Platelet-Leukocyte Cross Talk in Whole Blood

Platelet-Leukocyte Cross Talk in Whole BloodNailin Li, Hu Hu, Malin Lindqvist, Eva Wikström-Jonsson, Alison H. Goodall, Paul Hjemdahl

Abstract—Thrombosis and inflammation involve complex platelet-leukocyte interaction, the details of which are not fullyelucidated. Therefore, we investigated cross talk between platelets and leukocytes in whole blood, under the followingphysiological conditions: at 37°C, with normal calcium concentrations, and with shear force. Platelet P-selectin andleukocyte CD11b expression were used to monitor platelet and leukocyte activation, respectively, and platelet-leukocyteaggregation (PLA) was analyzed. The leukocyte-specific agonistN-formyl-methionyl-leucyl-phenylalanine (1026

mol/L) increased P-selectin–positive platelets from 2.560.1% to 5.160.6% (P,0.05). The increase was inhibited byeither the platelet-activating factor (PAF) antagonist SR27417, the superoxide anion scavenger superoxide dismutase,the 5-lipoxygenase inhibitor Zileuton, or the 5-lipoxygenase–activating protein inhibitor MK-886, suggesting theinvolvement of PAF, superoxide anion, and 5-lipoxygenase products in leukocyte-induced platelet activation. Theplatelet-specific agonist collagen (1mg/mL) increased leukocyte CD11b expression from 2.9460.52 to 3.8160.58(P,0.05); this was not inhibited by the thromboxane A2 receptor antagonist ICI 192.605 or the PAF antagonistSR27417. Platelet P-selectin expression induced byN-formyl-methionyl-leucyl-phenylalanine and leukocyte CD11bexpression induced by collagen could be suppressed by glycoprotein IIb/IIIa blockade or P-selectin blockade. This studydocuments platelet-leukocyte cross talk under conditions that mimic a physiological state and suggests that this involvesmultiple mediators and mechanisms. Furthermore, new evidence of integrin and selectin involvement in intracellular andintercellular signaling during platelet-leukocyte cross talk is provided.(Arterioscler Thromb Vasc Biol. 2000;20:2702-2708.)

Key Words: plateletsn leukocytesn platelet-leukocyte aggregatesn platelet-leukocyte cross talkn whole blood

Thrombosis and inflammation are closely related patho-physiological processes with multicellular activation in-

volving platelets, leukocytes, and endothelial cells. Evidenceis accumulating that there are complex interactions, or “crosstalk,” between these cells. Thus, the functional states of thecells involved may be influenced by a variety of stimulatoryor inhibitory mediators, and the end result will depend on thebalance between these influences.

On activation, leukocytes respond with degranulation,increased respiratory bursts, chemotaxis, and phagocytosis.All of these processes may be influenced by platelets.1 Forinstance, platelet-released adenine nucleotides and platelet-derived growth factor (PDGF) may induce leukocyte degran-ulation. Adherent platelets,2 platelet-derived microparticles,3

and platelet-released substances, such as PDGF, plateletfactor 4, and thromboxane A2 (TXA 2), may enhance leuko-cyte rolling and adhesion to the vessel wall. Platelets bound toneutrophils4 and platelet-released adenine nucleotides maypromote superoxide anion (O2

2) generation by neutrophils.Furthermore, platelet factor 4 and PDGF are chemotactic andmay enhance phagocytosis by neutrophils and monocytes.1

By contrast, intact platelets may inhibit neutrophil O22

generation and cytotoxicity, whereas leukocyte chemotaxis,

adhesion, and O22 generation may be inhibited by platelet-released NO5 and soluble P-selectin.6 Thus, platelets andplatelet-released products may influence leukocyte functionin a complex manner.

Similarly, platelet activation may be influenced by leuko-cytes.1 Leukocytes per se and leukocyte-released O2

2 mayenhance platelet adhesion. Furthermore, leukocyte-releasedsubstances, such as O2

2, platelet-activating factor (PAF),elastase, and cathepsin G, may induce platelet aggregationand secretion. Conversely, unstimulated or weakly activatedleukocytes may also attenuate platelet aggregation vialeukocyte-released NO7 and/or ADPase.8 Neutrophil-derivedelastase may bring about proteolysis of the GP Iba subunit,9

which contains the von Willebrand factor binding site, andthus influence platelet adhesion.

Transcellular metabolism also contributes to the cross talkbetween platelets and leukocytes. Thus, neutrophils can useplatelet-released arachidonic acid to synthesize metabolites,such as leukotriene (LT)B4,10 that are not produced byplatelets alone because they lack 5-lipoxygenase. Conversely,platelets can use neutrophil-derived precursors to synthesizeLTC4

11 and lipoxin A412 and thus greatly enhance their

production. Cell-cell adhesion via selectins and integrins may

Received April 10, 2000; revision accepted August 7, 2000.From the Department of Medicine (N.L., H.H., M.L., E.W.-J., P.H.), Division of Clinical Pharmacology, Karolinska Hospital, Stockholm, Sweden, and

the Division of Chemical Pathology (A.H.G.), University of Leicester, Glenfield Hospital, Leicester, UK.Correspondence to Paul Hjemdahl, MD, PhD, Professor, Department of Medicine, Division of Clinical Pharmacology, Karolinska Hospital, SE-171

76 Stockholm, Sweden. E-mail [email protected]© 2000 American Heart Association, Inc.

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also promote transcellular eicosanoid biosynthesis.13,14 Fur-thermore, platelets and leukocytes can produce PAF, butcoincubation of activated platelets and activated neutrophilsfurther enhances PAF-acether generation.15

As visualized previously,16 platelets and leukocytes mayform platelet-leukocyte aggregates or conjugates (PLAs),mainly via platelet-expressed P-selectin and its receptorsP-selectin glycoprotein ligand-1 (PSGL-1) and CD15, as wellas via fibrinogen bridging between glycoprotein (GP) IIb/IIIaand CD11b/CD18. The heterotypic conjugation may facilitateplatelet-leukocyte interaction. For example, platelet-monocyte conjugation may enhance thrombin generation, andconjugated platelets may facilitate leukocyte rolling, adhe-sion, and migration in vivo17 and in vitro.18

Previous studies have mostly been performed on isolatedcells19,20 and have thus neglected the possibly importantinfluences of red blood cells and plasma components on theseinteractions. Studies in whole blood21 were conducted in thepresence of citrate, ie, with subphysiological calcium concen-trations, which may alter platelet responses. Thus, previousresults may not have reflected the true physiological state.

Therefore, we investigated platelet-leukocyte cross talkunder conditions designed to mimic physiological conditions,ie, in whole blood, at 37°C, with physiological calciumconcentrations, and with stirring to induce a low shear force,which is likely to mimic the venous shear state. We used theleukocyte-specific agonistN-formyl-methionyl-leucyl-phenylalanine (fMLP) and the platelet-specific agonist colla-gen to induce leukocyte and platelet activation, respectively.We used a panel of antagonists to investigate possiblemediators involved in the cross talk and monoclonal antibod-ies (MAbs) that block platelet-leukocyte aggregation to elu-cidate the impact of heterotypic conjugation. Platelet-leukocyte cross talk was monitored by studies of plateletP-selectin expression and leukocyte CD11b expression by useof whole blood flow cytometry and methodology involvingminimal artifacts.

MethodsSubjectsTwenty-four healthy subjects (11 men and 13 women, aged 23 to 55years) gave informed consent to participate in the present study,which was approved by the ethics committee of the KarolinskaInstitute.

ReagentsThe platelet agonist used was equine collagen (Nycomed Arzneimit-tel GmbH). The leukocyte-specific agonist fMLP was from SigmaChemical Co. The PAF antagonist SR27417 was a gift from Dr J.-M.Herbert (Sanofi Recherche, Toulouse, France); the TXA2 analogueU-46619 and the TXA2 receptor antagonist ICI 192.605 were fromBIOMOL Research Laboratories Inc. The 5-lipoxygenase inhibitorZileuton was from Abbott Laboratories; the 5-lipoxygenase–activat-ing protein (FLAP) inhibitor MK-886, LTB4, LTC4, and LTD4 werefrom BIOMOL. Superoxide dismutase (SOD) was from Sigma. TheP-selectin–blocking MAb 9E1 was from R&D Systems. The GPIIb/IIIa–blocking MAb RFGP56 was from the laboratory of A.H.G.;the nonpeptide GP IIb/IIIa antagonist SR121566 was also a gift(J.-M.H.). CD11b (MAb 44)– and CD18 (6.5E)–blocking MAbswere gifts from Drs N. Hogg (Imperial Cancer Research Fund,London, UK) and M. Robinson (Celltech Ltd, London, UK), respec-tively. MAb MOPC21 (a gift from Dr Robinson) was used asnonspecific IgG control. HEPES and other chemicals were fromSigma.

Fluorescent antibodies for flow cytometric analysis were used atoptimal concentrations, as determined by titration. Platelets wereidentified with an FITC-conjugated anti-CD42a (GPIX) MAb Beb1(Becton Dickinson). Leukocytes were identified with anR-phycoerythrin (RPE)-conjugated panleukocyte, CD45 MAbT29/33 (Dakopatts AB). Platelet P-selectin expression was deter-mined by an RPE-conjugated anti–P-selectin MAb AC1.2 (BectonDickinson), and leukocyte CD11b expression was determined by anFITC-conjugated MAb, BEAR 1 (Immunotech). FITC- and RPE-conjugated isotypic MAb DAK-GO1 were used as negative controls.

Blood Collection and Sample PreparationBlood was collected by clean venipuncture with the use of sili-conized Vacutainer tubes containing 1/10 vol of 200mg/mL recom-binant hirudin (CIBA-Geigy). Within 3 minutes of collection, 200mL aliquots of blood were added to prewarmed siliconized cuvettes.Blood was incubated at 37°C for 5 minutes in the presence of vehicleor appropriately diluted antagonist(s) or blocking MAb(s). After-ward, collagen (1mg/mL) or fMLP (1026 mol/L) was added toinduce platelet or leukocyte activation, respectively, and the sampleswere further incubated for 5 minutes, with stirring at 900 rpm.Thereafter, 5mL blood was added to 45mL HEPES-buffered salinecontaining appropriately diluted fluorescent MAbs. The sampleswere incubated at room temperature for 20 minutes and then dilutedand mildly fixed with 0.5% (vol/vol) formaldehyde saline beforemeasurement with use of a Coulter EPICS XL-MCL flow cytometer(Coulter Corp), as described previously.22

Platelet-Poor Blood PreparationTo confirm that the collagen preparation does not activate leuko-cytes, platelet-poor blood was prepared by using Percoll (AmershamPharmacia Biotech) as described previously18 but omitting the stepof red blood cell lysis. The lower layer (granulocytes and red bloodcells) was resuspended with HEPES-buffered saline containing1.25 mmol/L CaCl2 and centrifuged at 800gfor 10 minutes. Thepellet was resuspended in the same buffer. Afterward, the platelet-poor blood was incubated with or without collagen as describedabove.

Flow Cytometric Analysis

Platelet P-Selectin ExpressionThe flow cytometric analysis of platelets in whole blood has beendescribed previously.23 RPE-CD62P fluorescence was monitored toobtain the percentage of P-selectin–positive platelets. The P-selec-tin–blocking MAb 9E1 did not interfere with platelet P-selectinmeasurements with the use of MAb AC1.2, as determined in separateexperiments with collagen-stimulated platelets.

Leukocyte CD11b ExpressionThe flow cytometric analysis of leukocyte CD11b expression inwhole blood has been described previously.24 CD11b expression wasdetermined as mean fluorescence intensity (MFI) in total leukocytesand leukocyte subpopulations and expressed in arbitrary units.However, the CD18-blocking MAb 6.5E interfered with CD11bmeasurements that made use of MAb BEAR 1.

Platelet-Leukocyte AggregatesPLA analysis has been described previously.22 The percentages ofplatelet-conjugated leukocytes in the total leukocyte population(PLA), lymphocytes (P-Lyms), monocytes (P-Mons), and poly-morphonuclear cells (P-PMNs) were obtained.

Statistical AnalysisData are presented as mean6SEM. Individual measurements werecompared with the Wilcoxon signed rank test (StatView 4.5, AbacusConcepts). A value ofP,0.05 was considered statisticallysignificant.

ResultsTitration of AntagonistsPAF (1026 mol/L, n54) increased P-selectin–positive plate-lets from 3.861.2% to 22.9615.3%, and this effect was

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completely blocked by SR27417 at$1028 mol/L. PAFincreased leukocyte CD11b MFI from 3.0260.51 to5.4460.74, and this effect was abolished by 1026 mol/LSR27417. Thus, 1026 mol/L SR27417 was chosen to blockthe effects of PAF.

The TXA2 analogue U-46619 (1026 mol/L, n54) increasedP-selectin–positive platelets from 1.860.5% to 95.560.7%and leukocyte CD11b MFI from 3.8560.51 to 6.2260.45.Both effects were abolished by the TXA2 antagonist ICI192.605 at$1026 mol/L.

fMLP-Induced Leukocyte ActivationThe leukocyte-specific agonist fMLP (1026 mol/L) increasedleukocyte CD11b expression (MFI) from 2.8860.38 to6.8960.51 (P,0.05, n57). This effect was predominantlyseen in polymorphonuclear leukocytes (PMNs; from3.6760.58 to 9.8160.48,P,0.05), with only small increasesamong monocytes (from 2.4360.38 to 2.7860.20,P,0.05)and no effect in lymphocytes. Neither the PAF antagonistSR27417 (1026 mol/L) nor the 5-lipoxygenase inhibitorZileuton (1026 mol/L) significantly influenced leukocyteCD11b expression in unstimulated samples (data not shown).The leukocyte responses to fMLP were partially inhibited bySR27417 (P,0.05) but not by Zileuton. Similar results were

found in the presence of the GP IIb/IIIa antagonist SR121566(data not shown).

fMLP-Induced Platelet ActivationStimulation with fMLP (1026 mol/L) increased P-selectin–positive platelets from 2.560.1% to 5.160.6% (P,0.05).This leukocyte-dependent platelet activation was markedlyinhibited by the PAF antagonist SR27417 (1026 mol/L) or the5-lipoxygenase inhibitor Zileuton (1026 mol/L), which pro-duced 87% and 94% inhibition, respectively (P,0.05). Thecombination of both inhibitors did not have additive effects(Figure 1A).

When the nonpeptide GP IIb/IIIa antagonist SR121566(1026 mol/L) was used to minimize influences of platelet-platelet aggregation (n57), fMLP failed to induce P-selectinexpression in single platelets (Figure 1B).

To clarify whether the effect of Zileuton was related to5-lipoxygenase inhibition, the FLAP inhibitor MK-886 (1025

mol/L) was also studied (n56). As with Zileuton, MK-886markedly inhibited fMLP-induced platelet P-selectin expres-sion (86% inhibition). However, LTB4, LTC4, and LTD4 (1027

mol/L) failed to increase platelet P-selectin expression inwhole blood (n55, data not shown).

fMLP-Induced PLA FormationPLA formation was increased by fMLP (1026 mol/L) as aresult of increases in P-PMNs (Figure 2). Neither the PAFantagonist SR27417 nor the 5-lipoxygenase inhibitor Zileu-ton influenced this response. Similar results were obtained inthe presence of the GP IIb/IIIa antagonist SR121566 (data notshown).

Effects of O22 in fMLP-Induced Leukocyte and

Platelet ActivationTo investigate the effects of O22 in leukocyte-induced plateletactivation, hirudinized blood was preincubated without orwith 100 U/mL SOD, which did not influence leukocyteCD11b expression or PLA formation in either resting orfMLP-stimulated samples. SOD did not influence plateletP-selectin expression in resting samples (2.260.3% without

Figure 1. Effect of fMLP (1026 mol/L) on platelet P-selectinexpression in whole blood (n57) in the absence (A) or presence(B) of the GP IIb/IIIa antagonist SR121566. Samples were prein-cubated with vehicle, PAF antagonist SR27417 (1026 mol/L),5-lipoxygenase inhibitor Zileuton (1026 mol/L), or both inhibitorsfor 5 minutes. Afterward, they were further incubated and stirredfor 5 minutes without or with fMLP. *P,0.05 compared withunstimulated samples; †P,0.05 compared with fMLP-stimulatedsamples.

Figure 2. Effect of fMLP (1026 mol/L) on PLA formation (n57).Experimental procedures were as in Figure 1. Heterotypicaggregates among PLAs, P-PMNs, P-Lyms, and P-Mons areillustrated in panels A, B, C, and D, respectively. *P,0.05 com-pared with unstimulated samples.

2704 Arterioscler Thromb Vasc Biol. December 2000



and 2.360.4% with SOD,P50.74; n57) but tended to inhibitfMLP-induced platelet P-selectin expression (3.160.3%without and 2.661.1% with SOD,P50.06).

Collagen-Induced Platelet P-Selectin ExpressionCollagen markedly increased platelet P-selectin expression(Figure 3A, n57) in the absence (from 2.960.2% to24.462.5%) and presence (from 1.660.3% to 59.668.3%) ofthe GP IIb/IIIa inhibitor SR121566. The enhancement bySR121566 is presumably due to blockade of platelet-plateletaggregation, which increases the numbers of single activatedplatelets. The effect of collagen was largely blocked by theTXA 2 antagonist ICI 192.605, inasmuch as P-selectin positiveplatelets fell to 8.061.5% in the absence and 2.960.7% in thepresence of the GP IIb/IIIa inhibitor.

Collagen-Induced Leukocyte CD11b ExpressionThe collagen preparation used did not increase CD11bexpression of isolated PMNs in platelet-poor blood(2.5860.17 in the absence and 2.3860.20 in the presence of1 mg/mL collagen,P50.11; n53). However, in stirred wholeblood, collagen increased CD11b expression in total leuko-cytes (Figure 3B, n57), predominantly because of an in-crease among PMNs (from 4.0160.54 to 5.5060.60 in theabsence and from 4.3960.67 to 5.5860.94 in the presence ofthe GP IIb/IIIa antagonist SR121566, respectively;P,0.05

for both). The TXA2 antagonist ICI 192.605 reduced CD11bexpression in unstimulated samples and attenuated the leuko-cyte response to collagen in the presence of the GP IIb/IIIaantagonist SR121566 (P,0.05). However, in the absence ofSR121566, ICI 192.605 reduced the basal CD11b expressionbut not the effects of collagen.

To investigate whether platelet-derived PAF mediatescollagen-triggered leukocyte activation, blood samples werepreincubated without or with the PAF antagonist SR27417(1026 mol/L) and then further incubated in the absence orpresence of 1mg/mL collagen (n55). SR27417 did notinfluence platelet P-selectin expression or leukocyte CD11bexpression in either resting or collagen-stimulated samples(data not shown).

Collagen-Induced PLA FormationCollagen increased PLA formation markedly in the absenceand in the presence of the GP IIb/IIIa antagonist SR121566(Figure 3C). Leukocyte subpopulation analysis showed thatP-PMNs increased more markedly with GP IIb/IIIa blockade(from 14.561.8% to 72.465.2% without and from22.267.8% to 95.361.2% with SR121566). The TXA2 an-tagonist ICI 192.605 partially inhibited collagen-inducedPLA formation; this effect was seen in all leukocyte subpopu-lations (data not shown).

Influence of P-Selectin BlockadeTo investigate the roles of direct cell-cell contact, wholeblood was preincubated with blocking MAbs before fMLP orcollagen stimulation. The nonspecific control antibody(MOPC21) did not influence any of the parameters studied(data not shown).

As expected, fMLP increased leukocyte CD11b expres-sion, platelet P-selectin expression, and PLA formation with-out blocking reagents (Figure 4). The fMLP-induced increaseof platelet P-selectin expression was not influenced by theanti–P-selectin MAb 9E1, although fMLP-induced PLA for-mation was markedly reduced.

Collagen markedly increased platelet P-selectin expressionand PLA formation and also caused a mild increase inleukocyte CD11b expression (Figure 5). MAb 9E1 com-pletely blocked collagen-induced PLA formation and attenu-ated collagen-induced leukocyte CD11b expression.

Effects of GP IIb/IIIa BlockadeThe GP IIb/IIIa MAb RFGP56 and the nonpeptide GP IIb/IIIainhibitor SR121566 did not significantly influence fMLP-induced leukocyte CD11b expression. fMLP-inducedP-selectin expression was inhibited by RFGP56 andSR121566 (Figure 4B), whereas fMLP-induced PLA forma-tion was enhanced, or not altered (Figure 4C).

Both GP IIb/IIIa inhibitors enhanced collagen-inducedplatelet P-selectin expression (Figure 5A) and PLA formation(Figure 5C), presumably because of reduced platelet-plateletaggregate formation, leading to the retention of more acti-vated platelets as single cells. Collagen-induced leukocyteCD11b expression was not significantly influenced by MAbRFGP56 but was inhibited by the nonpeptide GP IIb/IIIainhibitor SR121566 (Figure 5B).

Figure 3. Effect of collagen (1 mg/mL) on platelet P-selectinexpression (A), leukocyte CD11b expression (B), and PLA for-mation (C) in the absence (open bars) or presence (stippledbars) of the GP IIb/IIIa inhibitor SR121566 (n57). Whole bloodwas preincubated without or with the TXA2 receptor antagonistICI 192.605 (1026 mol/L) for 5 minutes, followed by further incu-bation and stirring for 5 minutes without or with collagen.*P,0.05 compared with unstimulated samples; †P,0.05 and`P50.06 compared with corresponding collagen-stimulatedsamples.




Ligand-Receptor Systems in fMLP- andCollagen-Induced PLA FormationIn the experiments shown in Figures 4 and 5, a blocking MAbcocktail containing 9E1, RFGP56, CD11b MAb44, and CD18MAb 6.5E was also used to block the major ligand-receptorsystems involved in PLA formation simultaneously. How-ever, CD18 MAb 6.5E interfered with the flow cytometricmeasurements of CD11b, which limited the use of thecocktail to PLA analysis. The cocktail markedly reduced PLAformation in unstimulated but stirred samples (from16.261.5% to 3.860.5%, P,0.05) and totally blockedfMLP-induced PLA formation (from 23.061.8% to4.360.5%, P50.40 compared with unstimulated sampleswith the cocktail). MAb 9E1 alone caused partial inhibition(8.161.8%,P,0.05; Figure 4C). Similarly, the inhibitorycocktail totally blocked collagen-induced PLA formation.With collagen stimulation, PLA formation was equallyblocked by 9E1 alone.

DiscussionThe present study demonstrates platelet-leukocyte interac-tions under conditions that mimic the physiological state, ie,in stirred whole blood and with normal extracellular calciumlevels. The results indicate that fMLP-activated leukocytescan induce platelet activation, ie, increased platelet P-selectinexpression, and that this effect may be related to the gener-ation of PAF, 5-lipoxygenase production, and superoxide

anions. Furthermore, collagen-induced platelet activation canlead to leukocyte activation, seen as increased leukocyteCD11b expression; this response was not inhibited by block-ade of TXA2 or PAF receptors. The present study alsoprovides new evidence that P-selectin and GP IIb/IIIa areinvolved in cellular signaling during platelet-leukocyte crosstalk.

Activated leukocytes may influence platelets via differentmediators in different experimental settings.19,21,25,26In thepresent investigation in stirred whole blood, fMLP-activatedleukocytes increased platelet P-selectin expression, and thiswas inhibited by blockade of PAF receptors by SR27417 andO2

2 scavenging by SOD, suggesting the involvement of PAFand O2

2. Blockade of 5-lipoxygenase by either the 5-lipoxy-genase inhibitor Zileuton or the FLAP inhibitor MK-886inhibited fMLP-induced platelet activation. However, neitherLTB4, LTC4, nor LTD4 increased platelet P-selectin expres-sion in whole blood. Thus, inhibition of leukocyte-plateletcross talk by 5-lipoxygenase blockade involves not onlyreduced leukotriene formation but also complex mechanisms.Several lines of evidence suggest that 5-lipoxygenase prod-ucts may contribute to the cross talk. For instance, 5-hydroxy-eicosatetraenoic acid and LTB4 have been shown to enhancegranulocyte PAF synthesis.27 Further investigation to clarifythe mechanisms involved would be of considerable interest.Other leukocyte-derived mediators not investigated in the

Figure 4. Effects of blocking agents (n57) on 1026 mol/L fMLP-induced leukocyte CD11b expression (A), platelet P-selectin(P-S’tin) expression (B), and PLA formation (C). Whole bloodwas preincubated without or with the P-selectin–blocking MAb9E1 or the GP IIb/IIIa–blocking agents RFGP56 and SR121566,followed by 5 minutes of fMLP stimulation and stirring. *P,0.05compared with unstimulated samples; †P,0.05 compared withfMLP-stimulated samples.

Figure 5. Effect of blocking agents (n57) on 1 mg/mL collagen–induced platelet P-selectin expression (A), leukocyte CD11bexpression (B), and PLA formation (C). Whole blood was prein-cubated without or with P-selectin–blocking MAb 9E1 or GPIIb/IIIa–blocking agents RFGP56 and SR121566. Afterward,samples were further incubated and stirred for 5 minutes in theabsence or presence of collagen. *P,0.05 compared withunstimulated samples; †P,0.05 compared with collagen-stimulated samples.




present study, such as neutrophil-released proteinases,19,26

may also be involved in the cross talk, albeit to a limitedextent. Furthermore, PAF antagonism also inhibited fMLP-induced leukocyte CD11b expression, suggesting an auto-crine effect of PAF.

Blockade of collagen-induced platelet P-selectin expres-sion by the TXA2 receptor antagonist ICI 192.605 confirmsthat collagen-induced platelet activation is dependent onTXA 2 synthesis. Our collagen preparation did not increaseleukocyte CD11b expression in platelet-poor blood but didenhance leukocyte CD11b expression in whole blood. TheTXA 2 receptor antagonist ICI 192.605 and the PAF antago-nist SR27417 reduced leukocyte CD11b expression in un-stimulated and collagen-stimulated samples. However, theleukocyte CD11b response to collagen was not reduced byICI 192.605 or SR27417. This suggests that collagen inducesleukocyte CD11b expression either via platelet-derived me-diators other than TXA2 or PAF (eg, platelet dense granule–released ADP20 and platelet-derived microparticles3) or viacell-cell contact, as discussed below.

In the present study, collagen induced marked plateletactivation and mild leukocyte activation, whereas fMLPinduced marked leukocyte activation and mild platelet acti-vation. Because collagen enhanced PLA formation moremarkedly than did fMLP, it seems as if PLA formation ismore dependent on platelet activation than on leukocyteactivation. This is quite reasonable, inasmuch as the majoradhesion molecule involved in PLA formation, P-selectin, isexpressed only on the surface of activated platelets, whereasits counterparts PSGL-1 and CD15 are constitutively ex-pressed on leukocytes. Differential blockade of PLA forma-tion with different stimuli and different blocking agentssuggests that multiligand-receptor systems are involved inPLA formation under the present conditions. Our results arecompatible with the idea that platelet activation–initiatedPLA formation is entirely dependent on ligation viaP-selectin, whereas leukocyte activation–initiated PLA for-mation involves ligation via P-selectin–PSGL-1/CD15 andGP IIb/IIIa–fibrinogen–CD11b/CD18. In contrast, GP IIb/IIIa blockade alone may enhance PLA formation, presumablybecause of the inhibition of platelet-platelet aggregation,which provides more activated platelets for heterotypicconjugation.

Enhancement or inhibition of PLA formation by blockingagents seemed to have only minor influences on fMLP-induced platelet P-selectin expression, indicating thatleukocyte-platelet cross talk is likely to depend on solublemediators rather than direct cell-cell contact. Collagen-induced leukocyte CD11b expression was attenuated byblockade of PLA formation by the P-selectin–blocking MAb9E1. Thus, direct cell-cell contact may contribute to platelet-induced leukocyte activation. However, this was apparentlycontradicted by the data obtained with the GP IIb/IIIainhibitor SR121566, which attenuated platelet-induced leu-kocyte CD11b expression despite enhanced collagen-inducedPLA formation. Therefore, platelet-leukocyte cross talkseems to involve complex mechanisms, and further investi-gation is warranted to define the mechanism(s) that maymediate collagen-induced leukocyte CD11b expression.

Cellular signaling involves integrins as well as selec-tins.28,29 Blockade of selectins and integrins not only severs

the ligations of cell-cell adhesion/conjugation but also affectsintercellular signaling. The present findings reinforce previ-ous evidence of selectin and integrin involvement in intercel-lular cross talk.13,14,30,31We showed that GP IIb/IIIa blockadeinhibited fMLP-induced platelet P-selectin expression andthat GP IIb/IIIa blockade by SR121566 and P-selectin block-ade by MAb 9E1 inhibited collagen-induced leukocyteCD11b expression. These findings indicate that GP IIb/IIIaoccupancy by antagonists may interfere with the inside-outsignaling of platelets and that P-selectin blockade (if notsimply by its inhibition of PLA formation) and GP IIb/IIIaantagonists may inhibit intercellular signaling between plate-lets and leukocytes. Our data add new evidence for theinvolvement of the integrins and selectins in intracellular andintercellular signaling during cellular activation and interac-tion.28,29 Integrin- and selectin-linked cell signaling is com-plex and has not yet been well defined, but interest in thisfield is expanding. Perhaps it will be possible to identifyintegrin- or selectin-proximal signaling proteins as futuredrug targets.

It is well worth stressing that although platelet-leukocytecross talk induces only mild platelet and leukocyte activation,the major physiological importance of this intercellular inter-action may be the priming of platelets and leukocytes, leadingto platelet and leukocyte hyperreactivity. Several antagonistsalso decreased platelet P-selectin expression, leukocyteCD11b expression, and PLA formation in unstimulated sam-ples, suggesting that there is spontaneous platelet and leuko-cyte activation during incubation with stirring and that suchspontaneous activation also involves multiple mediators.

In conclusion, the present study has provided strongsupport for the existence of platelet-leukocyte cross talkunder physiological conditions in whole blood. The presentstudy has demonstrated that several mediators are involved inthe cross talk and that blockade of GP IIb/IIIa or P-selectinmay inhibit platelet-leukocyte cross talk.

AcknowledgmentsThe study was supported by grants from the Swedish Medical

Research Council (5930), the King Gustaf V and Queen VictoriaFoundation, the Swedish Heart-Lung Foundation, and the KarolinskaInstitute. The authors are grateful to Maud Daleskog and Maj-Christina Johansson for their expert technical assistance.

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HjemdahlNailin Li, Hu Hu, Malin Lindqvist, Eva Wikström-Jonsson, Alison H. Goodall and Paul

Platelet-Leukocyte Cross Talk in Whole Blood

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Platelet-Leukocyte Cross Talk in Whole Blood