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(Journal of Leukocyte Biology. 2002;72:455-461.)
© 2002 by Society for Leukocyte Biology

Molecular mechanisms of platelet-mediated leukocyte recruitment during myocardial reperfusion

Christian Kupatt*, Reinhard Wichels*, Jan Horstkotte*, Fritz Krombach{dagger}, Helmut Habazettl{ddagger} and Peter Boekstegers*

* Internal Medicine I, Klinikum Grosshadern, Munich, Germany;
{dagger} Institute for Surgical Research, Ludwig—Maximilians-Universität, Munich, Germany; and
{ddagger} Department of Physiology, Free University and Deutsches Herzzentrum, Berlin, Germany

Correspondence: Christian Kupatt, M.D., Internal Medicine I, Klinikum Grosshadern, Marchioninistr. 15, 81377 Munich, Germany.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leukocyte interaction with platelets and endothelial cells as cause of myocardial stunning was investigated. Mice were anesthetized and, after thoracotomy, the LAD was ligated for 20 min. Where indicated, rhodamine 6G for leukocyte labeling, fluorescence-labeled platelets, and the GPIIb/IIIa antagonist Tirofiban were infused at the onset of reperfusion in vivo. After 15 min, hearts were quickly excised and analyzed by fluorescence microscopy or assessed for left ventricular developed pressure (LVDP). After in vivo ischemia and reperfusion, leukocyte retention in the heart was 55 ± 5/field in wild-type hearts, 38 ± 3/field in P-selectin-/- hearts, and 23 ± 4/field in P-selectin/intercellular adhesion molecule-1 (ICAM-1)-/- hearts. Postischemic LVDP (48±4 mmHg in wild-type hearts) improved in P-selectin-/- and P-selectin/ICAM-1-/- hearts (58±4 and 79±6 mmHg). Tirofiban reduced platelet adhesion (23±4/field vs. 61±2/field in wild-type hearts) and leukocyte recruitment (34±2/field), improving LVDP (63±4 mmHg). Whereas wild-type platelets displayed similar adherence to P-selectin/ICAM-1-/- hearts as platelets from the same genetic strain (63±3 vs. 61±4 platelets/field), wild-type platelet infusion restored postischemic leukocyte recruitment in P-selectin/ICAM-1-/- hearts (55±4/field vs. 23±4/field), an effect sensitive to Tirofiban inhibition (23±4 leukocytes/field, 22±3 platelets/field). We conclude that platelets contribute postischemic leukocyte adhesion in the heart via P-selectin and GPIIb/IIIa.

Key Words: PMN • adhesion molecules • Tirofiban • P-selectin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reperfusion of an occluded coronary artery, although an essential treatment of myocardial infarction, may induce arrhythmias [1 ], myocardial stunning [2 , 3 ], and microcirculatory obstruction [4 ]. As early studies demonstrated a beneficial effect of antibodies against MAC-1 (CD11b/CD18), an adhesion molecule of polymorphonuclear neutrophils (PMN), recruitment of PMN in the myocardium has been characterized as a contributing factor in the evolvement of cardiac reperfusion injury [5 6 7 ].

The interaction of PMN with coronary endothelial cells during early reperfusion most likely takes place in the postcapillary venules [8 9 10 ]. Once initial contact and deceleration of the blood-borne cells (rolling) have been achieved by selectin (P-, E-, and L-selectin) interactions with their ligands (PSGL-1, Sialyl LewisXmoieties), firm adhesion (sticking) may occur. The latter adhesion step is mediated by endothelial intercellular adhesion molecules 1 and 2 (ICAM-1, ICAM-2) and leukocyte ß2-integrins, i.e., MAC-1 and lymphocyte function-associated antigen-1 [11 , 12 ]. More recent reports have added a role of platelets in this adhesion process. In addition to activation of the endothelium [13 ] and leukocytes [14 ], platelets may directly interact with PMN [15 , 16 ], thereby exerting functional detriment during myocardial reperfusion [17 , 18 ]. In the intestinum, platelets are attracted to the reperfused endothelium by tethering on endothelial P-selectin (P-sel) [19 ]. In isolated hearts, P-sel was found to contribute to platelet-PMN interaction during reperfusion [17 ]. However, the role of platelets for postischemic leukocyte recruitment and myocardial dysfunction has not been assessed in vivo.

In the present study therefore we used a mouse model of ischemia and reperfusion in vivo to address the molecular mechanisms of early myocardial leukocyte recruitment. By the use of wild-type, P-sel, and P-sel/ICAM-1-deficient mice as well as infusion of donor platelets in recipient mice of the same background, we could demonstrate that P-sel and GPIIb/IIIa are involved in platelet-mediated postischemic leukocyte recruitment. Interestingly, P-sel-expressing platelets restored leukocyte recruitment in P-sel/ICAM-1-deficient mice, reflecting the potential role of platelets as adhesion interface between endothelial cells and leukocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
We used male mice of C57BL/6 background, displaying wild-type phenotype, P-sel deficiency, or combined ICAM-1/P-sel deficiency (Jackson Laboratory, Bar Harbor, ME), with P-sel deficiency on platelets and ICAM-1 deficiency on leukocytes being verified by flow cytometry. Tirofiban was purchased from Merck, Sharp and Dohme (Haar, Germany), rhodamine 6G and BCECF-AM were from Sigma Chemical Co. (Deisenhofen, Germany). Tirofiban was used at 0.36 µg/g bodyweight, a dose that was sufficient to inhibit adhesion of platelets on fibrinogen-coated microtiter plates. As depicted in Figure 1 , 5 µg/ml blocked platelet adhesion as much as a fivefold higher concentration. This concentration was used in mice of 20–25 mg bodyweight, assuming an intravasal volume of 0.75 ml/10 g bodyweight.



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  		Figure 1. Microtiter wells coated with mouse fibrinogen were exposed to washed, wild-type platelets (mouse) labeled with rhodamine 6G. After 5 min centrifugation (300 g), nonadherent platelets were removed, and adherent platelets were counted by fluorescence microscopy (n=3). #, P < 0.05 versus untreated controls; §, P < 0.05 versus 2.5 µg/ml treatment group.;9>

 
Heart preparation
The care of the animals and all experimental procedures conform with the German legislation for the protection of animals and were approved by the state Animal Protection Commission. Animals were initially anesthetized with ketamine (75 mg/kg) and xylazin (15 mg/kg). The intraperitoneal anesthesia was prolonged with ketanest when needed. After tracheotomy, animals were intubated and ventilated via a Hugo Sachs Harvard Apparatus respirator (100x200 µl/min; Hugstetten, Germany). A polyethylene catheter (280 µm inner-diameter) was placed in the external jugular vein. Thereafter, sternotomy was performed, and the pericardium was removed. The left anterior descending artery (LAD) was ligated in the proximal third using a 7–0 suture and a polyethylene tube as described [20 ]. After 20 min of ischemia (sublethal injury), the LAD was reperfused by opening the suture. In vivo reperfusion was allowed for 15 min.

Fluorescence microscopy
For leukocyte staining, mice (n=six per group) received rhodamine 6G (50 µl, 0.05%) prior to ischemia. Platelets of donor mice were isolated from whole blood samples drawn on ethylenediaminetetraacetate (50 µl 0.1%). After centrifugation (800 g, 5 min), the platelet-rich plasma was taken and washed in calcium-free phosphate-buffered saline (PBS) by centrifugation (2000 g, 5 min), discarding the supernatant. The resulting pellet was resuspended in 500 µl calcium-free PBS, and 25 µl BCECF-AM (1 mg/ml dimethyl sulfoxide stock solution) was added for leukocyte-platelet-coaggregate detection, or 50 µl rhodamine 6G (0.01% stock solution) was added for single-platelet detection. After 10 min, platelets were washed, resuspended in 200 µl PBS, and counted in a Coulter counter. Platelets (200x106) were infused into the recipient mouse at the onset of reperfusion.

After 15 min of in vivo reperfusion, the aorta ascendens was cannulated, while anesthesia was continued and the eart was beating, and ligated. Immediately thereafter, coronary arteries were perfused in a Langendorff mode for 3 min with isothermic Tyrode’s solution (at 2 ml/min). With this perfusion scheme, systolic perfusion pressure did not exceed 80 mmHg, as assessed by pressure monitoring (Statham transducer and Hugo Sachs Harvard Apparatus amplifier), mimicking the in vivo systolic pressure. After 3 min of retrograde saline perfusion, removing nonadherent leukocytes and platelets, hearts were placed on a microscopic stage. Analysis of leukocyte recruitment and leukocyte-platelet interaction was performed with the surface of the left ventricle being exposed to a microscope (Ploemopak, Leitz, Wetzlar, Germany) with a tenfold objective (L10, 0.22 aperture, Leitz) during epi-illumination with a H130 mercury light source. Images were generated by a charge-coupled device camera (COHU 4400, Prospective Measurements, San Diego, CA). The distribution of rhodamine-stained cells was studied under an N2 filter block (Leitz), and distribution of BCECF-AM-stained platelets was analyzed by a I2,3 filter block (Leitz).

This method allowed for quantitative analysis of rhodamine 6G-labeled leukocytes and their interaction with BCECF-labeled platelets in coaggregates (Fig. 2 ) in epicardial regions of the myocardium (0.5–1.5 mm depth). Single platelets were visualized by rhodamine 6G staining in parallel experiments. Quantitative results are given per microscopic field (=0.8 mm2).



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  		Figure 2. Examples of fluorescence microscopy of leukocytes (A) and thrombocytes (B) adhering in a wild-type heart after ischemia (20 min) and reperfusion (15 min). Interaction of leukocytes and platelets was detected (C).

 
Assessment of myocardial function
To assess left ventricular function, hearts (n=8 per group) were quickly excised after 15 min reperfusion. Thereafter, retrograde perfusion of modified Krebs-Henseleit buffer in the Langendorff mode was performed at constant pressure (80 mmHg). A pressure balloon was inserted through the mitral valve into the left ventricle and connected to a pressure monitor. Heart rate, left ventricular developed pressure (LVDP), dP/dtmax, and dP/dtmin were assessed after 15 min of equilibration.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Leukocyte and platelet adhesion during early reperfusion
Fluorescence microscopy was used to detect fluorescence-labeled leukocyte and platelet distribution in the epicardial vessels of wild-type mouse hearts (example in Fig. 2 ). Quantitative analysis revealed that ischemia/reperfusion induced a tenfold increase in leukocyte recruitment (from 5±1 to 55±5/field, Fig. 3 A ) and a ninefold increase in leukocyte-platelet coaggregate formation (from 0.9±0.5 to 8.0±0.8/field, Fig. 3B ). In parallel, platelet adhesion was increased (from 5±3 to 75±11/field). P-sel-deficient mice displayed significantly reduced leukocyte recruitment and leukocyte-platelet coaggregates after ischemia and reperfusion (Fig. 3A and 3B) . Additional ICAM-1 deficiency further reduced both parameters. In contrast, platelet adhesion after myocardial ischemia was not affected by the absence of P-sel and/or ICAM-1 (Fig. 3C) .



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  		Figure 3. Ischemia (20 min LAD occlusion) and reperfusion (15 min in vivo) induce leukocyte recruitment, leukocyte-platelet interaction, and platelet adhesion. Quantitative analysis of fluorescence-microscopy detection of leukocytes (A), leukocyte-platelet coaggregates (B), and platelets (C) at the epicardial surface of the left ventricles. Wild-type mice (WT) were compared with P-selectin (P-sel-/-) or double-deficient mice (P-sel/ICAM-1-/-; n=six per group). *, P < 0.05 versus sham; #, P < 0.05 versus ischemia and reperfusion (I/R) WT; ||, P < 0.05 versus I/R P-sel. n.d., Not determined;10>.

 
The differences in leukocyte recruitment and leukocyte-platelet coaggregate formation yielded functional changes of the left ventricular myocardium: Compared with sham-operated mice, hearts of mice subjected to ischemia and reperfusion displayed a substantial decrease of LVDP (from 110±11 mmHg to 48±6 mmHg), dP/dtmax (from 3280±459 mmHg/s to 1543±321 mmHg/s), and dP/dtmin (from -2520±546 to -1213±176 mmHg/s). Moreover, whereas P-sel-deficient hearts revealed only tendencies toward functional improvement, additional ICAM-1 deficiency improved LVDP, dP/dtmax, and dP/dtmin (Table 1 ).


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  		Table 1. Systolic Function (LVDP, dP/dtmax) and Diastolic Function (dP/dtmin) after Ischemia and Reperfusion (I/R) in Wild-Type and Transgenic Mice

 
Role of platelets for leukocyte recruitment
Tirofiban, a synthetic antagonist against the platelet- and megakaryocyte-restricted integrin GPIIb/IIIa, was administered intravenously directly before myocardial ischemia. Tirofiban blocked platelet adhesion and leukocyte-platelet interaction after ischemia and reperfusion (Fig. 4B and C). Concomitantly, it reduced leukocyte recruitment by 38% (Fig. 4A) . Conversely, LVDP increased by 31% (Table 1) . In the absence of P-sel and ICAM-1, Tirofiban decreased platelet adhesion and leukocyte-platelet coaggregate formation (Fig. 4B and 4C) . However, in these experiments, it did not influence leukocyte retention (Fig. 4A) or myocardial function (Table 1) . Thus, inhibition of GPIIb/IIIa decreased platelet adhesion, leukocyte recruitment, as well as platelet-leukocyte interaction, concomitantly improving myocardial detriment in wild-type but not P-sel/ICAM-1-/- mice. The latter finding points to an essential role of P-sel or ICAM-1 function for platelet mediation of postischemic leukocyte recruitment.



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  		Figure 4. Inhibition of GPIIb/IIIa decreases leukocyte recruitment, leukocyte-platelet interaction, and platelet adhesion in postischemic wild-type mice hearts. Quantitative analysis of fluorescence microscopy detection of leukocytes (A), leukocyte-platelet coaggregates (B), and platelets (C) at the epicardial surface of the left ventricles. The effect of Tirofiban was assessed in WT and P-sel/ICAM-1-/- hearts (n=six per group). *, P < 0.05 versus I/R WT; #, P < 0.05 versus all other groups.

 
Platelet P-sel suffices for postischemic leukocyte recruitment
To further elucidate the contribution of P-sel to postischemic leukocyte recruitment, we infused platelets derived from wild-type, P-sel-/-, or P-sel/ICAM-1-/- mice into P-sel/ICAM-1-/- hearts, which lacked direct leukocyte interaction with endothelial P-sel or ICAM-1. In hearts of the double-deficient genotype, postischemic-platelet adhesion did not differ between the wild-type, P-sel-/-, and P-sel-/-/ICAM-1-/- group (Fig. 5 B ). The leukocyte recruitment after ischemia and 15 min of reperfusion, however, increased with transfusion of wild-type platelets in contrast to P-sel-deficient platelets (Fig. 5A) . In addition, in the P-sel/ICAM-1-deficient hearts, Tirofiban was capable of reducing wild-type platelet retention and decreasing leukocyte retention to the level observed with P-sel-deficient or double-deficient platelets. Taken together, these experiments indicate that P-sel-expressing platelets, adhering to postischemic coronary endothelium, compensate for the leukocyte-adhesion deficit of P-sel/ICAM-1-/- mice.



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  		Figure 5. Platelets enhance leukocyte recruitment in P-sel/ICAM-1-/- hearts via P-sel and GPIIb/IIIa. A leukocyte recruitment and B platelet adhesion in P-sel-/- ICAM-1-/- hearts after ischemia and reperfusion and transfusion of homologous or P-sel-/- or WT platelets with or without Tirofiban. Leukocyte recruitment is enhanced after transfusion of P-sel expressing WT platelets, an effect blunted by concomitant inhibition of platelet adhesion by Tirofiban (n=six per group). *, P < 0.05 versus all other groups.

 
Contribution of leukocytes and platelets to myocardial reperfusion injury
The impact of leukocyte or platelet recruitment and myocardial function was assessed by correlating leukocyte or platelet counts/field with LVDP of corresponding experimental groups. As depicted in Figure 6 , a close correlation between leukocyte recruitment and impaired left ventricular function was found (r=0.93, P<0.01). In contrast, differences in platelet recruitment did not correlate significantly with LVDP of the various experimental groups (r=0.30, P=0.13). We conclude that leukocyte recruitment determines the residual left ventricular function during early postischemic reperfusion. Platelets, which do not appear to directly exert myocardial damage, might contribute to postischemic myocardial dysfucntion through modulation of leukocyte recruitment.



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  		Figure 6. Correlation of leukocyte recruitment (A) and platelet adhesion (B) with left ventricular function (LVDP). Mean values of postischemic left ventricular function correlated significantly (P<0.01) with mean values of postischemic leukocyte recruitment (A), but not platelet adhesion (B; n=eight per group).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we investigated the molecular mechanisms of platelet interaction with the endothelium and leukocytes and their impact on early reperfusion injury in vivo. Absence of P-sel, a platelet and endothelial-adhesion molecule, reduced leukocyte recruitment and left ventricular function moderately (Fig. 3 , Table 1 ). This effect was enhanced in the additional absence of ICAM-1. Although the function of P-sel and ICAM-1 is thought to mainly influence leukocyte adhesion, a distinct role of platelets in mediating leukocyte recruitment was found: Reduction of platelet adhesion via the GPIIb/IIIa inhibitor Tirofiban reduced leukocyte adhesion and LV dysfunction (Fig. 4 , Table 1 ). Moreover, in P-sel/ICAM-1-/- hearts, where direct adhesion of leukocytes is severly hampered, wild-type platelet infusion fully restored leukocyte adhesion, unless platelet adhesion was blocked by Tirofiban (Fig. 5) . These experiments demonstrate a role of platelet P-sel and GPIIb/IIIa in postischemic leukocyte recruitment and leukocyte-dependent reperfusion injury.

Of note, several studies have correlated increased platelet-leukocyte interaction with myocardial reperfusion injury in patients [21 22 23 ], as well as in animal models [17 ]. How do platelets mediate leukocyte recruitment? One possibility consists of formation of coaggregates of platelets with leukocytes, which may physically plug in capillaries. These PMN-platelet coaggregates may enhance postischemic myocardial dysfunction, as demonstrated in a xenotypic model using human PMN in guinea pig hearts, where direct PMN-endothelial interaction was excluded [18 ]. In the present model, which studies adhesion of homologous platelets and PMN, is capillary plugging still instrumental for platelet-dependent leukocyte recruitment? Our data do not support this view; e.g., P-sel-/- platelets did not increase leukocyte recruitment in P-sel/ICAM-1-/- mice in comparison with wild-type platelets treated with Tirofiban, although coaggregate retention in P-sel-/- and ICAM-1/P-sel-/- experiments (Fig. 3B) is higher than in Tirofiban-treated groups (Fig. 4B) .

As an alternative mechanism, platelet adhesion and subsequent leukocyte recruitment to platelets were found in isolated hearts subjected to ischemia and reperfusion: When a bolus of wild-type platelets was infused in P-sel/ICAM-1-/- hearts at the onset of reperfusion, prior to leukocyte infusion without overlap of both boli, leukocyte recruitment increased fourfold compared with experiments lacking platelet infusion (data not shown). These data fit into a two-step model of platelet-mediated leukocyte recruitment, where platelet adhesion at the endothelium precedes the leukocyte sticking at platelet-adhesion molecules.

In these experiments, postischemic perfusion pressure was similar in wild-type and transgenic mice, ruling out hemodynamic changes as the cause of differences in leukocyte recruitment (data not shown). Similarly, in vivo systolic blood pressure has been found identical in wild-type, P-sel-/-, and ICAM-1-/- hearts [7 , 24 ].

Which vascular segment is responsible for platelet-dependent leukocyte adhesion? This problem has not been followed in the present study. However, we have shown in isolated hearts that postischemic leukocyte adhesion preferentially occurs in small, postcapillary venules (15–50 µm diameter) and to some extent in larger venules, but not in arterioles [8 , 10 ]. The same vascular segment appears to be used, although not exclusively, by adherent platelets, as recently demonstrated in mesenteric circulation [19 , 25 ]. Moreover, platelet-dependent leukocyte adhesion has also been found at this vascular segment in the mesenteric microcirculation [25 ].

Platelet adhesion on the coronary endothelium, a prerequisite for the role as anchorage of leukocytes, involves the ß3-integrin GPIIb/IIIa, the inhibition of which blocked platelet adhesion in wild-type and transgenic mice (Figs. 4 and 5) . This pleiotropic molecule is a receptor for fibrinogen, fibronectin, and von Willebrand factor, in turn binding to endothelial ICAM-1 (fibrinogen), {alpha}vß3 (fibrinogen and fibronectin), and GPIb (von Willebrand factor). The role of these endothelial-platelet receptors has been demonstrated on resting human umbilical vein endothelial cells [25 , 26 ] and is most likely present on postischemically activated endothelium, as {alpha}vß3 [27 ] and GPIb [28 ] are up-regulated rapidly upon endothelial activation. Conversely, using P-sel/ICAM-1-/- mice, we showed that absence of endothelial ICAM-1, which serves as platelet receptor during mesenteric reperfusion [29 ], does not alter platelet adhesion in the heart in our model (Fig. 3C) . Additional absence of P-sel in the double-deficient mice also did not impair platelet adhesion (Fig. 3C) , similar to a study in mouse venules where platelet adhesion at low shear was sharply increased by von Willebrand factor, but not P-sel translocation [30 ].

Beyond endothelium-platelet interaction, GPIIb/IIIa inhibition decreased leukocyte recruitment and leukocyte-platelet coaggregate formation. A potential direct effect of Tirofiban on leukocyte-adhesion molecules has not been described, in contrast to another GPIIb/IIIa inhibitor, abciximab [31 ]. However, in vitro platelet GPIIb/IIIa has been described as the main receptor for polymorphonuclear granulocytes, which most likely use the ß2-integrin MAC-1 and fibrinogen to achieve adhesion on platelets under shear stress conditions [15 ]. The minor fraction of platelet-leukocyte interactions observed in our study in the presence of Tirofiban (Fig. 4B) might be a result of platelet ICAM-2 binding to leukocyte ß2-integrins [32 , 33 ] or platelet GPIba binding to the I domain of leukocyte MAC-1 [34 ].

Of note, the interaction of leukocyte ß2-integrins with thrombocyte GPIb, GPIIb/IIIa, or ICAM-2 mediates firm and definitive interaction of leukocytes and platelets. However, similar to endothelial-leukocyte contact formation, initial contact of leukocytes and platelets is characterized by a reversible tethering [15 ]. Importantly, platelet P-sel binding to PSGL-1 on leukocytes has been described in cell culture [35 , 36 ] and in vivo [16 ]. To dissect the interaction of platelet or endothelial P-sel with leukocyte receptors, we used mice devoid of P-sel and ICAM-1. Here, direct leukocyte adhesion and indirect leukocyte recruitment via platelets were severly reduced when circulating and infused platelets lacked P-sel (Fig. 5) . In sharp contrast, exogenous platelets expressing P-sel induced a more than twofold increase in leukocyte recruitment in hearts of P-sel/ICAM-1-/- mice. Importantly, the difference between platelets without or with expression of P-sel was obtained in mice of the same strain, with a defined number of exogenous platelets infused, excluding potential differences in leukocyte or platelet count. Thus, here we showed for the first time that platelet P-sel is an essential molecule for platelet-dependent leukocyte recruitment in the postischemic heart in vivo, resembling its role in the reperfused gut [25 ] and kidney [37 ].

In summary, platelets contribute indirectly to myocardial-reperfusion injury by enhancing postischemic leukocyte adhesion. This platelet-mediated leukocyte adhesion requires platelet but not endothelial P-sel expression. GPIIb/IIIa antagonism, which has proven effective in patient treatment with thrombolysis [38 ], percutaneous transluminal coronary angioplasty [39 ], and stent implantation [40 41 42 ], inhibits both platelet adhesion as well as platelet-mediated leukocyte recruitment. At least in our model of myocardial ischemia and reperfusion, GPIIb/IIIa antagonists are protective beyond platelet inhibition by reducing early reperfusion injury induced by platelet-mediated leukocyte adhesion.


   ACKNOWLEDGEMENTS
 
This study was supported by Deutsche Forschungsgemeinschaft. All experiments were conducted at the Institute for Surgical Research of the Ludwig-Maximilians-University of Munich.

Received November 17, 2001; revised April 20, 2002; accepted May 2, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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