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,
,2,3
* Hematological Research Institute, National Academy of Medicine, and
Laboratory of Immunopathology, Institute of Biology and Experimental Medicine (IBYME), National Research Council (CONICET), Buenos Aires, Argentina;
Biochemistry and Molecular Biology Institute, Department of Biological Sciences, National University of La Plata, La Plata, Argentina; and
Department of Biological Chemistry, Faculty of Exact and Natural Sciences, University of Buenos Aires, Buenos Aires, Argentina
3Correspondence: M.S., Instituto de Investigaciones Hematológicas, Academia Nacional de Medicina, Pacheco de Melo 3081, Buenos Aires (1425), Argentina. E-mail: mschattner{at}hematologia.anm.edu.ar; G.A.R., Instituto de Biologia y Medicina Experimental, CONICET, Vuelta de Obligado 2490, Buenos Aires (1428), Argentina. E-mail: gabyrabi{at}ciudad.com.ar
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: lectin • P-selectin • inflammation • hemostasis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. When platelets perceive activating signals through their cell surface receptors, they undergo dramatic structural and chemical changes, involving a complex interplay of cell adhesion and signaling molecules. Activated platelets rapidly bind circulating platelets, via membrane glycoprotein complex 
IIbβ3 (GPIIb/IIIa) and fibrinogen, to form a thrombus or a plug for preventing bleeding at sites of vascular injury (2)
. However, platelet aggregation can also occlude atherosclerotic arteries causing cardiac and cerebrovascular diseases (3
, 4)
.
Galectins, an evolutionarily conserved family of animal lectins, have emerged as regulators of immune cell homeostasis, inflammation, and cancer (5
, 6)
. Extracellularly, galectins can bind to galactose-containing glycoconjugates present on the cell surface and extracellular matrix and trigger a cascade of transmembrane signaling events leading to apoptosis, cytokine secretion, cell adhesion, and migration (7)
. In addition, galectins are engaged in intracellular processes that are essential for basic cellular functions, such as pre-mRNA splicing, regulation of cell growth, and cell cycle progression (8)
.
Galectin-1 (Gal-1), a 14.5-kDa member of this family, preferentially recognizes multiple Galβ1,4GlcNAc (LacNAc) units, which may be presented on the branches of N- or O-linked glycans on cell surface glycoproteins (7)
. Through binding and cross-linking specific glycoconjugates, Gal-1 regulates adaptive immune responses by controlling T cell survival, cytokine secretion and transendothelial migration (9
10
11
12
13
14
15)
. In addition, Gal-1 can act as a link between innate and adaptive immunity by modulating the physiology of neutrophils, monocytes, and dendritic cells (16
17
18)
. Furthermore, recent evidence indicates that Gal-1 contributes to the immunosuppressive activity of CD4+ CD25+ FOXP3+ regulatory T cells (19)
. In vivo, direct administration or genetic delivery of Gal-1 suppresses chronic inflammation in experimental models of autoimmunity by skewing the balance of the immune response toward a TH2 cytokine profile (20
21
22
23)
. Analysis of the mechanistic basis of this anti-inflammatory effect recently revealed that TH1 and TH17-differentiated cells share a common glycan motif, which can be specifically targeted by Gal-1, providing a novel link between differential glycosylation, susceptibility to cell death, and the regulation of the inflammatory response (24)
. In addition, expression of Gal-1 at sites of tumor growth and metastasis can influence tumor progression by regulating cell-cell and cell-matrix interactions, tumor cell invasiveness, and angiogenesis (25
26
27)
. Furthermore, Gal-1 can also function as a soluble mediator employed by tumor cells to evade the immune response (28)
.
Given the crucial role of platelets in immune cell homeostasis, inflammation and tumor progression, we investigated the impact of Gal-1 on platelet physiology. We demonstrate here that Gal-1 is a potent platelet activating agonist that triggers different effector responses, including F-actin polymerization, aggregation, P-selectin expression, shedding of microparticles and leukocyte-platelet aggregates. Moreover, endogenous Gal-1 appears to be a mediator of platelet aggregation induced by classical platelet agonists. Our results provide evidence of a novel unrecognized role for Gal-1 in the control of platelet physiology with potential implications at the crossroad of thrombosis, inflammation, and cancer.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-thrombin, annexin V/PI kit, and TRICT-phalloidin were from Sigma (St. Louis, MO, USA). The protein kinase C (PKC) inhibitor, Ro 32–0432, was from Biomol International L.P. (Plymouth Meeting, PA, USA). Prostacyclin (PGI2), and 1-propanamine-3-(2-hydroxy-2-nitroso-1-propylhydrazino) (PAPA/NO) were from Cayman Chemical (Ann Arbor, MI, USA). Stock solutions of the PKC inhibitor were stored in dimethyl sulfoxide, and dilutions were prepared in PBS. The following monoclonal antibodies: unlabeled-anti-CD29 (clone HUTS-21) and anti-CD41 (clone HIP8), phycoerythrin (PE)-labeled anti-CD61, fluorescein isothiocyanate (FITC)-labeled anti-CD45 and anti-PAC-1, and isotype-matched IgG1 and IgM were from BD Biosciences (San Jose, CA, USA). Unlabeled anti-CD42b (clone HIP1), FITC-labeled anti-CD62P, and isotype-matched IgG1 were from Beckman Coulter, Inc. (Fullerton, CA, USA). Unlabeled-anti-CD61 (clone Y2/51) was from Dako A/S (Glostrup, Denmark). Recombinant human Gal-1 was produced essentially as described previously (29)
. The recombinant protein was purified by affinity chromatography on an asialofetuin-agarose column and stored at –20°C. Lipopolysaccharide content of the purified samples was tested using a Gel Clot Limulus Test (Cape Cod., East Falmouth, MA, USA). Rabbit anti-human Gal-1 polyclonal antibody was obtained as described previously (20)
.
Blood collection
Blood samples were obtained from healthy donors, who had taken no medication for at least 10 days before sampling. This study was performed according to institutional guidelines (National Academy of Medicine, Buenos Aires, Argentina) and received approval of the institutional ethics committee and written consent from all the subjects. Whole blood was drawn directly into plastic tubes containing ACD (1/6). For the aggregation studies or PAC-1 binding, 3.8% sodium citrate (1:9 v/v) was used as anticoagulant.
Preparation of platelet-rich plasma (PRP) and washed platelets (WPs)
PRP was prepared by centrifugation of blood samples at 180 g for 10 min. After removal of the PRP, platelet-poor plasma (PPP) was obtained by centrifugation of the remaining blood sample for 20 min at 500 g. WPs were prepared by centrifugation of the PRP (200 g for 15 min) in washing buffer (140 mM NaCl, 2.5 mM KCl, 0.10 mM NaHCO3, 0.5 mM NaPO4H2, 1 mM MgCl2, 22 mM sodium citrate, 0.1% (w/v) glucose, 0.75 nM PGI2, 3.5% (w/v) BSA pH 6.5). Platelets were finally resuspended in Tyrode buffer containing 1 mM CaCl2. Samples of platelets prepared under this procedure typically contained <0.01% leukocytes as assessed on a Coulter Gen S System 2 Analyzer (Coulter Electronics, Hialeah, FL, USA). In all cases, platelet number was adjusted to 4 x 108/ml.
Platelet aggregation and ATP release
Platelet aggregation and ATP release were measured simultaneously in a Chrono-Log Lumi aggregometer (Chrono-Log, Havertown, PA, USA), as described previously (30)
. Briefly, aggregation was measured at 37°C under continuous stirring in 0.4 ml aliquots of PRP or WPs and monitored for 5–15 min. Aggregation was expressed as the percentage of maximal light transmission. ATP release was determined at the end of each experiment by adding known amounts of ATP. In some experiments, platelets were preincubated for 1 min with different inhibitors at 37°C before addition of Gal-1.
Flow cytometry analysis and binding assays
For detection of GPIIIa or P-selectin expression, WPs stimulated or not with Gal-1 were incubated with PBS containing 0.1% (v/v) FBS and saturating concentrations of PE-labeled anti-GPIIIa (CD61) and FITC-labeled anti-P-selectin (CD62P) or isotype-matched PE- or FITC-IgG1 as controls. After 30 min incubation in the dark at room temperature (RT), samples were fixed with 1% (w/v) paraformaldehyde and analyzed on a FACSCalibur flow cytometer using CellQuest (BD Biosciences) or WinMDI software (http://www.facs.scripps.edu).
To discriminate platelets from background, upper and lower forward scatter (FSC) thresholds were defined, including only the events that were positive for platelet GPIIIa. These events were operationally defined as single platelets while all GPIIIa-positive events below the lower FSC-threshold were defined as platelet microparticles, and all events above the upper threshold were considered as platelet microaggregates (31)
. P-selectin and GPIIIa expression were analyzed in the single platelet region. PAC-1 binding was evaluated by incubating WPs with saturating concentrations of PE-labeled anti-GPIIIa and FITC-labeled anti-PAC-1 antibodies. FITC- and PE-isotype matched antibodies were used as controls.
Gal-1 binding to the platelet cell surface was determined by incubation of platelets with biotinylated Gal-1 (bGal-1) for 30 min as described (24)
. After washing, samples were incubated with FITC-streptavidin for 15 min. In some experiments, fixed platelets were incubated with antibodies specific to CD29, CD41, CD42b, CD61, or isotype controls for 30 min, prior to bGal-1 incubation. Platelets incubated with FITC-streptavidin in the absence of bGal-1 were used as controls for nonspecific binding. Phosphatidylserine expression was determined by incubating WPs with FITC-annexin V and PE-labeled anti-GPIIIa.
Platelet-leukocyte mixed aggregate assays
Platelet-leukocyte aggregates were detected as described (32)
. Briefly, whole blood samples (5 µl) were incubated in the presence or absence of Gal-1 in PBS buffer (45 µl) containing leukocyte- and platelet-specific antibodies (FITC-anti-CD45 and PE-anti-GPIIIa monoclonal antibodies). After 10 min, samples were fixed with 1% (w/v) paraformaldehyde and analyzed by flow cytometry. The instrument was set to acquire 10,000 CD45+ events. Total leukocyte-platelet aggregates were categorized into subpopulations of monocyte-, polymorphonuclear (PMN)- and lymphocyte-platelet aggregates according to their specific light scatter profiles and the levels of CD45 expression. Platelet-specific events (GPIIIa+) occurring within these three separate regions were counted as platelet-leukocyte complexes. As a control, the PE-anti-GPIIIa antibody was substituted for PE-IgG1.
Immunoblot analysis, immunocytochemistry, and confocal microscopy
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed in a Miniprotean-II electrophoresis apparatus (Bio-Rad, Hercules, CA, USA). Samples were centrifuged and resuspended in 100 µl ice-cold lysis buffer containing a protease inhibitor cocktail (Sigma). Protein concentration was measured using the Micro-BCA kit (Pierce, Rockford, IL, USA). Equal amounts of protein (30 µg) of cell lysates obtained from platelets of different donors were loaded into each lane of the gel. Samples were electrophoresed on a 15% separating polyacrylamide slab gel, transferred onto nitrocellulose membranes, and probed with a 1:1000 dilution of the rabbit anti-human Gal-1 polyclonal antibody. Blots were then incubated with 1 µg/ml horseradish peroxidase-conjugated anti-rabbit IgG, developed using the ECL detection reagent (Amersham Biosciences, Piscataway, NJ) and exposed to Amersham Hyperfilm for 3 to 5 min. Equal loading was checked by incubation of the blots with a β-actin-specific monoclonal antibody (Sigma). For flow cytometry analysis, cells were fixed, permeabilized, and stained with rabbit anti-human-Gal-1 IgG (1/100 dilution) and a mouse anti-GPIIIa antibody (1/100 dilution) (Dako), followed by FITC- and Cy3-conjugated secondary antibodies, respectively. Nonspecific binding was determined by using control isotype antibodies.
For immunohistochemistry and confocal microscopy, WPs were fixed with 1% (w/v) paraformaldehyde for 15 min at RT and adhered to microscope slides. Cells were then treated with acid ethanol and blocked with 10% (v/v) goat serum, 2% (w/v) BSA for 1 h and incubated with a rabbit anti-human Gal-1 IgG (1/50 dilution) or a rabbit preimmune IgG (same dilution) for 1 h and mouse anti-CD41 in 10% (v/v) goat serum for 1 h at RT. Cells were then incubated with FITC-labeled anti-rabbit IgG (BD Biosciences) and Cy3-conjugated anti-mouse IgG (Jackson Immunoresearch, Baltimore, MD, USA).
To examine the ability of platelet agonists to induce F-actin polymerization, WPs were stimulated with thrombin (0.3 U/ml) or increasing concentrations of Gal-1 (2 to 6 µM) for different time periods. Cells were processed, as described above, and stained with TRICT-phalloidin (10 µM). Stained samples were finally analyzed under an Olympus fluorescent (Olympus, Tokyo, Japan) or Nikon laser confocal microscope (Nikon, Tokyo, Japan) in five randomly selected fields for each experiment, with at least 50 cells counted per condition.
Statistical analysis
All results are expressed as means ± SE. Student’s paired t test was used to determine the significance of differences between means, and values of P < 0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). These results indicate that human platelets express carbohydrate ligands specifically recognized by Gal-1. Remarkably, pretreatment of platelets with ADP or thrombin did not modify the binding of bGal-1 to the platelet surface (data not shown). In addition, incubation of platelets with anti-CD29 (β1 integrin), anti-CD41 (IIb integrin), anti-CD61 (β3 integrin), or anti-CD42b (GPIb) blocking antibodies did not interfere with bGal-1 reactivity (data not shown), suggesting that at least the epitopes recognized by these antibodies do not participate in Gal-1 binding to human platelets.
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To investigate whether binding of Gal-1 to human platelets can influence the activation state of these cells, we studied platelet aggregation by the traditional turbidimetric method. The addition of Gal-1 to WPs induced platelet aggregation in a concentration- and saccharide-dependent manner (Fig. 2
A). To further investigate whether Gal-1 primes platelet responses triggered by a second stimulus, platelets were pretreated for 1 min with 2 µM Gal-1, a concentration that does not induce aggregation per se. As shown in Fig. 2B
, Gal-1 significantly potentiated platelet aggregation and ATP release induced by suboptimal concentrations of thrombin. This priming effect was also observed in PRP, although the required concentrations of Gal-1 were higher than those required for WPs (Fig. 2C
). Interestingly, in Gal-1-pretreated platelets, the first wave of aggregation induced by ADP was converted into a second wave (Fig. 2C
). The sequence of ligand addition did not modify the synergism observed between Gal-1 and thrombin or ADP (data not shown). This synergistic effect of Gal-1 with ADP was completely blocked by pretreatment of platelets with EDTA (Fig. 2C
, right), indicating the requirements of Ca2+ in Gal-1-mediated platelet aggregation and the absence of an agglutination effect induced by this lectin. Thus, Gal-1 is capable per se of triggering human platelet activation, but it also primes human platelets for activation induced by other classical agonists.
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Gal-1 induces P-selectin and GPIIIa expression and triggers conformational changes in GPIIb/IIIa and F-actin polymerization on human platelets
Platelet activation triggered by a number of agonists, including thrombin or collagen results in cell surface expression of -granule proteins, including P-selectin and GPIIIa (1)
. Therefore, we analyzed the ability of Gal-1 to modulate expression of these molecules as reliable markers of platelet activation. Stimulation of WPs with Gal-1 resulted in a concentration- and time-dependent increase in GPIIIa and P-selectin expression on the platelet cell surface (Fig. 3
A–C). Furthermore, Gal-1-induced P-selectin and GPIIIa up-regulation was completely blocked by the specific disaccharide lactose (Fig. 3A, B
), but not by the nonrelated disaccharide sucrose (data not shown).
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Another distinctive feature of platelet activation induced by activating agents is represented by typical conformational changes in the GPIIb/IIIa molecule and the consequent exposure of the high-affinity binding site for fibrinogen; this resulting epitope is recognized by the monoclonal antibody PAC-1 (33)
. Exposure of human platelets to Gal-1 resulted in marked activation of GPIIb/IIIa, an effect that was reflected by higher PAC-1 binding (Fig. 3D
).
Platelet activation also involves critical morphological changes, including extension of filopodia and lamellipodia by F-actin polymerization (2)
. To evaluate the occurrence of these morphological alterations, human platelets were stimulated with thrombin, increasing concentrations of Gal-1, or vehicle control for 1 or 10 min. Cells were then fixed and labeled with TRICT-phalloidin to stain F-actin. Whereas platelets exposed to control vehicle maintained the characteristic discoid morphology of unactivated cells, treatment with increasing concentrations of Gal-1 or thrombin resulted in F-actin polymerization and extensions of filopodia and lamellipodia (Fig. 3E
).
Thus, treatment with Gal-1 results in dose-dependent up-regulation of P-selectin and GPIIIa expression, F-actin polymerization, and the occurrence of conformational changes in GPIIb/IIIa on the surface of human platelets.
Gal-1 promotes the generation of platelet-derived microparticles and microaggregates
As previously shown, when examined by cytofluorometric analysis, resting platelets appear as a single population according to their light-scattering profile (Fig. 4
A), whereas stimulation with activating agonists such as thrombin results in the appearance of two distinct particle populations expressing GPIIIa with different forward light-scattering profiles (31
, 34
, 35)
(Fig. 4A-C
). Interestingly, similar responses were observed when platelets were stimulated with Gal-1 (Fig. 4A-C
). The small particles, considered microparticles with lower light scatter profile (gate R4), expressed substantial amounts of P-selectin (70±10% of positive cells in Gal-1-treated platelets vs. 2±1% in vehicle-treated cells, n=3) and phosphatidylserine (30±3% positive cells in Gal-1-treated platelets vs. 1±0.8% in vehicle-treated cells, n=3). On the contrary, particles displaying higher light scatter profiles (gate R2) represented platelet microaggregates. Importantly, induction of both particle populations depended on the concentration of Gal-1 and was highly sensitive to inhibition by the specific disaccharide lactose (Fig. 4A-C
). Furthermore, Gal-1-induced GPIIIa expression and formation of microparticles and microaggregates were significantly increased by a suboptimal concentration of thrombin (Fig. 5
).
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Signaling pathways involved in Gal-1-mediated modulation of platelet responses
Platelet function is modulated by several activation pathways that are differentially turned on, according to individual agonists and the type of effector responses. Among these signaling pathways, the most relevant include a rise in intracellular Ca2+ levels, thromboxane A2 (TXA2) formation, and protein kinase C (PKC) activation (1)
. We analyzed the role of these intracellular pathways in Gal-1-mediated platelet activation by pretreating human platelets with EDTA, aspirin, and Ro 32–0432 as Ca2+, cyclooxygenase, and PKC inhibitors, respectively. As shown in Table 1
, EDTA completely suppressed the formation of microparticles and microaggregates, as well as the up-regulated expression of P-selectin. However, inhibition of cyclooxygenase or PKC activity did not show any effect on Gal-1-mediated modulation of platelet responses (data not shown). Therefore, Gal-1 elicits Ca2+-dependent mechanisms to modulate human platelet responses.
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Given that inhibition of platelet activation involves an increase in the levels of cyclic nucleotides (36)
, we evaluated the role of these second messengers by using cGMP and cAMP-elevating agents such as the nitric oxide (NO) donor PAPA/NO and PGI2, respectively. Incubation of platelets for 1 min with PAPA/NO or PGI2 before exposure to recombinant Gal-1 significantly prevented shedding of microvesicles and P-selectin up-regulation (Table 1)
. Remarkably, under similar experimental conditions, the formation of microaggregates was not inhibited by either NO or PGI2; however both compounds markedly suppressed the occurrence of microaggregates triggered by lower concentrations of Gal-1 (2–3 µM; data not shown). Our results indicate that an increase in cyclic nucleotide levels may prevent Gal-1-mediated platelet activation in vitro.
Gal-1 triggers the formation of mixed leukocyte-platelet aggregates
It is well established that P-selectin expression on the surface of activated platelets mediates the interaction of platelets with PMN and monocytes, thus triggering multiple intracellular events within leukocytes and platelets that promote vascular inflammation and facilitate atherosclerosis and thrombotic episodes (37)
. Given the up-regulated expression of P-selectin on Gal-1-treated human platelets, we further analyzed the ability of this protein to trigger platelet-leukocyte interactions. Exposure of whole blood to recombinant Gal-1 induced the formation of either PMN-platelet or monocyte-platelet aggregates (Fig. 6
). However, treatment with this lectin was not capable of inducing lymphocyte-platelet interactions (data not shown). Although monocytes appeared to be more sensitive than PMN to bind Gal-1-sensitized platelets (EC50=4.3 and 5.3 µM, respectively) (Fig. 6)
, the percentage of PMN-platelet mixed aggregates were significantly higher than those obtained with monocytes.
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Human platelets express Gal-1, which mediates ADP-induced platelet aggregation
To investigate the impact of endogenous Gal-1 in platelet physiology, we assessed the expression of Gal-1 in human platelets. Substantial amounts of Gal-1 were found on platelets purified from different subjects as shown by immunoblot analysis (Fig. 7
A). Expression of this lectin in platelets was further demonstrated by flow cytometry (Fig. 7B
) and immunofluorescence staining using optical or confocal microscopy (Fig. 7C
). Remarkably, targeted inhibition of endogenous Gal-1 using a blocking anti-Gal-1 serum (Fig. 7D
) or lactose (data not shown), was able to suppress ADP-induced platelet aggregation, suggesting a role for endogenous Gal-1 in human platelet activation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In the present study, we investigated the influence of Gal-1, an endogenous lectin overexpressed at sites of inflammation and tumor growth (7)
, on platelet physiology and examined the expression and functional relevance of this protein in human platelets. Our results indicate that Gal-1 triggers different platelet activation responses, including F-actin polymerization, increase in P-selectin and GPIIIa expression, exposure of fibrinogen-binding sites, shedding of procoagulant microparticles, and formation of homotypic and heterotypic aggregates. Additionally, we show that platelets express Gal-1, which contributes to agonist-induced platelet activation. Interestingly, direct platelet aggregation was observed at relatively high Gal-1 concentrations; these substantial amounts of Gal-1 may occur associated to the extracellular matrix and stromal cells at sites of inflammation and tumor growth (38)
. However, lower concentrations of Gal-1 were capable of priming the aggregation response induced by different physiological agents such as thrombin or ADP, supporting a role for this endogenous lectin in the control of platelet activation. In this regard, a screening of the aggregation properties of several galectins, including the Gal-1 homologues CG-16 (chicken galectin-16) and bovine Gal-1 in different cell types, revealed the ability of these lectins to induce aggregation of neutrophils, thymocytes, and platelets (39)
. Interestingly, Gal-1 treatment resulted in conformational changes in GPIIb/IIIa, which constitutes a prerequisite for platelet cross-linking (40
). This effect was accompanied by morphological changes of human platelets involving extension of filopodia and lamellipodia and F-actin polymerization. Furthermore, the observation that Gal-1-mediated aggregation was completely blocked by pretreatment of platelets with EDTA indicates that this effect is associated with platelet activation and not with a mere agglutination phenomenon.
The major role of P-selectin expression on the platelet surface is the interaction with P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes to form platelet-leukocyte aggregates. This interaction promotes the activation of both cell types, a crucial condition to trigger inflammation, vascular remodeling, and thrombosis (41)
. We found here that Gal-1 induces P-selectin externalization, which contributes to the formation of monocyte- and PMN-platelet aggregates. Similar to other agonists (32)
, platelet-lymphocyte aggregates were not observed following Gal-1 stimulation. Platelet-leukocyte aggregates represent an established link between inflammation and thrombosis in acute syndromes, including coronary diseases and related disorders (42)
. Furthermore, the interaction of P-selectin with PSGL-1 induces the up-regulation of tissue factor in the leukocyte membrane and the production of procoagulant microparticles, thereby contributing to a prothrombotic state (43)
. In addition, a role for P-selectin in platelet aggregation and the formation of arterial thrombi, has also been described (44)
. Thus, it is reasonable to propose that Gal-1-mediated P-selectin expression might play a relevant role in the pathogenesis of thrombus formation and the modulation of inflammatory responses. Interestingly, several reports demonstrated that Gal-1 possess anti-inflammatory properties (20
21
22
23)
. However, it has been recently suggested that according to its concentration, Gal-1 could exert different opposing functions in regulating inflammation (17
, 18)
. Whether anti-inflammatory effects occur at low concentrations of Gal-1, while proinflammatory effects prevail at high concentrations, still remains to be established. Our present findings showing that Gal-1 promotes platelet activation lend support to the notion that under certain circumstances, Gal-1 could also act as a proinflammatory factor.
Vesiculation of the platelet membrane and formation of microparticles are characteristic effects of certain agonists, such as collagen, thrombin, or C5b-9 complement fragment (45)
. Microparticles are released not only during platelet activation in vitro but are also detected in vivo (46
47
48)
. They are thought to provide catalytic surface for several enzyme complexes of the coagulation system and to underlie the procoagulant responses elicited by platelet activation (49)
. Moreover, microparticles might themselves evoke cellular responses in the immediate microenvironment, including platelet adhesion to the site of endothelial injury and angiogenesis (50
, 51)
. Our findings showing that Gal-1 induces platelet microparticles expressing phosphatidylserine suggest that Gal-1 would not only promote platelet activation but might also indirectly activate the coagulation cascade. This result is in agreement with the ability of Gal-1 to promote phosphatidylserine exposure in primed neutrophils, thus favoring leukocyte turnover (52)
. Formation of microparticles by Gal-1 appears to be a Ca2+-dependent event, as it was completely suppressed by pretreatment of platelets with EDTA. In contrast, activation of PKC or generation of TXA2 was not required for vesiculation of the platelet membrane. These results are also consistent with previous work showing that similar mechanisms may operate in microparticle formation triggered by C5b-9 complement fragment (53)
.
Most of the extracellular functions mediated by Gal-1 involve the interaction of this protein with cell surface glycoconjugates containing repeating units of N-acetyllactosamine [Galβ1,4GlcNAc] (6)
. However, there is increasing evidence that protein-protein interactions may also be involved in Gal-1-mediated responses (54)
. The observations that platelet responses elicited by Gal-1 were blocked by lactose, but not sucrose, indicate that these effects involve the interaction of this lectin with specific carbohydrate ligands on the platelet surface. Different cell surface glycoconjugates, as well as extracellular matrix glycoproteins appear to be primary receptors for Gal-1 (55)
. Besides, recent evidence shows that integrins are involved in Gal-1-mediated biological responses. While in vascular smooth muscle cells, the interaction of Gal-1 with the 1β1 integrin modulates attachment, spreading, and migration on laminin (56)
, the binding of Gal-1 to 7β1 integrin regulates myogenesis by inhibiting the association of laminin with integrin (57)
. Given the relevance of different integrins in platelet function, these molecules may represent potential binding partners for Gal-1. However, we found that blockade of IIb, β1, and β3 integrins or GPIb using specific monoclonal antibodies was not capable of interfering with the binding of bGal-1 to the surface of human platelets. Since these antibodies may recognize epitopes that are not implicated in Gal-1 binding, we do not completely rule out the participation of these molecules as potential Gal-1-binding partners. Further experiments are warranted to identify the platelet glycoprotein backbones bearing the discrete sets of oligosaccharide ligands required for Gal-1 binding.
Thrombosis and disseminated intravascular coagulation are common complications in cancer patients (58
, 59)
. A hypercoagulable or prothrombotic state of malignancy occurs due to the ability of tumor cells to activate platelets and the coagulation system. Prothrombotic factors in cancer include the ability of tumor cells to produce and secrete procoagulant/fibrinolytic substances and inflammatory cytokines and the physical interaction between tumor and vascular cells (monocytes, platelets, neutrophils) (60)
. However, the mechanisms allowing the occurrence of prothrombotic states in cancer patients are not completely understood. Given the pivotal role of Gal-1 in tumor progression (7)
, we might speculate that Gal-1-induced platelet activation might contribute to the pathogenesis of thrombosis in cancer patients.
It has been extensively shown that depletion or functional inactivation of platelets through a variety of genetic and pharmacological manipulations, markedly reduces tumor progression and metastasis (61
62
63
64)
. Platelets may influence the metastatic potential of tumor cells via several mechanisms: 1) through the expression of P-selectin, platelets may contribute to the stable adhesion to endothelium and/or transmigration of tumor cells outside of the vasculature (65)
, 2) through the formation of heterotypic aggregates of circulating cancer cells with platelets that may protect tumor cells against immune attack (66)
, and 3) through the release of a variety of inflammatory mediators that may influence tumor growth and stroma formation (1)
. Because Gal-1 expression in tumor or stromal cells is considered an independent predictor of tumor progression associated with immune cell evasion (28
, 55
, 67)
, it is conceivable that Gal-1-mediated platelet activation may represent an alternative mechanism by which platelets contribute to tumor progression and metastasis.
Finally, we provide the first experimental evidence showing that human platelets express substantial levels of Gal-1, which mediates agonist-induced platelet activation. Thus, platelet-derived Gal-1 may represent a novel therapeutic target to interfere with platelet activation in thrombotic processes, atherosclerosis, and cancer. Current studies are being conducted using Gal-1-deficient mice to elucidate the role of Gal-1 in the regulation of platelet functions in vivo under different physiological and pathological conditions.
In conclusion, the present study provides evidence of a novel unrecognized role of galectin-carbohydrate interactions in the regulation of human platelet physiology with potential implications in thrombosis, inflammation, and metastasis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 These authors jointly supervised this work.
Received for publication July 31, 2007. Accepted for publication October 11, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-granules. Blood 94,3791-37997β1 integrin with fibronectin and laminin by L-14 lectin during skeletal muscle differentiation. J. Cell Sci. 107,175-181[Abstract] supports experimental lung metastasis. Proc. Natl. Acad. Sci. U. S. A. 104,9024-9028This article has been cited by other articles:
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  L. V Norling, M. Perretti, and D. Cooper Endogenous galectins and the control of the host inflammatory response J. Endocrinol., May 1, 2009; 201(2): 169 - 184. [Abstract] [Full Text] [PDF]
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