A study of intravascular platelet aggregation by continuous platelet counting

A study of intravascular platelet aggregation by continuous platelet counting

TIPS - April 1981 1(~5 A study of intravascular platelet aggregation by continuous platelet counting G. M. Smith Sol e e l n f Pharmac.r, Robert Gor...

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TIPS - April 1981


A study of intravascular platelet aggregation by continuous platelet counting G. M. Smith Sol e e l n f Pharmac.r, Robert Gordon'.s In.st;tale o f Techtlnlot¢,; A b e r d e ,n .'wothlnd. I+' K.

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Studies on platelel aggregation Effects


Introduction The experimental study of arterial thrombosis was greatly advanced by the introduction of the Born aggregometer. This instrument enabled quantitative in vitro studies of blood platelet aggregation to be made in an ant;coagulated medium. Although the aggregometer is now a standard piece of equipment in any platelet laboratory, it has two fundamental disadvantages. Firstly it does not give information of the activity of platelets in v i m and secondl~ the platelet aggregation occurs in the absence of calcium. There are many published methods of studying platelet aggregation and thrombosis in experimental animals and it is obvious from the plethora of techniques that there is no't a reliable quantitative method that has been accepted for the in rive study of platelet aggregation. This article describes a technique for measuring platelet aggregation in anaesthetized experimental animals. The technique is based on the observation of Born and Cross' that a transient fall in the n u m b e r of circulating platelets occurred


during the infusion of adenosine diphmphate ( A D P ) into anaesthetized cats. Regoli and Clark ~ showed that thin ADPinduced fall in platelet count could be prevented by the simuhaneous infusion of adenosine. These findings have been confirmed by many o t h e r workers.

The technique of continuous platelet counting

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prostanoid, l_~44t~6'4. ~ ill also ~.au:,e a fall in the le~,el of circulati'~e r, latc!cts. \Vc shelved that A D P also ga,,. cons;stem and dine-dependent responses in dogs. pig,; and squirrel monke~.¢..,,DP ,lid not cause aggregation in cat'; and ., conlinuou,., platelet count could not t'~. obtained in guinea pigs. Recent ~ork has s h o ~ n th:lt consistent results ;ire ix~ssible in guinea pigs ~ h c n 10(10 units of heparin are injected ,veil+re the aorta ix callrlulatcd. Effet t¢

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in 1973 Franz Freuler and I introduced a technique for studying platelet aggregation in vivo that did not require blood samples to be taken and counted ~. O u r technique uses the Technicon Autocounter which is a completely automated system for counting platelets and white cells. Since it is a continuous flow system, the blood can be continuously sampled from a suitable after}. To avoid the injection of an anticoagulant. a special double cannula was designed to enable 3.8% trisodium citrate to be pumped to the tip of the cannuhc The eitrated blood was then removed al ILl ml min -~ and diluted in the manifold v, here the red cells ~ e r e Ivsed by 1% ammonium oxalate/0.002% saponin solution (Fig. 1).

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niquc can be u~-d to slud,, bath qimulantx a0d inhibitors of p!atclel aggregation. In the rabbit, infusilm~ ol 6.25-2110/.tg kg rain ~ of A D P fi,r 2.5 min at i 5 mm intervals will gi~e dosc-def~.rndent and reproducible falls in platelel count m ~ r a F~'riod ul up to 6 h (Fig. 2) ~. In the rat. mllagcn and A D P will similarh give c,n,i~tcnt results m c r 2 h IFig 3) ~. 5-hxdrox~-

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d.gt'nt.L ~,VC haxc u,;ed the t c c h n l q d e It', assess potential anti-thrombotic agents in rats arid rabbits. ]|olmt:set al. rc|'~rtcd the inhibitoly acti~.ib of PGE~, dipyridamole. two 79ho.phodiestcrase inhibitors. Stl N69 and VK +74 on ADP-induced aggregation in rabbit~ (Fig. 4) ~, Hohnes compared the activity uf two anti-inflammatory agents, aspirin :rod pmquazone, using collageninduced aggregation in rats ~ He also f t-I~rt N.rth.Hol!~nd B+~,mcdk¢atP-c,, I~Sl

TIPS - April 1981

106 R E P R O D U C I B I L I T Y OF C O L L A G E N - I N D U C E D P L A T E L E T AGGREGATIOI"J Platelet Count x 10 a mm "3 3 rain


lin infusion. When prostacyclin was infused into a guinea pig with an initial count of 4-5 x ! IP platelets m m - a a rise in platclet count could not be produced. T h e ~ results suggest that in ~ m e guinea pigs the anaesthetic or surgery will produce reversible thrombocytopaenia caused by platelets adhering and aggregating in the microvasculature. Clearly, if the cause of this effect could be. found, an excellent model for studying disaggregation would be available. Com~arison with other published models


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sh~wed thal adrenaline produced a dosed.:'pendent potentiation t,f ADP-induced a~zgregation in vivo in rabbits s. The adrenaline-induced potentiation was inhibited i,y dihydroergoloxine, an aadrenoceptor b!ocking drug. Gordon Duncan anti I reporled that iadomethacin and aspirin would only partially inhibit collagen-induced aggregalionL More recently we have shown that continuous platelet counting can be used to study the mechanisms involved in intravascular platelet aggregationL By comparing the activity of n-l-butylimidazole and phthalazinol in their ability to inhibit aggregation produced by collagen, arachidonic acid and PGH2 in rabbits and rats, we have shown that |he thromboxane pathway is not th,e main pathway involved in the mediation of ,:ollagcn-induced aggregation in the rat but is of importance in the rabbit. Effects o f prostacyelin ( P G h ) . Prostacyclin is the most potent known inhibitor of platelet aggregation and is formed in the walls of blood vessels from prostaglandin endoperoxide. The infusion of PGh (0.25-1 p.g kg -~ rain -~) in anaesthetized rats and rabbits produced a d o ~ dependent inhraition of the fall in platelet count produced by ADP and collagen. When 15-hydn~peroxyarachidonic acid, an inhibitor of prostacy, clin synthesis, was infused in rats. the fall in platelet count induced by collagen was significantly greater, l'his suggests that PGh is being continuously ffwmed by the rat vascular endothelium '~o antagonize the effects of collagen. By :,tudying the continuous platelet count in the guinea pig, the disaggregatory activity (~f pros,,acyclin can be studied. In a number of experiments with guinea pigs, a

count below !,5 x liP platelets mm -s was obtained. The reason for this low count is not known, but Tschopp and Baumgartner have shown mat guinea pig arterial tissue produces less prostacyclin than the rabbit t°. The success of the technique in rats may he due to their observation that rats produce more prostacyclin than e~ther rabbits or guine~ pigs. When a low count is obtained with a guinea pig preparation, an infusion of 0,125-2/zg kg -~ rain -~ for 10 rain of prostacyclin increased the count to 4-5 x l0 s platelets mm -s (Fig. 5). Further infusions at this level did not raise the platelet count any further. The platelet count returned to its previous low level within 30 rain of the start of the prostacyc-

It: vivo modeis to study potential antithrombotic agen :s can be divided into four groups. (I) Ex-vivo t~ethods. In these models, agents are injected into experimental animals and blood ,,amples taken before and after the application of the drug and #z vitro aggregation studies performed. These models require the presence of anticoagulants and reproducibility is a problem. (2) Prosthetic devices. Numerous models have been described where polythene tubing is inserted in blood vessels and a thrombotic response is assessed in the presence and absence of a drug. (3) Injury to blood vessels. Thrombus formation in arteries and veins can be induced by mechanical, electrical, chemical and physical stimuli directed at the vessel. The iontophoretic application of ADP on to a vessel wall will also produce thrombus formation. l'he greates~ difficulty with the models


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Fig. 4. Inhibition o f ADP-induced reduction in circulating platelet numbers in the rabbit oy PGE, (A), dipyridamole (II). $1"1-869 ( 0 ) and VK- 774 (v). lnhibitors were infused intravenously ( ! 0 min) after 2 -3 comrol A D P effects. A D P (50 t~g kg - ~rain -I) was infused intravenously for 2.5 rain, at 15 rain intervals, during and a~'er drug infusion. Inhibition is'expressed as maximum percentage inhibition o f control A D P effects observed up 1¢ 80 rain after the start o f drug infu.~i, ,n. Each point represents the mean o f three rabbiL¢. Each animal received only one dose o f i n h ibitor.

77PS - April 1981


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described in 2 and 3 above is the quantitation of both the thrombotic response and the drug effect. (4) In vivoplatelet aggregation. Only two methods have been published to measure platelct aggregation in vivo. Hornstra used the aortic loop which enabled heparinized blood to pass through a filter in an extracorporeal circuitL ADP will cause aggregation which will temporarily block the filter. This produces a pressure differential on either side of the filter which is measured by transducers. A D P produces a do,edependent response measured by the changes in pressure but the technique has some serious disadvantages. It can only be used in rats, collagen cannot be studied and heparin is required. Also, the insertion of the aortic loop is difficult and very time consuming. Continuous platelet counting is tht,' other technique for measuring platelet aggregation in vivo and this has a number of distinct advantages over the aortic goop technique and the other techniques that have been described. It can be used in a range of different animals. Many diKerent aggregating agents can be used and no anticoagulant is required. The surgery required is minimal and both the thrombotic response and the drug effect are easily and reproducibly quantitated. On the other hand, an ideal method should avoid the use of anaesthetics and be directly related to the clinical condition. The technique of contim~ous platelet counting has been shown to be a reliable, reproducible and versatile technique that will facilitate advances in ou c knowledge ol thrombosis and will enable the assessment of potential ant(thrombotic agents.

Acknowledgements I should like to acknowledge the support i received from Sandoz Ltd during the

development of this technique. ! also ~ish to thank Dr I, B. Holmes and Mr F. Freuler for their significant contributions to the work described in this article. The current support of the Welleome Trust and the British Heart Foundation is gratefully acknowledged.

Readinglist '~ Born. G. V. g. and Cro,,~,. M. J. (1%3) ~2uure (London) lq7. 974-976 2 Duncan,G. G. and Smith. G. M. 119%#) ~hcrovaac. Res. 17. $51 3 Duncan, G. G. and Smith. G. M. (198(1)Pint J. Phnrmacol. 69. 340P-341P 4 Holmes, I. B. (197~;)Arch. bu. Pharmarodvn Therap. 228. 136-152

( L I I Sp'uth t,,raduated from the .S~'hool ol Phar,n,a l .eMon t-?ni~er~it~. After 4 xt'ar~ in thc. ph¢lrr?!t.t t,ztllt I,[ lrtdtt~try, he ioined the Ot'ptlrfrPter~t o f ['h,irr;:,lt <~logt L'nlverstt~ (.2+lictor+ London, un,h'r the ~upt'rl :~t,*n ,~t Pro.rhino. H. 0 5~hiid. F R S. a,~d ~a~ a . arde,J a Ph.D. He then ipent 5 )ear~ in the pharma~,,h+~:u,;i ]tlbortglort~+~ 0.[ S a n d o z 1 t d tn 5~i!12t'r~d~ld. a tlt'r¢' lrt I~ida r~2"~ptItl~lDld ~ r tie't tO', +plr/e r-'li I]l~ ~ds fi ~ , :l~dl p+ )lt'?I nil( ilnl|-'h.roPllbl!tl~ ¢t~dtll~ 5t?l(~ + lug3, llt" Jitl~ +~'t'¢~l S('ntor I dt'rllrt'r l.~ lahar*~lat+~l,)~ In lilt" ~,1!.,oi ,,r ~htlrtn£lt ~. Rt~bc'rl I il)rdtwl, ~ IPL~t lit[( , '~, ~¢k hlrtq~.i~ Aberdeen

Denervation supersensitivity in salivary glands Francisco J. E. Stefano and Carlos J. Perec ht~tituto de Im'estiga¢i, me~ /Varmaco/0gira ~, ( ' O ~ ' l ( E I, and (atcdra de [i~<,&,~'ta. Iqzcuir, M ,; e ~hl,,n/, ,/,
Throughout the histoD of physiolog) and pharmacology, salivary gands have been useful as models for the study of the e~cnts which take place at the autonomic neuroeflector synapse. The response of these organs, secretion of saliva, is the result of the activation of secretory cells by neurotransmitters released from both the parasympathetic and sympathetic divisions of the autonomic nervous system. With the exception of some salivary glands, like the sublingual glands of some species which show a basal spontaneous secretion indc-





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stimuli; m o r e o v e r , h o r l n o n ~ ; r c g u l a l i o n o f sali',arx :-ecretion ~,CCIII>. tO p[:.l~, ;.I n l i r k , r role. Surgical approaches lO nerve pathways are ~ell known and relativel.~ ca*} to perform. The different portions of the reflex arc responsible for sali~a~ secretion are sensitive targets for pharmaculogical agents. The sum of all the~e advantage,, explain why it great proportion of the present know, ledge of the events which take EI~.l~l'Norlh-HoUand Rhlmedl~d] Prt.,, t '~ [