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THE INFLUENCEOF PLASMA PROTEINS ON HUMAN PLATELET METABOLISM M. Sandbjerg Hansen* and Nils U. Bang* (Lilly Laboratoryfor Clinical Research and the Departmentof Medicine, Indiana University School of Medicine, Indianapolis,Indiana,U.S.A.)

(Reciived 1.9.1978; in revised form 13.10.1978. Accepted by Editor M.I. Barnhart)

ABSTRACT This study, which focuses on certain essential parametersof platelet metabolic activity--oxgen consumption,lactate productionand cyclic AMP (cAMP1 content--indicatesthat platelet metabolism is significantlyinfluencedby the plasma protein environment. Thus, platelet oxygen consumptionin platelet-rich plasma was 88 per cent higher than oxygen consumptionof washed platelets. Oxygen consumptionof washed platelets could be restored to values comparable to those obtained for platelet-rich plasma by the addition to the washed platelet suspensionsof either autologous plasma, purified albumin or purified IgG imnunoglobulin. In contrast, the addition of fibrinogen and purified IgA myeloma protein from a patient with bleeding diathesis and platelet dysfunctionhad no effect on oxygen consumptionof washed platelets. Platelet lactate production was identical in platelet-richplasma and washed platelet suspensions. The addition to washed plateletsof autologous plasma, albumin or g-a globulin did not affect lactate production;however, when either fibrinogenor the purified IgA myeloma protein were added to suspensionsof washed platelets, lactate production Increased significantlyover the values observed in either platelet-richplasma or washed platelets. CAMP levels were considerablylower in washed platelets than in platelets in platelet-richplasma. When gamma globulin and albumin were added to suspensionsof washed platelets, CAMP levels further decreased; and this reduction in CAMP content was progressivewith time. In contrast, the *Present address: Departmentof Clinical Chemistry, University of Copenhagen,Hvidovre Hospital, 2650 Hvidovre, Denmark. **Reprint requests: Lilly Laboratoryfor Clinical Research, Wishard Memorial Hospital, 1001 West 10th Street, Indianapolis,Indiana 46202, U.S.A. 131

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addition of fibrinogen to suspensionsof washed platelets caused no change in platelet CAMP content. Thus, different plasma proteins affected different parametersof platelet metabolism in different ways. Although an understandingof the mechanisms whereby plasma proteins affect platelet membrane function and metabolic activity is lacking, our experimentsunderscore the need for a concise definition of the plasma protein environment in the assessment of human platelet metabolism.

INTRoWcTIoN Most studies on platelet metabolism have focused on platelets separated from their nonasl plasma protein environment,overlooking Roskams concept of a "plasmaticatmosphere"of proteins necessary for optimum platelet function fonsulated in 1923. It has been proposed from this laboratory that the equilibrium between cationic and anionic proteins in plasma and on the platelet mambrane surface is an important regulator for the rate of platelet aggregation (Bang et al, 1972). Based on these observations.we investigatedthe influence of the plasma protein environmenton platelet metabolic activity. This carrnunication suaanarires our evidence indicatingthat the plasma protein enviroment substantially influencesplatelet oxygen consumption,lactate production and cyclic AMP content. HATERIALS AND METHODS Platelet-Preparations Plateletswere obtained from venous blood from normal adult volunteers. All blood was withdrawn into siliconitedsyringes and siliconizedglassware was used throughout all preparetiveprocedures. Blood was collected into EDTA to a final concentrationof 4ui4. Platelet-richPlasma

In experiments focusing on cAMP levels, platelet-richplasma was prepared by centrifugationat 150 x g for 20 minutes. In experiments focusing on platelet-oxygenconsumptionand lactate productionrequiring more concentratedplatelet suspensionsto obtain meaningful values, platelet-richplasma was prepared by an initial centrifugationat 150 x g for 20 minutes followed by recentrifugationof the platelet-rich plasma supernatantat 5900 x g for 20 seconds. The platelet button was dispersed and resuspended in its cwn platelet-poorplasma to one-tenth of the original volume. Washing of plateletswas performed using a modificationof a method previously published from our laboratory (Bang et al, 1972). Procedures were carried out at room temperature. EMA blood was centrifugedat 5900 x g for 20 seconds; platelet-poorplasma was removed; and all formed elements were resuspendedin a modified Tyrode's buffer containing trisodium citrate to 10 mH, but containing no calcium chloride. A

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20-second 5900 x g centrifugationwas repeated, and all fomd elements were resuspendedfor a second time in modified Tyrode's buffer. Resuspended formed elements were now centrifugedat 170 x g for 20 lllinUkS, which effectivelyseparated platelets from red cells and white cells. The platelet-richsupematant was centrifugedfor 5900 X 9 for 20 seconds and the platelet button resuspendedin a volume Of modifi ~2T;:%ts,l. buffer adjusted to give a final platelet count of l-2 x 10 All platelet counts were performed electronicallyin a Coulter Counter (Davis and Green, 1967). Immediatelyupon completionof the washing procedures,the different oroteins under study were added to different aliquots of the same platelet suspension. The osmolalityof the suspendingbuffer and all protein susoensionswere measured by freezing point depression and varied within narrow limits of 285 to 290 mOsm in all experiments. All experimentsinvolvingoxygen consumptionand lactate productionwere performedwithin 2 l/2 hours of canpletionof the washing procedures. Control measurementsof platelets suspended in buffer with no protein added were routinely performed inmediatelyupon completionof the washing and toward the end of the experiment. Platelet oxygen consumptionand lactate production did not vary significantlywithin this time span. PLASMA MANIPULATIOE Autologous Platelet-PoorPlasma The supernatantplasma harvested from the first high-speedcentrifugation during the washing procedureswas recentrifugedat 12,100 x g for 5 minutes. Individualolatelet susoensionswere alwavs _ reconstituted with their own platelet-poorplasma: Feat-TreatedPlasma Platelet-poorplasma was incubated at 80' C for 20 minutes. Dialyzed Plasma Platelet-poor was dialyzed against two changes of modified . -_ - plasma _. Tyrode's buffer for 24 hours. PURIFIED PROTEINS Fibrinogenwas prepared according to the methods of Kazal et al (1963) and Mosesson and Sherry (1966). The resultant preparationswe 96-100 per cent clottable and homogeneousby sodium dodecyl sulphatelizDS) polyacrylamidegel electrophoresis(5 per cent SDS gels, unreduced system) (Weber and Osborn, 1969). When examined by agarose gel chromatography on 8io Gel A-5m (Bang et al, 1973), these protein preparationseluted at sharp symmetricalpeaks and were, therefore, consideredto be free of high molecular weight aggregates. The resultant preparations containedno demonstrablethranbin or procoagulantactivities,since they did not clot following incubationwith calcium chloride to 28 mM or with brain tissue thromboplastinand calcium for 24 hours. In a few experimentsa carmercialpreparation (human fibrinogen, Grade L, KABI, Stockholm, Sweden) was used. This preparationwas 95

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per cent clottable and displayed no high molecular weight aggregates by agarose gel chromatography. It did contain trace procoagulants, since this preparationclotted after several hours' incubationwith brain tissue thranboplastinand calcium. Results discussed in the following were identical, irrespectiveof the source of fibrinogen. Gansaa lobulfn was Cohn fraction II (Lederle Laboratories,Pearl River* n some experimentsthis material was further purified by OEAE cellulose chromatographyfollowed by repeated ammonium sulfate precipitation(33 per cent saturation)and'preparativeultracentrifugation to remove aggregates. Results in the following were identical,whether the camaercialCohn fraction II preparationor highly purified gaamta globulin was utilized. Albumin was Cohn fraction V obtained from Cutter Laboratories (Berkmalifornia) and was used without further modification.

IgA myelana protein was obtained from patient L.R., who had sfgnificant bleeding episodes in her history despite a normal platelet count. She also showed laboratoryevidence of platelet dysfunction including a prolonged bleeding time, poor clot retraction and diminished aggregation in response to collagen.ADP, and epinephrine. This patient's serum was first precipitatedtw(ce with ammonium sulfate (33 per cent saturation) and the IgA myeloma protein further purified by DEAE cellulose chromatography according to Fahey et al (1958). Protein Concentrations. The final concentration(Pmol/l) of proteins added to platelet suspensions in the various experimentswere albumin 580, IgG 21.3, IgA myeluna protein 15.6, fibrinogen 8.8. Autologous plasma was added to 35 g/l final concentration. Platelet oxygen consumptionwas measured polarographicallyutilizing the Clark oxygen electrode (Clark et al, 1953) (Yellow Springs Instruments, Inc., Yellow Springs, Ohio). In All experiments two electrodeswere used simultaneously. One was immersed into a cuvette containing platelets in suspensionwith or without protein added. The other electrodewas imnersed into a control solution which contained buffer with or without the appropriate protein but no platelets. In this manner we excluded the possibilitythat the protein under study had changed the apparent oxygen consumptionby changing membrane characteristicsand oxygen diffusion across the membrane. The contents of both cuvettes were stirred at 480 rpm with magnetic stirrers. Both cuvettes were jacketed and thennostatedat 37O C. The cuvettes and the magnetic bars were siliconized. For each polarograghic assay one ml of the original platelet suspension containing l-2 x 10 plateletswas diluted to exactly 2 ml by either buffer alone or buffer containingthe protein under study. All suspensionswere preincubated at 370 C for 10 minutes at which time the electrodeswere inserted and recording begun. Continuous recordings for 15 minutes or longer were used in all experiments. Calibration of the instrumentutilized NADH and rat liver submitochondrial particles essentiallyas described by Chappell (1964).

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Platelet Lactate Production. One-ml aliquots of plateletswere incubated at 37' C without stirring but with occasional agitation. At time 9 and at time 60 minutes, one ml of 0.4 M perchloric acid was added to individualone-ml suspension aliquots. The protein-freesupernatantwas neutralizedwith potassium carbonateand assayed for lactate enzymaticallyaccording to Marbach and Weil (1967). 31-5' Cyclic AMP Content. Following the completionof the washing procedures,one.,mlaliquots of platelet suspensionswith or without proteins added were incubatedat 37' C for different time intervals. The suspensionswere deproteiniredand CAMP extracted through the addition of 0.5 ml of ice-cold, 0.60 M trichloraceticacid. The mixture was left in an ice bath for 15 to 30 minutes. After centrifugationat 12,100 x g for 50 minutes at 4O C, the supernatant was removed and extracted three times with 5 ml water-saturatedethyl ether. The extracted aqueous phase was heated at 80' C for 2 minutes and evaporated to dryness under a stream of dry nitrogen. The residue was dissolved in 0.6 ml of 50 n@lsodium acetate buffer, pH 6.2 and assayed directly or stored at -200 C for assay at a later time. CAMP was quantitatedby the radioimnunoassayof Steiner et al (1969) utilizing a camnercialpreparation pf rabbit antiserum to succinyl cAMP. This antiserun,as well as additional reagents, required for the test were obtained from CollaborativeResearch, Inc., Waltham, Massachusetts. Platelet Aaaregationand Release Reaction.

In separate experimentswashed platelet suspensionswith and without proteins added were incubated at 37' C for one hour and then examined for aggregate formation by phase microscopy. Similarly,stirred and unstirred platelet suspensionswere incubated at 37' C for one hour; and platelet factor 4 release was quantified according to Niewiarwski and Thomas (1969). 18 additionalexperimentssuspensionsof washed platelets prelabelled with C-serotoninwere incubatedunstirred but with occasionalagitation at 37O C for one hour in the presence and absence of proteins and serotonin release determined (Hirschmanand Shulman, 1973). rformed according to the techniques of Concentrationsof specific proteins on washed platelet suspensions, lysed by repeated freezing and thawing,mre determinedby radial i-nodiffusionor electroinnmuio assay (Laurell,1972) utilizingmono-specific antisera raised in rabbits in our laboratoryor obtained carnwrcially (Dako issnunoglobulins, Copenhagen,Denmark). Total protein concentrations were determined by the Folin-Ciocalteumethod. RESULTS Effectivenessof the Washing Procedure The washing proceduresused in .thisstudy effectivelyremoved plasma proteins from platelet surfaces.

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Direct imunochemical determinationsrevealed concentrationsof individualproteins remaining in washed platelet preparationsto be as follows: albumin ~1 umol/l, fibrinogen eO.14 ~mol/l, IgG eO.3 ~mol/l, IgA ~0.4 umol/l and IgM ~0.04 pmol/l. The final platelet suspension contained less than one erythrocyte/20,000 platelets,as evidenced by phase microscopy. When platelets from these suspensionswere prepared for transmissionelectrohmicroscopyaccording to the technique of White (1968). the resultant preparationscontained an average of 25 per cent disc shaped platelets and 75 per cent of platelets showing sphering and some pseudopod formation. No degranulationor aggregate formation was observed. We have previouslydemonstratedthe effect of our washing procedure in a series of electron micrographs of whole mount platelet preparations negativelystained with PTA (Bang et al, 1975). The platelets in plateletrich plasma are surounded by an intense cloud of PTA stained proteins. The washing procedure removes this colloidal cloud completely. Upon resuspensionof platelets in different protein solutions, protein again accumulatedaround platelets, but this accumulationtook different forms for different proteins. A uniform cloud of PTA-staining material was regularly seen when platelets are resuspendedin albumin, while resuspensionof platelets in either gwna globulin or fibrinogen resulted in a spotty, patchy attachment of protein to the platelet membrane leaving many areas of membrane uncovered by protein. Results in the following represent the mean values of 6-8 different experiments involving 6-8 different platelet preparationsin each series of experiments. Uxygen Consumption. As can be seen fran Figure 1, the oxygen consumptionof washed platelets averaged 27.1 f 0.9 natans/ml/l09platelets (Mean f SEM). This figure is in fair agreementwith values published in the literature. This value is substantiallylower than the levels observed in unwashed platelets in platelet-richplasma of 50.9 f 4.1 (Hean f SEMI. The differencesare significantat the 0.1 per cent level. The addition of autologous plasma to washed platele suspensionsrestored oxygen uptake to 4 platelets, a value in close agreement with a mean value of 57 natans/minute/lO the oxygen uptake of unwashed platelets in their native plasma protein envirofxnent.Preliminaryevidence that plasma proteins were responsible for this effect is also shown in Figure 1. Oxygen uptake did not differ significantlyfrom the buffer control when heat-treatedplasma was added to the washed platelets. On the other hand, when extensivelydialyzed plasma was added to suspensionsof washed platelets,there was a marked.statisticallysignificantincrease in platelet oxygen uptake as canpared to the buffer control. Figure 2 summarizes studies on the influenceof purified plasma proteins on platelet oxygen uptake. For comparison,this figure depicts, in addition to the control values for oxygen consumptionof washed platelets suspended in buffer, also the values for platelets in platelet-richplasma already depicted in Figure 1. The addition of albumin or gamma globulin to the washed platelet suspensionsresulted in an increase in oxygen

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Comparison of oxygen consumption (Mean ?rSEM) of washed platelets in buffer (thereforein protein-freemedium) with similarlywashed platelets resuspendedin either dialyzed, heated or autologousplasma.

FIG. 2 Platelet oxygen consumption (Mean t SEM) of platelets in platelet-richplasma compared with washed platelet suspensions in either albumin or normal IgG, IgA myeloma protein, fibrinogen,or buffer without protein.

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consumptionto mean values of 49 and 42 natoms/minute/lOgplatelets, respectively. The differences in oxygen uptake between platelets suspended in buffer and platelets resuspended in either albumin or gm globulin containingsolutions are significantat the 0.1 per cent level. Values for oxygen consumptionof washed platelets resuspendedin either albumin or gamma globulin were no different from the values observed for platelets in platelet-richplasma.

In contrast, the addition of fibrinogen to washed platelets produced oxygen consumption levels identical to the buffer control. The IgA myeloma protein from the patient with platelet dysfunction in contrast to regular IgG imnunoglobulindid not normalize oxygen consumptionwhen added to suspensionsof washed platelets. Lactate Production.

I

FIG. 3 Lactate production (Mean f SEM) of platelets in plateletrich plasma and washed platelet suspensions in solutions containing albumin, gamma globulin, fibrinogen,an IgA myeloma protein, autologous plasma or no protein. Lactate productionof platelets in platelet-richplasma was 2.2 f 0.3 micromol/hour/lOgplatelets. $actate productionof washed platelets was 1.8 +,0.3 micromol/hour/lO platelets (Mean * SEM) These two values were not different by statisticalanalysis, and both values were in good agreementwith figures already presented in the literature. (Corn, 1966, Karpatkinand Langer, 1968). lYeanlactate productionfollowing the addition of albumin, gy globulin and autologous plasma was 2.8, 3.8 and 1.9 micranol/hour/lO platelets, respectively. None of these differenceswere significant

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when caspared to either the washed platelet or the platelet-richplasma values. In contrast, the addition of fibrinogen to washed platelet suspensionssubs ntially enhanced mean lactate productionto 7.0 k platelets. The difference in lactate production micromol/hour/lO between fibrinogen containingsuspensionsand buffer controls were significantat the 0.1 per cent level. The same IgA myeloma protein which produced no increase in oxygen consumptionwhen added to suspensions of washed platelets produced n increase in lactate productionto a Mean Ifplatelets. The difference between myeloma level of 6.4 micromol/hour/lO protein containing suspensionsand buffer controls was also significant at the 0.5 per cent level. 3' - 5' Cyclic AMP Content.

FIG. 4 3’ - 5’ cyclic AiiPcontent (Mean + SEM) of platelets in platelet-richplasma and washed platelets.

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The experimentsdepicted in Figure 4 compare CAMP levels of washed platelets and platelets from platelet-richplasga. The Mean content of CAMP in platelet-richplasma was 145 picomol/lO plgtelets, and the mean CAMP content of washed plateletswas 112 piuxnol/lO platelets. These differencesare significantat the 0.1 Per cent level. The experimentssunmuHzed in Figure 5 examine CAMP content as a function of time following the addition of different proteins to washed platelet suspensions. In the controls representedby the'first group of

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5

3' - 5' cyclic AMP platelet content (Mean 2 SEM) in suspensions containingno protein compared with similar washed platelet suspensionsafter additions of either albumin, IgG or fibrinogen.

bars in Figure 5 in which no proteins were added, CAMP levels were determined inmediatelyupon completion of the washing procedure and 2 and 5 minutes later. The differences In CAMP levels were not significant. The addftlon of albumin produced a progressivedecrease in CAMP levels reaching significanceat 2 minutes and at 5 minutes. Similarly, the addition of ganna globulin produced significantdecreases in CAMP content at 2 and at 5 minutes. In contrast, the addition of fibrinogen to washed platelets gave values for CAMP which were not significantlydifferent from control levels at 15 seconds, 2 minutes and 5 minutes. Platelet Aggregation and Release. To ascertain whether these effects of plasma proteins on platelet metabolism were associatedwith platelet aggregationand/or release of intracellular constituentswe conducted additionalexperiments. Aliquots of washed platelets suspended in buffer and washed platelets suspended in buffer containing either gamma globulin, albumin, autologous plasma or fibrinogenwere stirred at 370 C for 1 hour and the suspenjionsat that time examined for aggregates by phase microscopy. In no instance did we detect platelet aggregates under these conditions. Table I smmnarizeslgxperiments which examined the release of two platelet constituents-- C-serotonin and platelet factor 4--ln washed platelets suspended in buffer or suspended in buffer containingeither autologous plasma, ganmnaglobulin, albumin or fibrinogen following one hour's incubation at 370 C. Although detectable quantities of both platelet factor 4 and 14C-serotoninwere released during one hour's incubation,nearly identical figures were obtained in the controls In which no protein was added and in experimentswhere different proteins were added to the platelet suspensions. Thus, we concluded that the observed effect of plasma proteins on platelet metabolic activity was not the consequenceof aggregationand release of platelet constituents.

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TABLE I Release of Platelet Factor 4 (PF4) and 2-14C-5erotonin(5HT) from Washed Plateletswith and without Proteins Release in Per Cent of Calculated Maximum Release PF4

5HT

No Protein

6

22

Albumin

6

14

Ganmm Globulin

6

25

Fibrinogen

6

27

DISCUSSION It has been recognizedfor some time that the necessity of isolating platelets from other blood componentscan pose major problems in studying platelet metabolism. Mustard and Packham (1970) pointed out that the time required to isolate the platelets from blood, the washing procedures and the suspendingmedia all significantlyinfluence platelet structure and function making it "virtuallyimpossibleto study platelet metabolism in what could be described as the,restingstate." In the past, several references have been made to indicate that washing procedures can substantially affect many aspects of platelet function and metabolism.

Rock and Nemerson (1969) observed a fall of ATP levels in washed platelets to about 50 per cent of initial values in 3 ours. This was accompaniedby a decreasing rate of incorporationof 3BPi. The authors suggested that uncouplingof oxidative phosphorylation occurs in plateletseither as a consequenceof washing and centrifuging the cells or due to the anticoagulantused. In this work it was also observed that resuspensionof washed platelets in ACD plasma resulted in better maintenanceof ATP and higher phosphorylationrates than did resuspensionin buffer irrespectiveof the substrates added, leading the authors to suggest the existence of plasma factors that stabilize the metabolism of platelets. Rossi (1972) noted a progressiveloss of lactate dehydrogenase, adenosine triphosphataseand adenylate kinase into protein-freeartificial media, as plateletswere repeatedlywashed and resuspended. The addition of albumin to the resuspensionmedia diminished enzyma loss from platelets suggesting to this author that albumin may provide the platelets with a protectiveprotein coat. Doery et al (1973) studied the effect of albumin and apyrase on the metabolic rate of suspensionsof washed platelets and noted that albumin

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significantlyreduced the leakage of platelet lactate dehydrogenase,beta glucuronidase,serotonin and nucleotideswhich othemise occurred during the resuspensionprocedure. Assays of levels of key glycolytic intermediates indicated that the washing procedures stimulated platelet Alctabolfsm. Return to resting metabolfc rates occurred more rapidly when albumin and apyrase were present in the resuspendingmedium. Our own experiments indicate that separating platelets fran their nonnal plasma protein environment substantiallyreduces oxygen consumption. We also noted that the reduction in platelet respirationcould be restored to nonal platelet-richplasma values upon the addition of normal plasma to suspensionsof washed platelets. Gamma globulin or albumin was just as effective in nonnaliring oxygen consumptionas was normal plasma, but purified fibrinogen had no effect. Platelet glycolysfs,as reflected by lactate production.was no different in platelets in their normal plasma protein milieu and in washed platelet suspensions. The addition to washed platelet suspensions of either autologous plasma, normal IgG imnunoglobulinand albumin produced no significantchanges in platelet lactate production. On the other hand, fibrinogenwhich had no influence on platelet respiration,produced statisticallysignificant increases in lactate production to values far exceeding the values observed in platelet-richplasma. Our findings that albumin produced no changes in platelet lactate production conflict with the results of Ooery et al (1973) who reported that albumin in the suspendingmedium resulted in a decrease in gl colysis rates of washed platelets. The small differencesbetween our resuzts and those reported by Ooery could be accounted for by the different washing procedures,different anticoagulantsand different protein concentrations used in their experiments,as compared to ours. In addition, we investigatedthe effects of a purified IgA myeloma protein from a patient with platelet dysfunction. We included this protein in the investigationbecause of our previously reported finding (Bang et al, 1972) that a similar IgA myeloma protein from another patient who had platelet dysfunctionand bleeding problems affected platelet aggregation in a manner principallydifferent from nonal IgG inanunoglobulin.Whereas, normal IgG Mnunoglobulin restored aggregationpotentialwhen added to suspensionsof washed platelets,this IgA myeloma protein had no such effect. It is of interest to note in the present series of experiments that the IgA myeloma protein utilized here also behaved differentlyfrom normal IgG imnunoglobulinwith respect to platelet metabolism in that the IgA myeloma protein, when added to a suspension of washed platelets,did not produce the enhancementof oxygen consumptionnoted for the normal ganInaglobulin preparation. In contrast, it produced significant increases in lactate production. Different plasma proteins also influenced the levels of platelet cyclic AMP content in differentways. The central role for cyclic AMP, as a regulator of platelet functional activities,has been emphasized in several studies in recent years. Thus, it has been shown conclusively that measures which increase platelet intracellularcyclic AMP will tend to maintain platelets in their nomml non-aggregatedstate (Salzman and

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Levine, 1971, Cole et al, 1971). while substancescupable of inducing platelet aggregationreduce platelet cyclic AMP content (Chiang et al, 1975, Salzman, et al, 1976). In our experimentsalbumin and gwm&globulin, when added to suspensions of washed platelets, produced a significantdecnase in platelet cyclic AHP content; whereas, fibrinogenhad no d~nstrable effect. Our results also establishedthat cyclic AMP content is signlflcantlylower in washed platelets than in platelets contained in their normal plasma protein environment. These latter observationsagree with previous reports indicatingthat even minor manipulationsof platelets,such as centrifugation, tend to reduce cyclic AMP content (Salzmanand Levine, 1971, and Salanan et al, 1970). It is unlikely that the observed metabolic effects of plasma proteins on washed platelet suspensionsare the results of concomitantchanges producing release and aggregation. In our studies we found little or no release of platelet factor 4 in stirred preparationsof washed platelets or platelets suspended in the different protein solutions. Significant quantitiesof labelled serotoninwere released; however, the cwnmt released was Identical In all platelet suspensions,irrespectiveof their protein content. In addition, we observed no evidence for aggregate fonsation by phase contrast microscopy. . One may speculate that the observed effects of dlffennt plarna proteins on intracellularplatelet events are mediated through a modificationof the localizationor conformationof certain membrane receptors. Nicolson (1973) has shorm that anionic sites (N-acetyl-neuramlnic acid) in erythrocytemambrana can redistributeunder certain stimuli consistent with his Fluid Mosaic Model. This concept has been substantiatedfor gelfilteredhuman platelets by Hawiger and Tbmaons (1975) by using a fluorescentprobe known to interact with hydrophobicmembrane regions. These authors demonstratedthat more hydrophobicsites were exposed when fibrinogenwas added to the suspendingbuffer. InvestigatingimmunologicreactSons involving platelets, Pfueller et al (1977) found the binding of IgG to platelet membranes to be Influenced by plasma proteins other than IgG. A possible relationshipbetween the plasma membrane structure and the intracellularconcentrationof cyclic AMP was demonstratedby Carley et al (1976). These authors, working with rat epitheloid kidney cells, found a lower concentrationof cyclic AMP In cells with plasma membrane microvilli. A connectionbetween the cyclic AMP concentration and the metabolic integrityof rabbit platelets has been demonstrated by Hashimoto et al (1975) using differentmetabolic inhibitors. This, of course, is not surprising,but underscoresthe close relationshipbetwaen the differentmolecular events in the cells. Still, the mechanisms behind the translationof diffennt surface stimuli caused by the adsorptionof different plasma proteins to the cell surface remains largely unknown. However, our data indicate that a meticulous definition of the plasma protein environmentis essential in platelet metabolic studies.

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Karpatkin,S. and Langer, R. M. Biochemicalenergeticsof simulated platelet plug formation. Journal of Clinical Investigations2158, 1968.

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Kazal, L. A., Ansel, S., Miller, 0. P. and Tocantins, L. M. The preparationand some propertiesof fibrinogenprecipitatedfrom human plasma by glycine. Proceedingsof the Society of Experimental Biology and Medicine 113:989, 1963.

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analysis Laurell. C. B. Electrophoreticand electro-imnunochemical of proteins. ScandinavianJ. Clinical and Lab. Invest.29 (Suppl 24): 21, 1972.

20.. Marbach, E. P. and Weill, M. R. H. Rapid enzymatic measurementof blood lactate and pyruvate. Clinical Chemistry (New York) 13:314, 1967. 21.

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Mustard, J. F. and Packham.M. A. Factors influencingplatelet function: adhesion, release and aggregation. PharmacologicalReviews 22_:97,1970.

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Niewiarowski,S. and Thomas, D. P. Platelet factor 4 and adenosine diphosphaterelease during human platelet aggregation. Nature 222: 1269, 1969.

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Pfueller, S. L., Weber, S. and Luscher, E. F. Studies of the mechanism of the human platelet release reaction induced by immunologicstimuli. III. Relationshipbetween the binding of soluble IgG aggregates to the Fc receptor and cell response in the presence and absence of plasma. J. of Immunology118:514, 1977.

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Rock, R. C. and Nemerson, Y. Energeticsof human blood platelets: Uncoupling of oxidative phosphorylationin intact cells. Journal of Laboratory and Clinical Medicine 73_:42,1969.

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