1015

Thrombin Receptor 14-Amino Acid PeptideMediates Endothelial Hyperadhesivity

and Neutrophil Adhesion byP-Selectin-Dependent Mechanism

Yasuo Sugama and Asrar B. Malik

Thrombin cleaves its receptor at arginine-41, resulting in the generation of a new receptor NH2-terminuswith the sequence SFLLRNPNDKYEPF. This peptide (TRP-14) may signal a variety of thrombin'sresponses. We examined the effects ofTRP-14 in inducing endothelial cell hyperadhesivity and neutrophil(PMN) adhesion to endothelial cell monolayers. Human umbilical vein endothelial cells (HUVECs)challenged with TRP-14 (10-4 to 10-5 M) produced concentration-dependent increases in endothelialadhesivity to PMN. In contrast, position 1 to 2 inverted peptide (FSLLRNPNDKYEPF) did not induce theresponse. The adhesion response was transient; that is, PMN adhesion increased within 15 minutes anddecreased by 75 minutes after TRP-14 challenge of HUVECs. The transient endothelial adhesivenessparalleled the time course of P-selectin expression. TRP-14-induced release of P-selectin from intracel-lular stores may be a critical determinant of the response since treatment of endothelial cells withanti-P-selectin monoclonal antibody (mAb) Gl prevented the increase in PMN adhesion. Controlnonneutralizing anti-P-selectin mAb S12 and mAb RR1I/1 directed against intercellular adhesionmolecule-1 (ICAM-1) on HUVECs were ineffective. The results indicate that the "tethered ligand" of thethrombin receptor created by the proteolytic action of thrombin on its receptor (i.e., TRP-14) signalsincreased endothelial adhesiveness by a P-selectin-dependent mechanism. Thrombin-induced PMNadhesion may involve formation of a new NH2-terminus of the endothelial thrombin receptor with thesequence SFLLRNPNDKYEPF followed by activation of endothelial second messenger pathways and thetransient expression of P-selectin. (Circultion Research 1992;71:1015-1019)KEY WoRDs * thrombin * neutrophils * human umbilical vein endothelial cells * endothelial

adhesivity * P-selectin * thrombin receptor * tethered ligand

T hrombin mediates neutrophil adhesion to endo-thelial cells by promoting endothelial hyperad-hesiveness.1-3 Thrombin-induced intravascular

neutrophil sequestration has been implicated as animportant factor in tissue inflammatory responses suchas the adult respiratory distress syndrome.4 The mech-anism of thrombin's action may involve binding ofthrombin to a receptor on endothelial cell membrane5and subsequent activation of second messenger path-ways that in turn can induce the expression of endothe-lial adhesive molecules such as P-selectin.6-8 P-Selectin,a constituent of the electron-dense Weibel-Palade bod-ies, is rapidly released in response to thrombin.79 Stud-ies have shown that the rapid component of the throm-bin-induced increase in neutrophil adhesion (occurringwithin 15-30 minutes) is associated with P-selectintranslocation to the plasma membrane and that the

From the Department of Physiology and Cell Biology, TheAlbany Medical College of Union University, Albany, N.Y.

Supported by grants HL-27016, HL-45638, and HL-46350 fromthe National Heart, Lung, and Blood Institute, National Institutesof Health, Bethesda, Md.Address for reprints: Dr. A.B. Malik, The Albany Medical

College, 47 New Scotland Avenue, Albany, NY 12208.Received March 6, 1992; accepted June 18, 1992.

adhesion event can be inhibited by anti-P-selectinantibodies.7,9The recent cloning of a thrombin receptor indicates

that thrombin binds to its receptor, which it cleavesafter arginine 41 in the receptor's N112-terminal portion,and thereby exposes a new NH2-terminus that has beenproposed to function as a "tethered ligand" for thereceptor.5 The binding of this "ligand" containing theterminal 14-amino acid peptide to an as yet undefinedregion of the thrombin receptor reproduces some ofthrombin's actions.5 In the present study, we investi-gated the role of the 14-amino acid peptide (which canmimic thrombin's effect such as platelet aggregation5and fibroblast proliferation10) in inducing increasedendothelial adhesivity to neutrophils.

Materials and MethodsMaterials

Delbecco's modified Eagle's medium (DMEM), fetalbovine serum, and Hanks' balanced salt solution(HBSS) were obtained from GIBCO, Grand Island,N.Y. The 14-amino acid thrombin receptor peptide(TRP-14) and a control peptide with positions 1 and 2inverted were synthesized by Dr. Thomas T. Andersen,Peptide Synthesis Core, Albany Medical College, Al-

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1016 Circulation Research Vol 71, No 4 October 1992

bany, N.Y. The anti-P-selectin monoclonal antibody(mAb) Gl and a control nonneutralizing anti-P-selectinmAb S12 were kindly provided by Dr. Rodger P.McEver, Department of Medicine, University of Okla-homa.11,12 An anti-intercellular adhesion molecule(ICAM-1) mAb RR1/1 was provided by Dr. RobertRothlein, Boehringer-Ingelheim, Ridgefield, Conn.13All mAbs were directed against the human antigens.

Peptide SynthesisThe 14-amino acid peptide SFLLRNPNDKYEPF

was synthesized as the C-terminal amide on a Biosearch9500 peptide synthesizer using the solid phase methodof Merrifield.14"1 The control peptide, in which theN-terminal position 1 and 2 amino acids were reversed(FSLLRNPNDKYEPF), was also similarly synthesized.tert-Butyloxycarbonyl (tBOC)-derived amino acids(Advanced ChemTech, Louisville, Ky.) were used, andthe side-chain protection was as follows: Ser (Bzl), Arg(Tos), Asp (OBzl), Lys (ClZ), Tyr (BrZ), and Glu(OBzl). The completed peptides were removed from theresin using the low-high method of Tam et al,16 washedwith ethyl acetate, extracted into acetic acid, and lyoph-ilized. The peptides were purified by reverse-phasechromatography on Sep-Pak Cartridges (Waters/Milli-pore, Bedford, Mass.). High-performance liquid chro-matography and amino acid analysis'7 indicated pep-tides of >98% purity and proper composition. Thepeptides were sequenced on a Porton 2090E enhancedgas phase sequencer, and the identified sequence wasthat expected for the synthetic peptides.

Isolation of NeutrophilsHuman polymorphonuclear leukocytes (PMNs) were

isolated from blood of normal volunteers that wassupplied by the American Red Cross, Albany, N.Y.PMNs were isolated within 3 hours of obtaining theblood. The blood was sedimentated with the samevolume of 4% dextran HBSS without Ca2' and Mg2+with 20 mM EDTA (disodium ethylenediamine tetraac-etate) for 40 minutes at room temperature. After thesedimentation, the leukocyte-rich plasma layer was di-luted in the same volume of HBSS with EDTA. TheFicoll-Paque (10 ml) (Pharmacia LKB Biotechnology,Inc., Piscataway, N.J.) layered with diluted leukocyte-rich plasma was centrifuged for 30 minutes at roomtemperature at 1,350 rpm (400g). The pellet containingred blood cells and PMNs was resuspended, and afterhypotonic lysis of red blood cells, the PMNs suspendedin 1 mg/ml bovine serum albumin (BSA), 20 mMEDTA, 25 mM HEPES/HBSS were counted.

Neutrophil LabelingThe isolated PMNs were labeled with 51-sodium

chromate (5"Cr) (New England Nuclear, Boston). ThePMNs were incubated with 1 rCi of `Cr per 106 cells ina 5% CO2 incubator at 37°C for 60 minutes, washedthree times with HBSS, and resuspended on DMEM(2x107 cells/ml).Endothelial CulturesHuman umbilical vein endothelial cells (HUVECs)

were cultured as described.'8 HUVECs were seededonto gelatin-coated 24-well plates (Corning, Corning,N.Y.) and grown to confluency.

Adherence AssayHUVECs were incubated (37°C, 5% C02, and 98%

humidity) for various times in DMEM without serumand with TRP-14. At the end of the incubation period,the endothelial monolayers were fixed with 1% parafor-maldehyde/phosphate-buffered saline at room temper-ature for 15 minutes, and then washed three times withDMEM without serum. The 5"Cr-labeled human PMNs(2x 106 cells/mI DMEM) were distributed at 1 ml perwell over the confluent HUVECs and coincubated for60 minutes at 37°C, 5% CO2, and 98% humidity. Theendothelial monolayers were then gently washed threetimes with DMEM without serum to remove nonadher-ent PMNs. The endothelial monolayers were kept over-night under 1 ml of 1N NaOH at 4°C. The cell lysateswere scraped, collected in polypropylene test tubes, andcounted for radioactivity in a Tm Analytical gammacounter.

Anti-P-Selectin Monoclonal Antibody Binding AssayThe time course of TRP-14-induced expression of

GMP-140 on HUVECs was determined by the bindingof 1"5I-labeled mAb Gl to HUVECs after TRP-14challenge for varying durations. The anti-P-selectinmAb was iodinated using the chloramine-T method.HUVECs grown to confluency in 96-well plates werewashed with DMEM without serum and incubated witheither a-thrombin (10`8 M) (supplied by Dr. JohnFenton, Albany Medical College, Albany, N.Y.) orTRP-14 (10`4 M) for 15, 30, 45, 60, 75, 90, or 120minutes. At the end of the incubation periods, the cellswere fixed with 1% paraformaldehyde/phosphate-buff-ered saline at room temperature for 15 minutes. TheHUVECs were then washed in DMEM containing 10%serum, `'I-labeled mAb Gi (10 ,g/ml) was added withDMEM containing 10% serum (75 1.l per well), and thecells were allowed to incubate for 60 minutes at 4°C.The specific activity of "'I-labeled Gl was 3.75 ,uCi/,ug.The cells were gently washed three times with DMEMwith serum to remove nonbinding 125I-Gl. The cellswere kept overnight in 150 ,ujl of 1N NaOH at 4°C, afterwhich the cell lysate was scraped, collected in test tubes,and counted for radioactivity in a Tm Analytical gammacounter. The specific binding of Gl was determined byadding 30-fold excess unlabeled Gl. The "1`-G1 bindingdata were normalized to cell protein measured in con-trol cells and after either thrombin or TRP-14challenge.

Data AnalysisStatistical analysis was performed with the Student's t

test, andp<0.05 was considered significant. The resultsare given as mean+SEM.

ResultsFigure 1 shows the effects of TRP-14 (the 14-amino

acid peptide that comprises the new NH2-terminus orthe ligand portion of the thrombin receptor) on theexpression of endothelial adhesiveness. The endothelialcells were treated with varying concentrations ofTRP-14 and then fixed with 1% paraformaldehydebefore applying 51Cr-labeled neutrophils. The resultsindicate an increase in endothelial adhesiveness occur-ring between TRP-14 concentrations of 10-' M and

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Sugama and Malik Thrombin-Receptor Peptide 1017

50-

40-

8

2. 30-

.C

z

P 20-0.

10-

0m

Control 1X105M 5x10-5M 1x10-4M

TRP-14

FIGURE 1. Bar graph shows concentration-dependent ef-fects of thrombin receptor peptide (TRP-14) on neutrophiladhesion. Effects of three concentrations of TRP-14 are

shown. Human umbilical vein endothelial cells were treatedwith TRP-14 for 30 minutes and then fixed with 1% parafor-maldehyde after which neutrophil SlCr-labeled neutrophilswere layered onto endothelial cells for 1 hour, and adhesionwas measured as described in 'Materials and Methods."Values are mean ±SEMfrom three experiments. PMN, poly-morphonuclear leukocyte.

5 x 10` M (Figure 1). The response at 10` M TRP-14showed a 12-fold increase in adhesion from basal val-ues. TRP-14-induced expression of endothelial adhe-sivity persisted in the presence of 5% serum (i.e.,TRP-14 [10-4 M for 30 minutes] increased neutrophiladhesion from a basal value of 5.6±1.1% to 49.9±4.1%[n=4]). In contrast, the control peptide FSLLRNPND-KYEPF (10-4 M) did not induce neutrophil adhesion(Table 1).The time course of the increase in endothelial adhe-

siveness is shown in Figure 2. Endothelial cells weretreated with TRP-14 for the indicated periods and fixedwith 1% paraformaldehyde before applying neutrophils.Endothelial adhesiveness increased within 15 minutesafter TRP-14 challenge and decreased to basal levelsafter treatment of endothelial cells for periods longerthan 75 minutes (Figure 2), indicating the reversibilityof the response. Figure 2 also shows the effects oftreatment of endothelial cells with the mAb Gl directedagainst human P-selectin. MAb Gl inhibited neutrophiladhesion to the TRP-14-challenged endothelial cells(Figure 2). In contrast, the anti-P-selectin control mAb

TABLE 1. Comparison of Neutrophil Adhesion Induced WithTRP-14 and a Control Peptide in Which the N-terminal AminoAcids Are Reversed

Peptides PMN adhesion (%)

Basal 0.9+0.1

SFLLRNPNDKYEPF (5 x 10-5 M) 29.1+2.6*

FSLLRNPNDKYEPF (10-4 M) 1.5+0.2

Values are shown as mean+-SEM; n=8 for each group. Humanumbilical vein endothelial cells were fixed with paraformaldehydebefore applying neutrophils (see "Materials and Methods"). Sim-ilar results were obtained in three experiments.

*Different from basal adhesion.

130

z

20

10

Basal 15 30 45 60 75 90

L-TRP-14 (Duration of Exposure in Min)iFIGURE 2. Bar graph shows time course of increase inadhesion after thrombin receptorpeptide (TRP-14) treatmentfor 15, 30, 45, 60, 75, and 90 minutes. After each treatmentperiod, human umbilical vein endothelial cells (HUVECs)were fixed and neutrophils were layered onto the monolayersfor 60 minutes (see "Materials and Methods"). Studies alsoexamined effects of treatment ofHUVECs with the anti-P-selectin monoclonal antibody (mAb) G] (10 pg/ml). GJprevented the TRP-14-induced neutrophil adhesion at eachtime point. Values are shown as mean+SEM; n=8 for eachcondition. PMN, polymorphonuclear leukocyte. *Differencefrom basal values.

S12 and the anti-ICAM-1 mAb RR1/1 were ineffectivein preventing neutrophil adhesion to TRP-14-treatedendothelial cells (Table 2).

Figure 3 shows the specific binding of `PI-labeledanti-P-selectin mAb Gl after challenge with a-throm-bin (a positive control) or TRP-14. An increase inspecific binding of `PI-G1 was evident within 15 minutesafter either a-thrombin or TRP-14 challenge, indicatingrapid P-selectin expression. In both cases, the bindingdecreased to control levels within 75 minutes afterTRP-14 challenge.

TABLE 2. Effects of Anti-GMP-140 Monoclonal Antibody Gl,Control Anti-P-Selectin Monoclonal Antibody S12, and Anti-ICAM-1 Monoclonal Antibody RR 1/1 on PolymorphonuclearLeukocyte Adhesion to Human Umbilical Vein Endothelial CellsTreated for 15 Minutes With Either TRP-14 or

a-Thrombin

PMN adhesion (%)TRP-14 a-Thrombin

Monoclonal antibody (5xl-5 M) (10-8 M)Control 23.9+3.3 21.3±3.2mAb Gl (10 gg/ml) 3.3+0.5* 2.0±0.5*mAb S12 (10 ,g/ml) 22.7± 1.1 18.2± 1.1mAb RR1/1 (10 ,ug/ml) 21.5±+ 2.7 ...

Values are shown as mean±SEM; n=8 for each condition.Human umbilical vein endothelial cells (HUVECs) were fixed with1% paraformaldehyde, and then treated with these antibodies 15minutes before applying polymorphonuclear leukocytes (PMNs).Basal PMN adhesion to fixed non-activated HUVECs was2.3+0.3%. Similar results were obtained in three experiments.TRP-14, thrombin receptor peptide; mAb, monoclonal antibody.

*p<0.05 from control.

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1018 Circulation Research Vol 71, No 4 October 1992

with paraformaldehyde before assessment of P-selectinT -.-- TRP-14 expression and neutrophil adhesion, the TRP-14-in-

e -Thrombin duced increase in endothelial adhesiveness cannot beexplained by secondary mediators released by endothe-lial cells.The neutrophil adhesion response to TRP-14 oc-

curred rapidly (the maximum response was evidentwithin 15 minutes) and was short-lived (the responsedecayed by 75 minutes). The time course of the re-sponse paralleled the expression of P-selectin on thesurface of endothelial cells. Incubation with TRP-14 for

-; ' periods longer than 75 minutes significantly diminishedr . . , , , , , , neutrophil adhesion from its peak value consistent with0 15 30 45 60 75 90 105 120 downregulation of P-selectin. Thrombin is known to

rapidly induce the release of P-selectin from Weibel-Duration of Exposure (min) Palade bodies in endothelial cells resulting in translo-

k Line graph shows binding of '251-labeled anti-P- cation of this adhesive molecule to the plasma mem-ionoclonal antibody GJ to human umbilical vein brane where it promotes neutrophil adhesion.6,7,22 Sincezl cells. Cells were treated with either a-thrombin P-selectin expression occurred within the same time(positive control) or thrombin receptor peptide course as thrombin's response and treatment of the4 . . . paraformaldehyde-fixed endothelial cells with mAb Gl(10- M) for the indicated periods (see 'Materials prevented the TRP-14-induced neutrophil adhesion,

tOds'). Values are shown as mean +-SEM; n-=4 forprvne th R'4idcdnurpi dein

ods"). n TRP-14 and thrombin may activate P-selectin release in(ition. the same manner. The second messengers responsible

for P-selectin upregulation are not known. Increase inDiscussion cytosolic Ca2+ signaled by TRP-14 binding to endothe-

yve previously shown that thrombin is a potent lial cells may be a critical event.23 In the present study,of neutrophil sequestration in the microcircu- the increase in endothelial adhesivity could not berhrombin may mediate its effects by two likely ascribed to ICAM-1 since an anti-ICAM-1 mAb RR1/1sms. Thrombin causes deposition of fibrin in did not alter TRP-14-induced neutrophil adhesion.sels, which provides a substrate for neutrophil The transient neutrophil adhesion response to TRP-14:nt to the intravascular fibrin.19 The "entrap- observed in the present study is different from longer-f neutrophils in the fibrin meshwork may be lasting increase in endothelial adhesivity observed with1 by binding of the neutrophil CD18 integrin to thrombin challenge.1,3 This suggests that thrombin acts byiterreceptor" on fibrin.19 The second mecha- mechanisms, in addition to thrombin's proteolytic activityolves expression of endothelial adhesiveness on this thrombin receptor. There may be other as yety to the upregulation of endothelial cell sur- undefined thrombin receptors or the proteolytic activity ofyecules, P-selectin2-7 and ICAM-1.3 Although thrombin may activate additional endothelial adhesionecules,owPtseaetinthrom andICAM-egAlathouh molecules such as ICAM-1,3 which can sustain the endo-Lave shown that thrombin can upregulate these thelial adhesion response to thrombin.nmolecules,'9 the mechanisms of the response In conclusion, we have shown that TRP-14 (whichplresent study we examined the role of the contains the amino acid sequence of NH2-terminus ofpresent study, we examined the role of the the thrombin receptor after its cleavage at arginine-41)

*acid thrombin receptor peptide (TRP-14), increases endothelial adhesivity and mediates transientitains the sequence of the NH12-terminus of the neutrophil adhesion to endothelial cells by a P-selectin-receptor created by proteolytic cleavage by dependent mechanism. The increase in endothelial ad-of its receptor after arginine-41.5 TRP-14 has hesiveness is associated with upregulation of P-selectinmist properties similar to thrombin such as its on the endothelial cell surface. The results suggest thatWinduce platelet aggregation,5 fibroblast prolif- receptor antagonist peptides directed against TRP-14),20 and prostacyclin generation from endothe- binding sites on the endothelial cell membrane may be21 The present results indicate that this peptide potentially useful anti-inflammatory agents in prevent-apable of inducing an increase in endothelial ing neutrophil adhesion and sequestration in they to neutrophils secondary to the expression of microcirculation.

P-selectin. The control 14-amino acid peptide in whichthe NH2-terminal amino acids in positions 1 and 2 werereversed (FSLLRNPNDKYEPF) did not cause neutro-phil adhesion.The time course of P-selectin expression after chal-

lenge of endothelial cells with TRP-14 or a-thrombinwas similar in that both caused an increase in thespecific binding of the anti-P-selectin mAb Gl within 15minutes of challenge, and the P-selectin expression wasdownregulated within 75 minutes. The latter processmay reflect internalization or "shedding" of the P-se-lectin antigen.7,9 Since the endothelial cells were fixed

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FIGURE 3selectin mendothelia(10-8 M)(TRP-14)and Metheach cond

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Y Sugama and A B Malikneutrophil adhesion by P-selectin-dependent mechanism.

Thrombin receptor 14-amino acid peptide mediates endothelial hyperadhesivity and

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 1992 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

doi: 10.1161/01.RES.71.4.10151992;71:1015-1019Circ Res. 

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