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Am J Physiol Cell Physiol 292: C1323-C1330, 2007. First published November 29, 2006; doi:10.1152/ajpcell.00510.2006
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RECEPTORS AND SIGNAL TRANSDUCTION

Modifications of cellular responses to lysophosphatidic acid and platelet-activating factor by plasma gelsolin

Teresia M. Osborn,1,2 Claes Dahlgren,2 John H. Hartwig,1 and Thomas P. Stossel1

1Translational Medicine Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts; and 2Department of Rheumatology and Inflammation Research, Göteborg University, Gothenburg, Sweden

Submitted 2 October 2006 ; accepted in final form 23 November 2006


   ABSTRACT
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METHODS
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Gelsolin is a highly conserved intracellular actin-binding protein with an extracellular isoform, plasma gelsolin (pGSN). Blood concentrations of pGSN decrease in response to diverse tissue injuries. Depletion of pGSN to critical levels precedes and often predicts complications of injuries such as lung permeability changes and death. Administration of recombinant pGSN ameliorates such complications and reduces mortality in animal models. One proposed mechanism for pGSN's protective effects is that it inhibits inflammatory mediators generated during primary injuries, since pGSN binds bioactive mediators, including lysophospatidic acid (LPA) and endotoxin in vitro. However, no direct evidence in support of this hypothesis has been available. Here we show that recombinant pGSN modestly inhibited LPA-induced P-selectin upregulation by human platelets in the presence of albumin (P < 0.0001). However, physiologically relevant pGSN concentrations inhibit platelet-activating factor (PAF)-mediated P-selectin expression by up to 77% (P < 0.0001). pGSN also markedly inhibited PAF-induced superoxide anion (O2–) production of human peripheral neutrophils (PMN) in a concentration-dependent manner (P < 0.0001). A phospholipid-binding peptide derived from pGSN (QRLFQVKGRR) also inhibited PAF-mediated O2– generation (P = 0.024). Therefore, pGSN interferes with PAF- and LPA-induced cellular activation in vitro, suggesting a mechanism for the protective role of pGSN in vivo.

P-selectin; neutrophils; superoxide production; peptide

GELSOLIN IS A UNIQUE PROTEIN that is expressed as both cytoplasmic and plasma isoforms (pGSN) originating from the same gene on chromosome 9 (30). The 87-kDa pGSN is identical to the cytoplasmic form except for an additional 23-amino acid sequence at the NH2 terminus (30) and is secreted by a processed signal peptide. Cytoplasmic gelsolin is involved in the actin remodeling that drives cell motility. It severs actin filaments (F-actin) in the presence of calcium, tightly caps actin filament fast-growing (barbed) ends, and promotes actin nucleation and elongation in the slow-growing (pointed) direction (1, 27). The gelsolin-actin filament complex can be disrupted at the plasma membrane by binding of D3- and D4-polyphosphoinositide to two distinct sites on gelsolin (17, 28), and the barbed filament ends generated by uncapping in this manner are intimately involved in receptor-mediated actin assembly reactions.

The role of pGSN, present in mammalian blood at very high concentrations (~200 mg/l), is much less clear. One proposed hypothesis is that pGSN, together with Gc-globulin, another extracellular actin-binding protein, functions as an "extracellular actin scavenger system" responsible for the removal of actin released by tissue injury (33). By severing F-actin, pGSN reduces blood viscosity by promoting polymer disassembly, allowing Gc-globulin to sequester free monomeric actin released from actin filament ends (8) for ultimate clearing in the liver (14, 35). Consistent with an actin scavenger role, plasma gelsolin levels diminish at the onset of disease states that involve or result in tissue damage (6, 10, 13, 25, 31, 34, 42, 50), and circulating actin concentrations in excess of pGSN or Gc-globulin have prothrombotic or cytotoxic activities (15, 22).

A second hypothesis relates to pGSN's binding to bioactive mediators, including lysophosphatidic acid (LPA) (40), amyloid beta-protein (Abeta) (4), lipopolysaccharide (LPS) (3), and diadenosine-5',5'''-P1,P3-triphosphate (Ap3A) (55). pGSN may protect against mediator-induced cell activation by acting as a buffering agent. During inflammatory and tissue-damaging conditions, actin exposed to the extracellular environment accumulates pGSN at the site of injury, leading to a decrease in circulating pGSN levels and loss of buffering activity. At sites of local injury, this loss could be beneficial, since these mediators contribute to host defense and repair responses. However, following catastrophic tissue injury, with significant amounts of actin being released to the blood, large quantities of inflammatory mediators no longer subject to buffering by pGSN may induce secondary injury. In support of this idea, the observed decreases in pGSN levels precede and predict more severe complications (31, 42), and pGSN replacement ameliorates such secondary injury in animal models (5, 32, 45).

Although pGSN influences the responses of cardiomyocytes to LPA in vitro (20), it did not significantly diminish the bioactivity of LPA. pGSN also binds to and inhibits certain effects of endotoxin in vitro (3), but recent studies documenting protective effects of pGSN against LPS in vivo showed no inhibitory effect of pGSN on LPS's ability to induce TNF-{alpha} secretion from human monocytes in vitro (32). In this study, we provide evidence that pGSN in combination with albumin can inhibit LPA-induced platelet activation. In addition, we investigated whether pGSN interferes with cellular responses to another bioactive lipid, platelet-activating factor (PAF). PAF is a potent lipid mediator, with biological responses detectable at subnanomolar levels (48). In contrast to LPA, PAF does not circulate as a normal constituent of blood in healthy individuals but is easily detected in plasma from septic and trauma patients, where its concentration can reach 60 nM (39, 58). PAF concentrations might be even higher during these conditions at sites of local inflammation (58). The PAF receptor (PAFR) is expressed on many different cell types, including platelets and neutrophils (26). PAF induces platelet activation and secretion as judged by expression of surface P-selectin (57) and induces a variety of responses including oxygen radical generation in neutrophils. We show that pGSN has a strong inhibitory effect on PAF-mediated activation of human platelets and neutrophils in vitro.


   METHODS
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Materials. The following were used: sodium citrate, NaCl, KCl, and CaCl2 from Fisher Chemicals (Fair Lawn, NJ); trisodium citrate from Mallinckrodt (Paris, KY); Tris from American Bioanalytical (Natick, MA); methanol from VWR Scientific Products; MgCl2·6H2O from EM Science (EM Industries, Cherry Hill, NJ); dextran and Ficoll-Paque from Amersham Biosciences (Pittsburg, PA); Nycodenz from Accurate Chemical and Scientific (Westbury, MA); protease-free, fatty acid-free bovine albumin (faf-BSA) from Serologicals Proteins (Kankakee, IL); TRAP from Bachem Bioscience (King of Prussia, PA); FITC-conjugated anti-human CD62P monoclonal IgG from BD Biosciences (San Diego, CA); horseradish peroxidase (HRP) from Boehringer-Mannheim (Mannheim, Germany); and LPA (18:1; 1-oleoyl-2-hydroxy-sn-glycero-3-phosphate), C16 PAF (1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine), and dioctanoylglycerol pyrophosphate (DGPP; 08:0) from Avanti Polar Lipids (Alabaster, AL). All other chemicals were from Sigma-Aldrich (St. Louis, MO). Recombinant human pGSN was produced in Escherichia coli, refolded with glutathione disulfide (56), formulated in 0.1 M NaCl and 1 mM CaCl2 by Biogen-Idec (Cambridge, MA), and kept at –70°C before use. Gelsolin-derived non-cell-permeant phospholipid-binding 10-mer peptide (9) (QRLFQVKGRR) was a gift from Dr. Paul Janmey (Department of Physiology, University of Pennsylvania, Philadelphia, PA).

Preparation of washed platelets. Blood was obtained by venipuncture from healthy male and female donors into 0.1 vol of Aster-Jandl anticoagulant (85 mM sodium citrate, 69 mM citric acid, 111 mM glucose, pH 4.6). Approval was obtained from the Institutional Review Boards of Brigham and Women's Hospital and Harvard Medical School, and informed consent was obtained from the blood donors in accordance with the Declaration of Helsinki and Title 45, United States Code of Federal Regulations, Part 46, Protection of Human Subjects (effective December 13, 2001). The blood was centrifuged at 150 g at room temperature for 20 min. The platelet-rich plasma was collected, and platelets were concentrated on a gradient consisting of 10% Metrizamide (or Nycodenz) layered over 25% Metrizamide (or Nycodenz) and centrifuged at 1,100 g for 15 min, as previously described (16). The platelets were washed in buffer (140 mM NaCl, 5 mM KCl, 12 mM trisodium citrate, 10 mM glucose, 12.5 mM sucrose, pH 6.0), and the gradient centrifugation was repeated. The cells were resuspended in a buffer solution (140 mM NaCl, 3 mM KCl, 0.5 mM MgCl2, 5 mM NaHCO3, 10 mM glucose, and 10 mM HEPES, pH 7.4) at a concentration of 2 x 108/ml and left standing for 1 h at 37°C before the start of the experiment.

Platelet P-selectin upregulation. Fifty microliters of 2 x 108/ml human platelets were incubated with LPA or PAF for 5 or 2 min, respectively. LPA and PAF were reconstituted in methanol (evaporated before use) at a concentration of 10 mM; 1 mM stock solutions of LPA in PBS containing 0.3 mM faf-BSA were used for further dilutions in the inhibition studies. PAF was resuspended in PBS without faf-BSA. For test of gelsolin inhibition, LPA or PAF were incubated 5–20 min in PBS with or without pGSN in borosilicate vials at room temperature. PAF and pGSN were also added separately to cells (pGSN was added first) to determine whether preincubation of the two is required. In all experiments, lipids were given from a 10x concentration stock to cells at a 1:10 volume ratio. Inhibition with the LPA3 receptor antagonist DGPP was accomplished by preincubating DGPP with platelets for 5 min before addition of LPA. Reactions were stopped by dilution of platelets with 150 µl of 1:250 FITC-conjugated mouse anti-human CD62P antibody in PBS. Cells were labeled for 20 min at room temperature before further dilution with 200 µl of PBS. Each experiment was done in triplicate. P-selectin exposure was quantified by flow cytometry using a Becton Dickinson FACSCalibur with Cellquest version 3.3 software (BD Biosciences, San Jose, CA). TRAP (25 µM), a protease-activated receptor 1 (PAR1) agonist, was used to elicit maximal P-selectin upregulation responses. For analysis, an overlay of the histogram of TRAP-activated cells over that of the resting condition was used to determine P-selectin-positive cells. Cells to the right of where the two histograms overlapped were considered positive for P-selectin. Phospholipid-induced exposure of P-selectin is reported as the percent P-selectin-positive cells. This value was >80% for TRAP and ~60–80% for the highest concentrations of PAF used in the inhibition studies after subtracting the resting values. The percent P-selectin-positive cells in the presence of pGSN was divided by PAF- or LPA-induced (0.5 or 1 µM) P-selectin upregulation in the absence of pGSN x 100 to calculate the relative P-selectin expression.

Isolation of human neutrophils. Neutrophils were isolated by dextran sedimentation and Ficoll-Paque centrifugation (2) of buffy coats obtained from healthy blood donors. The isolated neutrophils were washed and resuspended in a modified Krebs-Ringer phosphate buffer (KRG) composed of 120 mM NaCl, 5 mM KCl, 1.7 mM KH2PO4, 8.3 mM Na2HPO4, 10 mM glucose, 1 mM CaCl2, 1.5 mM MgCl2, pH 7.3, at a concentration of 5 x 106/ml. Cells were maintained at ice bath temperature before experiments and used within 2 h of isolation.

Neutrophil NADPH oxidase activity. An isoluminol (6-amino-2,3-dihydro-1,4-phtalazinedione)-enhanced chemiluminescence assay was used to measure neutrophil production and release of superoxide anions (11). The chemiluminescence activity was monitored over 3–6 min in a six-channel Bioluminat LB 9505 apparatus (Berthold, Wildbad, Germany). Reaction mixtures (900 µl) containing 5 x 105 neutrophils, HRP (4 U), and isoluminol (20 µM) in KRG with glucose were prepared in disposable 4-ml polypropylene tubes. The reaction mixture was equilibrated for 5 min at 37°C before addition of stimuli (100 µl) at concentrations indicated (see Fig. 3), including PAF, phorbol 12-myristate 13-acetate (PMA), N-formyl-Met-Leu-Phe (fMLF) or WKYMVm ± pGSN or 10-mer gelsolin peptide (QRLFQVKGRR). Gelsolin inhibition studies were performed both with and without preincubation with PAF. Six conditions (with or without pGSN) were analyzed simultaneously.


Figure 3
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  		Fig. 3. pGSN decreases PAF-induced superoxide production from human neutrophils. A: effect of pGSN on PAF-induced (1 µM) superoxide production from human neutrophils after preincubation with PAF [solid lines; n = 4 (4 donors)] or no preincubation [dotted lines; n = 4 (one donor)]. Data are expressed as %superoxide production relative to control. PAF in the absence of pGSN is the control. B: representative experiment showing the effect of pGSN on PAF-induced superoxide production. C: effect of 1 µM pGSN on fMLF-, WKYMVm-, and PMA-induced superoxide anion production; n = 3. D: inhibition of the PAF-induced (1 µM) superoxide anion production by a 10-mer peptide (QRLFQVKGRR) containing the phosphatidylinositol 4,5-bisphosphate- and LPA-binding site of gelsolin. The peptide was incubated at a 1:1 ratio with PAF before being added to cells. No inhibition was seen without preincubation; n = 4. Data are means (SD) in A, C, and D. Data in B are raw data. *P < 0.05, **P < 0.01, and ***P < 0.001.

 
pGSN-induced actin nucleation. Pyrene-actin was prepared by derivatizing actin with N-pyrenyliodoacetamide (Invitrogen Molecular Probes, Eugene, OR) following the procedure of Kouyama and Mihashi (29) but using a buffer with MgCl2 (2 mM) instead of CaCl2. Pyrene-actin was depolymerized in buffer A (0.5 mM ATP, 5 mM beta-mercaptoethanol, 0.2 mM Tris, 0.2 mM CaCl2, pH 7.4) at 20 µM at 37°C for 1 h to reach steady state before centrifugation at 250,000 g at 4°C for 30 min in an Optima TL Ultracentrifuge (Beckman). The supernatant fluid was stored at 4°C until use. The effect of different concentrations of PAF on 25 nM pGSN-induced polymerization of 1 µM pyrene-actin in buffer B (0.1 M KCl, 2 mM MgCl2, 1.5 mM CaCl2, 0.5 mM ATP, 10 mM Tris, 0.5 mM beta-mercaptoethanol, pH 7.4) was determined after 5-min preincubation of PAF with pGSN in a 96-well tissue culture-treated polystyrene plate at 100x concentration in buffer F (0.15 M KCl, 2 mM MgCl2, 0.2 mM CaCl2, 0.5 mM ATP, 10 mM Tris, 0.5 mM beta-mercaptoethanol, pH 7.4). Increases in pyrene-actin fluorescences were recorded for 5 min in 6 x 50 mm borosilicate glass culture tubes (Kimble) at excitation and emission wavelengths of 366 and 386 nm, respectively, using a luminescence spectrometer (Perkin Elmer, LS50B). A slope of the rate of the fluorescence increase was obtained, and a ratio of nucleation rate in the presence and absence of phospholipid calculated.

Statistics. Data are expressed as means ± SD. Paired-sample two-tailed Student's t-test was used to evaluate differences between platelet phospholipid responses with and without pGSN or gelsolin peptide. Unless otherwise stated, the n-value represents number of experiments performed and equals number of cell isolations tested. Each experiment was done in duplicate or triplicate. Kaleida Graph version 3.6.4 (Synergy software) was used for statistical calculations. A P value < 0.05 was considered statistically significant. P values < 0.05, <0.01, and <0.001 are indicated (see Figs. 2–4).


Figure 2
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  		Fig. 2. Effects of plasma gelsolin (pGSN) on LPA-, PAF-, and TRAP-induced P-selectin expression by human blood platelets. Data are means (SD). A: effect of pGSN (solid squares) and dioctanoylglycerol pyrophosphate (DGPP; open squares) on P-selectin expression of human blood platelets induced by 1 µM LPA in the presence of 0.3 µM faf-BSA; n = 20 and 7, respectively. Control is LPA in the absence of pGSN. B: effect of pGSN on P-selectin expression of human blood platelets induced by LPA in the absence of faf-BSA; n = 3. Data are raw data. Resting values are subtracted. C: effect of pGSN on P-selectin expression of human blood platelets stimulated with 25 µM TRAP (open diamonds; n = 4), 1 µM PAF (open circles; n = 4), or 0.5 µM PAF (solid circles) after preincubation with pGSN (solid line; n = 6–9) or no preincubation with pGSN (dotted line; n = 7). Control is respective activator in the absence of pGSN. D: effect of calcium on 1 µM PAF (open circles)- and 1 µM LPA (solid squares)-induced P-selectin expression; n = 3. In the inhibition studies (A, C, and D), the resting values were subtracted, and percent P-selectin-positive cells in the presence of pGSN was divided by the PAF (0.5 or 1 µM)-, LPA-, or TRAP-induced P-selectin upregulation in the absence of pGSN x 100 to calculate the relative P-selectin expression. *P < 0.05, **P < 0.01, and ***P < 0.001.

 

Figure 4
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  		Fig. 4. Effect of PAF (solid circles) and LPA (open squares) on 25 nM pGSN-induced pyrene-actin nucleation. Data are shown as ratio of PGSN-induced nucleation in the presence of PAF or LPA over pGSN-induced nucleation in the absence of phospholipid; n = 5 – 9 and 4 for PAF and LPA, respectively. Data are means (SD). Significance of increase in nucleation by PAF is indicated over pGSN alone. *P < 0.05, **P < 0.01, and ***P < 0.001.

 

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Effect of faf-BSA on LPA-induced platelet P-selectin expression. The presence of faf-BSA at a molar ratio of 30:1 to 1:10 LPA to faf-BSA increased LPA-induced (1 µM) P-selectin expression on human platelets (fig. 1A). However, faf-BSA concentrations >30 µM decreased responses. faf-BSA had no effect on TRAP-mediated responses and produced a significantly smaller biphasic effect than that observed with LPA on PAF-mediated (1 µM) responses. At PAF concentrations of <100 nM, the presence of faf-BSA increased the response compared with a no faf-BSA condition (data not shown). On the basis of these data, experiments with pGSN were performed in the presence of faf-BSA (0.3:1 ratio of faf-BSA to LPA) when activating with LPA but in the absence of faf-BSA when activating with PAF.


Figure 1
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  		Fig. 1. Dose responses of lysophospatidic acid (LPA)- and platelet-activating factor (PAF)-induced P-selectin expression analyzed by flow cytometry. PAR1 activation by TRAP was used to elicit maximal P-selectin upregulation responses. Resting values are subtracted. Data are means (SD). A: effect of fatty acid-free bovine albumin (faf-BSA) on 1 µM PAF (open circles)-, 25 µM TRAP (open diamonds)-, and 1 µM LPA (solid squares)-induced P-selectin expression by human blood platelets. Arrow indicates faf-BSA concentration chosen for subsequent LPA studies; n = 4, 4, and 6 for PAF, TRAP, and LPA, respectively. B: effect of LPA (in the presence of BSA) on P-selectin expression by human blood platelets. LPA induces responses of variable magnitudes among different donors; n = 4, 8, and 3 for high (open squares), medium (solid squares), and low (half-open squares) responders, respectively. C: effect of PAF on P-selectin expression by human blood platelets; n = 6. No significant individual variation in the response to PAF was observed in our blood donor population.

 
Individual variation in P-selectin expression in response to phospholipid. We observed three distinct groups of responders to LPA-induced P-selectin expression on human platelets and classified them as high, medium, and low (Fig. 1B). Such individual variations in LPA-induced platelet aggregation have previously been reported (24). The inhibition experiments were performed on the largest group, the medium responders, whose platelets at a concentration of 2 x 108/ml showed ~40% P-selectin upregulation when subjected to 1 µM LPA (in the presence of 0.3 µM faf-BSA). Most subjects consistently responded well to PAF, and a maximum PAF-induced P-selectin response could be observed at 100 nM PAF (Fig. 1C).

Effect of pGSN on LPA-induced P-selectin expression. pGSN inhibited the platelet response to LPA (1 µM) as monitored by P-selectin expression in the presence of faf-BSA (Fig. 2A); 1 µM pGSN had a modest inhibitory effect, and 10 µM pGSN, which is about four times the normal circulating level, decreased the response to 63% (SD 13) (n = 20; P < 0.0001). The LPA3 receptor antagonist DGPP (8:0) (18) incubated at a concentration of 30 µM with platelets for 5 min reduced the response of 1 µM LPA to 47% (SD 22), and at 100 µM the response was 22% (SD 14) (n = 7; P < 0.0001). As previously reported (20), pGSN slightly enhanced the presentation of LPA to cells in the absence of faf-BSA (n = 3, P = 0.046; Fig. 2B).

Effect of pGSN on PAF-induced P-selectin expression. Compared with LPA-induced P-selectin expression, pGSN exerts a much greater dose-dependent inhibition of PAF-induced P-selectin expression (Fig. 2C). One or five micromolar PAF-induced P-selectin upregulation was inhibited with 20 µM pGSN by 70% (SD 12) (n = 4; P = 0.0013) or 77% (SD 16) (n = 8; P < 0.0001), respectively. Without preincubation of PAF with pGSN, the inhibition was 42% (SD 23) for 0.5 µM PAF at the highest pGSN concentration (n = 7; P = 0.002). pGSN had no effect on TRAP-induced P-selectin expression (n = 4). Because LPA may aggregate in the presence of Ca2+, and the stock pGSN solution (200 µM) contains CaCl2 (1 mM), we tested the effect of CaCl2 on LPA- and PAF-induced P-selectin expression. The inhibition of phospholipid-induced P-selectin expression seen in the presence of pGSN was not due to the presence of Ca2+ in the pGSN buffer, since no inhibition was observed after incubation with the doses of CaCl2 used during the experiments (n = 3; Fig. 2D).

Effect of pGSN on PAF-induced O2– production by neutrophils. Neutrophils were used to further investigate the importance of the interaction between PAF and pGSN. pGSN also effected a concentration-dependent inhibition of PAF-induced superoxide production by human neutrophils as assayed by chemiluminescence. Physiologically relevant concentrations (~1.5–3 µM) of pGSN, approximately equimolar to the added PAF, caused marked inhibition of chemiluminescence (n = 4, P < 0.0001; Fig. 3, A and B). Without preincubation of pGSN with PAF, pGSN inhibited PAF-induced O2– production (n = 4; one donor), albeit less effectively. The fMLF (1 µM)-, WKYMVm (1 µM)-, or PMA (100 nM)-induced superoxide production was not significantly inhibited by 1 µM pGSN (n = 3; P = 0.80, P = 0.55, and P = 0.42, respectively; Fig. 3C), but 10 µM pGSN induced an ~50% decrease in the PMA response (data not shown).

The gelsolin-derived 10-mer peptide, QRLFQVKGRR, containing the phosphoinositide-binding (9) and LPA-binding (40) region in segment 2 of gelsolin also inhibited PAF-induced superoxide production at a 1:1 molar ratio (n = 4, P = 0.024; Fig. 3D).

Effect of PAF on pGSN-induced actin severing and nucleation. Phospholipids such as LPA (40), PIP2 (17, 28), and endotoxin (3) interfere with gelsolin's actin-severing function. No effect of PAF on pGSN's actin-severing activity was observed in our studies (data not shown). However, PAF increased the pGSN-induced nucleation of pyrene-actin. The increase was dependent on the amount of PAF and saturated between a 5- and 10-fold excess of PAF over pGSN (n = 5–9, P = 0.00018; Fig. 4). LPA did not have such an effect (n = 4; P = 0.36). PAF had no effect on actin polymerization in the absence of pGSN (data not shown).


   DISCUSSION
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Our findings show that pGSN can inhibit physiological in vitro responses of human platelets and neutrophils to two bioactive phospholipids associated with inflammation, LPA and PAF (21, 47, 49), and therefore represent the first empirical evidence in support of the mediator buffer hypothesis to explain beneficial effects of pGSN on inflammation and injury in vivo (fig. 5).


Figure 5
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  		Fig. 5. pGSN mediator buffer hypothesis. Tissue injury 1) disrupts membrane barriers between cytoplasmic actin filaments and the extracellular environment and 2) elicits production of bioactive mediators such as LPA and PAF. 3) pGSN, albumin, and presumably other proteins bind these mediators, inhibiting their ability to engage receptors of inflammatory cells and activate them. 4) Local pGSN depletion due to its entrapment on actin filaments in cells permeabilized by injury releases this inhibition. In massive injury with extensive tissue actin exposure or in sepsis where actin-containing microvesicles accumulate in the circulation, pGSN depletion takes place to a critical level such that unbuffered mediators inflict catastrophic secondary injury, including death.

 
LPA and PAF interact with heterotrimeric GTPase-coupled cell surface receptors (49, 54), and the lack of inhibitory effects of pGSN on responses to unrelated stimuli (TRAP and fMLF) indicates that the suppressive actions of pGSN result from a diversion of the ligands from their cellular receptors. The documented high-affinity binding of LPA to pGSN (20, 40) is consistent with this conclusion. As reported by others attempting to investigate interactions of PAF and proteins (7), we were unable to document saturable binding of PAF to pGSN by equilibrium dialysis or association with pGSN-coated beads, presumably because of solubility difficulties of the lipid. However, a decapeptide encompassing a phospholipid-binding site of pGSN also inhibited PAF-mediated activation of neutrophils, and PAF promoted the actin filament nucleation activity of pGSN, consistent with direct protein-lipid interactions. Previous studies with gelsolin-binding lipids have shown little effect on this nucleating function, documenting instead a marked inhibition of actin filament severing (3, 28, 40, 41). Although it is unlikely that the nucleating effect has a physiological relevance, it does indicate a direct interaction between PAF and human recombinant pGSN.

These differences between the functional biochemical actions of various phospholipids on pGSN-actin interactions may parallel variations in the effects of pGSN on cellular responses produced by these lipid mediators. Whereas the pGSN inhibition of P-selectin upregulation by LPA was modest and detectable only in the presence of albumin, inhibition of increased P-selectin expression in response to PAF was nearly complete and did not require albumin. Several possible reasons could explain these discrepancies. First, a universal problem with lipid mediators is their solubility, meaning that their composition, preparation, and ultimate environment can affect results. Second, platelets contain more than one class of LPA receptors, each responding differently to the presence of albumin (23), but only one type of PAF receptor (49). We observed interindividual variation in the extent of pGSN-mediated inhibition of PAF-induced P-selectin expression, although we did not investigate this phenomenon further.

PMA (43) or fMLF induces PAF production by neutrophils in the presence of calcium (38). However, in the absence of albumin (37) and within 5 min of stimuli, as in our assay, it is not likely that this PAF is contributing significantly to the superoxide production; thus the lack of inhibition on these mediators was expected.

In conclusion, pGSN, at levels found in human blood, caused marked inhibition of PAF-mediated platelet and neutrophil inflammatory responses in vitro, implying that this inhibition could be physiologically important. This inhibition might be more pronounced in vivo where PAF concentrations are well below the concentrations used in this study (39). The partial inhibition of LPA-induced platelet activation by pGSN, much weaker than that produced by the small molecule receptor antagonist (DGPP), is more difficult to assess. The albumin requirement to bring out this inhibition, however, consistent with the well-characterized interaction between LPA and albumin (52, 53), suggests that the inhibition may be accentuated at physiological but experimentally impractical albumin concentrations.

In contrast to LPA, PAF does not circulate as a normal constituent of blood in healthy individuals because of strict regulation of its production and rapid hydrolysis in plasma by the enzyme PAF-acetylhydrolase. In sepsis and trauma, however, PAF becomes detectable in plasma (39, 58), and platelets isolated from patients with sepsis have been reported to have reduced PAF-binding capacity, which may result from downregulation of the receptors (36). Although evidence that PAF is important in numerous models of severe inflammation is abundant (58), direct molecular intervention has thus far been unsuccessful in human trials (12, 44, 46, 51). In this respect, the modest clinical benefits of PAF inhibition in human sepsis resonate with a similar lack of success of therapies targeted to other individual mediators such as TNF-{alpha} (19). Because of its ability to bind several inflammation inducers, pGSN replacement might represent a more promising approach to prevention of secondary injury and death in sepsis and other severe injuries.


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T. M. Osborn was a recipient of the Göteborg University Jubileumsfond's stipend.


   ACKNOWLEDGMENTS
 
We thank Drs. Kurt Barkalow, Po-Shun Lee, Eric Osborn, Neal Pinckard, Gabor Tigyi, and Susan Weintraub for valuable help and discussions and Dr. Huamei Fu for help with chemiluminescence control experiments.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. M. Osborn, Translational Medicine Division/Dept. of Medicine, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115 (e-mail: tmagnuson{at}rics.bwh.harvard.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.


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 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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