Leukocyte-platelet interaction. Release ofhydrogen peroxide by granulocytes as amodulator of platelet reactions.

P H Levine, … , K L Scoon, N I Krinsky

J Clin Invest. 1976;57(4):955-963. https://doi.org/10.1172/JCI108372.

Because of the many potent biological capabilities of the blood granulocytes, and theircontact with platelets in various physiologic and pathologic states, a possible interactionbetween granulocytes and platelets was investigated. Platelets were purified by gel filtrationand via a dialysis membrane were separated from suspensions of autologous granulocytesprepared by dextran sedimentation and resuspended in modified Tyrode's buffer. After 20min at 37 degrees C platelet aggregation was shown to be diminished by such exposure, ascompared to the aggregation of platelets incubated with dialysates of buffer only. Whengranulocytes were stimulated by the addition of 1.1-muM latex spheres as target particles forphagocytes, the dialysate of these cells exhibited greatly enhanced platelet-inhibitoryproperties. The addition of catalase to the platelets abolished the effect of exposing thesecells to the dialysate of resting granulocytes and markedly inhibited the effect of exposingthe platelets to the dialysate of phagocytosing granulocytes. Catalase treated with 3-amino-1,2,4-triazole had no platelet-protective capacity. Purified suspensions of lymphocytesreleased no platelet-inhibitory principle under these experimental conditions. Hydrogenperoxide in the dialysate of granulocytes was measured directly with an assay involving anH2O2-induced decrease in the fluorescence of scopoletin catalyzed by horseradishperoxidase. The dialysate of phagocytosing granulocytes contained 0.86 +/- 0.55 nmolH2O2/2.5 X 10(7) granulocytes when sampled at 20 min. By an alternate measurementtechnique […]

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Leukocyte-Platelet Interaction

RELEASEOF HYDROGENPEROXIDEBY GRANULOCYTES

AS A MODULATOROF PLATELET REACTIONS

PETERH. LEVINE, RONALDS. WEiNGER, JOANNSIMONKmINEL. SCOON,and NORMANI. KRINSKY

From the Blood Coagulation Laboratory and Medical Division, the MemorialHospital; the Department of Medicine, University of Massachusetts MedicalSchool, Worcester, Massachusetts 01605; the Blood Coagulation Laboratoryand Hematology Service, Department of Medicine, NewEngland MedicalCenter Hospital, and the Department of Medicine and the Department ofBiochemistry and Pharmacology, Tufts University School of Medicine,Boston, Massachusetts 02111

A B S T R A CT Because of the many potent biologicalcapabilities of the blood granulocytes, and their contactwith platelets in various physiologic and pathologicstates, a possible interaction between granulocytes andplatelets was investigated. Platelets were purified by gelfiltration and via a dialysis membrane were separatedfrom suspensions of autologous granulocytes preparedby dextran sedimentation and resuspended in modifiedTyrode's buffer. After 20 min at 37TC platelet aggrega-tion was shown to be diminished by such exposure, ascompared to the aggregation of platelets incubated withdialysates of buffer only. When granulocytes were stim-ulated by the addition of 1.1-ILM latex spheres as targetparticles for phagocytes, the dialysate of these cellsexhibited greatly enhanced platelet-inhibitory proper-ties. The addition of catalase to the platelets abolishedthe effect of exposing these cells to the dialysate ofresting granulocytes and markedly inhibited the effectof exposing the platelets to the dialysate of phagocytos-ing granulocytes. Catalase treated with 3-amino-1,2,4-triazole had no platelet-protective capacity. Purified sus-pensions of lymphocytes released no platelet-inhibitoryprinciple under these experimental conditions.

Hydrogen peroxide in the dialysate of granulocyteswas measured directly with an assay involving an H202-induced decrease in the fluorescence of scopoletin cata-lyzed by horseradish peroxidase. The dialysate of pha-gocytosing granulocytes contained 0.86+0.55 nmol

Received for publication 5 May 1975 and in revised form27 October 1975.

H202/2.5 x 107 granulocytes when sampled at 20 min.By an alternate measurement technique in which scopo-letin and horseradish peroxidase were present in thedialysate from time zero, the mean amount of H202 inthe dialysate reached 4.0±+1.3 nmol/2.5 X 107 granulo-cytes at 20 min. This discrepancy suggested the con-sumption of H202, possibly mediated by the granulo-cytes themselves. This possibility was investigated bythe addition of exogenous H202 to the test system. Bothgranulocytes and platelets enhanced the disappearanceof H202 from the dialysate, and the amount consumedwas proportional to the amount of H202 added to thesystem.

Glucose oxidase at 12 M U/ml plus glucose in ex-cess resulted in the production of H202 at a rate andfinal amount comparable to that produced by phagocy-tosing granulocytes. This mixture, when substituted forphagocytosing granulocytes in the standard dialysismembrane experiment, induced an inhibition of plateletaggregation similar to that caused by the granulocytes.

The observation that the release of H202 by the bloodgranulocyte influences platelet function suggests a po-tential role for the granulocyte in the regulation ofhemostasis or thrombosis.

INTRODUCTIONThe platelet plays the central role in the initial hemo-static response and in the genesis of arterial thrombo-sis (1). Many studies have demonstrated interaction ofthe platelet with other platelets (2), with elements of

The Journal of Clinical Investigation Volume 57 April 1976 *955-963 955

the vessel wall (3), or with components of the bloodcoagulation system (4).

Although arterial thrombi are composed primarily ofaggregated platelets, they are also rich in granulocytes(5-7). Despite intimate contact between these two celltypes, almost nothing is known about how they interact.Given the many potent biological functions of the poly-morphonuclear leukocyte (PMN),' it would be sur-prising if it was passive at the site of platelet reactions.

In the course of their phagocytic activity, PMNun-dergo significant metabolic alterations. These alterationsinclude a respiratory burst (8), increased hexose mono-phosphate shunt activity (9), increased NADHoxidaseactivity (10), and H202 production (11). In additionto these intracellular events, phagocytosing granulo-cytes also release various agents, among which are H202(12) and 02-, the superoxide radical (13). Baehneret al. have demonstrated that granulocyte-released H202has the capacity to effect neighboring cells (14). Theseworkers mixed activated PMN with glucose-6-phos-phate dehydrogenase-deficient erythrocytes and observedHeinz body formation attributable to the elaboration andrelease of H202 by the granulocytes.

We have previously shown that brief exposure ofplatelets to micromolar concentrations of H202 cancause significant inhibition of platelet function (15, 16).The present study deals with two questions. First, cangranulocytes influence platelet function in vitro? Second,could such an interaction be mediated, at least in part,by granulocytic production of H202 ?

METHODSVenous blood from normal individuals who had abstainedfrom all medications, including aspirin, for at least 10 dayswas collected in plastic syringes and mixed immediately(5: 1) with acid citrate anticoagulant (15) (one part 5%Na citrate and one part 2.7% citric acid plus 4 g/100 mldextrose) in plastic tubes. The blood was centrifuged at200 g for 10 min at room temperature, and the platelet-richplasma (PRP) was removed with a siliconized Pasteurpipette.

Platelet purification. The platelets were separated fromthe PRP by a modification of the gel filtration technique ofTangen et al. (17). Plexiglas columns, 2.5 X 25 cm, werefilled with Sepharose 2B (Pharmacia Fine Chemicals, Inc.,Piscataway, N. J., lot 4326) so that the column bed con-tained 50 ml of the Sepharose gel. The top of the columnhad a sample applicator whose bottom was a fine nylonmesh; the column base had coarse and fine nylon mesh.Before each experiment the column was washed three timeswith 100 ml of eluting buffer. This eluting buffer was amodification of Tyrode's albumin solution and contained129 mMNaCl, 9 mMNa HCO3, 6 mMdextrose, 11 mMNa citrate, 11 mMTris, 1 mMKH2PO4, 3 mMKC1, 1 mMMgC12, 2 mMCaCl2, and 5 g/100 ml salt-poor albumin per

'Abbreviations used in this paper: 3-AT, 3-amino-1,2,4-triazole; GFP, gel-filtered platelets; PMN, polymorpho-nuclear leukocytes; PRP, platelet-rich plasma.

100 ml, which had previously been dialyzed against thissame buffer. The final pH was 7.35.

The volume of PRP applied to the top of the columnvaried from 6 to 10 ml. The PRP was gently layered ontothe gel and the run was started. The platelet peak appearedwell before the plasma and could easily be detected visually.All eluants were collected into plastic tubes, and the re-sultant gel-filtered platelets (GFP) were adjusted to acount of 250,000/mm3 by dilution with fresh eluant buffer.Apyrase (18) was immediately added to the GFP at a finalconcentration of 0.5 mg/ml.

Platelet aggregation. Aggregation studies were done in theGFP by the turbidometric method of Born (19), as modi-fied by Mustard, et al. (20). 0.4 ml of GFP were placedin the siliconized cuvette of a Chronolog aggregometer(Chrono-Log Corp., Havertown, Pa.) at 370C and stirredat 1,200 rpm with a Teflon-coated stir bar. The light trans-mittance was recorded on a moving strip chart recorderand increased as a function of the number and size ofplatelet aggregates. The degree of aggregation was ex-pressed as a percent, with the light transmittance of thesupernate of a sample of GFP spun on a Beckman 152 Mi-crofuge (Beckman Instruments, Inc., Spinco Div., Palo Alto,Calif.) for 2 min regarded as 100% transmission and thelight transmittance of the GFP as 0% transmission.

Granulocyte suspensions. Venous blood, collected in plas-tic syringes and mixed immediately with acid citrate anti-coagulant (as previously described) in plastic tubes, waspoured into a 100-ml plastic graduated cylinder flask. Dex-tran (Sigma Chemical Co., St. Louis, Mo., lot 112-1070,average mol wt 254,000) and 5 g/100 ml of 0.154 M NaCl,was added at a ratio of 1 ml of dextran solution for each2 ml of whole blood, as described by Babior, et al. (13).The blood was allowed to sediment for 45 min, after whichthe supernate was transferred to plastic tubes and centri-fuged for 15 min at 500 g. The resulting leukocyte buttonwas exposed to three parts iced distilled water for 30 sto lyse erythrocytes, and then one part 0.6 M NaCl wasadded. The suspension was spun at 500 g for 4 min, and theleukocyte button was again exposed to distilled water, cen-

trifuged, and finally washed with 0.154 M NaCl. A leuko-cyte suspension containing 50,000 leukocytes/mm' was madewith eluting buffer as the suspending fluid. Differentialcounts showed that 90-95% of these leukocytes were PMN.

Lymphocyte suspension. Blood was drawn into plasticsyringes, placed into Ehrlenmeyer flasks containing glassbeads, and mixed gently until defibrinogenated. Lympho-cytes were isolated by the Ficol-Hypaque method of Boyum(21) and suspended in the modified Tyrode's buffer so thatthe final count was 40,000-50,000 mononuclear cells/mm3.Differential counts of these cells demonstrated them to be95-98% lymphocytes.

Dialysis bag experiments. A system was required to ex-

pose platelet suspensions to the products of granulocyte me-tabolism without direct addition of granulocytes to theplatelets. This separation was achieved via a dialysis mem-brane. In the standard experiment leukocytes or biochemicalsubstances were placed in the dialysis bag, which containeda final volume of 0.55 ml, and the bag was immersed in 1.0ml of platelet suspension contained in a plastic test tube.After a 20-min incubation at 370 C the bag was removed,and samples of the platelet suspension were studied.

Basic leukocyte-platelet protocol. Blood was drawn forleukocyte preparation at approximately 45 min before bloodwas drawn for preparation of GFP, so that all studies wereperformed with freshly prepared GFP.

Eight plastic tubes were divided into two sets of four

956 P. H. Levine, R. S. Weinger, J. Simon, K. L. Scoon, and N. I. Krinsky

tubes each (Fig. 1). To each tube of one set was added0.90 ml of the GFP plus 0.10 ml of the stock catalase solu-tion (see below), to inactivate any H202 formed. Dialysisbags (Fisher Scientific Co., Pittsburgh, Pa.; 1.5 cm insidediameter) were suspended in each tube of GFP. Bags con-taining 0.55 ml buffer, in tubes 1 and 4 of each set, servedas controls for bags containing 0.5 ml leukocyte suspensionsplus 0.05 ml buffer (resting leukocytes, tube 2) and bagscontaining 0.5 ml leukocyte suspension plus 0.05 ml latexparticles (activated leukocytes, tube 3). The ratio of latexparticles to leukocytes in these studies was 1,000: 1.

Additional experiments were run with 0.50 ml of H202in dialysis bags, likewise placed into 1.0 ml GFP with orwithout catalase. After a 20-min incubation at 370C, thesebags were removed, and aggregation studies were performedon the GFP.

In each group of experiments statistical significance wasdetermined by Student's t test with paired data (22).

Generation of HO, by glucose/glucose oxidase. Themethod of Cohen and Hochstein (23) was used to generatevarious amounts of H202. Glucose oxidase (Sigma ChemicalCo.), 1,200 U/ml, was dialyzed overnight against distilledwater and then diluted 1: 2,000 with distilled water. Additionof this solution to the modified Tyrode's buffer containing1.25 mMglucose yielded H202, as described below.

Measurement of hydrogen peroxide. Direct measurementof H202, either produced by stimulated granulocytes or gen-erated by a glucose/glucose oxidase system, was made byassaying the decrease in the fluorescence of scopoletin cata-lyzed by horseradish peroxidase. This reaction, originallydescribed by Andreae (24), has been used by Root (25) todetermine the rate of release of H202 from activated granu-locytes. Fluorescence was determined in an Aminco-Bowmanspectrofluorometer (American Instrument Co., Silver Spring,Md.) with an excitation wavelength of 350 nm and anemission wavelength of 460 nm.

Reagents. Disodium ADP (Sigma Chemical Co.) wasdissolved in barbital buffer (pH 7.35) at a concentrationof 10 mg/100 ml, adjusted to pH 6.8, and frozen in 1-mlaliquots at -40'C. Addition of 30 ,l of this solution to 0.4ml of GFP yielded a final concentration of 13 1M ADP.

Apyrase (Adenosine-5'-triphosphatase, Sigma ChemicalCo., grade 1) had the approximate activities: 5'-ATPase,1.58 U/mg; 5'-ADPase, 0.57 U/mg, and 5'-AMPase, 0.03U/mg.

Dow latex particles obtained from Pitman-Moore Div.,Dow Chemical Co., Indianapolis, Ind., having a uniformdiameter of 1.10 ,um in a 10% solid suspension, were dia-lyzed against 0.9% NaCl and used as target particles forphagocytosis (26).

Hydrogen peroxide, 30% solution (J. T. Baker ChemicalCo., Phillipsburg, N. J.) was diluted in the modified Ty-rode's buffer. This was prepared fresh before each experi-ment and kept at 4°C until used.

Purified lyophilized catalase (Worthington BiochemicalCorp., Freehold, N. J., lot 5651-4), activity 3,000 U/mg,was dissolved in the modified Tyrode's buffer to a concen-tration of 10,000 U/mI. This solution was made fresh eachday and when used was added to GFP to a final concentra-tion of 1,000 U catalase/ml GFP.

3-Amino-1,2,4-triazole (3-AT), (Sigma Chemical Co., lot122C-0840) was dissolved in modified Tyrode's buffer to aconcentration of 1 M. This solution was made fresh eachday and when used was added to the GFP to a final con-centration of 100 mM.

Scopoletin (Sigma Chemical Co.), 1 mM, in distilledwater was stored at 4°C. Peroxidase, type II from horse-

BufferOnly

GFP +

BufferOnly

GFP +

Cotls

WBC

GFP +

Buffe r

2

BC

GFP +CotaI

(WBC

Lteo

GFP +Buf fer

3

WBC

GFP +

Caals

BufferOnly

GFP +

4

BufferOnly

GFP +

CatlsFIGURE 1 Basic granulocyte-platelet protocol. Dialysis bagscontaining 0.55 ml modified Tyrode's buffer (numbers 1 and4), 0.50 ml granulocytes plus 0.05 ml buffer (number 2),or 0.50 ml granulocytes plus 0.05 ml latex particles (number3), were suspended in 1.0 ml of autologous platelets. In thebottom set of four samples, catalase has been added to theplatelet suspension. After 20 min at 37°C, the bags wereremoved and discarded, and 0.4-ml samples of platelets wereremoved for aggregation studies. In each set tube 1 wasthe control for tube 2, and tube 4 was the control for tube 3.For all studies, the aggregating agent was ADP at 13 ,uMfinal concentration in the platelet suspension.

radish (Sigma Chemical Co.), 100-150 purpurogallin U/ml,was made up in distilled water at a concentration of 1.0mg/ml and was stored at - 200 C.

RESULTS

Effect of H.O.. To determine whether H202 coulddiffuse across a dialysis membrane and significantlyalter platelet function, a series of seven experimentswas performed. H202, at a concentration of 0.6 mMinmodified Tyrode's buffer, was placed in dialysis bagsthat were then suspended in GFP as described in Meth-ods. After 20 min at 370C there was a pronounced in-hibition of ADP-induced aggregation of the platelets incomparison to platelets exposed to buffer only (P <0.001). This is shown in column A of Fig. 2.

In a parallel set of seven experiments catalase wasadded to the GFP, and the above protocol was repeated.As shown in column B of Fig. 2, catalase abolishedthe H202 effect (P < 0.001). In the presence of catalase,the aggregation of platelets exposed to H202 was not

Granulocyte-Produced H.Ot and Platelet Reactions 957

50-

40-

20-

k 0-

- 10-

-20,FIGURE 2 Effect of H202 on platelet function. Plateletshave been exposed to dialysis bags containing buffer onlyor buffer plus 600 /AM H202 for 20 min at 370C. Each pointrepresents the maximum percentage of aggregation of plate-lets exposed to buffer minus the maximum percentage ofaggregation of platelets exposed to buffer plus H202, as ameasure of inhibition attributable to H202 exposure. Incolumn B, catalase (1,QOO U/ml GFP) has been added tothe GFP.

significantly different from that of platelets exposed tobuffer only (P > 0.05).

Effect of granulocyte products. The aggregation ofplatelets exposed to dialysis bags containing restinggranulocytes was significantly different from the aggre-gation of platelets incubated with bags containing bufferonly. The aggregation curves obtained from a typicalexperiment following the protocol illustrated in Fig. 1are shown in Fig. 3. As can be seen (Fig. 3A), restinggranulocytes inhibited platelet aggregation, and the

k-1%I

0.

A.

ADP / BUFFER I' BUFFER2

NO CATALASE -ACTIVE PMN

effect of phagocytosing granulocytes was much morepronounced. The addition of catalase to the platelets(Fig. 3B) largely eliminated this granulocyte-inducedinhibition of platelet function.

A statistical analysis of this phenomenon in nine ex-periments is shown in Fig. 4. Whereas resting granulo-cytes produced some inhibition of aggregation (columnA, Fig. 4, P <0.05), exposure of platelets to bags ofactivated leukocytes (Fig. 4, column B) produced astriking inhibition of aggregation, significant at theP < 0.001 level. (In numerous trials no effect on plate-let aggregation was seen when latex particles alonewere placed into bags.)

Since the platelet-inhibitory effect of externally addedH202 had been abolished by catalase (Fig. 2), similarexperiments were performed with respect to the platelet-inhibitory effects of granulocytes. In the presence ofcatalase, the aggregation of platelets exposed to restinggranulocytes (Fig. 4, column C) was not different fromthe aggregation of platelets exposed to buffer only (P> 0.05). On the other hand catalase-treated plateletsexposed to activated granulocytes (Fig. 4, column D)were significantly protected against the inhibitory effectof such exposure (P <0.001). It must be noted, how-ever, that even in the presence of catalase, there was aresidual inhibition of platelet aggregation after exposureto activated granulocytes (P <0.05).

The specificity of catalase as an inhibitor of thegranulocyte's effect on platelets was further evaluatedby use of the specific catalase antagonist (27), 3-AT.In a series of six consecutive experiments we againdemonstrated that catalase diminished the effect of acti-vated granulocytes on platelet aggregation (Fig. 5,column A). Addition of 3-AT to the catalase-treatedplatelets resulted in the reappearance of inhibition ofplatelet aggregation after exposure to activated granu-locytes (Fig. 5, column B, (P <0.01).

Effect of lymphocyte products. With an identicalprotocol, purified lymphocytes both with and withoutlatex spheres were studied. The platelets exposed to

B.

8BUFFER I;5.1RESTING PMN- q-~BUFFER 2

"-ACTIVE PMNADP

CATALASE.I I

0 I 2 3 0 1 2 3TIME IN MINUTES

FIGURE 3 Effect of granulocyte products on platelet aggregation. A typical set of aggregationcurves following the standard protocol outlined in the legend to Fig. 1 is shown. Data on ninesuch experiments are provided in Fig. 4.

958 P. H. Levine, R. S. Weinger, J. Simon, K. L. Scoon, and N. I. Krinsky

control dialysis bags had a maximum aggregation of50.8±10.4% (mean+SD); those exposed to lympho-cyte-containing bags had maximum aggregation of 50.4±+10.8%, and those exposed to lymphocytes plus latexbeads had maximum aggregation of 51.2±10.0%.

Quantitation of H*02 production. H202 concentrationwas measured directly in the dialysate of stimulatedgranulocytes after a 20-min incubation at 370C, in theabsence of platelets. Scopoletin, 4 IM, and horseradishperoxidase, 16 ,ig/ml, were added to the dialysate, andfluorescence was measured. The mean final amount ofH202 found in 10 experiments was 0.86+0.55 nmol(Fig. 6). These measurements were then repeated as afunction of time as outlined below.

Scopoletin at a final concentration of 4 /M was addedto the granulocyte suspension in a dialysis bag justbefore the addition of latex particles. The bag was im-mediately placed into a fluorometer cuvette that con-tained 2.5 ml of Krebs-Ringer bicarbonate buffer towhich had been added 4 AM scopoletin and horseradish

ACr/VArED PMN

k-

15-

90Qg-

IK

1 5-

5.

CATALASE CATALASE+ONLY 3-AT

A_, B

_

2 X

Q_

CATALASENO CATALASE added

Resting Active Resting ActivePMN PMN PMN PMN

FIGURE 4 Inhibition of ADP-induced platelet aggregationafter exposure to dialysis bags containing granulocytes.Protocol is outlined in legend to Fig. 1, and each pointrepresents the maximum percentage of aggregation of plate-lets exposed to dialysis bags containing buffer only, minusthe maximum percentage of aggregation of platelets ex-posed to dialysis bags containing granulocytes. The pointsconnected by each line are the results of studies on samplesof platelets and granulocytes from one donor. Note thatcatalase completely blocks the effect of exposing plateletsto resting granulocytes (column C vs. column A) but doesnot completely block the effect of exposure of platelets tophagocytosing granulocytes (column D vs. column B, P<0.01).

FIGuRE 5 Specificity of catalase as an inhibitor of thegranulocyte-platelet interaction. Six sets of studies arepresented following the protocol of Fig. 4, column D, ex-cept that 3-AT was added to prevent the destruction ofH202 by catalase. Column A: prevention by catalase ofgranulocyte-induced inhibition of platelet aggregation. Col-umn B: addition of 0.1 M 3-AT inhibited this protectiveeffect of catalase. (It should be noted that in a series ofsix experiments, 0.1 M 3-AT itself produced mild inhibitionof platelet aggregation. For this reason, the control foreach point in column A is the aggregation of platelets pluscatalase exposed to a dialysis bag of buffer only, and thecontrol for each point in column B is the aggregation ofplatelets plus catalase plus 3-AT exposed to a dialysis bagof buffer only.)

peroxidase, 16 /Lg/ml, previously warmed to 370C. Atregular intervals the bag was removed, and the fluores-cence of the scopoletin in the dialysate was determined.The concentration of H202 in the dialysate is shown inFig. 6. At the end of the 20-min incubation, the meanamount of H202 in the dialysate had reached 4.0±)1.3nmol. Resting granulocytes were similarly studied, andwithin the limits of the sensitivity of the method noreproducible change in fluorescence could be found.

Consumption of HsO.. The experiments described inthe above two paragraphs revealed a discrepancy inthe amount of H202 measured in the dialysate of acti-vated PMN. The final amount was considerably greaterwhen scopoletin and horseradish peroxidase were pres-ent in the dialysate. This suggested that some of theH202 produced was disappearing during the course ofthe 20-min incubation. Could the activated leukocytesthemselves be responsible for degradation of H202 ?To answer this question, activated granulocytes in di-alysis bags were exposed to either buffer or H202-con-

Granulocyte-Produced H201 and Platelet Reactions 959

2 4 6 8 10 12 14 16 A8 20Incubation Time in Minutes

FIGuRE 6 Appearance of H202 in the dialysate of phago-cytosing granulocytes. A dialysis bag containing 0.5 mlgranulocyte suspension and 0.05 ml latex particles was sus-pended in a fluorometer cuvette containing 2.5 ml Krebs-Ringer bicarbonate buffer to which has been added 4 uMscopoletin and 16 tsg/ml horseradish peroxidase. The bagwas removed at regular intervals, and fluorescence was readwith an excitation wavelength of 350 nm and an emissionwavelength of 460 nm. Decrease of scopoletin fluorescencewas proportional to H202 present. Results shown are themean and SD of eight sets of duplicate determinations. Thetriangle represents the mean and SD of the final amountof H202 measured by another method, as follows: thedialysis bag is suspended in Krebs-Ringer bicarbonate bufferwithout scopoletin or horseradish peroxidase and left un-disturbed for 20 min. An aliquot of the dialysate is thenremoved and added to the scopoletin and horseradish per-oxidase in a fluorometer cuvette. Hydrogen peroxide ismeasured as above.

taining solutions, and the H202 consumption was deter-mined by the following equation: H202 consumed =(H202 added + H202 produced) - H202 observed. Tocontrol for dilution and the spontaneous disappearanceof H202 at each concentration of H202 added, bags con-taining only buffer were handled similarly. As shownin Fig. 7, there was an apparent consumption of H202by the phagocytosing granulocytes. The amount con-sumed was proportional to the amount of H202 added tothe system.

Since the platelet is responsive to H202, studies wereperformed to determine whether this cell can also con-sume H202, as does the granulocyte. GFP were placedin dialysis bags at a concentration of 200,000/mm3, andthe bags were suspended in fluorometer cuvettes con-taining Krebs-Ringer bicarbonate buffer to which vary-ing amounts of H202 had been added. To control fordilution and spontaneous disappearance of H202, bagscontaining only buffer were added to cuvettes andhandled similarly. The consumption of H202 mediatedby platelets was determined by the following equation:H202 consumed = H202 added - H202 observed. (In aseries of six experiments no H202 production could beobserved from either resting or ADP-aggregated GFP.)The consumption of Ha20 by GFP is shown in Fig. 8.

Use of HsOs-generating system. H202 was generatedby the incubation of various amounts of glucose and

glucose oxidase in dialysis bags, and the rate of H202release was measured in a manner analogous to thatused for intact granulocytes. By serial dilution of glu-cose oxidase in the presence of excess glucose a con-centration was selected (0.012 U/ml) that resulted inthe production of H202 at both a rate and a final con-centration comparable to that produced by intact, acti-vated granulocytes. This mixture of glucose and glucoseoxidase was placed in dialysis bags and immediatelysuspended in tubes containing GFP. Control GFP wereexposed to dialysis bags containing only buffer. Asshown in Fig. 9, the exposure of GFP to bags contain-ing glucose plus glucose oxidase resulted in a significantinhibition (P <0.001) of ADP-induced platelet aggre-gation.

DISCUSSIONThe formation of aggregates of platelets at a site ofhemorrhage constitutes the primary hemostatic response(28). In addition aggregates of platelets are the mainconstituent of arterial thrombi (29). Therefore, factorsthat alter platelet function may have profound effects onhemostasis or thrombosis (30). The experiments re-ported here are an attempt to define a potential inter-action between the platelet and the blood granulocyte,since these cells have the opportunity to make closephysical contact with one another under a variety ofcircumstances. Our results indicate that granulocytescan release a factor or factors capable of crossing adialysis membrane and inhibiting the platelet aggre-gation response.

Phagocytosing granulocytes were shown to exert amarked inhibitory effect on platelet function, while rest-

7.0o

" 6.0.

i.g 5.0-

4.0.Z* Qt 3.0.

ct c 2.01..

it1.0

* 0

0

0 *

0

2 4 6 8 10 12 14 16 18 20 22nmol H202 in Dialysate (Exogenous + Granulocyte Produced)

FIGURE 7 Consumption of H202 by phagocytosing granulo-cytes. Activated granulocytes in dialysis bags are suspendedin 2.5 ml buffer only or in 2.5 ml buffer containing vary-ing amounts of H202. The vertical axis shows H202 con-sumed, determined by the following equation: H202 con-sumed = (H202 added + H202 produced by granulocytes)- H202 observed. Note that consumption increased withincreasing amounts of H202 present. The incubation periodwas 20 min.

960 P. H. Levine, R. S. Weinger, J. Simon, K. L. Scoon, and N. I. Krinsky

ing granulocytes had a small but significant inhibitoryeffect. The latter may simply reflect the inability toprepare a truly "resting" population of granulocytes.

Treatment of GFP with catalase abolished the inhibi-tory -effect of resting granulocytes, but did not com-pletely block the inhibitory effect of activated granulo-cytes. The inhibitory activity attributable to activatedgranulocytes in the presence of catalase was still sig-nificant (P <0.05 when compared to GFP in the ab-sence of catalase. The inability of catalase to completelyblock the effects of phagocytosing granulocytes could bedue to the elaboration of additional inhibitory factors bythe granulocyte. For example, stimulated granulocytesrelease superoxide into the surrounding medium (13).Studies are in progress to determine whether super-oxide has a direct effect on platelet function. An alter-native explanation for the inability of catalase to elimi-nate completely the inhibitory effects of phagocytosinggranulocytes is that platelets may successfully competewith catalase for the available H202.

The specificity of the catalase effect was shown bythe experiments with 3-AT, which specifically inhibitsthe degradation of H202 by catalase (27). In the pres-ence of 3-AT there is a significant decrease in theaction of catalase as an inhibitor of leukocyte-platelet in-teraction. This is further evidence that the inhibitoryprinciple released is, at least in part, hydrogen peroxide.

As a further control for these experiments, GFPwere exposed to dialysis bags containing autologouslymphocytes with or without latex particles. These cells

go 3.5-35.

R 3.0-

bc 2.5

2.0-

'1.5.

% Q5.

0

00

00

.

2.5 5.0 7.5nmol H202 in Dialysate

FIGURE 8 Consumption of exogenously added H202 byGFP. Experimental design is as in Fig. 7, except that 0.5ml of GFP at 200,000/mm3 is substituted for granulocytesin the dialysis bags. H202 consumed = H202 exogenouslyadded - H202 measured. (The platelets themselves releasedno measurable amount of H202).

50.

cijc

40

tUg30.

20.

10.

t ).

BUFFERONLY

GLUCOSE+GLUCOSEOXIDASE

A B

FIGURX 9 Effect of a cell-free H202-generating system onplatelet function. Glucose oxidase at 0.012 U/ml and excessglucose, when placed in a dialysis bag, release H202 into thedialysate at a rate and final concentration analogous tophagocytosing granulocytes. In an experiment analogous tothat in Fig. 3, aggregation after exposure to such bags (col-umn B) was inhibited, as compared to the aggregation seenafter exposure to bags of buffer only (column A vs. columnB, P < 0.01).

did not affect platelet physiology under these experi-mental conditions.

If H202 is the major diffusable platelet-inhibitoryfactor elaborated by activated granulocytes, it shouldhave been possible to mimic the phenomenon with a cell-free H202-generating system. This first required deter-mination of the amount of granulocyte-produced H202actually diffusing across the dialysis membrane. Wefound a mean concentration of 0.86 nmol H202 in the2.5-ml dialysate of activated granulocytes after a 20-min incubation (Fig. 6). Since this was considerablylower than expected from the kinetic measurement ofH202 generation by activated granulocytes (25), wedetermined the rate of appearance of H202 in the di-alysate. Granulocyte-produced H202 diffused across thedialysis membrane at a fairly steady rate over the 20-min incubation period. This rate averaged 0.20 nmol/min/2.5 X 107 granulocytes and yielded a final H202concentration of 4.0 nmol/2.5 ml in our experimentalsystem.

The difference between the appearance of H202 inthe dialysate of stimulated PMIN when monitored con-tinuously (4.0 nmol) and that observed by analyzingthe dialysate at 20 min (0.86 nmol) indicates eitherdegradation or consumption of the generated H202.

Granulocyte-Produced H3O0 and Platelet Reactions 961

Since H202 was shown to be consumed by the activatedPMN, our value of 0.86 nmol in the dialysate of acti-vated PMNafter a 20-min incubation reflects only thatH202 generated but not subsequently consumed by thePMN.

In addition to PMN, GFP were present in the ex-perimental set-up described in Fig. 1. These cells werealso found to have the capacity to consume H202. Again,the consumption appeared to be related to the initialconcentration of H202 surrounding the GFP.

Knowing the rate and amount of H202 diffusing fromthe dialysis bag of phagocytosing granulocytes, we couldnow design a simple H202-generating system of corre-sponding rate and concentration. An appropriate con-centration of glucose-glucose oxidase was substitutedfor granulocytes (Fig. 9), and this mixture was capableof causing significant inhibition of platelet aggregationwhen H202 was generated at a rate comparable tothat produced by phagocytosing granulocytes. It mustbe noted that this effect was less intense than thatseen after exposure to intact granulocytes.

Our previous studies on the addition of single dosesof H202 to platelet suspensions had led us to believe thatconsiderably higher doses of H202 than those measuredhere were necessary to alter platelet function. Whenadded as a single dose, a minimum concentration of 20,MM H202 was necessary (15). However, when H202was generated continuously, from either activated PMNor glucose oxidase plus glucose, much lower concen-trations were capable of altering platelet function. Ata rate of H202 production of 0.2 nmol/min/2.5 X 107PMN, the concentration at 20 min would be only 1.6 t&M(4.0 nmol/2.5 ml). This observation on the increasedsensitivity of platelets to low levels of constantly gen-erated H202 is in keeping with that of Cohen and Hoch-stein (23), who observed an increased oxidation oferythrocyte glutathione and increased osmotic fragilitywhen these cells were exposed to a low-level, steady-state infusion of H202.

In summary, the data presented here indicate that aproduct or products of blood granulocytes can exertsignificant effects on platelet-physiology. Moreover,there is clear evidence that granulocyte-produced H202is responsible for a significant portion of the alterationin platelet function. Biochemical alterations broughtabout by H202 that might explain the inhibition ofplatelet function include membrane lipid peroxidation(31), oxidation of free sulfhydryl groups (23), andalteration of important soluble aggregating agents re-leased by platelets, such as prostaglandins or their pre-cursor endoperoxides (32, 33). Such changes might bedirectly attributable to H202, or they could be broughtabout by the hydroxyl radical (OH. ) that can beformed from H202 and another granulocyte product,superoxide, in the following reaction (34): H20 + 02-

>OH. + OH- + 02. These various possibilities arenow under investigation in our laboratory.

The incorporation of large numbers of granulocytesinto freshly formed thrombi has been documented (5).In the organization phase of thrombosis, large numbersof neutrophils invade the surfaces of the thrombusclosest to the blood (7). These neutrophils have beenassumed to serve only a phagocytic function (6). Couldsuch activated granulocytes, via liberation of H202 orother potent compounds, inhibit propagation at thethrombus-blood interface? Could granulocytes involvedin earlier stages of the initial hemostatic response (35,36) alter or help to regulate platelet reactions in vivo?In 1966, Harrison et al. (37) showed that varying de-grees of contamination of PRP by leukocytes alteredthe in vitro aggregation response of the platelets. Ourfindings may explain their observation. Perhaps the lossof platelet function during storage of platelet concen-trates is attributable in part to products the granulocytesrelease in these concentrates.

If the granulocyte can indeed influence the course ofhemostasis and thrombosis by modulating platelet func-tion, a new avenue of approach to the therapy of hemor-rhagic and thrombotic diseases could conceivably beopened.

ACKNOWLEDGMENTSThe authors are indebted to Drs. Robert S. Schwartz andJane F. Desforges for reviewing the manuscript, to Dr.Richard K. Root for his advice in the development of theH202 assay, and to Eileen M. O'Brien and Judy C. Hardinfor their invaluable assistance in the completion of thiswork.

This investigation was supported in part by NationalInstitutes of Health grant S01 RR 05598.

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