelet adhesion is significantly increased in postcapillary venules of the small bowel after longer periods (hours) of reperfusion. Our findings indicate that while both endothelial- and platelet-derived P-selectin mediate platelet adhesion in the postischemic vasculature, platelet P-selectin makes a greater contribution to the observed adhesion, probably through its interactions with PSGL-1.
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MATERIALS AND METHODS |
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Animals. Wild-type (C57BL/6J) and P-selectin-deficient (B.6129S7-SELPTMLBAY) mice were obtained from Jackson Laboratory (Bar Harbor, ME). Male mice between 8 and 12 wk of age were used in the experiments.
Surgical procedure. The animals were anesthetized with a mixture of ketamine hydrochloride (150 mg/kg ip) and xylazine (7.5 mg/kg ip). The right carotid artery was cannulated for blood pressure measurements with the use of a disposable pressure transducer (Cobe Laboratories) attached to a pressure monitor (model BP-1, World Precision Instruments) and was recorded on a MacLab/8e computer with Chart version 3.5.2. The right jugular vein was cannulated for platelet infusion. A midline laparotomy was performed, the animal was placed in a supine position, and a loop of the small bowel was exteriorized and superfused with a warm bicarbonate-buffered saline solution.
Blood sampling and platelet preparation. Approximately 0.9 ml of blood was harvested via a catheter placed in the carotid artery. The blood was collected in polypropylene tubes containing 0.1 ml acid-citrate-dextrose buffer (Sigma; St. Louis, MO) and centrifuged at 120 g for 10 min. Platelet-rich plasma was obtained by two sequential centrifugations (120 g for 8 min and 120 g for 3 min). The platelet-rich plasma was removed and spun again at 550 g for 10 min, and the pellet was resuspended in phosphate-buffered saline (PBS; pH 7.4). Platelets were then incubated for 10 min (room temperature) with the fluorochrome carboxyfluorescein diacetate succinimidyl ester (Molecular Probes, Eugene, OR, final concentration: 90 µm). The fluorescently labeled platelet solution was then centrifuged, resuspended in 500 µl of PBS and protected from light. Manual blood cell counts yielded 0.01% leukocytes in the platelet suspension. Platelets were derived from wild-type mice for all experiments, except in one experimental group, where platelets were obtained from P-selectin-deficient mice.
Flow cytometry. To determine whether the platelet isolation procedure influenced platelet activation status, flow cytometry was used to measure P-selectin expression on isolated and washed platelets. Platelets were isolated as described above for analysis by flow cytometry. Before being stained with antibodies, isolated washed platelets were suspended at a concentration of 1 × 108 cells/ml in fluorescence-activated cell sorter (FACS) buffer (2% fetal calf serum in PBS), and divided into nonstimulated and thrombin-stimulated samples. Human thrombin was added at a final concentration of 1 U/ml for 20 min. Nonstimulated and thrombin-stimulated samples were incubated with either a FITC-conjugated anti-GPIIb/IIIa antibody (BD Pharmingen; San Diego, CA) for identification of platelet populations, or FITC-conjugated anti-P-selectin (BD Pharmingen) antibody to assess platelet activation, for 20 min at room temperature. Cells were then washed twice with FACS buffer and analyzed on a FACSCalibur flow cytometer using CellQuest software (BD Biosciences; San Jose, CA).
Intravital fluorescence microscopy. Platelets were visualized with a Nikon Diaphot upright microscope equipped with a 75-W XBO xenon lamp. Carboxyfluorescein diacetate succinimidyl ester visualization (excitation: 490 nm, emission: 518 nm) required a Nikon filter block with an excitation filter (470-490 nm), a dichroic mirror (510 nm), and a barrier filter (520 nm). With a ×20 objective (0.4 numerical aperture; Nikon), the magnification on the video screen (Sony Trinitron, PVM-2030, diagonal 50.6 cm) was ×740. The microscopic images were received by a charge-coupled device (CCD) video camera (model C2400, Hamamatsu) and optimized by a CCD camera controller (model C2400, Hamamatsu). The images were then recorded on a video recorder (model BR-S601MU, JVC) for off-line evaluation. The intestinal loop was scanned for 3-5 venules, which were each recorded for 1 min.
Video analysis. Venular diameter was measured and venular length set at 200 µm. Platelets were classified according to their interaction with the venular wall as either free flowing or adherent. Firmly adherent platelets were classified according to the duration of their immobility on the venular wall: 1) saltation >2 s <30 s and 2) adhesion >30 s. Platelet adherence was expressed as the number of cells per square millimeter of venular surface, calculated from diameter and length, assuming cylindrical vessel shape (21). Data from the 3-5 venules recorded per mouse were averaged (n = 1).
Experimental protocols. All animals except platelet donors were fasted for 24 h. In the I/R group, the superior mesenteric artery was clamped for a period of 45 min. Sham animals were subjected to an identical protocol without actual clamping of the vessel. Platelet adhesion was measured following the specified period of reperfusion. In some experiments, mice were pretreated with a blocking monoclonal antibody (mAb) directed against PSGL-1 (2 mg/kg; 2PH1, BD Pharmingen). The mAb was administered intravenously 15 min before platelet infusion. Fluorescently labeled platelets from one donor mouse were used in two recipient mice. Platelets (100 × 106) were infused over 5 min with the use of an infusion pump (Harvard Apparatus; S. Natick, MA), yielding ~5% of the total platelet count. The platelets were allowed to circulate for a period of 5 min before being recorded. Platelet infusion commenced at the end of the specified reperfusion time. In those mice subjected to 45 min of ischemia and 0 h of reperfusion, platelets were infused after 40 min of ischemia.
Estimates of pseudo-shear rate in venules were obtained using measurements of venular diameter (Dv) and the maximal velocity of flowing platelets (Vplt) according to the following formulation: pseudoshear rate = (Vplt/1.6)/Dv × 8 (32). The experimental procedures described above were reviewed and approved by the LSU Health Sciences Center-Shreveport Institutional Animal Care and Use Committee and performed according to the criteria outlined in the National Institutes of Health guidelines.Statistics. Data were analyzed using unpaired t-test or ANOVA and Scheffé's (post hoc) test and were reported as means ± SE from 4-9 mice per group. Statistical significance was set at P < 0.05.
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RESULTS |
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Figure 1 is a histogram
depicting P-selectin expression in unstimulated (gray) and
thrombin-stimulated (black) washed platelets. Flow cytometric analysis
of platelets isolated for intravital microscopy revealed that the
isolation procedure did not result in activation of platelets, with
only 1.55 ± 0.54% of platelets positive for P-selectin. These
isolated platelets were still functional as indicated by a 54-fold
increase in the percentage of P-selectin positive platelets (83.65 ± 6.06%) after stimulation with thrombin.
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Previous studies (22) have demonstrated a significant
increase in platelet-endothelial adhesion immediately after I/R in murine small intestine. Figure 2
summarizes quantitative data of platelet-endothelial adhesion after 45 min of ischemia and up to 8 h reperfusion in
post-capillary venules of the small intestine. Minimal platelet
adhesion was observed immediately (5 min) after I/R; however, 4-h
reperfusion induced a ninefold increase in platelet adhesion
(144.12 ± 16.07 platelets/mm2), compared with sham
controls (15.71 ± 6.74 platelets/mm2). Platelet
adhesion then declined, although levels remained significantly increased compared with sham controls for up to 8 h of reperfusion (89.70 ± 21.38 platelets/mm2). Figure
3 presents representative
microfluorographs of the peak platelet adhesion response observed in
animals after 4-h reperfusion and the absence of platelet adherence in
a sham-treated animal.
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The second major objective of this study was to define the molecular
determinants that promote the recruitment of platelets in the
intestinal microcirculation at 4 h of reperfusion. Figure 4 summarizes the data from experiments
designed to assess the role of both platelet and endothelial cell
P-selectin and the P-selectin ligand PSGL-1. In the absence of either
platelet or endothelial P-selectin, platelet-endothelial adhesion was
significantly decreased by 93 and 65%, respectively. Platelet adhesion
was reduced to levels comparable to those observed in sham-treated
animals when P-selectin deficient platelets were infused into wild-type recipient animals. The administration of a blocking mAb against PSGL-1
also significantly decreased adhesion by 57%. Platelet adhesion in
P-selectin-deficient or PSGL-1 mAb-treated recipient mice was not
significantly different to that observed in sham-treated mice or
wild-type mice receiving P-selectin-deficient platelets.
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Table 1 presents hemodynamic data from
the mice and intestinal vessels used to generate the data in the
aforementioned figures. No significant differences were observed with
regard to mean arterial blood pressure, venular diameter, or
pseudoshear rate between any of the experimental groups examined.
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DISCUSSION |
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I/R induces both a proinflammatory and a prothrombogenic state in the intestinal microvasculature. This condition is created by the enhanced production of chemical mediators (e.g., reactive oxygen species, cytokines, and chemokines) that activate endothelial cells and circulating blood cells that course through the postischemic vasculature. The activated vascular and blood cells respond by expressing surface glycoproteins that mediate blood cell-endothelial cell adhesion. While the kinetics and molecular determinants of leukocyte-endothelial cell adhesion in venules exposed to I/R have been extensively characterized, relatively little is known about the same issues for platelets. The limited amount of published information on I/R-induced platelet adhesion addresses the very early adhesion response of platelets, despite circumstantial evidence in the literature supporting a more intense adhesion response at later times after reperfusion, possibly involving different underlying mechanisms. Hence, the overall objective of the present study was to characterize the time dependency of platelet adhesion in the postischemic microvasculature and to assess potential roles of P-selectin and its ligand PSGL-1 as molecular determinants of this adhesion process.
To assess the time course of platelet adhesion in the postischemic microvasculature, mice were exposed to 45 min of ischemia and up to 8 h of reperfusion. In contrast to previous studies (22, 23), no significant increases in either saltating or adherent platelets were observed immediately (5 min) on reperfusion, and while the number of saltating platelets was significantly increased at 2 h of reperfusion, a significant increase in firmly adherent platelets (>30 s adhesion) was not observed until 4 h of reperfusion. Levels of adherent platelets remained significantly elevated compared with sham-treated animals for up to 8-h reperfusion. These findings are in contrast to the aforementioned reports (22, 23) that describe an early phase of platelet adhesion. Another notable difference was the high incidence of platelet adhesion observed within arterioles in these previous studies (22, 23), a phenomenon that was not encountered in free-flowing vessels in any of the animals studied here. The intensity and time course of platelet adhesion observed after 4-h reperfusion corresponds with that observed in the small bowel after 4 h LPS treatment (2), implying that similar mechanisms may underlie both these responses. Such mechanisms may include the transcription-dependent upregulation of cell adhesion molecules (e.g., P-selectin), and/or the accumulation of pro-inflammatory mediators within the microcirculation.
P-selectin has been implicated in mediating the platelet adhesion observed in the ischemic small bowel (22). This early phase of platelet adhesion was demonstrated to be dependent on endothelial, rather than platelet, P-selectin because adhesion was significantly attenuated when wild-type platelets were infused into P-selectin-deficient recipient mice. Such an increased adhesion immediately after ischemia indicates it is likely due to mobilization of the preformed pool of P-selectin within endothelial cells (22). In contrast to the present study, a role for platelet P-selectin could not be demonstrated because P-selectin-deficient platelets adhered to the endothelium in the same manner as wild-type platelets. This early endothelial cell P-selectin-dependent response is consistent with our previous study (3) showing a rapid expression of P-selectin in the intestinal vasculature of mice exposed to I/R. In the same study, a slower more intense expression of P-selectin that correlated with P-selectin mRNA, suggesting transcription-dependent expression was also detected. This raised the possibility that endothelial P-selectin could play an even greater role in mediating platelet adhesion hours after reperfusion.
In the current study, the platelet adhesion observed hours after reperfusion was reliant on both endothelial and platelet P-selectin expression. However, whereas our studies strongly implicate P-selectin in this late phase of I/R-induced platelet adhesion, the data indicate that platelet rather than endothelial P-selectin is more important. Flow cytometry data do not support the possibility that our findings reflect platelet activation during isolation/purification. A likely explanation for the larger role of platelet P-selectin is that the profile of chemical mediators generated within the postischemic intestine is likely different hours (vs. minutes) after reperfusion and these mediators may exert a greater influence on platelets coursing through intestinal venules. These chemical mediators may be released either by activated endothelial cells and/or circulating blood cells (e.g., leukocytes and platelets). During reperfusion, both endothelial cells and leukocytes are known to release considerable amounts of reactive oxygen species (8, 17). Reactive oxygen species are capable of causing platelet activation and P-selectin expression (18, 20). Furthermore, activated platelets per se are also a source of reactive oxygen species (18, 19). The platelet agonists platelet-activating factor and thrombin are also known to mediate I/R-induced leukocyte adhesion (14, 27). Thrombin in particular is a potent activator of platelets leading to increased P-selectin expression (10). Once activated, platelets may serve to potentiate the inflammatory response via the release of cytokines such as interleukin-1 (9), indeed platelet-derived interleukin-1 stimulates chemokine release and adhesion molecule expression in cultured endothelial cells (7).
Interactions between platelets and leukocytes have also frequently been observed in the postischemic microcirculation (16, 22) and appear to be mediated by platelet P-selectin (13, 22). Interactions between these two cell types can result in a reciprocal activation, resulting in an increased cell adhesion molecule expression, superoxide generation and phagocytic activity of leukocytes (25, 28) and increased P-selectin expression by platelets (20). Such cell-cell interactions are likely to increase hours after reperfusion due to the increased expression of transcription-dependent mediators of inflammation.
Although P-selectin has previously been implicated in I/R-induced platelet adhesion, the ligand used by P-selectin to mediate this response has not been addressed. In the present study, we addressed the possibility that PSGL-1 is the counter-receptor for P-selectin that mediates I/R-induced platelet adhesion. This seemed plausible because 1) PSGL-1 has been shown to mediate other platelet adhesion induced by calcium ionophore (4) and 2) PSGL-1 is expressed on both leukocytes (24) and platelets (4) and therefore may be involved in platelet-endothelial and platelet-leukocyte adhesion. Treatment of ischemic mice with a PSGL-1 blocking antibody significantly decreased platelet adhesion in the intestinal microvasculature, suggesting that some of the observed platelet adhesion is mediated by P-selectin binding to PSGL-1. Because platelet-associated P-selectin, rather than the endothelial form, is the major mediator of I/R-induced platelet adhesion, it is unlikely that interactions between platelet-associated PSGL-1 and endothelial cell P-selectin account for a significant proportion of the observed platelet adhesion. Furthermore, greater platelet recruitment would have been expected when P-selectin-deficient platelets were infused into wild-type mice if this were the case. A more likely scenario is that platelet P-selectin is interacting with leukocyte-associated PSGL-1. Because the PSGL-1 blocking MAb did not reduce platelet adhesion to levels comparable to those observed with the P-selectin deficient platelets, it is unlikely that this interaction accounts for all of the observed platelet adhesion. The remaining adhesion may result from platelet-P-selectin interacting with an as-yet unidentified ligand on either leukocytes or the endothelium.
In conclusion, this study demonstrates a significant increase in platelet adhesion hours after reperfusion of the ischemic small bowel. This adhesion is predominantly reliant on platelet P-selectin with endothelial P-selectin and PSGL-1 also playing a role. Such a time course of platelet adhesion and the importance of platelet P-selectin in mediating this adhesion suggests that the accumulation of circulating inflammatory mediators may elicit the increased adhesion via transcription-dependent processes. The increased leukocyte-endothelial cell adhesion that occurs in the microcirculation after I/R may also contribute to the observed platelet adhesion. Further studies are needed to identify both the putative mediators that may induce platelet adhesion and the role of leukocytes, if any, in the induction of this prothrombogenic phenotype in venules.
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ACKNOWLEDGEMENTS |
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This study was supported by National Heart, Lung, and Blood Institute Grant HL-26441.
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. N. Granger, Dept. of Molecular and Cellular Physiology, LSU Health Sciences Center, 1501 Kings Hwy., Shreveport, LA 71130-3932 (E-mail: dgrang{at}lsuhsc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpgi.00457.2002
Received 24 October 2002; accepted in final form 8 February 2003.
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