0074_platelet adhesion & aggregation in thrombosis.pdf

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Platelet Adhesionand Aggregation in Throm bosisCountermeasures

Transactions of the Eighteenth An nual Symposium on Blood,Wayne State University School of Medicine, Detroit, Michigan,held on January 16 and 17, 1970

E.F. MAMMEN G. F.ANDERSONE D ITORS

M. I. BARN HART

203 Figures, 50 Tables

19 ~

F. K. SCHATTAUER VERLAG STUTTGART - NEW YORK


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SUPPLEMENTUM XXXXII AD THROM BOSIS ET DIATHESIS HAEMORRPIAGICA


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Anti Adhesive Drugs in Throm bosisI-I. I. BICHE~

The causal participation of blood cell aggregation in thrombus formation hasbeen desoribed by several investigators under clinical (5) and experimental (6, 34,36) conditions. Of the blood cells involved, special attention has been given to theerythrocytes and platelets. These ceils seem to possess the ability to start andaccelerate the chain of events that leads to organic vessel occlusion.Knisely et al. (29) and Bloch (5) observed long ago the pathological significanceof intravascular red cell aggregation (sludge). Borgstrom et al. (9) were able todemonstrate a direct etiological relation between experimentally induced sludgeand venous thrombosis in the rabbit. In previous work (2) we suggested that intra-vascular red cell aggregation may be a factor in the induction of anoxic myo-cardial damage.

The importance of platelet aggregation for hemostasis and for the pathogenesisof thrombotic processes is well established (6, 40). The adherence of platelets toeach other and to other surfaces is considered a fundamental step in the formationof hemostatic plugs and thrombi (34).

The purpose of this paper is to assess the therapeutic value of a group of drugsthat share the common property of preventing both red cell and platelet aggrega-tion, in vitro and in vivo, demonstrated with several objective methods devised oradapted to evaluate this action on a quantitative basis. Three different compoundsshowing these effects are introduced, ~n d their possible action discussed, both at theplatelet membrane function level and for the mechanism of their potential anti-thrombotic usefullness. These substances are called anti-adhesive drugs.

MaterialsAdenosine diphosphate (ADP), Sigma Chemical Corporation, U.S.A. High molecular weightdextran, Pharmacia, Upsalla, Sw eden. 2 Methyl 2 tert butyl-3-5-6 tetrioxotetrahydropyran (sub-stance ,~86~) w as synthetized according to Taubs m ethod (46). 5 (5-hydroxindol-3-indoleyl-methyl)tetrazol (BLR-74 3), Bristol Laboratories, Syracuse, U.S.A.~~ 4-Isobutylcarboyl-5-carbobenzoxy-2,3 dioxo-~/-lactone (substance ~,86-B~), supplied by Dr. M . Cais, Dr. W. Taub, and Dr. L. Vrom an,Technion, Haifa, Israel.** (Fig. 1 ).

Department of Anatomy, Medical University of South Carolina, Charleston, South Carolina.


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H.I. Bicher

N~N

NIC=O, , BLR- 743"5-- [1- - (Z,- -Chlorobenzoyl) -3-indoly .methy~ tetrazole

C1

i so -C 4 H 9 - -C O- - C ~ C / OH8 6 B

4 - Isobutylcarboy[- - 5 - - car bobenzoxy 2,3.dioxo - - gamma-- lactone

o R

2 u ~O 8 6

1= Methy[R 2 = Te r t - b u ty l

2 M e th y l, 2 T e r t -- b u t y l- - 3 , 5, 6-tr ioxotetrahyd ropyranFig. 1. Ch emical structure of the anti-adhesive drugs.

MethodsA. Platelet and red cell aggregation

1. Testing of anti-platelet aggregation properties of chemical compoundsa) The >> Roll ing Tube c plate le t adhesiveness tes tThis method is a modification of the procedure described by W right (48) for clinical investiga-tion.Blood was ob tained with a plastic syringe by venipuncture of the antecubitalvein in the ex-periments carried out with human platelets, or from the fem oral artery through a polyethylenecanula in Nembutal anesthetized cats. Sodium citrate 3.8 %, in a proportion of 1 : 10 was used asanticoagulant.0.4 m l of blood was placed in a 10 ml, 1.2 cm diameter non-siliconized test tube containing0.1 ml of the added solutions (solvents tested drug). The tube was slowly rotated on its longeraxis, 16 times per m inute, for 2 minutes.The adhesive platelets adhered to the wall of the test tube. Those remaining in the b lood werecounted. The difference between platelet counts of control (blood only test tubes) and those con-* Thanks are given to Dr. M. Pindell, of Bristol Laboratories, for supply of this drug andvaluable discussion.** The autor is indebted to all three members of this successful group of chemists, for supply ofthe described drugs and invaluable scientific advice.


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Anti-Adhesive D rugs in Thrombosis 19 9taining increasing concentrations of the tested drug indicated the percentage of platelet adhesive-ness prevention.A second set of test tubes was run as described, but adenosine diphosphate (ADP) was added ata concentration of 0.5 ~g/ml. This induced increased platelet adhesiveness, and a further drop inthe number of remaining platelets. Here again, the difference between test tubes containing ~iiDPalone, or ADP plus the tested drug indicated the percentage of adhesiveness prevention.b Screen filtration pressure

The screen filtration pressure (SFP) technique has been described by Swank et al. (44, 45)as a suitable procedure for the determination of the forces that hold together the formed elementsof agglutinated blood.By this method, the pressure required to force blood at a constant rate through a screen withmultiple 20 micron-square pores is measured. When the blood elements are separated they passthrough .the pores without any appreciable resistance. An increase in pressure indicates an increasein the number of aggregated cells in the blood.

Swank demonstrated that the addition of ADP to the tested blood induced a sharp rise in theSFP, which was probably dependent on an increased platelet aggregation, although other cells mayhave participated in this action.

In order to clarify the effect of the studied compounds on platelets in a simplified system, ADPwas added to PRP (platelet-rich plasma) at a concentration of 1 ~g/ml, and then increasing dosesof the tested drug were added at successive runs. A d~crease in the SFP indicated prevention of theADP induced platelet aggregation.

c) Photoelectric methodThe photoelectric method to determine platelet aggregation in platelet-rich plasma (PRP) hasbeen described by Born (6). Continuously stirred PRP is placed in a transilluminated test tube, andthe amount of light transmitted is measured with a photocell. Platelet aggregation, induced byADP or collagen changes the amount of light received by the photocell and can, thereby, be

recorded. Pretreatment of the PRP with increasing doses of anti-adheslve drugs allowed for thedetermination of the minimum effective concentration of the tested drug that prevented plateletaggregation.

Fig. 2. Simultaneous recording of the membrane capacitance aggregometer (upper tracing) andBorns photoelectric method (lower tracing). A. Aggregation induced by collagen, note goodresponse in both tracings. B. Lack of response to collagen after pretreatment of PRP with~BLR-74 3


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2 O O H. I. Bicher

d) ~ ,M embr ane C apac i tance ~ aggr egometerThis method allows for the determination of platelet aggregation in whole blood, without thesomewhat cumbersome technical procedures involved in SFP readings. It is based on the assumptionthat the changes in platelet membrane function and ion fluxes to and from these cells that occurduring aggregation, as will be described later in this paper, should create detectable changes inblood capacitance during the active phases of the process.

A sensitive capacitance meter was built as designed by Haapnen (20). A total of 2 ml of oxalatedblood were placed between two sensitive plates facing each other on both sides of a 5 mi siliconizedtest tube. The blood was continuously stirred and its temperature maintained at 37 o C in the sameway as is usually done in Borns method (6, 7). The output of the capacitance meter was recordedon a model 5 Grass polygraph.Platelet aggregation was induced by adding either ADP, 0.2 [tg/ml, or collagen to the testedblood. The minimum effective dose of the evaluated drugs that prevented aggregation was deter-mined using the same procedure as in methodIn preliminary series of experiments, methods ?~c** and ~d~ were used in parallel, from the samePRP, and the simultaneous records compared (Fig. 2).In vivo platelet experimentsIn vivo experiments were performed for all four platelet procedures a, b, c, and d on Nembutalanesthetized cats and dogs, a~er arterial, venular and tracheal cannulation. Blood samples wereobtained from the artery while all solutions were injected into the vein. Polyethylene cannulaewere used.

2. Testing of anti-red cell aggregation properties of chemical compoundse) Erythrocyte sedimentation rate (ESR) Thorsen and Hint method.

Quantitative in vitro studiesThis procedure, first published by Thorsen and Hint (47) provides for a simple quantitativeestimation of the erythrocyte aggregation properties of colloids or plasma. It has been adapted forthe determination of the anti-aggregation properties of drugs, as a screening method that lends itselfto the numerical comparison of the relative strength of action of the different compounds. Theprinciple involved is that, when red cells are suspended in a medium of fixed aggregation power,their sedimentation rate is directly related to the degree of aggregation.This method is based on the measurement of the ESR in Westergren tubes, using a series ofdilutions of plasma or artificial colloids. One part of washed human red cells was suspended in twoparts of each dilution of the suspension fluid consisting of progressive dilutions of the tested colloid,diluted in saline. In this way a fixed hematocrit value was obtained.The log of the one hour sedimentation rates was plotted against the log of the colloid concen-trations in the respective tubes. The points formed straight lines with nearly the same slopes. Thepoints where lines cut the abscissae gave the concentration of colloid which caused a sedimentationrate of 1 mm per hour. This parameter, called the critical point by Thorsen and Hint, was depend-ent on the colloid concentration in individual tubes and could, therefore, be considered as a quan-titative measure for their erythrocyte aggregation power. When the colloid concentration was lowerthan critical, the erythrocyte aggregation disappeared; when the concentration wa~ raised, aggre-gation and sedimentation rate rose very rapidly.For the determination of the anti-aggregation properties of substance ~86~ and related com-pounds, a fixed aggregation force was chosen, giving a sedimentation rate of 40 ram/hour (in thepresent experiments this was obtained by using dextran, M. W. 150,000, at 1% concentration).Increased concentratio.n of the tested substances were added, and the inhibition of aggregation asrepresented by the decrease in the sedimentation rate, was measured as percentages of the control.

D Vital microscopy o~ the peripheral circulation.In vivo experimentsMicroscopic observations of the microcirculation were performed on the transilluminared omen-turn of the cat. Using a Leitz dissection microscope, the observations were made at magnification


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Anti-Adhesive Drugs in Thrombosis 20 1of 180 and never continued for more than 10 minutes at a time. Subsequent observations weremade occasionally, but not more than twice in the same animal.

Intravascular red cell aggregation was induced by injection of high molecular weight dextranintravenously at a concentration of 1 g/kg. The flow improving effect of the anti-adhesive drugswas followed a~er their intravenous administration. The effect of dextran infusion and anti-adhesive drugs on the erythrocyte sedimentation rates were measured using a standard Westergrentechnique.

B. Potassium flux at the platelet membrane during aggregationAll glassware with which platelets had contact was coated with silicone. Rabbit blood wasobtained under Nembutal anesthesia, through adequate plastic tubing, and collected in test tubes

containing an aqueous solution of the disodium salt of EDTA as an anticoagulant (1 part of EDTA:9 parts of blood). The final concentration of EDTA in blood was 0.005 M. Platelet-rich plasma(PRP) was obtained by differential centrifugation. Unless specified otherwise, platelets from 3 mlportions of PRP were sedimented and washed twice with, and finally resuspended in 1.8 ml of trisbuffered saline, pH 7.5, containing potassium chloride (0.004 M), glucose (0.0055 M), and EDTA(0.0013 M). The washings were performed at 4 o C and required approximately 30 minutes. Thefinal concentration of platelets was approximately 250,000 per mm3. The washed platelets wereincubated without agitation at 37 o C under various experimental conditions. After incubation, theplatelets to be analyzed for potassium were washed once with tris buffered saline containing EDTAbut no potassium or glucose. This final washing procedure was performed at 4 o C and requiredapproximately 10 minutes. The platelets were then sedimented by centrifugation at 22,000 g for5 minutes and lysed ~ 3 ml of a 3 % solution of trichloracetic acid. Potassium in the lysates wasmeasured by use of a flame photometer employing an internal lithium standard. Radioactive potas-sium (K42) was measured by use of a Welb scintillation detector.

C. Electron microscopic studiesThe effects of auti-adhesive drugs on the ultrastructure of rabbit platelets were studied by in-cubating aliquotes of platelet-rich plasma (PRP) with each of these agents at different concentrationsin serial dilution. Batches of platelets so treated were fixed in phosphate buffered 4 glutaraldehyde

at room temperature for two to five hours. After rinsing in sucrose and post fixation in 1% OsO4,platelets were dehydrated and embedded in Epon 812 or Araldite 506.The drugs were incubated with platelets at concentrations ranging from 0.1 mg/ml to 30 mg/ml.

An Hitachi electron microscopetype HU-11E was used, at a magnification of 10,000 .

ResultsPrevention of platelet aggregation and adhesiveness

Substance ,~86,~, substance ,,86B,,, and r~BLR-743,, were active in preventingplatelet aggregation and adhesiveness in all four tests performed in vitro (methodsa, b, c, and d). These results are summarized in Table 1. Using substance ~86,,-asreference unit of activity, it can be conclud ed that substance ,~8 6 B,, was five tim esmore active than su bstance ,~ 8 6,, in preventing platelet aggregation in all tests, and,>BLR-743,~ twice as active in preventing ADP induced platelet aggregation andten times more active in preventing collagen induced platelet aggregation andplatelet glass adhesiveness (Rolling Tube Test).


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2 2 H. I. Bicher

Table 1. Relative potency of action of the anti-adhesive drugs in vitro experiments. Figures re-present mean effective doses preventing blood ceil aggregation in lzg/mI.

BORN MCA ROLLINGDRUG ESR SF P (ADP) (collagen) TUBE~86~ 250 2,000 2,000 1,000

2,000 2,000~86B~ 25 40 0 40 0 40 0 40 0~BLR-743 ~ 100 1,000 100 1,000 10 0

ESR = Erythrocyte sedimentation r~te-modified Thorsen and Hint Method.SF P = Screen Filtration pressure method.BORN Photoelectric method-Platelet aggregation induced by ADP or collagen.MCA = Membrane capacitance aggregometer -- ADP induced platelet aggregation.ROLLING TUBE = Platelet to glass adhesiveness.

In vivo,the mean i.v. effective doses preventing platelet aggregation and ad-hesiveness were 200 mg/kg for substance ,,86~ and 30 mg/kg for substance ,,86B(,BLR -743 ~< prevented collagen induced aggregation and platelet adhesiveness at adose of 30 m g/kg, i.v.

Prevention of red cell aggregationThe effective doses of the studied substances preventing red cell aggregation

in vitro are presented in Table 1 (ESR column). It should be noted that substance>, 86 B>BLR-74 3,,was only two to three times more active. Another fact to be considered is thatonly one fourth to one tenth as m uch of these drugs w as required to prevent red cellaggregation than to prevent platelet aggregation.In vivo, all three substances were tested at the same doses as for platelet aggre-gation (see above). Af[er the intravenous injection of the drugs, ;he erythrocytesedimentation rates of the studied animals were inhibited for a period of four tosix hours.Microscopic observations of the microcirculation aPcer infusion of high molecularweight dextran revealed a slow circulation of blood. The red cells were aggregatedinto large masses (sludge) with spaces of cell free plasma separating them (plasmaskimming). The sludge formation was especially heavy in venules, but was presentalso in capillaries and arterioles. In many places, red cell impaction of small postcapillary venules could be discerned (Fig. 3A).Aiier the intravenous administration of anti-adhesive drugs the circulationbecame faster, red cell aggregates separated, and the plasma skimm ing disappeared,especially in the arterioles. V enular sludge remained in m ost cases. This effect lastedfor one hour, alter which time observations were discontinued. The most strikingimprovement occurred during the first 15 minutes after injection (Fig. 3B).


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Anti-Adhesive D rugs in T hrombosis 2O3

Fig. 3. Prevention of sludge formation in vivo by substance ,,86~. Omentum microcirculation in acat after bleeding and injection of high molecular weight d4xtran (for description of methods seetext). A. Sludge in a venule and arteriole. Note plasma skimming at arrow. B. Fifteen minutes afterthe injection of substance ~,86~ (200 mg/kg i.v.). Same area plasma skimming diappeared, red celflow is continuous.

A. Effect of ADP and anti-adhesive drugs on potassiumrelease from platelets1. I~latelets incubated in plasma

The normal content of potassium in rabbit platelet~ was found to be 93 micromoles per l011 platelets. There were no significant changes in the potassium contentof platelets incubated in plasma for periods of up to two hours. This experimentwas reproduced 5 tim es and served as control.Platelets incubated with 1, 2, or 3 8g/ml of ADP under the same conditions,showed a consistent decrease in their potassium concentration a~er 40 minutes (seeFig. 4). The mean potassium content (in 10 experiments) was 68 moles per 1011platelets. The platelets were coarsely aggregated during the incubation period, theplatelet aggregates discernible with the unaided eye.

The addition of anti-adhesive drugs completely prevented the loss of plateletpotassium induced by ADP. The minimal concentration of these drugs requiredwas 2.5 mg/ml. Platelet aggregation during the incubation period was also pre-vented. Anti-adhesive drugs incubated with platelets in plasma did not induce anypotassium release.


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2 O 4 H. I. Bicher

A D P alone.... AD P end anti adhes =redrugs

Time of incubation(min)Fig. 4. A DP induced loss of potassium from platelets incubated in plasma.

2. I~latelets incubated in potassium free bufferWashed platelets incubated in bui~er containing EDTA and glucose but no potas-

sium were depleted of 25 to 50 % of their potassium within i0 minutes and 80 %of their potassium within 2 hours; A marked increase in the rate of potassiumrelease from the platelets was induced by the addition of anti-adhesive drugs, at thesame concentrations as in the previous experiment (Fig. 5). This experiment wasrepeated t 0 times. The action of ADP on this system could not be estimated becauseof the erratic responses obtained. Usually, a slight increase in the rate o~ potassiumrelease was obtained.

\

\ \ \ \-- Control ptatelets Platelets treatedwith onti adhesivedrugs oT i m e o f i n c u b a t io n ( m i n i

Fig. 5. Increased loss of p o t s s iu m f r o m platelets incubated in potassium free buffer, induced byanti-adhesive drugs.


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Anti-Adhesive Drugs in Thrombosis 20 5

B. Effect of ADP and anti-adhesive drugs on the uptakeof radioactive K42 by plateletsIn 5 experiments, twice washed platelets from 3 ml portions of PRP were in-

cubated at room temperature in tris buffered saline containing radioactive potas-sium (K 42) in addition to stable potassium, EDTA and glucose. The concentrationof stable potassium in the buffer differed slightly in each experiment in order tomaintain the amount or radioactivity (counts per minute) sufficiently high foraccurate counting. Th is variation did not influence the results. A~e r varying periodsof time the platelets were separated from the buffer, washed once, and the radio-activityfrom the total potassium of the buffer as well as lysates of platelets wasmeasured. The K42 in the platelets reached an apparent maximum within 60 minutes(Fig. 6), and the average specific activity (K = counts per minute / total K - mmoles) of the potassium in the platelets was 54 % of the .specific activity of thebuffer.In 6 experiments when A DP (1 , 2, or 5 ~g/ml) was added to the same mixture, theuptake of radioactive potassium was inhibited by 40 %, while the total amount ofplatelet potassium was only slightly diminished (3 experiments), or unchanged(3 experiments). This was reflected by a diminished specific potassium activity,during all the time of the experiment, in relation to that of controls (see Fig. 6).

This ADP effect was again reversed by the addition of anti-adhesive drugs, inthe same concentrations as in the previous experiments. Both the specific activityand potassium contents of these p latelets was in the same range as those of controls(Fig. 6).

~000- Specific octivity in buffer Control.... ADP ADP and anti adhesive drugs

7 o o o .6 ~ o o5 o o o3 , o o o -2 0 0 0~.ooo.

0Time{rain}

S A = K ~2 {counts per min)/total Kimitiimoles)Fig. 6. Activity of ADP and anti-adhesive drugs on the uptake of radioactive potassium by platelets.


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206 H. I. Bicher

Electron microscopic studiesSubstance ~> 86~ and ~BLR-74 3 ~ were studied with this method, at concentrationsranging from i to 30 mg/ml. At the lowest concentration employed (0.1 mg/ml),both substance ~>86~ and BLR induced a spherical shape to platelets. At a higher

concentration (1 mg/ml), BLR treated platelets were spherical in overall shapeand contained a preponderance of spherical shaped vesicles.These vesicles appearedto be part of the surface connected system which in untreated platelets showsvesicles of odd and variable shape (Fig. 7). Other cytoplasmic components in- cluding mitochondria, dense granules, and microtubules appeared to be unaffected.While overall sphericity of platelets treated with higher concentrations of sub-stance ~86~ was apparent, no significant amount of sphericity was induced in thesurface connected vesicles of platelets so treated.

Fig. 7. Electron micrographs of normal platelet (A) and platelet pretreated with 1 mg/ml of sub-stance ,~BLR-743~. Note spherical shape of the drug treated platelet and rounding up of the surfaceconnected system, pointed out by the arrows. (X 10,000.)Disscussion

A variety of methods have been developed to study the adhesive characteristicsof platelets (23, 33, 48). In Wrights technique PRP is rotated in a specially designedglass bulb and the percentage change in the platelet count is determined. Mooltenet al. (33) passed wh ole blood through a glass woo l filter and estimated the ratio ofthe platelet to the red cell loss. Hellem (23) developed a method in which wholeblood plasma was passed through a column containing a known weight of glassbeads of standard size. This test provided a good system for the evaluation of theadhesive action of ADP on platelets.


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Anti-Adhesive D rugs in T hrombosis 207

The more widely used procedure for the evaluation of the ADP induced plateletaggregation is that developed by Bo rn (6) as described above. It is ,noteworthy thatRozenberg and Firkin (40) using this method were unable to show increased ten-dency to platelet aggregation in several diseases, as has been described using otherprocedures (13, 33).

Correlation between different methods has not always been found. Horli& (24)reported that the results of tests whi& he performed according to the Wright tech-nique, differed from those obtained when the Moolten te&nique (33) was used.Reber and Studer (38) also pointed out that under certain circumstances the Bornand Wright techniques gave false positive results when drugs were tested for theiranti-aggregative action, and concluded that both tests were not specific for thesame prop erty of blood platelets.

Taking these facts ~nto consideration, we decided to run our platelet tests inparallel using four different procedures both in vitro and in vivo, as describedabove. The results obtained demonstrated the following facts:

1. All three anti-adhesive drugs tested were active in all tests for platelet ad-hesiveness and aggregation.2. They acted in a similar way under the different conditions imposed by eachprocedure. The only outstanding effect seemed to be the relatively low doses of,,BLR-743~ required to prevent collagen induced aggregation. This may be because

of the anti-inflammatory properties of the compound. The pharmacology of thiscompound, related to its specific effect on platelets, has been described by Flem-ming et al. (15).

3. The relative potency of action of the different compounds remained fairlystable. These results can be interpreted as an indication that these drugs interferedwith a physiological process at the membrane level of the platelet, thus renderingthe cell less responsive to different agglutinating stimuli, such as foreign andwettable surfaces, collagen, or adenosine diphosphate.

Our modification of the Thorsen and Hint method (41) provided an accuratetool for the easy and quick quantitative evaluation of the anti-adhesive power ofnew chemical compounds. The results obtained, summarized in Table 1 show a goodparallelism, both in respect to the type of activity, and relative potency of actionof the different drugs when tested using this method, or the methods previouslydescribed for platelet agglutination. The concentrations of the drugs needed to dis-perse the red cell aggregates were less than those required to disperse platelets,probably because the forces holding the erythrocytes together (mainly a matter ofcoating or physical attraction [3, 10]) are not as strong as those linking plateletaggregates (an active chemical process requiring energy consumption [ 8, 1 7]).

The fact that all three compounds acted in the same way in preventing both redcell and platelet aggregation indicated that this property of preventing blood cells


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20 8 H I B i c h e r

from sticking to each other was more generalized and affected not only the bloodplatelets. OBrien (37 ) described the ab ility of certain anti-malarials, local anesthet-ics, and imiprami~ne derivatives, to inhibit the ADP induced platelet aggregation.Constantine ( 1) reported that histamine possessed a similar effect and that thisproperty was also shared by some antihistamines and anti-inflammatory com-pounds. OBrien proposed the name of ,,anti-adhesive drugs


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Anti-Adhesive Drugs in Thrombosis 209

been shown to contain Ca++ ions and to act in a similar way to the tub ular systemofthe muscle, in coupling contraction of thrombosthenin, the contractile protein ofthe platelets (30).

These results are highly suggestive of a sim ilar mechanism in platelet aggregationand muscle coupling ~nd contraction. Both systems use high energy phosphates,ionized Ca++, and induce K + ion fluxes, however, mo re extensive studies will benecessary before any final ~onclussions can be draw n.

These changes, both in membrane structure and ionic strength served as theo-retical basis for the development of our capacitance ag gregometer. The results herereporte.d proved its usefullness as a simple method to determine platelet aggrega-tion in whole blood.

Recently (1) we. have presented a concept that might explain the in vivo anti-thrombotic effect of the anti-adhesive drugs. This is summarized in Figure 8, andbasically consists of the following steps:Ph a se A: In tra va sc u la r red ce i l a g g reg a t io n , combined with atherosclerosis ortransient hypoxia.

The absence of intravascular red cell aggregation during health has been con-firmed in man (28). Based on microscopic observations of microthrombotic occlu-sions in capillary vessels, showing marked sludging of blood cells, a relation be-tween sludge and thrombosis has been suggested. Experimental studies (9) andclinical observations (4, 22) confirmed this relationship.Since sludge is presen t in thrombotic disease, the obvious qu estion is: Is it a con-sequence or the beginning of the thrombotic process?

Gelin (18) summarized the possible pathogenetic mechanisms by which sludgecould induce tissue injury. Our experiments (1, 2, 3) clearly showed that whensludge is combi,ned with atherosclerosis or a relatively short period of transienthypox ia, anoxic tissue damage is a conseq uence, both in the brain and heart.

Phas e B: Anoxic damage to vas cu lar endo the I ium.In the post capillary venules and the venous limb of the capillary, the delivery ofoxygen is normally very precarious. It is in this region that hypoxic damage tovessel walls due to restricted blood flow is m ore easily evidenced alter vasoconstric-tion or intravascular agglutination, or by the combined effects of both and athero-sclerosis. Knisely (28) described this damage that leads to endothelial lesion andprotein leakage.

Recently, Reneau et al. (39) developed a mathematical model in which the fac-tors influencing oxygen diffusion to tissue were analyzed, and predicted the for-mation of minute hypoxic areas at the venous ends of brain capillaries under dif-ferent pathological conditions.

Ph a se C: P la te le t to ves se l w a l l r ea c t io n (platelet adhesiveness phase).Contact is established through a ~leaky,, endothelium between platelets and


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210 H.I. Bicher

tissue factors (collagen, etc.) that enhance platelet adhesiveness, thus initiating amore active chain of events in the thrombotic process.Platelet adhesiveness has been correlated with collagen induced platelet aggre-

gation. Native collagen fibrils aggregate platelets in citrated plasma. The fibrilsrelease considerable amou nts of ADP from the platelets, and the aggregation is in-hibited by AMP (25). Evidence has also been obtained that collagen releases sero-tonin from platelets and renders platelet factor 3 available (43).The electron microscopic studies of Hovig (25) indicated that the platelet mem-brane remains intact during interaction with collagen while the intracellulargranules disappear. The release is therefore probably either due to an increasedmembrane permeability to ADP and serotonin, or to an extrusion of plateletgranular material without membrane rupture. Recent work by Johnson (26) hasprovided further details of this complicated process.The release of these factors leads to the next steps of throm bus formation.

Phase D: Pla telet aggrega t ion w~th release o f more endog enou s p la telet aggrega-t io n fa c to rs ep a f ) , p la te le t ~a c to r 3, min o r red ce i l t ra p p in g , a n d en h a n ced lo ca lnoxi

The importance of platelet aggregation for hemostasis and for the pathogenesisof thrombotic processes is well established. Changes in platelet reactivity of ADP,the chemical agent most frequently considered to be the natural platelet aggrega-tion-inducer has been determined in a number of hemorrhagic and thrombotic con-ditions (13, 34) with the aid of different methods that lend themselves to a quanti-tative determination of platelet function. Platelet aggregate embolism is also con-sidered to be part of the m echanism leading to atherosclerosis, according to Duguidstheory (12).Once the process is initiated, more .endogenous ADP and other aggregation fac-tors are liberated, and the process tend s to perpetuate itself,

Our experiments (1) demonstrated that red cells are trapped in the platelet ag-gregates, becoming a maior factor to increase the mass of the microemboli a,ndprobably increase the local hypoxic condition.At this point, coagulation factors liberated from tissue (thromboplastin), plate-lets (factors 3, 5), and red cells probably initiate changes in plasm a which lead to:Phas e E: ~Cas cade* or ~Th rombin Ac t i va t ion* proces s o f F ibr in Format ion .The well known theories (MacFarlane [311 and Seegers et al. [42]) are not dis-cussed in this paper which mainly tends to demonstrate the manner in which theneeded coagulation factors are made available by the hypoxia-aggregation pro-cess, which can, being reversible in nature, regulate the amounts and availabilityof these factors.Phase F: Major red ce l l trapp ing. Formation of the >>red thrombus~.French (16) has described how the actual length of a thrombus is determined by


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Anti-Adhesive D rugs in Thrombosis 21 1the mass of red cells trapped in it. This similar to the trapping of Phase ~>C~,,but in a major proportion, the cells being incorporated into the fibrin meshformed.

The site of activity of the anti-adhesive drugsAccording to this scheme of thrombus formation we propose that the anti-

adhesive drugs exert their anti-thrombotic action on phases A, B, C, D, and F Ofthe thrombotic sequence, (marked with X in Fig. 8). This concept establishes therationale for the use of anti-adhesive drugs in the treatment of thrombotic disease,and also suggests combining their use with the classical anticoagulant therapy,aimed at preventing the events occuring in phase ,>E,~.

SummaryBoth platelet and red cell aggregation hav e been related to the etiology of throm-

bosis. This paper describes a new category of drugs which prevent both types of.aggregation a,nd are devoid of any other pharmacological activity. These substan-ces are called ,,anti-adhesive~ drugs.

A photoelectric method, a new ,,membrane capacitance,, aggregometer, thescreen filtration pressure method, and a new sim ple glass contact method were usedto measure platelet aggregation and adhesiveness. A sedimentation rate techniqueand microcirculation observations determined red cell aggregation. The anti-ad-

X Phase A

X Phase BX Phase CX Phase D

Phase E

X Phase F

Intravascular red cell aggregation combined with atherosclerosis and/or tran-sient hypoxiaAnoxic damage to vascular endotheliumPlatelet to vessel wal reaction-ADP serotonin release (epaf)Platelet aggregation -- further epaf, plat,let factor 3 liberation, minor bloodcell trappingFibrin formationMajor red cell trapping

X-Phases prevented by the action of anti-adhesive drugs.

Fig. 8. A current concept of the thrombotic process, showing the proposed mechanism of action ofthe anti-adhesive drugs.


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212 t-I. I. Bicher

hesive drugs counteracted blood cell aggregation in vitro and in vivo, and also pre-vented experimental thrombosis and anoxic myocardial damage.The effect of the anti-adhesive drugs on platelet membrane mechanisms was de-termined using physiological and morphological techniques. It was found that K"permeability changes during ADP induced aggregation, leading to decreased intra-cellular potassium levels. This effect is prevented by the anti-adhesive drug s. Elec-tron microscopic studies revealed that swelling and rounding of the surface con-nected system of the platelet occurs after incubation w ith anti-adhesive drugs.A possible me chanism for the anti-thrombotic action of these drugs is discussed.

References(1) Bicher, H. I.: A concept of anti-thrombotic therapy based on the pharmacological effects ofdrugs preventing platelet and red cell aggregation. Proc. II. Int. Congress on Atherosderosis.

(In press.)(2) Bicher, H. I., A. M. Beemer: The role of sludge in the production of experimental ischemicmyocardial damage. BibL Anat. 9:116 (1967).(3) Bicher, H. I., M. H. Knisely: Brain tissue reoxygenation time, demonstrated with a newultra micro oxygen electrode. J. appl. Physiol. (In press.)(4) Bloch, E. H.: Visual changes in the living microvascular system in man and experimentalanimals as they are related to thrombosis and embolism. Angiology 10:6 (1956).(5) Bloch, E. H., A. Powell, H. T. Meryman, L. Warner, E. Kafig: A comparison of the surface ofhuman erythrocytes from health and disease by in vivo light microscopy and in v i t r o electronmicroscopy. Angiology 10:6 0956).(6) Born, G. V. R.: Quantitative investigations into aggregation of blood platelets. J. Physiol.(Lond.) 162:67 (1962)(7) Born, G. V. R., M. J. Cross: The aggregation of blood platelets. J. Physiol. (Lond.) 168:178(1963).(8) Born, G. V. R., M. f. Cross: Effect of adenosine diphosphate in the concentration of plateletsin circulating blood. Nature (Loud.) 197:174 (1963).

(9) Borgstr~m, S., L. E. Gelin, B. Zederfeldt: The formation of vein thrombi following tissue in-jury. Acta. chit. scan& Suppl. 247 (1959).(10) Castaneda, fl. R., E. Bernstein, F. Gangstadt, R. k. Varco: Effect of P~P, dextrose and dex-tran on red blood cell charge. Israel J. exp. Med. 11:128 (1964).(11) Constantine, ]. W,: Inhibition of ADP induced platelet aggregation by histamine. Nature(Lond.) 207:91 (1965).(12) D u g u i d , H . B .: The arterial lining. Lancet If: 207 (1952).(13) Eastham, R. D.: Adhesive platelets and myocardial infarction. Geriatrics 21:182 (1966).(14) F a h re u s , R . : The suspension stability of the blood. Acta reed. scand. 55: (1921).(15) Flemming, ]. S., M. E. Bierwagen, M. Rosada, M. H. Pindell: Effect of BLR-743, a newanti-inflammatory agent, on platelet aggregation. Microvascular Res. (In press.)(16) French, H. E., R. G. MacFarlane, A. G. Sanders: The structure of haemostatic plugs and ex-

perimental thrombi in small animals. Brit. J. exp. Path. 45:2167 (1964).(17) Gaardner, A., ]. Jonsen, S. Leland, A. Hellem, P. A. Owren: Adenosine diphosphate in redcell as a factor in the adhesiveness of human blood platelets. Nature (Lond.) 192:531 (1961).(18) Gelin, L. E.: The significance of intravascular aggregation following tissue injury. Bull. Soc.Intern. Chir. 18:4 (1959).(19) Greiter, K.: Studies on the mechanism of thrombin catalyzed hemostatic reactions in platelets.Acta physiol, scan& Suppl. 195:83 (1962).


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Anti-Adhesive Drugs in Thrombosis 213(20) H a a p a n e n , . E . : A high-sensitivity capacitance meter. J. electr. Engin. 34:183 (1962).(21) Hampton, J. R., J. R. Mitchell: A transferable factor causing abnormal platelet behaviour in

vascular disease. Lancet. H: 764 (1966).(22) Harriers, H.: Intravascular agglutination of erythrocytes. Schweiz. reed. Wschr. 87:11 (1957).(23) Hellem, A.: The adhesiveness of human blood platelets in vitro. Scand. J. clin. Lab. Invest.

12: Suppl. 51 (1960).(24) Horlick, L.: Platelet adhesiveness in normal persons and subjects with atherosclerosis. Amer.J. Cardiol. 8:459 (1961).(25) Hovig, T.: The effects of various enzymes on the ultrastructure, aggregation and clot retrac-tion ability of rabbit blood platelets. Thrombos. Diathes. haemorrh. (Stuttg.) 13:84 (1965).

(26) Johnson, S. A.: Platelets in hemostasis. In: Blood C otting Enzymology. (W. S. Seegers, Ed.)p. 380. Academic Press, New York 1967.(27) Judah J. D.: Ciba foundation symposium on enzymes and drug action, p. 339. Churchill, Lon-don i947.(28) Knisely, M. H.: Intravascular erythrocyte aggregation. In: Handbook of Physiology, (P. Dow,Ed.), Washington, Section 2, vol. III, p. 2249 (1965).(29) Knise~y, M. H., E. H. Bloch, T. S. Elliot, L. Warner: Sludged-blo0d. Science 106:431 (1947).

(30) kiischer, E. F.: Biochemical basis of platelet function. Proc. 58th Annual Meeting Int. Acad.Pathol., San Francisco. (In press.)(31) M a c F a rl a n e , R . G.: The basis of the cascade hypothesis of blood clotting. Thrombos. Diathes.haemorrh. (Stuttg.) 15:591 (1966).(32) M a rkwa rd t , F . , W . B a r t h ~ l , E . Gl u sa : Changes in the histamine and serotonin content of bloodplatelets due to the effect of local anesthetics. Naunyn Schmiedebergs Arch. exp. Path. Pharm.253:336 (1966).(33) Moolten, S. E., L. Vroman, G. M. S. Vroman, B. Goodman: Role of blood platelets inthromboembolism. Arch. int Med. 88:667 (1949).(34) Mustard, H. F., H. C. Rowsell, F. Lotz, B. Hegardt: The effect of adenine nucleotides onthrombus formation, platelet count and blood coagulation. Exp. tool. Path. 5:4 3 (1966).(35) Nordoy, A., A. B. Chandler: Platelet thrombosis induced by adenosine diphosphate in therat. Scand. J. I-Iaemat. 1:25 (1964).(36) Nordoy, A., T. O. Ravick: Some effects of adrenaline on rat platelets in vitro and in vivo.Stand. J. clin. Lab. Invest. Suppl. 84:151 (1964).(37) OBrien, J. R.: Platelet aggregation. Part. II. Some results from a new method of study. J. clin.Path. 15:452 (1962).(38) Reber, K., A. Studer: Influence of certain drugs on the adhesiveness of human, rat and rabbitblood platelets in vitro. Thrombos. Diathes. haemorrh. (Sruttg.) 13:248 (1965).(39) Reneau, D. D., D. F. Bruley, M. H. Knisely: A mathematical simulation of oxygen release,diffusion, and corsumption in the capillaries, and tissue of the human brain, in chemicalengineering in medicine, and biology. Plenum Press 1967.(40) Rozenberg, M. C., B. G. Firkin: The rate of platelet aggregation in haemorrhagic disease andthrombosis. Scand. J. Haemat. 3:5 (1965).(41) Seaman, R. W., R. G. Mason, R. H. Wagner, K. M. Brinkhous: Studies on thrombin inducedplatelet aggregation. J. exp. Med. 114:905 (1961).

(42) Seegers, W. H., H. Schr6er, E. Marciniak: Activiation of prothrombin. In: Blood ClottingEnzymology. (W. I-I. Seegers, Ed.) p. 103. Academic Press, New York 1967.(43) Spaet, T. H., M. B. Zucker: Mechanism of platelet aggregation by formation and role of ADP.Amer. J. Physiol. 206:1267 (1964).(44) Swank, R. L.: Adhesiveness of platelets and leukocytes during acute exanguination. Amer. J.Physiol. 202:261 (1962).(45) Swank, R. L., ]. Roth, J. Jansen: Screen filtration pressure method and adhesiveness and ag-gregation of blood cells. J. appl. Physiol. 19:340 (1%4).(46) Taub, W.j M. Cais: The synthesis of ketolactones with potential pharmacodynamic properties.Bull. Res. Counc. Israel 114:18 (1962).


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(47) Thorsen, G., H. Hint: Aggregation, sedimentation and intravascular sludging of erythrocytes.Interrelation between suspension stability and colloids in suspension .fluid. Acta. chit. scand.154 (1950).(48) Wright, H. P.: Changes in the adhesiveness of blood platelets following parturition and sur-gical operations, J. Path. Bact. 54:461 (1942).

(49) Zieve, P. D., J. L. Gam~,Ie, Jr., P. J. Dudley: Effects of thrombin on the potassium and ATPcontent of platelets. J. din. Invest. 43:2063 (1964).

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