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Circulation. 1996;93:2152-2160

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(Circulation. 1996;93:2152-2160.)
© 1996 American Heart Association, Inc.

Articles

Plasmin Promotes an Endothelium-Dependent Adhesion of Neutrophils

Involvement of Platelet Activating Factor and P-Selectin

G. Montrucchio, MD; E. Lupia, MD; A. De Martino, PhD; L. Silvestro, MD; S. Rizea Savu, MD; G. Cacace, PhD; P.G. De Filippi, MD; G. Emanuelli, MD; G. Camussi, MD

From the Dipartimento di Fisiopatologia Clinica (G.M., E.L., A.D., G.E.), Istituto di Nefro-Urologia (G.C., S.R.S.), Università di Torino, Italy; IBMP-Institut fur Biomedizinische und Pharmazeutische (L.S.), Heroldsberg, Germany; Servizio di Medicina Nucleare (G.C., P.G.D.), Ospedale Molinette, Torino, Italy; and Cattedra di Nefrologia, Dipartimento di Scienze Cliniche e Biologiche (G.C.), II Facoltà di Medicina, Università di Pavia, Varese, Italy.

Correspondence to Dr G. Camussi, Cattedra di Nefrologia, Dipartimento di Scienze, Cliniche e Biologiche, Viale Borri 57, 3100 Varese, Italy.


*    Abstract
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Background The adhesion of leukocytes to the endothelium and the edema of vessel wall may cause vascular reocclusion after thrombolytic therapy. The aim of this study was to evaluate the role of platelet activating factor (PAF) and P-selectin on the adherence of polymorphonuclear neutrophils (PMN) to the endothelium and of PAF on the increased vascular permeability induced by tissue-type plasminogen activator, streptokinase, and plasmin.

Methods and Results We studied (1) the adhesion of 111In-labeled PMN to human umbilical cord vein–derived cultured endothelial cells (HUVEC), (2) the transfer of 125I-labeled albumin across HUVEC monolayers, and (3) the adhesion of PMN to isolated bovine coronary arteries under flow conditions. It was found that the adhesion of PMN, induced by tissue-type plasminogen activator, streptokinase, and plasmin, correlated with the synthesis of PAF by HUVEC and was inhibited by WEB 2170, a PAF receptor antagonist. The adhesion of PMN was also inhibited by the treatment of HUVEC with anti–P-selectin antibodies or of PMN with soluble P-selectin or with anti-CD18 monoclonal antibodies. Plasmin also increased the permeability of HUVEC monolayers, an effect that was partially prevented by WEB 2170. Moreover, plasmin promoted the synthesis of PAF from isolated bovine coronary arteries and the adherence of PMN to the endothelium under flow conditions. The pretreatment of PMN with WEB 2170 or with soluble P-selectin prevented adhesion.

Conclusions The synthesis of PAF by endothelial cells at the site of plasmin generation and the endothelial expression of P-selectin may render the endothelial cell surface proadhesive for neutrophils and may favor a local increase in vascular permeability.


Key Words: leukocytes • platelet-derived factors • endothelium • cells


*    Introduction
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 *Introduction
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Thrombolytic therapy in patients with myocardial infarction induces intravascular generation of PAF,1 a phospholipid mediator that may limit the beneficial effect of thrombolysis.2 PAF synthesized by endothelial cells is in fact a potential candidate for local reocclusion as the result of its action on platelets and leukocytes. PAF is an acetylated ether lipid mediator that possesses a number of biological activities relevant in the development of inflammatory reactions.3 4 Several evidences indicate that this mediator is also involved in ischemia-reperfusion injury,5 6 7 8 9 a condition in which the recruitment of PMN is critical.10 11 12 Recently, PAF was shown to act as a mediator of the rapid and transient adhesion of the PMN to the endothelium induced by several stimuli such as thrombin, histamine, and elastase.13 14 15 16 17 It was proposed that in these experimental conditions, PAF is instrumental in the functional upregulation of adhesion molecules.18 In addition, PAF, which is known to enhance vascular permeability,19 20 21 may contribute to the increase in vascular permeability caused by thrombolytic therapy.22

The aim of this study was to evaluate whether the synthesis of PAF induced by TPA, SK, and plasmin and the endothelial expression of P-selectin may promote the adherence of PMN to the endothelium and may enhance the permeability of endothelial cell monolayers.


*    Methods
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 *Methods
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Materials
Human plasmin, thrombin, {alpha}2-AP, and lysophosphatidyl-choline palmitoyl (C16-LPC) were obtained from Sigma Chemical Co. {alpha}1-PI was purchased from Athens Research and Technology. TPA (Actilyse) was provided by Boehringer Ingelheim. SK (Streptase) was obtained from Behringwerke AG. Synthetic alkyl-PAF C16:O (1-hexadecyl-2-acetyl-sn-glyceryl-3-phophorylcholine), alkyl-PAF C18:O (1-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine), and 2-lyso-PAF C16:O (1-hexadecyl-glyceryl-3-phophorylcholine) were obtained from Bachem Feinchemikalien. 1-O-[3H]-Alkyl-PAF C16:O (1-O-[3H]hexadecyl-2-acetyl-sn-glyceryl-3-phophorylcholine)1-O-[3H]-alkyl-PAF C18:O (1-O-[3H]octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) was purchased from Du Pont-NEN. 1-Palmitoyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (acyl-PAF C16:O) and [14C]-palmitoyl-2-acetyl-sn-glyceryl-3-phosphorylcholine ([14C]-acyl-PAF C16:O) were obtained by acetylation of 1-palmitoyl-sn-glyceryl-3-phosphorylcholine and [14C]-palmitoyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (55 mCi/mmol, Amersham International) with acetic anhydride and dimethyl-aminopyridine as previously described.23 1-O-Hexadecyl-2-[d3]acetyl-sn-glyceryl-3-phophorylcholine ([d3]alkyl-PAFC16:O) and 1-palmitoyl-2-[d3] acetyl-sn-glyceryl-3-phosphorylcholine ([d3] acyl-PAF C16:O) were synthesized, respectively, by acetylation of 2-lyso-PAF C16:O and 1-palmitoyl-sn-glyceryl-3-phosphorylcholine with deuterated acetic anhydride (Aldrich Chemie) in the presence of pyridine as previously described.24 WEB 2170 (Boehringer) was used as PAF receptor antagonist.25 mAb anti–P-selectin (anti–GMP-140), anti–ICAM-1, and anti-CD18 were from Becton Dickinson. P-selectin (GMP-140) was purified from washed human platelets as described by Johnston et al.26 Rabbit anti-human factor VIII was from Sigma. Fluorescein-labeled phalloidin (F-PHD) was from Sigma. Mouse monoclonal antivinculin IgG was from Chemicon International. Fluorescein-conjugated rabbit anti-mouse IgG and anti–HLA DR antibodies were purchased from Beckton Dickinson. TLC plates (60F254) were obtained from Merck. Culture media were obtained from Flow Laboratories. 111In-oxine and [3H]-acetic anhydride were purchased from Amersham International. 125I-labeled human serum albumin (HSA) was obtained from Sorin Biomedica.

Cell Preparations
HUVEC were cultured and characterized as previously described.27 Confluent monolayers of HUVEC at the second to third passages were used for adhesion and PAF synthesis. In selected experiments, bovine aortic endothelial cells (BAEC) were prepared, cultured, and characterized according to Yannariello-Brown et al.28

Human PMN were prepared from healthy donors by differential centrifugation and gelatin sedimentation, followed by osmotic shock as previously described.29 Smears of cells stained with May-Gruenwald-Giemsa showed 85% to 90% PMN. The viability of PMN, determined by trypan blue exclusion, was always >95%. Approximately 55 µCi of 111In-oxine was added drop-wise to 2 mL of PMN suspension and allowed to incubate at room temperature for 10 minutes. After centrifugation at 1400g for 10 minutes, PMN were resuspended in Tris-buffered Tyrode's solution containing 0.5% HSA. The labeling efficacy was {approx}90%.

PMN Adhesion to HUVEC
HUVEC were washed with M199 medium containing 0.5% HSA and incubated at 37°C with control buffer or an agonist for variable periods of time. The incubation medium was then removed, the monolayers were washed twice, and 111In-labeled PMN were added. Adhesion was studied in static and nonstatic conditions. To perform the adhesion assay in nonstatic conditions, the plates with HUVEC were placed on a platform rotator (80 rpm) and 50-µL aliquots of the cell suspension were added to the wells at 37°C for 8 minutes, as described by Spertini et al.30 After the incubation periods, nonadherent PMN were removed, HUVEC were washed twice with the incubation medium, the adherent radiolabeled PMN were solubilized for 10 minutes with 1N NaOH and 1% sodium dodecyl sulfate, and net percent adhesion was determined by counting radioactivity of the adherent PMN fraction and calculating the percentage of total radioactivity added. The enhanced PMN adherence is expressed as percentage variation over net percent adhesion values of control untreated cells (relative adhesion-to-control ratio). In some experiments, HUVEC or PMN were preincubated with WEB 2170 (5 µmol/L) before the coincubation. When indicated, HUVEC were preincubated with anti–P-selectin mAb (10 µg/mL) or anti–ICAM-1 (10 µg/mL) and PMN with anti-CD18 mAb (20 µg/mL) or with soluble P-selectin (8 µg/mL).

Immunofluorescence Studies
Fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG (Cappel) were used to detect binding of mAb anti–P-selectin and anti-CD18 by indirect immunofluorescence performed as previously described.17 For study of the cell cytoskeleton, coverslip-attached HUVEC were fixed for 5 minutes in 3% paraformaldehyde in PBS (pH 7.6) containing 2% sucrose and permeabilized with HEPES–Triton X-100 buffer (20 mmol/L HEPES, pH 7.4, 300 mmol/L sucrose, 50 mmol/L NaCl, 3 mmol/L MgCl2, and 0.5% Triton X-100).20 F-actin was then stained with 2 µg/mL of F-PHD for 30 minutes at 37°C.31 Vinculin was detected by indirect immunofluorescence with mouse monoclonal antivinculin IgG and a rabbit FITC-conjugated anti-mouse IgG as secondary antibody.20

Permeability Studies
HUVEC were grown to confluence on polycarbonate filters (pore size, 0.4 µm) of Transwell chamber assemblies (Costar) coated with fibronectin. Permeability of HUVEC monolayers was measured by diffusion of 125I-albumin.20 The upper chamber was filled with 0.5 mL of Iscove's medium containing 0.1 µCi 125I-albumin. Fluid volumes were selected to avoid hydrostatic pressure gradient across monolayers. The chambers were incubated at 37°C in 5% CO2 with continuous agitation with trypan blue–albumin complex, prepared as described by Rotrosen and Gallin.32 Monolayers with no leakage of the dye after 5 minutes of incubation were used for the experiments. The transport of albumin across HUVEC monolayers was determined by sampling aliquots and measuring the radioactivity in the outer and inner wells in duplicate.

Preparation of Bovine Coronary Arteries
The bovine hearts were removed within 10 minutes after death, and the circumflex coronary arteries were dissected and immersed in cold HEPES-buffered Tyrode's solution (pH 7.4, NaCl 137 mmol/L, KCl 4.0 mmol/L, NaH2PO4 1.8 mmol/L, MgCl2 0.5 mmol/L, CaCl2 2.0 mmol/L, HEPES 5.0 mmol/L, glucose 5.5 mmol/L). The arteries were placed in a bath filled with warmed (37°C), oxygenated buffer and then perfused with oxygenated buffer for 60 minutes at a constant flow (0.5 mL/min) with a syringe infusion pump (Perfusor Secura, B. Braun, Melsungen AG). Vessels were then perfused for 15 minutes with buffer (with and without 1 U/mL plasmin), washed with buffer alone for 5 minutes at a constant flow of 1 mL/min, and exposed to 107/mL 111In-labeled PMN at a constant flow (0.5 mL/min) for 20 minutes. In some experiments, the arteries were perfused with buffer containing PMN pretreated with 10 µmol/L WEB 2170 or 8 µg/mL soluble P-selectin. After perfusion with PMN, the lumen of vessels was washed again with buffer at a constant flow (1 mL/min) for 5 minutes to remove nonadherent PMN. Total vessel radioactivity (in counts per minute, cpm) was normalized by vessel wet weight (grams). Data are expressed as percentage of the cpm values of vessel perfused with buffer alone (relative adhesion-to-control ratio).

Assay and Quantification of PAF
PAF was quantified by bioassay on washed rabbit platelets after extraction and purification on TLC (silica gel 60, F254, Merck; solvent system: chloroform/methanol/water 65:35:6 vol/vol/vol) and HPLC (µPorasil Millipore Chromatographic Division, Waters; mobile phase, chloroform/methanol/water 60/55/5 vol/vol/vol; flow rate, 1 mL/min) as previously described.27 The recovery of radioactive standards submitted to the same procedures of TLC and HPLC purification of biological samples was, respectively, 95% to 97% for 1-O-[3H]-alkyl-PAF C16:O, 96-98% for 1-O-[3H]-alkyl-PAF C18:O, and 79% to 82% for[14C]-acyl-PAF C16:O. The specificity of platelet aggregation was inferred from the inhibitory effect of 3 µmol/L WEB 2170.25 PAF bioactivity was not inhibited by phospholipase A1, thus suggesting a relation to alkyl-PAF rather then to acyl-PAF, which is known to be more than 1000 times less active than the alkyl-species of PAF.4 The bioactive material was further characterized as PAF on the basis of TLC and HPLC behavior and of physicochemical characteristics1 27 33 such as inactivation by strong bases and phospholipase A2 but resistance to phospholipase A1 and acidic treatment. After TLC and HPLC purification, PAF-bioactive material also was analyzed by a recently developed technique based on HPLC (reverse phase column Spherisorb C18, 5 µm, 100x1-mm internal diameter; mobile phase, methanol/isopropanolol/hexane/0.1 mol/L aqueous ammonium acetate 100/10/2:5 vol/vol/vol; flow rate, 75 µL/min)-MS/MS.24 33 Mass spectrometric analysis was performed under MS/MS conditions by parent ion scan or by MRM. Fragmentation was obtained by collision with argon at a collision gas concentration of 2.7x1012 atm/cm3 and at an impact energy of 70 eV. Parent ion spectra, positive mode, were obtained from daughter ions with m/z 184 corresponding to the phosphocholine fragment; the scanning range was m/z 100 to 600. In the MRM analysis, acquired in positive mode, the study of different PAF molecular species was performed using the following reactions (parent ions->daughter ions): alkyl-PAF C16:O, 524->183.8; alkyl-PAF C18:O, 552->183.8; and acyl-PAF C16:O, 538->183.8. The analysis of samples was compared with data obtained by analysis of the following standards: alkyl-PAF C16:O, alkyl-PAF C18:O, and acyl-PAF C16:O. In selected experiments of plasmin-stimulated HUVEC (n=3), [d3] alkyl-PAF C16:O and [d3] acyl-PAF C16:O were added as internal standards to verify the recovery after extraction and TLC purification.34 The fragmentations 527->184.8 and 541->184.8 were monitored to detect, respectively, [d3] alkyl-PAF C16:O and [d3] acyl-PAF C16:O. The comparison of peak areas obtained by MRM chromatograms obtained before and after extraction and TLC purification allowed estimation of recovery of 92.66±1.45% for [d3] alkyl-PAF C16:O and 81.66±1.40% for [d3] acyl-PAF C16:O.

Statistical Analysis
All data are expressed as mean±SEM. Statistical analysis was performed by Student's t test, by one-way ANOVA for repeated measures, or by ANOVA with either Dunnett's or Newman-Keuls multiple comparison test where appropriate. Correlation between PAF synthesis by HUVEC and PMN adherence was evaluated by linear regression analysis.


*    Results
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 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
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Plasmin stimulated the synthesis of PAF by HUVEC and the adhesion of PMN to HUVEC monolayers as tested in static conditions (Fig 1Down). The amount of PAF synthesized by HUVEC and the percentage of PMN adhesion were dose-dependent. Moreover, a correlation between the amount of PAF synthesized by HUVEC and the adherence of PMN was observed (Fig 1Down, inset). Fig 2Down shows that the time course of PMN adhesion paralleled that of plasmin-induced PAF synthesis by endothelial cells. A slight increase in PMN adhesion was also induced by SK and TPA (Fig 3Down); both were able to stimulate PAF synthesis by HUVEC.1 PAF remained almost completely associated with cells, since no significant release of PAF was detected in the supernatants. PAF bioactivity detected by washed rabbit platelet aggregation and inhibited by WEB 2170, a specific PAF receptor antagonist, should be attributed to the 1-alkyl derivatives of PAF, since the platelet aggregation bioassay was relatively insensitive to acyl derivatives.4 Moreover, PAF-bioactive material was insensitive to treatment with phospholipase A1, which cleaves the acyl-PAF but not alkyl-PAF.4 33 35 To evaluate the efficiency of phospholipase A1 treatment, three samples containing PAF-bioactive material were added with [14C]-acyl-PAF before treatment with phospholipase A1.35 The amount of [14C]-acyl-PAF hydrolyzed (recovered as a free fatty acid) was 89±3.05, whereas the biological activity was not significantly reduced (90±2.51% recovered activity). These results suggest that the aggregation of rabbit platelets in the biological samples was due to the presence of alkyl-PAF rather than acyl-PAF. When added to HUVEC, PAF-bioactive material, extracted from plasmin-stimulated endothelium (4 ng/mL), induced an enhanced adhesion of PMN (183±7%) that was not inhibited by treatment with phospholipase A1. Bioactive PAF produced by HUVEC stimulated with plasmin, SK, and TPA was studied by HPLC-MS/MS. Fig 4Down shows the MS/MS parent ion spectra obtained from the more relevant peaks observed in a sample of bioactive PAF extracted from HUVEC stimulated with plasmin. In the first spectrum (Fig 4ADown), the main relevant parent ion detected was m/z 538, which corresponded to acyl-PAF C16:O. In the second spectrum (Fig 4BDown), it detected the parent ions with m/z 524 corresponding to alkyl-PAF C16:O. In the last spectrum (Fig 4CDown), the only relevant parent ion had a m/z of 552, corresponding to alkyl-PAF C18:O. Fig 5Down shows the HPLC-MS/MS chromatograms obtained by MRM from standards and samples of HUVEC stimulated with plasmin, SK, and TPA, respectively. The chromatograms shown correspond to alkyl-PAF C16:O, alkyl-PAF C18:O, and acyl-PAF C16:O. The analysis of all samples of stimulated HUVEC showed chromatographic peaks, with MS/MS fragmentation and retention time corresponding to the standards alkyl-PAF C16:O, alkyl-PAF C18:O, and acyl-PAF C16:O. The results reported in Fig 6Down were obtained by analysis of a sample of HUVEC stimulated with plasmin and added to deuterated alkyl-PAF C16:O and acyl-PAF C16:O as internal standards. The MRM chromatograms showed a good recovery and detectability of these molecules. Since previous studies indicated that {approx}82% to 90% of PAF synthesized by endothelium was acyl-PAF,36 37 we evaluated whether the acyl-PAF exogenously added may interfere with the adherence of PMN to the endothelium. Synthetic alkyl-PAF C16:O (2x10-8 mol/L), acyl-PAF C16:O (2x10-7 mol/L), and a mixture of alkyl-PAF C16:O and acyl-PAF C16:O in a 10-fold greater amount (alkyl-PAF C16:O, 2x10-8 mol/L; acyl-PAF C16:O, 2x10-7 mol/L) was added to HUVEC before PMN adhesion. As shown in Fig 7Down, acyl-PAF neither stimulated significant PMN adhesion nor interfered with adhesion induced by alkyl-PAF. Moreover, we found that pretreatment of PMN with WEB 2170 inhibited PMN adhesion induced by alkyl-PAF. The PAF receptor antagonist WEB 2170 also significantly reduced the adherence of PMN to the endothelium treated with SK, TPA, (Fig 3Down), or plasmin (Fig 8Down). As shown in Fig 8Down, plasmin-induced PMN adhesion was inhibited by pretreatment of HUVEC with monoclonal antibodies to P-selectin. Moreover, pretreatment of PMN with soluble P-selectin almost completely blocked the adhesion of PMN to HUVEC prestimulated with plasmin. These results suggest that expression of P-selectin on the cell surface may act with PAF in the stimulation of PMN adherence. The expression of P-selectin on the surface of plasmin-treated HUVEC was confirmed by immunofluorescence (Fig 9Down). PMN adhesion also was inhibited by pretreatment of HUVEC with anti–ICAM-1 mAb and by pretreatment of PMN with anti-CD18 mAb. Thus, the adhesion of PMN required the interaction of PMN CD18 integrins with the correspondent endothelial counterreceptors. Clustering with formation of small patches of CD18 antigen on the surface of PMN was observed during adhesion to HUVEC pretreated with plasmin, and it was prevented by WEB 2170 (Fig 10Down). The ability of plasmin to stimulate the adhesion of PMN to the endothelial cells was significantly reduced also by preincubation of HUVEC with {alpha}2-AP (100 µg/mL), a specific inhibitor of plasmin enzymatic activity, and to a lesser extent with {alpha}1-PI (10 µg/mL), a serine proteinase inhibitor (Fig 8Down). Both {alpha}2-AP and {alpha}1-PI reduced the synthesis of PAF triggered by plasmin (1 U/mL plasmin alone, 4.09±0.69 ng PAF; {alpha}2-AP + plasmin, 0.85±0.38 ng PAF; {alpha}1-PI + plasmin, 1.23±0.52 ng PAF). In contrast, when {alpha}2-AP or {alpha}1-PI was added to HUVEC after stimulation with plasmin immediately before the addition of PMN, no significant inhibition of adhesion was observed (Fig 8Down). Because PMN adhesion assayed under static conditions may not reflect the situation under flow conditions, experiments were performed by a rotational endothelial leukocyte attachment assay.30 Also in these experimental conditions, plasmin was shown to induce PMN-endothelium adherence, which was inhibited both by pretreatment of PMN with WEB 2170 and with soluble P-selectin (Fig 11Down).



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  		Figure 1. A, 111In-labeled PMN adhesion to HUVEC unstimulated (C) or stimulated at 37°C with thrombin (THR, 2 U/mL for 5 minutes) or different doses of plasmin (PLN, 0.01 to 1 U/mL for 15 minutes). The enhanced PMN adherence is expressed as percentage variation over net percent adhesion values of control untreated cells (8.44±1.49). B, PAF synthesis by HUVEC unstimulated (C) or stimulated with thrombin (2 U/mL for 5 minutes) or different doses of plasmin (0.01 to 1 U/mL for 15 minutes). PAF was expressed as nanograms of PAF associated with 5x105 cells. Data are mean±SEM of six experiments. ANOVA with Dunnett's multiple comparison test was performed (*P<.05 vs control). Linear regression analysis between the amounts of PAF synthesized and the percentage of PMN adherence for different doses of plasmin at 15 minutes is shown in the inset (r=.827, P<.01).



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  		Figure 2. Time course of 111In-labeled PMN adhesion to HUVEC (A) and of PAF synthesis by HUVEC (B) unstimulated (open columns) or stimulated (shaded columns) for various periods of time with plasmin 1 U/mL. The enhanced PMN adherence is expressed as percentage variation over net percent adhesion values at time 0 (relative adhesion-to-control ratio). PAF was expressed as nanograms of PAF associated with 5x105 cells. Data are mean±SEM of five experiments. ANOVA for repeated measures between stimulated and unstimulated HUVEC was significant (P<.01).



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  		Figure 3. Adhesion of 111In-labeled PMN to HUVEC stimulated with SK (100 U/mL) or TPA (1000 ng/mL) for 15 minutes at 37°C in the absence (open columns) or the presence (shaded columns) of 5 µmol/L WEB 2170. The enhanced PMN adherence is expressed as percentage variation over net percent adhesion values of control untreated cells (8.18±1.27). Data are mean±SEM of five experiments. Student's t test vs cells treated with SK or TPA in the absence of WEB 2170 was performed (*P<.05).



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  		Figure 4. Mass spectra, parent-ion mode, obtained during HPLC separation of TLC-purified PAF samples obtained from HUVEC stimulated with plasmin. Confluent HUVEC (150-mm diameter Petri dish) were stimulated for 15 minutes at 37°C with 1 U/mL plasmin in M199 containing 0.25% BSA. After extraction, PAF was purified by TLC and analyzed by HPLC-MS/MS as described in "Methods." Similar spectra were obtained in three separate experiments. Rel.Int.% indicates percentage of relative intensity.



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  		Figure 5. Analysis by HPLC-MS/MS of synthetic standards (A) and of PAF molecular species synthesized by HUVEC stimulated with plasmin (B), SK (C), and TPA (D). The following standards were used: alkyl-PAF C16:O (10 ng/mL), alkyl-PAF C18:O (10 ng/mL), and acyl-PAF C16:O (10 ng/mL). HUVEC were stimulated for 15 minutes at 37°C with 1 U/mL plasmin, 100 U/mL SK, or 1000 ng/mL TPA in M199 containing 0.25% BSA. After extraction, PAF was purified by TLC and analyzed by HPLC-MS/MS. Chromatograms were performed at the following MRM conditions (parent ion->daughter ion): (1) alkyl-PAF C16:O, 524->183.8; (2) alkyl-PAF C18:O, 552->183.8; and (3) acyl-PAF C16:O, 538->183.8. The absolute ion count is obtained according to absolute ion count=(value of relative intensity % of the chromatogram/highest value relative intensity % in the y-axis)xvalue of ion count. Similar results were obtained in three individual experiments. Rel.Int.% indicates percentage of relative intensity.



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  		Figure 6. MRM chromatograms obtained by analysis of a sample of HUVEC stimulated with plasmin (1 U/mL) for 15 minutes at 37°C. Three nanograms of [d3] alkyl-PAF C16:O and [d3] acyl-PAF C16:O was added immediately before the extraction. Besides the chromatographic traces corresponding to alkyl-PAF C16:O and acyl-PAF C16:O, the fragmentations 527->184.8 and 541->184.8 corresponding to [d3] alkyl-PAF C16:O and [d3] acyl-PAF C16:O were detected.



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  		Figure 7. 111In-labeled PMN adhesion to HUVEC unstimulated (C) or stimulated at 37°C for 5 minutes with alkyl-PAF C16:O (2x10-8 mol/L), acyl-PAF C16:O (2x10-7 mol/L), or alkyl-PAF C16:O (2x10-8 mol/L) and acyl-PAF C16:O (2x10-7 mol/L) and effect of pretreatment of PMN with WEB 2170 (5 µmol/L) on adhesion induced by treating HUVEC with alkyl-PAF C16:O (2x10-8 mol/L). The enhanced PMN adherence is expressed as percentage variation over net percent adhesion values of control untreated cells (5.05±1.44). Data are mean±SEM of four experiments. ANOVA with Newman-Keuls multiple comparison test was performed (*P<.05 vs C, #P<.05 vs alkyl-PAF C16:O).



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  		Figure 8. Effect of pretreatment of HUVEC with anti–P-selectin mAb (10 µg/mL), anti–ICAM-1 mAb (10 µg/mL), {alpha}2-AP (100 µg/mL), or {alpha}1-PI (10 µg/mL) and effect of pretreatment of PMN with WEB 2170 (5 µmol/L), soluble P-selectin (8 µg/mL), or anti-CD18 mAb (20 µg/mL) on the adhesion of 111In-labeled PMN to HUVEC stimulated with plasmin (PLN) 1 U/mL for 15 minutes. The enhanced PMN adherence is expressed as percentage variation over net percent adhesion values of control untreated cells (5.10±1.03). Data are mean±SEM of six experiments. ANOVA with Dunnett's multiple comparison test was performed (*P<.05 vs plasmin 1 U/mL). In the experiments indicated as {alpha}2-AP+PLN and {alpha}1-PI+PLN, the {alpha}2-AP and {alpha}1-PI were added to HUVEC 5 minutes before the stimulation with plasmin. In the experiments indicated as PLN+{alpha}2-AP and PLN+{alpha}1-PI, {alpha}2-AP and {alpha}1-PI were added to HUVEC after the stimulation with plasmin.



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  		Figure 9. Surface expression of P-selectin on HUVEC unstimulated (A) or stimulated with plasmin 1 U/mL for 15 minutes (B). Magnification x400.



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  		Figure 10. Immunofluorescence staining with anti-CD18 mAb of paraformaldehyde-fixed PMN. A, Linear distribution of the antigen on the surface of PMN incubated with unstimulated HUVEC; B, clustering of CD18 antigen with granular distribution in PMN incubated with HUVEC prestimulated with plasmin 1 U/mL; and C, linear distribution of CD18 antigen in PMN treated with 5 µmol/L WEB 2170 before incubation with plasmin (1 U/mL) stimulated HUVEC. Magnification x800.



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  		Figure 11. Adhesion in nonstatic conditions of 111In-labeled PMN to HUVEC unstimulated (C) or stimulated at 37°C for 15 minutes with 1 U/mL plasmin (PLN) with or without addition of 5 µmol/L WEB 2170 or 8 µg/mL soluble P-selectin. HUVEC plates were placed on a platform rotator (80 rpm) and 50-µL aliquots of 111In-labeled PMN were added to the well at 37°C for 8 minutes, as described by Spertini et al.30 The enhanced PMN adherence is expressed as percentage variation over net percent adhesion values of control untreated cells (5.74±1.19). Data are mean±SEM of six experiments. ANOVA with Newman-Keuls multiple comparison test was performed (*P<.05 vs C, #P<.05 vs plasmin).

As shown in Fig 12Down, plasmin increased the passage of albumin across HUVEC monolayers. Pretreatment with WEB 2170 inhibited, at least in part, the increased permeability induced by plasmin. The alteration of barrier function of endothelial cells may depend on changes in cytoskeletal organization leading to cell retraction and formation of intercellular gaps. As shown in Fig 13Down, after stimulation with plasmin, the peripheral F-actin bands became indistinct and central stress fibers contracted into dense microfilaments; finally, they tended to disappear. Moreover, vinculin streaks, corresponding to areas of focal contact of the ventral membrane with adhesion substratum, disappeared after treatment with plasmin and the vinculin staining became more diffuse in the cytoplasm. Pretreatment with WEB 2170 prevented the cytoskeletal changes induced by 0.1 U/mL plasmin. However, WEB 2170 had no evident effect on the morphology of HUVEC (data not shown) when 1 U/mL of plasmin was used to stimulate cells, even though the reduction of albumin transfer across the monolayer was statistically significant (Fig 12Down). To evaluate PMN adhesion to coronary endothelium, bovine circumflex coronary arteries were used. In preliminary experiments, the ability of BAEC to synthesize PAF after plasmin stimulation was established (unstimulated BAEC, 0.2±0.1 ng/5x105 cells; plasmin 1 U/mL–treated BAEC, 4.1±0.6 ng/5x105 cells). Moreover, bovine coronary arteries perfused ex vivo with 1 U/mL plasmin synthesized PAF (0.8±0.1 ng/g dry wt), which all remained associated with the endothelial cell surface and could be eluted after perfusion with methanol. No PAF was detected in control bovine coronary arteries perfused with the buffer alone. As shown in Fig 14Down, the pretreatment of coronary arteries with 1 U/mL plasmin increased adherence of 111In-labeled PMN to the luminal surface of the coronary arteries compared with the control perfused with the buffer without plasmin. WEB 2170 and soluble P-selectin significantly reduced the increase of PMN adherence induced by plasmin.



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  		Figure 12. Effect of the PAF receptor antagonist WEB 2170 on the percent transfer of 125I-albumin across HUVEC monolayers untreated (C) or treated at 37°C with plasmin (PLN, 0.1 and 1 U/mL for 15 minutes) or PAF (10 nmol/L for 5 minutes). Open columns indicate HUVEC untreated with WEB 2170; shaded columns, HUVEC pretreated with 5 µmol/L WEB 2170. The transfer of labeled albumin across the monolayer was evaluated as described in "Methods." Mean±SEM of three experiments performed in triplicate is shown. ANOVA with Newman-Keuls multiple comparison test was performed (*P<.05 vs control; #P<.05 for HUVEC treated with WEB 2170 vs the untreated).



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  		Figure 13. Distribution of F-actin and vinculin in fixed and permeabilized HUVEC. Control HUVEC show an elaborate array of microfilament bundles of the stress fiber type (A) and numerous vinculin-containing streaks (D). After 15-minute treatment with plasmin (0.1 U/mL), HUVEC progressively lose their network of stress fibers with accumulation of F-actin at the periphery of the cells and in correspondence to ruffles; arrows indicate areas of lateral cell margin retraction with formation of intercellular gaps (B). Treatment with plasmin makes vinculin streaks disappear, and a diffuse perinuclear distribution of vinculin is seen (E). Pretreatment with WEB 2170 (5 µmol/L, 5 minutes) inhibited the redistribution of F-actin (C) and vinculin (F) induced by plasmin. Magnification x400.



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  		Figure 14. Adhesion of 111In-labeled PMN to isolated bovine coronary artery treated with plasmin (PLN; 1 U/mL, 15 minutes) in absence or presence of WEB 2170 (5 µmol/L) or soluble P-selectin (8 µg/mL). Data are expressed as percentage of the cpm values of vessel perfused with buffer alone (6066±1343 cpm/g wet wt). Data are mean±SEM of four experiments. ANOVA with Newman-Keuls multiple comparison test was performed (*P<.05 vs C; #P<.05 vs plasmin).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The present study shows that PAF may, at least in part, mediate the adhesion of PMN to the endothelium and the enhancement of vascular permeability induced by plasmin. This interpretation is based on the observation that the adhesion of PMN to the endothelium was correlated with the amount of PAF synthesized by stimulated HUVEC. Moreover, the pretreatment of PMN with a PAF receptor antagonist significantly inhibited the adhesion of PMN to the endothelial cells. Previous studies have shown that endothelial cells activated by thrombin or other agonists induce a rapid and transient adherence of PMN that requires the concomitant expression on the endothelial surface of PAF and P-selectin and the activation of CD11/CD18 integrins on the PMN surface.13 18 It was suggested that PAF and P-selectin would concur to functional activation of CD11/CD18 adhesion molecules.18 Furthermore, it has been shown that PAF synthesized by or added to endothelial cells has a dual action: first, it may stimulate, via PAF receptors, the endothelial cells themselves by inducing surface expression of P-selectin and loss of sulfated proteoglycans from the glycocalyx17 ; second, it may contribute to the stimulation of functional upregulation of CD11/CD1818 by promoting the clustering of these molecules in the PMN plasma membrane, leading to an increased focal density of binding sites.17

The present study demonstrates that plasmin acts on endothelial cells with a mechanism similar to that described for thrombin,18 ie, it induces an early endothelial cell–mediated adhesion of PMN. The expression of an endothelial cell–dependent adhesion occurs in the first 15 minutes and decreases thereafter. However, while the effect of thrombin is exhausted within 30 minutes, the plasmin-induced synthesis of PAF and the adhesion of PMN persist, significantly enhanced, for up to 240 minutes. Our study was focused on the role of endothelium in inducing PMN adhesion. However, it should be considered that in vivo plasmin also may act directly on PMN. Indeed, Lo et al38 have demonstrated in coincubation experiments a PMN-endothelial cell adhesion dependent on the action of plasmin on PMN. Therefore, plasmin may induce an adhesion dependent on the activation of endothelial cells that peaks at 15 minutes and persists up to 240 minutes and an adhesion dependent on the activation of PMN that peaks at 60 minutes and persists up to 30 minutes after removal of plasmin.

Moreover, our results indicate that plasmin can stimulate both PAF synthesis and surface translocation of P-selectin, a membrane glycoprotein constitutively present in endothelial cell vesicles. Previous studies demonstrated that about 82% to 90% of PAF synthesized by HUVEC stimulated with thrombin or A23187 was acyl-PAF.36 37 However, we observed that synthetic acyl-PAF did not enhance PMN adhesion to the endothelium and that phospholipase A1 treatment, which is known to hydrolyze acyl-PAF, did not significantly inhibit PMN adhesion induced by biological samples. This suggests that the alkyl-PAF was, in our experimental conditions, the main effector of PMN adhesion to the endothelial cells. However, adding exogenous alkyl-PAF or acyl-PAF to endothelial cells to promote PMN adherence does not necessarily mimic all the biological potential of endogenously synthesized PAF, which can both act as an intracellular mediator and be expressed on the endothelial cell surface to activate PMN. It is therefore possible that endogenously synthesized acyl-PAF may contribute to the activation of adherent PMN, as it has been shown that it increases cytosolic calcium concentrations.39 In our experiments, pretreatment of PMN with WEB 2170 inhibited the adhesion of PMN to the endothelium stimulated with alkyl-PAF, thus suggesting that the exogenously added alkyl-PAF either remained associated with the endothelial cell surface to activate PMN or stimulated, as previously reported, an endogenous synthesis of PAF.40

As demonstrated by Loran et al,18 P-selectin, which is coexpressed with PAF on the endothelial cell surface, acts with this phospholipid mediator in the activation of CD11/CD18 on the PMN surface. In fact, anti–P-selectin mAb or WEB 2170 alone induced only partial inhibition of PMN adhesion to plasmin-stimulated HUVEC, whereas together they almost completely prevented the phenomenon. P-selectin may serve to bind PMN to the endothelial surface, thus allowing a close contact between PAF exposed on the cell surface and the receptors on PMN. Indeed, it has been demonstrated that soluble P-selectin inhibits PMN adhesion to plasmin-treated HUVEC as previously described for thrombin or histamine.18 Moreover, P-selectin and PAF have been implicated in the leukocyte-induced vasoconstriction and endothelial cell dysfunction of isolated cat coronary arteries41 and in the recruitment of PMN during ischemia-reperfusion injury.9 42 43 These observations are consistent with an active role of endothelial cells and of PAF in the recruitment of PMN in ischemia-reperfusion injury. Thrombolytic therapy by generation of plasmin44 and thrombin45 may stimulate both the synthesis of PAF and the expression of P-selectin, inducing an endothelial cell–dependent early adhesion of PMN that may favor local endothelial cell injury and reocclusion. Indeed, we found that the plasmin-induced PAF synthesis and the PAF-dependent and P-selectin–dependent adhesion of PMN occur not only in static conditions but also under conditions of flow in isolated bovine coronary arteries as well as in a rotational endothelial leukocyte attachment assay. During thrombolytic therapy, besides plasmin, transient generation of thrombin45 may stimulate the synthesis of PAF by the endothelium and the adhesion of leukocytes.13 Moreover, the reperfusion that follows successful thrombolysis may promote the generation of oxygen radicals.46 They may in turn induce synthesis of PAF15 and endothelial cell injury, thus promoting the adhesion of PMN to vessels.15 47 Furthermore, it has been reported that thrombolytic therapy increases vascular permeability.22 The experiments reported herein demonstrate that plasmin induces changes in cytoskeletal organization and loss of barrier function similar to that previously described for PAF.20 The inhibitory effect of WEB 2170 suggests that these phenomena are at least in part dependent on an autocrine effect of PAF produced by the endothelial cells.

Conclusions
PAF synthesized by endothelial cells at the site of plasmin generation may render proadhesive the endothelial cell surface, thus favoring an early recruitment of inflammatory cells, which may be instrumental either in the coronary reocclusion after thrombolysis or in the myocardial injury occurring after ischemia-reperfusion.


*    Selected Abbreviations and Acronyms
 
{alpha}2-AP = {alpha}2-antiplasmin
{alpha}1-PI = {alpha}1-proteinase inhibitor
HPLC = high-performance liquid chromatography
HPLC-MS/MS = HPLC–tandem mass spectrometry
HUVEC = human umbilical cord vein–derived cultured endothelial cell(s)
mAb = monoclonal antibodies
MRM = multiple reactions monitoring
PAF = platelet activating factor
PMN = polymorphonuclear neutrophil(s)
SK = streptokinase
TLC = thin-layer chromatography
TPA = tissue-type plasminogen activator


*    Acknowledgments
 
This work was supported by the National Research Council (CNR), targeted project "Prevention and Control of Disease Factors," subproject "Causes of Infective Diseases" (CT 9500778.PF41 to G. Camussi); and by the Associazione Italiana per la Ricera sal Cancro (to G. Camussi).

Received August 10, 1995; revision received December 4, 1995; accepted January 2, 1996.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

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