Vitronectin Receptor (αvβ3) Mediates Platelet Adhesion to the Luminal Aspect of Endothelial Cells
Implications for Reperfusion in Acute Myocardial Infarction
Background Platelet interaction with endothelium plays an important role in the pathophysiology of coronary microcirculation. We assessed the role of the vitronectin receptor (integrin αvβ3) in platelet/endothelium adhesion.
Methods and Results We investigated the effect on platelet/endothelium adhesion of plasma obtained from patients with acute myocardial infarction during reperfusion (before and 8, 24, 48, and 72 hours and 5 to 7 days after direct angioplasty) and with pretreatment with α-thrombin (2 U/mL) and recombinant human interleukin-1β. Platelet/endothelium adhesion was significantly enhanced by ≈20% after pretreatment of endothelium with patient plasma for 4 hours (P<.05) compared with endothelium treated with pooled control plasma. Plasma-induced platelet/endothelium adhesion was, in part, RGD peptide dependent. Pretreatment of endothelial cells with α-thrombin or recombinant human interleukin-1β enhanced platelet/endothelium adhesion and surface expression of αvβ3 on the luminal aspect of endothelium (P<.05). The adhesion of platelets, isolated platelet microparticles, and Chinese hamster ovary cells bearing human recombinant αIIbβ3 (platelet glycoprotein IIb-IIIa) to activated endothelial cells was inhibited by antiadhesive peptides GRGDSP and c(RGDfV) and monoclonal antibodies 4F10, LM609, and 7E3.
Conclusions The expression of vitronectin receptor exposed on the luminal aspect of activated endothelium is enhanced and mediates platelet/endothelium adhesion. Vitronectin receptor–mediated platelet attachment to activated endothelium during reperfusion may contribute to reperfusion injury and could be a target for antiadhesive therapy.
In AMI, the benefit from timely reperfusion may be compromised by reperfusion injury.1 2 Among the cellular interactions involved in reperfusion injury, those between platelets and endothelium are the least well understood so far. Platelet-derived mediators have been shown to promote endothelial activation, coronary artery vasoconstriction, and reduction in coronary blood flow.3 4 5 6
Recently, we found that plasma obtained from patients with AMI during reperfusion promotes platelet/endothelium adhesion in vitro.7 Previously, we showed that recombinant human β3 integrins αvβ3 and αIIbβ3 expressed in nuclear cells mediate heterotypic cell adhesion in the presence of fibrinogen.8 9 Thus, fibrinogen bridging platelet GPIIb-IIIa (αIIbβ3) to endothelial αvβ3 could have a pathophysiological relevance in platelet/endothelium adhesion during reperfusion.
αvβ3 is the major integrin expressed on endothelial cells, and it binds RGD-containing glycoproteins.10 αvβ3 operates in concert with other adhesion receptors to promote endothelial cell adhesion to the vascular matrix10 11 12 as well as white blood cell adhesion to and transmigration across endothelial cells.13 Recently, αvβ3 has been a subject of considerable interest because experimental and clinical studies suggest that αvβ3 is involved in mechanisms of angiogenesis,14 apoptosis of proliferating vascular cells,11 15 smooth muscle cell migration,11 16 neointimal hyperplasia,16 and restenosis.17 It has been suggested that αvβ3 is involved in disturbances of endothelial barrier function and promotes edema in lung tissue.18
In the present study, we focused on the role of αvβ3 in platelet adhesion to activated cultured endothelium. Specifically, we investigated the effect of plasma obtained from patients with AMI during reperfusion on platelet adhesion and αvβ3 surface exposure on cultured endothelial cells. Moreover, we assessed the role of αvβ3 for interaction of platelets with activated HUVECs.
Patients, Study Design, and Specimen Collection
The studies were performed in 10 patients who presented with AMI (Table 1⇓). All patients gave written informed consent. All patients were successfully treated with direct coronary angioplasty (PTCA) for an occluded coronary artery, achieving TIMI 3 flow. Before PTCA, an intravenous bolus injection of 15 000 IU unfractionated heparin, 500 mg acetylsalicylic acid, and 5 to 20 mg metoprolol, depending on the individual response, was administered. During the observation period (7 days after PTCA), all patients received a combined antiplatelet therapy of 2× 100 mg aspirin and 2× 250 mg ticlopidine PO, 2.5 to 10 mg enalapril, and 50 to 150 mg metoprolol. Ten milliliters of citrate/phosphate/dextrose/adenine–anticoagulated blood were taken after puncture of a forearm vein before and 8, 24, 48, and 72 hours and 5 to 7 days after direct PTCA and processed as previously described.7
Monoclonal Antibodies, Peptides, and Reagents
Anti-CD51 (IgG1 mouse, clone AMF7, Immunotech) recognizes the αv subunit, and anti-CD61 (IgG1 mouse, clone SZ21, Immunotech) recognizes the β3 subunit of the vitronectin receptor αvβ3. mAb LM609 (IgG1 mouse, generously provided by Dr David Cheresh, Scripps Clinic, La Jolla, Calif) is a blocking antibody directed against αvβ3 and binds to the receptor only in its complexed form.10 mAb 7E3 (IgG1 mouse, a gift from Dr Barry Coller, Mount Sinai Hospital, New York, NY) blocks the fibrinogen binding site on GPIIb-IIIa and also recognizes the complexed form of αvβ3.19 mAb 4F10 (IgG1 mouse) inhibits fibrinogen binding to GPIIb-IIIa (αIIbβ3) (a gift from Dr Virgil Woods, University of California San Diego).20 mAb anti-LIBS1 (IgG1 mouse, kindly provided by Dr Mark Ginsberg, Scripps Clinic) is a conformation-dependent antibody that induces a high-affinity state of platelet GPIIb-IIIa.21 Anti-CD42b binds to platelet GP-Ib (IgG1 mouse, clone SZ2, Immunotech), and anti-CD54, a phycoerythrin-conjugate (IgG1 mouse, clone 84H10, Immunotech) binds to ICAM-1. In all immunostaining studies, an irrelevant isotype-matched control antibody was used (anti-αM, IgG1 mouse, clone Bear1, Immunotech). For flow cytometric analysis, all mAbs were conjugated with FITC to obtain fluorescence-to-protein ratios of 2.5 to 3.0.22
The peptides GRGDSP and GRGESP were purchased from Calbiochem. GRGDSP is recognized by both GPIIb-IIIa and αvβ3, whereas GRGESP is biologically inactive and was used as a control peptide. c(RGDfV) (kindly provided by Prof Dr J. Kessler, Institut für Biochemie, Technische Universität München, Germany) is a cyclic pentapeptide with high selectivity for αvβ3.23 Fibronectin-depleted fibrinogen was purified according to described methods. The RGD-containing polypeptide echistatin (Sigma Chemical) binds with high affinity to αvβ324 and was conjugated for flow cytometric binding studies with FITC according to a standard protocol to obtain a fluorescence-to-peptide ratio of 3.4.22
Primary HUVECs were harvested using collagenase digestion (Worthington) and cultured as previously described.25 ECV-304, an immortal HUVEC cell line (American Type Culture Collection), was cultured in complete medium M199 (10% FCS, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 mg/L streptomycin). The stable CHO cell line, cotransfected with human β3 and αIIb cDNA, expresses functional recombinant human platelet GPIIb-IIIa (kindly provided by Dr Mark Ginsberg, Scripps Clinic) and was cultivated as previously described.9 26
Immunostaining of Endothelial Cells
Confluent monolayers of endothelial cells were stimulated as indicated with human α-thrombin (Sigma; 2 U/mL) for 20 minutes or rhIL-1β (R & D Systems; 100 pg/mL) for 4 hours, respectively. After a washing step, cell layers were incubated with saturating concentrations of FITC-conjugated mAbs (30 μg/mL) and PE-conjugated anti–ICAM-1 mAb (20 μg/mL) for 20 minutes at 37°C. After the addition of 2 mL M199 containing 1.67 mg/mL LDS-751 (Styry 18, Exciton Inc), the mAb solution was diluted 1:20. Endothelial cells were then mechanically detached and separated into single-cell suspension by repetitive pipetting. For flow cytometric analysis, endothelial cells were identified by LDS-751 and ICAM-1 fluorescence; 10 000 events were evaluated, and mean intensity of FITC immunofluorescence was used as a parameter of antigen expression. Immunoelectron microscopy was performed as previously described.27 28 After incubation with anti-αv mAb, the endothelial monolayer was incubated for 30 minutes with a second gold-conjugated goat anti-mouse antibody (gold particle size, 6 nm; Aurion). Fixation was then performed in 2.5% glutaraldehyde followed by silver enhancement (Aurion R-Gent) of the conjugates.
Effect of Patient Plasma on Platelet/Endothelium Adhesion
After incubation of endothelial monolayers for 4 hours with patient plasma diluted 1:2 in M199, the supernatant was aspirated, and 100 μL of gel-filtered platelet suspension that contained 300 μg/mL fibrinogen, 2 mmol/L Ca2+, and 10 μmol/L ADP (final platelet concentration, 108/mL) was added to each well. After a 60-minute incubation at 37°C without agitation in culture condition atmosphere, the nonadherent platelets were removed by centrifugation of the inverted culture plate (500g for 5 minutes). Integrity of cell monolayer was verified by direct microscopy. Thereafter, samples were fixed for 1 hour at room temperature by the addition of freshly prepared 4% paraformaldehyde/PBS solution to each microtiter well. The fixative was washed off, and 50 μL of platelet-specific mAb anti-CD42b was then added in saturating concentration (20 μg/mL) to each well. Specific antibody binding was detected by use of a secondary peroxidase-conjugated mAb (Sigma).
Nonactivated or stimulated (α-thrombin, rhIL-1β) endothelial monolayers were incubated (2 hours at 37°C) with various concentrations of FITC-echistatin (0.1, 0.0625, 0.25, 0.5, 0.75, 1.0 μmol/L) diluted in M199 that contained 1 mmol/L Ca2+. Binding experiments were performed in the presence and absence of 1 mmol/L GRGDSP. Thereafter, endothelial cells were stained with LDS-751 and anti–ICAM-1 as described above. After mechanical detachment and separation into a single-cell suspension, binding of FITC-echistatin to endothelial cells was evaluated by flow cytometry; 10 000 events were counted that were positive for LDS-751 and ICAM-1.
Platelet adhesion to monolayers of cultured endothelial cells (24-well plates) was evaluated by the addition of 200 μL gel-filtered platelets (final platelet concentration, 108/mL) resuspended in Tyrodes-HEPES buffer (2.5 mmol/L HEPES, 150 mmol/L NaCl, 12 mmol/L NaHCO3, 2.5 mmol/L KCl, 1 mmol/L MgCl2, 5.5 mmol/L d-glucose, and 1 mg/mL BSA, pH 7.4) that contained 1 mmol/L Ca2+, 300 μg/mL fibrinogen, 10 μmol/L ADP, and additional compounds as indicated. After a 60-minute incubation at 37°C without agitation in culture condition atmosphere, supernatant was aspirated, and 200 μL of M199 containing saturating concentrations of mAbs anti-CD42b and PE-anti-CD54 was added to each well. After 20 minutes of incubation, the cells were mechanically detached through repetitive pipetting, and a single-cell suspension was evaluated by flow cytometry as described above. In experiments with activated endothelium, the adherent monolayers were conditioned for the indicated time with α-thrombin (2 U/mL) or rhIL-1β (100 pg/mL) and washed three times with M199 until the addition of platelet suspension. In experiments with α-thrombin, activation was stopped by the addition of excess (10 U/mL) of hirudin to neutralize thrombin activity (Sigma). Platelet-endothelium adhesion was verified by laser scanning and raster electron microscopy as previously described.7 27 28
Platelet microparticles were purified and characterized as previously described.29 Platelet microparticles (100 μg/mL) were resuspended homogeneously in Tyrodes-HEPES buffer supplemented with 1 mmol/L Ca2+ and 300 μg/mL fibrinogen. Monolayers of cultured endothelial cells were incubated after the addition of 100 μL membrane suspension to each well for 60 minutes at 37°C. Thereafter, microparticles bound to endothelial cells were detected as described above for intact platelets.
Adhesion of αIIbβ3 Transfectants to HUVECs
Preparation of cell suspensions and fluorescent labeling of the transfected CHO cells were performed as previously described.8 CHO transfectants were labeled with the red fluorochrome hydroethidine (Polysciences), and HUVECs were labeled with the green fluorochrome sulfofluorescein diacetate (Molecular Probes). Equal volumes of labeled cells (4×106 cells/mL) in suspension were mixed, and coadhesion was evaluated as previously described.8 9 The appearance of two-color particles (red-green) was used as parameter of coadhesion between αIIbβ3 transfectants and HUVECs.
The Kolmogorov-Smirnov test showed that the data were not normally distributed. Differences were tested by Friedman’s test followed by Wilcoxon’s matched-pairs signed rank test. Differences between unpaired samples were tested by the Mann-Whitney-Wilcoxon rank sum test. A value of P<.05 was regarded as statistically significant.
Effects of Plasma Obtained From Patients With AMI on Platelet Adhesion to Cultured Endothelial Monolayers
Preincubation of endothelial cells (ECV-304) with patient plasma for 4 hours resulted in increased platelet/endothelium adhesion compared with endothelium that has been treated with pooled control plasma (P<.05) or untreated endothelial cells (not shown) (Table 2⇓). Plasma-induced adhesion of ADP-activated platelets to endothelium was enhanced throughout all tested time points of reperfusion (Table 2⇓).
In the presence of the synthetic peptide GRGDSP (500 μmol/L), induced adhesion of ADP-activated platelets to endothelium was markedly reduced with maximal inhibition 48 and 72 hours and 5 to 7 days after direct PTCA (Table 2⇑).
Activation of Endothelium and Surface Expression of Vitronectin Receptor αvβ3
We tested whether activation of endothelium affects expression of αvβ3 on the luminal aspect of cultured endothelial cells. α-Thrombin (2 U/mL) and rhIL-1β (100 pg/mL) significantly enhanced surface exposure of both subunits of the vitronectin receptor by ≈10% to 40%, as demonstrated by enhanced binding of mAb anti-β3 or anti-αv (P<.05) (Fig 1⇓). Similarly, binding of both mAb LM609, which recognizes exclusively the functional and complexed form of the receptor αvβ3, and of mAb LIBS1, which recognizes conformational changes in β3 integrins, was enhanced on activated endothelium compared with untreated cells (P<.05) (Fig 1⇓). No significant changes in binding of an isotype-matched control mAb, anti-αM, was found on activated endothelium (Fig 1⇓). Activation-dependent expression of αvβ3 was observed on both cultured HUVECs and ECV-304 monolayers (Fig 1A⇓ and 1B⇓). The presence of significant amounts of vitronectin receptor on the luminal surface of endothelial cells was verified by transmission electron microscopy (Fig 2⇓).
To assess functional aspects of αvβ3, we performed binding experiments with echistatin, a polypeptide that contains the RGD recognition motif and that binds to αvβ3.24 Binding of echistatin to cultured endothelium was saturable (Fig 3⇓) and was inhibited through GRGDSP (1 mmol/L). Pretreatment of endothelial cells with α-thrombin or rhIL-1β resulted in significantly enhanced GRGDSP-dependent echistatin binding (P<.05) (Fig 3⇓).
Platelet Adhesion to Nonstimulated and Activated Endothelium
Adhesion of ADP-activated platelets to α-thrombin–or rhIL-1β–pretreated endothelial monolayers was significantly enhanced compared with nonactivated endothelium (P<.05) (Fig 4A⇓). Platelet adhesion to both α-thrombin– and rhIL-1β–pretreated endothelial cells was reduced in the presence of 500 μmol/L GRGDSP (P<.05) but not in the presence of the biologically inactive GRGESP peptide (500 μmol/L) (Fig 4A⇓). The cyclic peptide c(RGDfV), which has a high selectivity for αvβ3, also considerably reduced platelet adhesion to activated endothelium (P<.05) (Fig 4A⇓). As shown by direct microscopy, primarily single platelets underwent significant morphological changes and adhered to endothelial cells (Fig 5⇓).
Similar to intact platelets, adhesion of microparticles to activated endothelium was significantly enhanced (P<.05) (Fig 4B⇑). Platelet membrane/endothelium adhesion was inhibited by GRGDSP and c(RGDfV) but not by GRGESP (500 μmol/L) (P<.05) (Fig 4B⇑).
Heterotypic Adhesion of CHO Cells Expressing Recombinant Human Fibrinogen Receptor αIIbβ3 (GPIIb-IIIa) and HUVECs
CHO cells bearing the recombinant human platelet fibrinogen receptor αIIbβ3 (GPIIb-IIIa) adhered to HUVECs in the presence of soluble fibrinogen (300 μg/mL), Ca2+ (2 mmol/L), and mAb anti-LIBS1 (20 μg/mL) (Fig 6⇓). The adhesion was specific for αIIbβ3 because it was inhibited by blocking anti–GPIIb-IIIa mAb 4F10 (50 μg/mL) (Fig 6⇓). Heterotypic adhesion of transfectants and HUVECs was also αvβ3 specific because it was inhibited by mAb LM609 (50 μg/mL) (Fig 6⇓). mAb 7E3 (50 μg/mL), which inhibits ligand recognition of both β3 receptors, also reduced adhesion of transfectants to HUVECs (Fig 6⇓). No inhibition of heterotypic coadhesion was found in the presence of mAb anti-αv (anti-CD51) (30 μg/mL) or of an irrelevant isotype-matched control antibody anti-αM (30 μg/mL) (Fig 6⇓).
As described above for platelets, adhesion of CHO cells bearing αIIbβ3 to activated (α-thrombin, rhIL-1β) HUVECs was enhanced compared with nonstimulated HUVECs (P<.05) (Fig 7⇓). Heterotypic adhesion was inhibited by αIIbβ3 -specific mAb 4F10, αvβ3-specific mAb LM609, or mAb 7E3 (P<.05) (Fig 7⇓). GRGDSP (500 μmol/L) but not GRGESP inhibited adhesion of αIIbβ3 transfectants to activated HUVECs (Fig 7⇓).
The major findings of the present study are (1) plasma obtained from patients with AMI during reperfusion promotes adhesion of ADP-activated platelets to cultured endothelium that is, in part, mediated by an RGD-dependent mechanism; (2) activation of cultured endothelium by α-thrombin or rhIL-1β enhances adhesion of ADP-activated platelets to endothelial cells and surface expression of vitronectin receptor on the luminal aspect of cultured endothelium; and (3) nuclear cells expressing recombinant human platelet GPIIb-IIIa (αIIbβ3) adhere to monolayers of HUVECs in the presence of soluble fibrinogen. Coadhesion of αIIbβ3 transfectants can be inhibited through antagonists of β3 integrins.
These findings imply that platelet/endothelium adhesion is enhanced during reperfusion. Inhibition of vitronectin receptor function by specific antiadhesive compounds during reperfusion may be beneficial for patients with AMI.
Surface Expression of Vitronectin Receptor αvβ3 on the Blood-Facing Endothelial Surface
The function of the luminal-facing integrin is still poorly understood. αvβ3 is a receptor for a variety of plasmatic adhesive glycoproteins such as fibrinogen, vitronectin, or thrombospondin. Soluble forms of these proteins do not bind to endothelial β3 integrins.11 29 In contrast, vitronectin immobilized on microbeads binds significantly to endothelium in an RGD-dependent manner.31 Luminal surface expression of αvβ3 has been shown to support monocyte adhesion to endothelium.13
Our present study shows that expression of αvβ3 is enhanced on the luminal aspect of activated endothelial cells, as demonstrated by increased binding of αvβ3-specific mAbs and of echistatin after pretreatment of endothelial cells with α-thrombin or rhIL-1β. These results are in concert with studies that show enhanced vitronectin binding and RGD-dependent tumor cell adhesion to Il-1β–treated endothelial cells.32 Large amounts of αvβ3 are stored intracellularly, and αvβ3 receptors are translocated to the membrane surface on activation.31 32 33 34 Thus, luminal surface expression of endothelial αvβ3 is activation dependent and might contribute substantially to altered adhesive properties.
Platelet/Endothelium Adhesion in Reperfusion
In the present study, we found that plasma obtained from patients during reperfusion promotes adhesion of ADP-activated platelets to cultured endothelial cells. Plasma-induced adhesion could be inhibited, in part, through use of the antiadhesive peptide GRGDSP throughout reperfusion.
Numerous mediators (eg, complement factors, cytokines, reactive oxygen) are released during reperfusion.1 2 39 Because these mediators alter endothelial function,40 it seems likely that patient plasma contains compounds that are released during reperfusion. The presence of proinflammatory substances in plasma derived from patients during reperfusion may activate endothelial cells in culture and change their phenotype into a proadhesive one.
To evaluate the role of activation on αvβ3-mediated platelet adhesion, endothelial cells were stimulated with α-thrombin or rhIL-1β and the effect of antiadhesive RGD peptides on platelet adhesion was studied. In the presence of GRGDSP, a peptide that binds to a variety of integrins, including αvβ3 and platelet fibrinogen receptor GPIIb-IIIa (αIIbβ3), adhesion of ADP-activated platelets to pretreated endothelium was significantly decreased. Similar effects were shown for the cyclic synthetic peptide c(RGDfV), which is characterized by high selectivity for the αvβ3 receptor.23 This indicates that αvβ3 mediates platelet adhesion to the activated endothelial cell surface.
Adhesion to endothelium was not limited to intact platelets because isolated platelet membranes that lack intracellular structures also adhered to activated endothelium in an RGD-dependent manner. This suggests that the mechanism of platelet/endothelium adhesion is independent of endothelial-derived products (eg, nitric oxide, prostacyclin) that are released into the extracellular compartment and that can modulate activation-dependent processes within the intact platelet. Moreover, enhanced interaction of platelet membranes with endothelium may be of pathophysiological importance. Platelet-derived membrane fragments contain procoagulative compounds and are formed during reperfusion.7 Entrapment of these particles may support procoagulant activities on the endothelial monolayer.41
To further analyze the mechanisms of platelet adhesion to endothelial cells, we used a CHO cell line that expresses functional recombinant human platelet GPIIb-IIIa (αIIbβ3).8 26 We found that in the presence of fibrinogen, CHO cells adhered to cultured HUVECs. This interaction was specifically mediated by both β3 integrins; it could be inhibited by blocking mAbs directed to αIIbβ3 (4F10, 7E3) or αvβ3 (LM609). Activation of HUVECs by α-thrombin and rhIL-1β promoted coadhesion of αIIbβ3 transfectants and HUVECs. Again, this effect was dependent on enhanced surface expression of αvβ3 in that it was blocked by specific mAb LM609. Thus, fibrinogen bridging between platelet GPIIb-IIIa and endothelial αvβ3 is a likely mechanism involved in αvβ3-mediated platelet adhesion to activated endothelium.
Mechanisms other than the one described above may also contribute to platelet/endothelium adhesion. Platelets expose significant amounts of thrombospondin on their activated surface, which is able to bind αvβ3.11 12 Activated platelets also degranulate fibronectin, which is recognized by fibronectin receptor α6β1 on endothelial cells.42 It has been shown that the presence of ICAM-1 on endothelium binds fibrinogen that is immobilized on cell surfaces.43 Endothelial cells synthesize large amounts of von Willebrand factor that, in part, remain associated with the cell surface.37 Thus, interaction of platelet GPIb with cell-surface–immobilized von Willebrand factor might be another candidate for platelet/endothelium adhesion.
Studies on platelet adhesion to endothelium have been criticized for the possibility of platelet contact to extracellular structures after damage of the confluent monolayer. For example, thrombin induces retraction of cultured endothelial cells, which results in loosening cell contact of the monolayer.36 In our study, in all experiments the integrity of the endothelial layer was verified by direct microscopy. Moreover, flow cytometric analysis of detached cells allowed us to directly study platelet adhesion to single endothelial cells.
Endothelial cells derived from different blood vessel districts have specific adhesive properties.42 Endothelial cells isolated from the coronary circulation may also be functionally different. In our experiments, we used primary cultures of HUVECs and the stable endothelial cell line ECV-304. Both cell types are widely used to study endothelial cell function. Integrin exposure on the apical aspect of the membrane, however, is not a specific property of HUVECs or ECV-304 because cultured endothelium from saphena magna and adrenal gland microvessels also showed these characteristics.32 33 In situ staining of human tissues showed that αvβ3 is present in large vessel endothelium and, to a lesser extent, expressed in the microcirculation.32 Further experiments with endothelial cells derived from coronary macrovessels and microvessels will clarify whether αvβ3 is differentially surface exposed in response to activation and whether it promotes adhesion of platelets.
Pathophysiological Considerations and Therapeutic Implications
The adhesion of platelets to endothelium has been described in a variety of pathophysiological conditions.7 28 36 37 38 44 The biological relevance of platelet adhesion is of interest. Adhering platelets might (1) facilitate attachment and transmigration of leukocytes to and across the endothelial layer, (2) change the endothelial cell surface into a prothrombotic and proadhesive phenotype, and (3) induce inflammatory reaction within the endothelium. Activation and adhesion of platelets result in release of P-selectin (CD62P) on the surface, a central receptor for leukocyte tethering.45 Adherent platelets to the vessel wall have been shown to promote leukocyte-dependent fibrin deposition via P-selectin.46 Moreover, vitronectin receptor has been shown to promote in concert with P-selectin monocyte migration across endothelium.13 Thus, platelet attachment to endothelium might be a potent mechanism of targeting leukocyte sequestration toward the tissue of interest. Platelets release a variety of mediators that support platelet aggregation and coagulation.47 Moreover, platelet membranes that are sheered off platelets during activation are generated during reperfusion7 and possess significant procoagulant activities by activating the prothrombinase complex.41 In the present study, we found that microparticles are able to bind to endothelium and thus may promote fibrin generation localized at the site of adhesion. In addition to their role in adhesion, integrins act as outside-in signaling receptors.48 Integrins have been shown to mediate increases of cellular Ca2+, tyrosine phosphorylation of cell proteins, and induction of gene expression.48 49 Thus, anchorage of platelets to endothelial cells might induce inflammatory responses via integrins. The importance of platelet adhesion in inflammatory reactions is supported by experiments showing that P-selectin enhances production of monocytic-chemotactic peptide 1 (MCP-1) in neutrophils.45 Moreover, platelet-associated IL-1 activity induces ICAM-1 surface expression and IL-6 and IL-8 production50 51 ; all cytokines have been suggested to be involved in pathophysiological mechanisms of reperfusion.39 We (the present study) and others52 showed that IL-1β stimulates endothelial surface expression of αvβ3, which may further support platelet adhesion.
Although in the present study we could not address the potential clinical relevance of platelet/endothelium adhesion in reperfusion, results showed mechanisms that may stimulate clinical studies in this field, with the goal of improving therapy during reperfusion. The availability of reagents that modulate vitronectin receptor function, such as mAb 7E319 53 or LM60912 or αvβ3-specific peptides such as c(RGDfV),23 may be of interest in upcoming studies.
Selected Abbreviations and Acronyms
|AMI||=||acute myocardial infarction|
|CHO||=||Chinese hamster ovary|
|HUVEC||=||human umbilical vein endothelial cell.|
|ICAM-1||=||intercellular adhesion molecule–1|
|PTCA||=||percutaneous transluminal coronary angioplasty|
|rhIL-1β||=||recombinant human interleukin-1β|
The study was supported in part by grants from the Deutsche Forschungsgemeinschaft (Ga 381/2-1) and the NIH (HL-48728). The authors appreciate the excellent technical assistance of Caroline Bogner, Margit Huber, and Kirsten Langenbrink.
Presented in part at the 69th Scientific Sessions of the American Heart Association, New Orleans, La, November 1996.
- Received November 21, 1996.
- Revision received April 8, 1997.
- Accepted April 18, 1997.
- Copyright © 1997 by American Heart Association
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