platelet aggregation - journal of clinical pathology · j. r. o'brien the effect of time and...

J. clin. Path. (1962), 15, 446

Platelet aggregationPart I Some effects of the adenosine phosphates, thrombin, and

cocaine upon platelet adhesiveness

J. R. O'BRIEN

From the Portsmouth and Isle of Wight Area Pathological Service

SYNOPSIS Platelets in native blood adhere spontaneously to glass independently of temperature:if adenosine diphosphate is added to the blood the adhesiveness of the platelets is increased and thiseffect is largely independent of temperature. The mono- and triphosphates decrease adhesivenessat 20°C. and 37°C. but have no effect at 0°C.; cocaine inhibits adhesion at 37°C. and at 0°C.

Aggregation and viscous metamorphosis of platelets in native plasma is induced at 37°C. byadenosine diphosphate or by thrombin; these reactions do not occur at 0°C. Cocaine and all theother anti-adhesive drugs inhibit thrombin or adenosine diphosphate-induced aggregation. Themono- and tri-phosphates appear to compete with adenosine diphosphate and inhibit aggregation;they also inhibit thrombin-induced aggregation. Aggregation induced by adenosine diphosphate or

thrombin is not prevented by any of the usual enzyme inhibitors or uncoupling agents at the appro-

priate strength. At 37°C. aggregation and viscous metamorphosis induced by adenosine diphosphateor thrombin are reversible, and the addition of more adenosine diphosphate or of thrombin againproduces aggregation and viscous metamorphosis.

Platelets incubated with adenosine diphosphate but not agitated lose their power to aggregate butwhen more adenosine diphosphate is added with agitation, then aggregation is again produced. Theseobservations are presumably explained by the finding that intact platelets, but not fragmentedplatelets, can inactivate adenosine diphosphate. From these results it is tentatively concluded thatadhesion may involve intrinsic adenosine diphosphate in the platelet which may be activated bythrombin and inhibited by the added mono- or triphosphate. The anti-adhesive drugs act in a

different manner. These phenomena have a remarkable similarity to those concerning mito-chondrial swelling.

It is not known why a platelet sticks, but Hellem(1960) reported that a factor R isolated from redcells caused platelets to stick to glass; 0llgaard(1961) also studied a non-protein extract fromplatelets and red cells that caused platelet aggre-gation. Gaarder, Jonsen, Laland, Hellem, andOwren (1961) showed that their factor is adenosinediphosphate, that it is highly specific, and that itinduces platelet aggregation in citrated platelet-richplasma. O'Brien (1961) showed that the adhesion ofnative platelets to glass and to damaged cells in vitroand in vivo was inhibited by many anti-malarial,anti-histaminic and local anaesthetic and some otherdrugs which will be called the 'anti-adhesive' drugs.These findings stimulated the present study of the

Received for publication 5 February 1962.

effects of adenosine diphosphate and thrombin onplatelet adhesiveness to glass and on platelet aggre-gation and viscous metamorphosis. The effect of theanti-adhesive drugs and adenosine monophosphateand the triphosphate and enzyme inhibitors werealso studied in an attempt to understand the pro-cesses involved in adhesion, aggregation, andviscous metamorphosis.

METHODS

ADHESION OF PLATELETS TO GLASS Native blood collectedin a plastic tube was used at once or after cooling forfive minutes in ice-water; the blood was mixed withbarbitone buffered saline as a control or with the materialunder study and was passed immediately through 'filterunits' consisting of a standard quantity of glass beads in a

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Platelet aggregation

plastic tube (O'Brien, 1961) at room temperature or at0°C. By counting the platelets in the blood before andafter passing them through the filter the percentage of theplatelets lost can be calculated; those had presumablystuck to the glass during their passage through the filter.

PLATELET AGGREGATION AND VISCOUS METAMORPHOSISPlatelet-rich plasma was prepared by slow centrifugationof native, citrated (9 parts of blood to 1 of 3-8% sodiumcitrate) or heparinized (4 units of heparin/ml. blood)blood. This platelet-rich plasma with or without additionswas examined within two hours of collection on a slideunder a coverslip so supported that it could be rocked.A 1/6 microscope objective and phase illumination wasused. With constant rocking, platelet aggregation andviscous metamorphosis could be observed and roughlytimed. Platelet aggregation was also studied in tubes of7 mm. internal diameter in which usually 0-2 ml. ofplatelet-rich plasma was mixed with 01 ml. of the sub-stance under investigation. The tube was continuouslyagitated in a 37°C. water-bath, and frequently inspectedthrough a powerful lens with strong transverse illumina-tion.

Viscous metamorphosis is used here to mean the changewhen a clump of discrete platelets that have stuck to-gether is observed under the microscope to change into ahyaline amorphous mass with a clearly defined, smooth,refractile edge and complete loss of platelet identity.The reagents used were diluted with 0 9% saline

buffered with barbitone buffer to pH 7 35. The adenosinedi- and triphosphate and uridene diphosphate were over99% pure and adenosine monophosphate was 95% pure,and were obtained from the Sigma Chemicals Co.Human thrombin was obtained from the Lister Institute,Ro 3/0837 from Dr. J. Marks of Roche Products, Ltd.,and N. butyl 3-5-di-iodo-4-hydroxybenzoate from Dr. R.L. Greif.

RESULTS

From Table I, which reports the mean figure fromthree to six experiments in each situation, it will beseen that native platelets (saline control) stuck to theglass as well at 0°C. as at room temperature ineither the long (8-8 cm.) or short (5 cm.) units.Adenosine triphosphate at a final concentration of0-2 jug./ml. decreased platelet adhesion at roomtemperature but had no effect if the experimentwas carried out at 0°C. Adhesion was also decreasedby adenosine monophosphate, 200 ,ug./ml., and0-2 ,tg./ml. probably still had some effect. Cocaineprevented platelet adhesion equally in the cold andat room temperature. Using the shorter units (fewerplatelets stick spontaneously to the reduced glasssurface area) it will be seen that adenosine di-phosphate at a final concentration of 0-2 ,ug./ml.greatly increased platelet adhesiveness at roomtemperature and also when the whole procedure wascarried out at 0°C. If adenosine diphosphate wasadded and the adhesiveness not tested till five

TABLE IEFFECT OF ADDITIONS AND TEMPERATURE ON THE

ADHESIVENESS OF PLATELETS TO GLASS

Filter Final Concentration of Addition Percentage PlateletsLength (sg.iml.) Adhering(cm.)

At Room At 0°C.Temperature

8-88-88-88-88-8

SalineAdenosine triphosphate, 0-2Adenosine triphosphate, 0-02Adenosine monophosphate, 200Cocaine 3,000

711655436

7873

10

5 0 Saline 25 495-0 Adenosine diphosphate, 0-2 82 -

5 0 Adenosine diphosphate, 2-0 - 805-0 Adenosine diphosphate, 0-2 (5 min.)' 8 -

5-0 Adenosine diphosphate, 0-02 16 -

5 0 Adenosine triphosphate, 200 +adenosine diphosphate, 0-2 35 -

5-0 Adenosine monophosphate, 200 +adenosine diphosphate, 0-2 14 -

5-0 Cocaine 3,000 + adenosinediphosphate, 0-2 32 -

Native blood at room temperature or after cooling in ice-water forfive minutes was mixed with the addition and immediately passedthrough the filter.'Adenosine diphosphate was mixed with the blood and the mixtureleft for five minutes before it was passed through the filter.

minutes later, the platelets had then become lessadhesive than normal. The immediately increasedadhesiveness of platelets produced by adenosine di-phosphate was inhibited by mono- and triphosphatesand by cocaine. It was also shown that when acompound with anti-serotonin activity (referred tohere as Ro 3/0837) at a final concentration of 1 mg./ml. blood was added to native blood it reducedplatelet adhesion to glass from 73% (saline control)to 22%.

ADENOSINE DIPHOSPHATE-INDUCED PLATELET AGGRE-GATION AND VISCOUS METAMORPHOSIS Platelets innative plasma, in citrated plasma, and in heparinizedplasma aggregated equally when small quantities ofadenosine diphosphate were added and the mixturewas immediately agitated in a water-bath at 37°C.The speed with which platelet clumps were formedand their size were dependent on the amount ofadenosine diphosphate added. The minimum aggre-gating concentration was found by preparing serialtenfold dilutions of adenosine diphosphate andadding 0-1 ml. of each dilution to 0-2 ml. platelet-rich citrated plasma with a platelet count of 700,000per c.mm. These mixtures were continuously agitatedfor one minute and then transferred to a coverslippreparation and inspected for platelet clumps.Adenosine diphosphate at a final concentration of0-007 ,ug./ml. produced significant clumping. It canbe calculated that approximately 100,000 moleculesadenosine diphosphate per platelet produced thesechanges.

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The effect of time and temperature on plateletaggregation was investigated in tubes as follows:To 01 ml. of adenosine diphosphate, 2 ,ug./ml.,

was added 0-2 ml of platelet-rich citrated plasmaand gross aggregation occurred in 15 seconds at37°C. if agitation was continuous. If this experimentwas carried out at 0°C. no aggregation occurredwithin five minutes but if the tube was then warmedand agitation continued, good aggregation was

rapid. If the adenosine diphosphate and platelet-rich plasma were mixed and incubated at 37°C.without agitation for five minutes and then agitatedno aggregation occurred: if more adenosine di-phosphate was then added and the tube agitated,aggregation was rapid. Furthermore, it could beshown by the following experiment that the adeno-sine diphosphate originally added had been in-activated during the five minutes' stationary incuba-tion.

After adding adenosine diphosphate in the strengthsA, B, and C shown in Table II to platelet-rich plasma, thetubes were incubated for five minutes: agitation thenconfirmed that there was no aggregation in tubes B andC. Excess adenosine diphosphate in A had causedaggregation. The platelets in the reaction tubes A, B, andC were centrifuged and 0-2 ml. of each supernatantplasma was transferred into a subsample tube containing0-2 ml. of the platelet-rich plasma to test for the presenceof adenosine diphosphate surviving in the plasma. Onagitation, no aggregation occurred in the subsampletubes B and C. As controls, adenosine diphosphate wasincubated with platelet-poor plasma and with platelet-rich plasma at 0°C.; subsamples from these mixturesproduced aggregation, indicating that the adenosinediphosphate had not been inactivated. If platelets werefragmented by freezing and thawing twice or by ultrasonicagitation, no adenosine diphosphate was inactivated. Itis concluded that 1 ml. of this platelet-rich plasma whichcontained 400,000 platelets per c.mm. could almostinactivate 1,000 jig. adenosine diphosphate, and hasinactivated at least 100 jig., or one platelet inactivates4 x 108 molecules.

The following experiments showed that aggre-gation and viscous metamorphosis are reversiblephenomena.

TA]INACTIVATION OF ADENOSINE ]

One drop of adenosine diphosphate, 2 ,ug./ml., wasadded to one drop of platelet-rich citrated plasma undera coverslip which was rocked. In 30 seconds plateletshad visibly clumped and in 15 minutes viscous meta-morphosis was marked with few recognizable plateletsremaining. The slide was then incubated in a moistchamber at 37°C. for two hours and inspected. Many ofthe hyaline masses had disappeared and clumps ofrecognizable, if distorted, platelets were seen. Agitationcaused many platelets to float away. The addition ofmore adenosine diphosphate again rapidly caused com-plete aggregation and viscous metamorphosis.

INHIBITION OF ADENOSINE DIPHOSPHATE-INDUCEDPLATELET AGGREGATION Uncoupling agents separ-

ate the normally interdependent processes ofoxidation and phosphorylation in cells or mito-chondria. A number of these uncoupling agents andenzyme inhibitors were tested for their ability toprevent adenosine diphosphate-induced aggregation(Table III). It will be seen that none inhibitedaggregation when used at the concentration requiredto inhibit enzyme activity in other systems but at ahigher concentration some prevented aggregation.Table III also reports the inhibitory effect of theanti-adhesive agents. In these experiments theweakest final strength of adenosine diphosphate toproduce aggregation was 0 5 ,tg./ml. and a finalconcentration of 1 mg. of cocaine per ml. preventedaggregation. If 500 ,tg. adenosine diphosphate/ml.was added to platelet-rich plasma the concentrationof cocaine had only to be increased to 10 mg./ml. toprevent aggregation. Adenosine triphosphate in-hibited adenosine-diphosphate-induced aggregationof platelets in citrated plasma at 37°C. but theamount of the triphosphate required varied accord-ing to the amount of adenosine diphosphate present.An approximately equimolar solution did not preventaggregation but a solution of adenosine triphosphate10 times stronger than the adenosine diphosphateusually prevented aggregation over the whole con-centration range studied (Table IV). On the otherhand, the addition of 2 mg./ml. adenosine tri-phosphate to native or heparinized plasma at 20°C.caused aggregation and did not inhibit adenosinediphosphate-induced aggregation. It was also shown

BLE II

DIPHOSPHATE BY PLATELETS

Shaken after five minutes: Supernatant Added to Platelet-Aggregation rich Plasma Aggregation

A 0-4 ml. platelet-rich plasma + 400 lAg. adenosine diphosphate Trace GrossB 0-4 ml. platelet-rich plasma + 40 ,ug. adenosine diphosphate Nil NilC 0 4 ml. platelet-rich plasma + 4 lAg. adenosine diphosphate Nil Nil

0-4 ml. platelet-poor plasma X 400 ug. adenosine diphosphate Gross0 4 ml. platelet-poor plasma + 40 ,ug. adenosine diphosphate Gross0-4 ml. platelet-poor plasma + 4 ug. adenosine diphosphate Trace

The mixtures were incubated undisturbed at 37°C. for five min. and then agitated. After centrifuging, 0-2 ml. of each supernatant was agitatedwith platelet-rich plasma.

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TABLE IIIINHIBM ON OF PLATELET AGGREGATION

The following compounds prevent platelet aggregation induced bythrombin (0 4 u./ml.) or adenosine diphosphate (4M x 10-'/ml.) inthe final concentration shown:-

TABLE IVADENOSINE TRIPHOSPHATE CONCENTRATION NEEDED TOINHIBIT ADENOSINE DIPHOSPHATE-INDUCED AGGREGATIONAdenosine triphosphate + Adenosine diphosphate Aggregation(pg.) (ug.)

QuininePaludrineCocaineProcaineXylocaineNupercaine'BenadrylPhenerganChlorpromazineImipramineRo 3/0837Reserpine

Sodium citrateE.D.T.A.

Adenosine monophosphateAdenosine triphosphate

1-65M x 10-22-5M x 10-'2-5M x 10-'SM x 10-'8M x 10-23M x 10-'M x 10-'2M x 10-27M x 10-'7M x 10-'6M x 10'M x 10-'

M x 10-'1-25M x 10-'

1-7M x 10-'1-3M x 10-'

The following compounds in the final concentrations shown do notprevent platelet aggregation induced by thrombin or adenosinediphosphate:-

Di-isopropyl phosphofluoridate2.4 Dinitro-phenolPotassium cyanideSodium fluorideSodium iodoacetateSodium amytalMercuric chloride2Sodium arsenatelAresenious oxide2Sodium azideCaffeine citrate'Nicotinamide'N. Butyl 3-5 di-iodo4-Hydroxy benzoateStrophanthin GPhloridzinSerotoninUridine di-phosphateSodium malonate

M x 1O-35M x 10-'2-5M x 10-42-5M x 10-'2-5M x 10-'4-5M x 10-'2 5M x 10-'2-5M x 10-'25M x 10-'2-5M x 10-41-25M x 10-42-5M x 10-'

2-5M x 10-'1-5M x 10-'M x 10-'6M x 1O-41-25M x 10-'25M x 10-l

'This compound was inhibitory when used at 10 times the concen-tration indicated.'These compounds were inhibitory when used at 100 times theconcentration indicated.

that the monophosphate quantitatively inhibitedadenosine-diphosphate-indiced aggregation in nativeand heparinized and citrated plasma. The inhibitoryeffect of minimal quantities of the mono- and tri-phosphates increased if they were incubated with theplatelet-rich plasma before adding adenosine di-phosphate.

Platelet-rich plasma + saline + 2Platelet-rich plasma + 2 + 2Platelet-rich plasma + 20 + 2Platelet-rich plasma + 20 + 20Platelet-rich plasma + 200 + 20Platelet-rich plasma + 200 + 200Platelet-rich plasma + 2,000 + 200

GrossGrossNilGrossNilGrossNil

To 0 2 ml. of platelet-rich citrated plasma was added the amount ofadenosine triphosphate indicated contained in 0-1 ml. saline and thenadenosine diphosphate also in 0-1 ml. and the tube was agitated for30 seconds and inspected.

fibres, but they remained adherent to the fibrin evenwhen the coverslip was rocked. The addition ofmore thrombin again produced viscous meta-morphosis. If thrombin was added to platelet-richplasma from a case of congenital afibrinogenaemiano fibrin threads formed but with agitation plateletsclumped and viscous metamorphosis occurred, fewplatelets remaining free. Two hours later the plateletswere again seen to be in loose clumps and withagitation could be separated, yielding large numbersof free, floating platelets. All the compounds testedfor their ability to prevent adenosine-diphosphate-induced aggregation were also tested using thrombin(Table III). None of the enzyme inhibitors or un-coupling agents prevented thrombin-inducedaggregation, and all the anti-adhesive drugs wereinhibitory. Adenosine mono- and triphosphate alsoinhibited thrombin-induced aggregation in nativeblood or plasma or in citrated plasma.A comparison of the concentrations of thrombin

and adenosine diphosphate required to inducesimilar degrees of platelet aggregation in platelet-richcitrated plasma is reported in Table V. It will be seenthat a fourfold increase in the concentration ofthrombin covers the whole range of effects from novisible aggregation to rapid and complete aggre-

TABLE VSENSITIVITY OF AGGREGATION TO CONCENTRATION

OF THROMBIN OR ADENOSINE DIPHOSPHATE

Final Concentration Aggregation

THROMBIN-INDUCED AGGREGATION AND VISCOUS META-MORPHOSIS If thrombin was added to citratedplatelet-rich plasma in a slide preparation or ifnative platelet-rich plasma was used alone, firstclumping, then clotting, and finally viscous meta-morphosis could be observed under the microscope.If such slides were incubated for two hours, discreteplatelets in masses were again seen among the fibrin

Thrombin(units/ml.)

Adenosine Diphosphate(g./ml.)

0080-110 170-33

0070770700

No effectTraceModerateMaximal

A range of concentrations of either thrombin or adenosine diphos-phate was added to aliquots of the same platelet-rich plasma andagitated. The concentration of each substance added to producesimilar degrees of aggregation is reported.

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gation. On the other hand, the concentration ofadenosine diphosphate has to be increased a thousandfold to produce similar results.

DISCUSSION

Platelets in native blood stick to glass spontaneouslyand the number sticking is the same at 0°C. as at20°C.; the addition of adenosine diphosphatemarkedly increases the number of platelets that stickand this result is also independent of temperature.When either the mono- or triphosphate is added tonative blood fewer platelets stick; they also inhibitthe effect of the added diphosphate, but are effectiveonly at room temperature. Cocaine prevents spon-taneous or adenosine diphosphate-induced adhesionat room temperature and at 0°C.

Since the tri- and monophosphates inhibit theincreased adhesiveness induced by the diphosphate,these three compounds may be acting competitively;and as the mono- and triphosphates also reduce theadhesiveness of platelets in native blood, it is possiblethat normal adhesiveness is due to a mechanisminvolving intrinsic adenosine diphosphate. Thecompetitive situation must however be complex inview of the different effects of temperature.The adhesion of one platelet to another, aggre-

gation, differs from platelet adhesion to glass in thataggregation in fresh plasma does not occur in theabsence of thrombin or adenosine diphosphate andthese substances are effective only at 20°C. and 37°C.The mono- and triphosphates appear to interactstoichiometrically with the diphosphate in citratedplasma at 37°C. while cocaine and the other anti-adhesive drugs are acting in a different manner. Sinceadhesion to glass and platelet aggregation areinhibited by so many different anti-adhesive drugs(0llgaard, 1961, also finds inhibition in the presenceof quinine) and since they are effective in the cold, itmay be suggested that these drugs all act in a non-specific way. It has been shown that cocaine isincorporated into a monolayer of stearic acid(Shanes, 1958) and of nerve tissue lipoids (Skou,1954), thereby increasing the molecular packing andsurface pressure. Shanes was concerned primarilywith the permeability of living membranes and hasevolved the concept of 'stabilizers' and 'labilizers'.On the other hand, Judah (1961a), working with adifferent system, suggests that anti-histamines inter-fere with a phosphoprotein kinase. Accordingly it ispossible that the anti-adhesive drugs are incorporatedinto the lipids at or near the surface of the platelet,the properties of which would then be altered.

It is remarkable that we do not know the natureof the forces holding a platelet stuck to a glass surfaceor to another platelet, nor do we know if the forces

are similar to these two situations: clearly they arenot identical. The following tentative suggestion mayserve as a working model. Adhesion occurs as aresult of attractive forces between 'adhesive sites' onone or both of the two adhering surfaces and fewadhesive sites are needed for platelet adhesion toglass: added adenosine diphosphate even in the coldincreases the number or strength of the adhesivesites, thereby encouraging adhesion to glass and atroom temperature it produces even more adhesivesites which permit platelets to stick to each other.

Platelet aggregation induced by thrombin re-sembles adenosine diphosphate-induced aggregationin that it is inhibited by the mono- and triphosphatesand also by the anti-adhesive drugs; on the otherhand the concentration of thrombin added toplatelet-rich plasma has to be increased fourfold tochange the result from no aggregation to a maximalresponse but the concentration of adenosine di-phosphate has to be increased by a thousand fold toproduce the same results. The dynamics of the twosituations are clearly different, and might suggestthat thrombin is acting as an enzyme, and possiblythat adenosine diphosphate is a substrate in a dif-ferent system with it and the mono- and tri-phosphates, all competing for the same site. Theobservation that the triphosphate at 20°C. in nativeor heparinized platelet-rich plasma induces aggre-gation is unexplained. Whether the nature ofadhesivesites is the same in thrombin-induced as in adenosinediphosphate-induced aggregation is unknown.Zucker and Borrelli (1961) report that 15 minutesafter adding thrombin to washed platelets theadenosine triphosphate concentration decreased byhalf. Perhaps thrombin converts the tri- into the di-phosphate.Calcium ions and or possibly magnesium ions

(Zucker and Borrelli, 1958) seem essential for alltypes of adhesion since adhesion is prevented byE.D.T.A. and by strong citrate. The concentration ofcalcium ions required for adhesion is considerablyless than that needed for coagulation, as reported bySharp (1958). A final concentration of 0.38% citratepermits adhesion and aggregation but preventsclotting; 1I0% citrate prevents both phenomena.It has been suggested that calcium acts by kationicbonding between two negatively charged surfaces(O'Brien, 1961). It has recently been shown bymicroelectrophoresis that platelets in native plasmacarry a negative charge and that cocaine does notalter this overall net charge (Bangham and O'Brien,1961). If cocaine is, in fact, incorporated at thesurface it could have replaced other positivelycharged ions, leaving the net charge unaffected;indeed Bangham (1961) has shown that cocainecompetes with calcium for adsorption at a glass

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surface, and the antagonism between calcium andcocaine has also been shown to occur in relation toion migration and the electric potential in frogmuscle (Karzel, Draper, and Friebel, 1961). It seemsunlikely that adenosine monophosphate and tri-phosphate inhibit sticking by chelating calcium,since the diphosphate, which can also chelatecalcium, has a directly opposite effect on adhesion.

Five minutes after adding adenosine diphosphateto platelets they will not stick to glass or to eachother, and in time viscous metamorphosis is rever-sible as reported by Ollgaard (1961). These findingspresumably are related to the observation that intactplatelets at 37°C. can inactivate large quantities ofadenosine diphosphate but fragmented platelets orintact platelets at OC. cannot. Indeed 0llgaard(1961) has reported that platelets and white cellscan inactivate his aggregating factor, which waspresumably adenosine diphosphate. If it is involvedin some metabolic activity leading to stickiness it isremarkable that in the reported experiments none ofthe usual enzyme poisons or uncoupling agents at theappropriate strength prevented its effects. Thesituation is probably complex, particularly since0llgaard (1961) and Hellem (1962) report thatmono-iodo-acetate can under some circumstancesprevent adenosine-diphosphate-induced aggregationor adhesion. Fig. 1 summarizes some of the observedinteractions and puts forward some speculativesuggestions.These results also show that Ro 3/0837 has con-

siderable anti-adhesive activity in many tests in vitro.Its effect in vivo is at present being investigated.The drugs with anti-adhesive properties have

many other important properties. They make redcells in plasma swell (O'Brien, 1961) and decreasethe osmotic fragility of red cells in saline (Judah,1961b). Serotonin uptake by platelets is stimulated byadenosine triphosphate (Sano, Kakimoto, andTaniguchi, 1958) and inhibited by cocaine andchlorpromazine (Stacey,- 1961) and by reserpine(Hardisty, Ingram, and Stacey, 1956). Anti-histamines, local anaesthetics, and some anti-malarials inhibit both ion transport in red cells anda Na and K activated adenosine triphosphatase inred cells (Judah, 1962). Mitochondrial swelling('damage') is influenced by many of the same drugs.Grief (1961) reports that adenosine triphosphatereverses thyroxine-induced mitochondrial swellingand Judah (1961a) states that swelling is producedby calcium ions and is prevented by adenosine tri-phosphate and by E.D.T.A. Swelling is also pre-vented by anti-histamines, quinine, pro^aine, and bychlorpromazine (Judah, 1961b). Judah thinks thatthese drugs may interfere with a phospho-proteinkinase that couples one phosphate group from

INTRINSIC A.D.P.-

A.D.P.I NACTIVATING

SYSTEM

INTRINSIC A.D.P.-

L A.M.P OR ATP.

A.D.P.-INDUCED AGGREGATION

INTRINSIC A.D.P.-

I TTHROMBIN

THROMBIN-INDUCED AGGREGATION

FIG. 1. Diagram of the possible mechanisms of plateletadhesion and its inhibition. The inside of the platelet is onthe left. The hatched area represents the platelet membrane.Adhesive sites, according to their frequency or strength,are marked JLYL or FLFLF-LFL; inhibitors ofadhesion are underlined, substances promoting adhesionare not.

adenosine triphosphate onto the protein to producea phospho-protein and the diphosphate. At the sametime Bangham, Rees, and Shotlander (1962) showthat many anti-adhesive drugs protect liver cellsagainst damage caused by carbon tetrachloride andthat their protective power runs parallel to theirphysical effects at a lipid-water interface. Thesefindings and the overlap of pharmacological pro-

$ % COCAINE

CA.

OE.D.T A.

A.M.P. OR AXT P.

SPONTANEOUS ADHESION TO GLASS

COCA I NE

~2.CA.

: -E. D.T. A.

EXTRINSIC A.D.P.

COCAINE

CA.

A AE.DOT AA.

VIA -----lA.M. P. OR A.T. P.

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perties indicate some basic and general effect of allthese drugs on cellular or organelle membranes.

REFERENCES

Bangham, A. D. (1961). Personal communication.-, and O'Brien, J. R. (1961). Unpublished observations.

Rees, K. R., and Shotlander, V. (1962). Nature (Lond.) 193754.

Gaarder, A., Jonsen, J., Laland, S., Hellem, A., and Owren, P. A.(1961). Nature, 192, 531.

Grief, R. L. (1961). In Advances in Thyroid Research, Trans. 4th int.Goitre Conf., London, 1960, p. 515. Pergamon Press, Oxford.

Hardisty, R. M., Ingram, G. I. C., and Stacey, R. S. (1956).Experientia, 12, 424.

Hellem, A. (1960). Scand. J. clin. Lab. Invest., 12, suppl. 51.(1962). Biological Aspects of Occlusive Vascular Disease (in thepress). University Press, Cambridge.

Judah, J. D. (1961a). Biochim. biophys. Acta (Amst.), 53, 375.(1961b). Personal communication.(1962). Ciba Foundation Symposium on Enzymes and DrugAction, p. 339. Churchill, London.

Karzel, K., Draper, M. H., and Friebel, H. (1961). Naunyn Schmiede-berg's Arch. Exp. Path. Pharmak., 241, 172.

O'Brien, J. R. (1961). J. clin. Path., 14, 140.0ligaard, E. (1961). Thrombos. Diathes. haemorrh. (Stuttg.), 6, 86.Sano, I., Kakimoto, Y., and Taniguchi, K. (1958). Amer. J. Physiol.,

195, 495.Shanes, A. M. (1958). Pharmacol. Rev., 10, 59.Sharp, A. A. (1958). Brit. J. Haemat., 4, 28.Skou, J. C. (1954). Acta pharmacol. (Kbh.), 10, 325.Stacey, R. S. (1961). Brit. J. Pharmacol., 16, 284.Zucker, M. B., and Borrelli, J. (1958). J. appl. Physiol., 12, 453.-,- (1961). Blood Platelets, p. 383. Churchill, London.

Part II Some results from a new method of study

SYNOPSIS The light transmitted through a suspension of platelets increases if the platelets aggregate.A method derived from this observation has been used to study the kinetics of platelet aggregation.Aggregation is more rapid at 37°C., with rapid stirring, with high concentrations of adenosinediphosphate, and a high platelet concentration; a decrease in any of these conditions slows theprocess.

The study of platelet aggregation, that is the ad-hesion of one platelet to another, has been handi-capped by the methods used, which include theexamination of a few platelets under the microscopeor the macroscopic observation of platelet suspen-sions agitated in a small test tube. This paper brieflyreports a new method of study and amplifies theobservations made in Part I.

METHOD

Human citrated platelet-rich plasma, 3 ml., was placed ina cuvette with a cross section of 1 x 1-5 cm. in a simplephotoelectric colorimeter. A small stirrer with a variablespeed control was so placed that the blades were justsubmerged in the plasma. Using a green filter, the lightintensity was adjusted till the reading on the opticaldensity scale was about 0 40. When stirred at 1,000 r.p.m.without additions the optical density of the suspensionremained practically constant. When 20 ug. adenosinediphosphate in 0-1 ml. of barbitone buffered saline wasintroduced into the stirred plasma at room temperaturethe optical density decreased and was read at five-secondintervals; at intervals of 0, 10, 20, 40, and 80 secondsafter the addition, 0-1 ml. of the stirred plasma waswithdrawn and placed in 1-0 ml. of formol saline. Thenumber of individual platelets and the number of clumpsof platelets containing two to 10 platelets were countedin these subsamples. Technical difficulties prevented thecounting of larger particles. Few if any platelets werebroken up. The plasma in the cuvette finally containedmany easily visible coarse platelet clumps; if cocainewas added, the platelets separated and could then becounted and this count was similar to the initial count.

RESULTS

From Fig. 1 it will be seen that for about the first50 seconds the optical density decreased apparentlylinearly with time, that the free platelet count de-creased rapidly and exponentially, and that smallaggregates containing two to 10 platelets increasedfor 10 to 20 seconds and then decreased again.After about 30 seconds it was established visuallythat the bulk of the platelets were in large clumpseach containing 20 to 500 platelets. The opticaldensity decreased because the light scattering powerof the comparatively few coarse particles present atthe end of the experiment is less than that of thesmall particles, free platelets, whose diameterapproaches the wavelength of light. The analysis ofwhy the optical density initially decreased in alinear fashion is complicated and was not pursued.The addition of adenosine diphosphate withoutstirring did not alter the optical density, so it wasconcluded that the change in optical density withstirring was a faithful mirror of platelet aggregationfrom single platelets to dimers, to clumps of five andto clumps of 100 or more. Since the initial rate offall in optical density is linear and maximal fromzero time, i.e., from the moment the adenosinediphosphate was added, it is suggested that all theplatelets had very rapidly become maximally 'sticky'and that thereafter adhesion depended solely on thechance of one platelet colliding with another or oneclump with another. It was now possible with this

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