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Platelet Adhesion and Aggregation in Thrombosis: Countermeasures


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

E.F. MAMMEN

G. F.ANDERSON E D ITORS

M. I. BARN HART

203 Figures, 50 Tables

19 ~ 70

F. K. SCHATTAUER VERLAG STUTTGART - NEW YORK

SUPPLEMENTUM XXXXII AD THROMBOSIS ET DIATHESIS HAEMORRPIAGICA

Anti-Adhesive Drugs in Throm bosis


I-I. I. BICHE~

The causal participation of blood cell aggregation in thrombus formation has been desoribed by several investigators under clinical (5) and experimental (6, 34, 36) conditions. Of the blood cells involved, special attention has been given to the erythrocytes and platelets. These ceils seem to possess the ability to start and accelerate the chain of events that leads to organic vessel occlusion. Knisely et al. (29) and Bloch (5) observed long ago the pathological significance of intravascular red cell aggregation (sludge). Borgstrom et al. (9) were able to demonstrate a direct etiological relation between experimentally induced sludge and venous thrombosis in the rabbit. In previous work (2) we suggested that intravascular red cell aggregation may be a factor in the induction of anoxic myocardial damage. The importance of platelet aggregation for hemostasis and for the pathogenesis of thrombotic processes is well established (6, 40). The adherence of platelets to each other and to other surfaces is considered a fundamental step in the formation of hemostatic plugs and thrombi (34). The purpose of this paper is to assess the therapeutic value of a group of drugs that share the common property of preventing both red cell and platelet aggregation, in vitro and in vivo, demonstrated with several objective methods devised or adapted to evaluate this action on a quantitative basis. Three different compounds showing these effects are introduced, ~nd their possible action discussed, both at the platelet membrane function level and for the mechanism of their potential antithrombotic usefullness. These substances are called anti-adhesive drugs.
Materials Adenosine diphosphate (ADP), Sigma Chemical Corporation, U.S.A. High molecular weight dextran, Pharmacia, Upsalla, Sweden. 2 Methyl 2 tert butyl-3-5-6 tetrioxotetrahydropyran (substance ,~86~) was synthetized according to Taubs method (46). 5 (5-hydroxindol-3-indoleyl-methyl) tetrazol (BLR-743), 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. Vroman, Technion, Haifa, Israel.** (Fig. 1).
Department of Anatomy, Medical University of South Carolina, Charleston, South Carolina.

H.I. Bicher

N~N

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


C1

iso-C4H9--CO-- C~ C/OH

o BR R2 u--~O "86"

R1= Methy[ R2= Tert- butyl

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

2 Methyl, 2 Tert-- butyl--3,5,6 -trioxotetrahyd ropyran

Fig. 1. Chemical structure of the anti-adhesive drugs. Methods


A. Platelet and red cell aggregation 1. Testing of anti-platelet aggregation properties of chemical compounds

a) The >>Rolling Tube(c platelet adhesiveness test This method is a modification of the procedure described by Wright (48) for clinical investigation. Blood was obtained with a plastic syringe by venipuncture of the antecubitalvein in the experiments carried out with human platelets, or from the femoral artery through a polyethylene canula in Nembutal anesthetized cats. Sodium citrate 3.8 %, in a proportion of 1 : 10 was used as anticoagulant. 0.4 ml of blood was placed in a 10 ml, 1.2 cm diameter non-siliconized test tube containing 0.1 ml of the added solutions (solvents tested drug). The tube was slowly rotated on its longer axis, 16 times per minute, for 2 minutes. The adhesive platelets adhered to the wall of the test tube. Those remaining in the blood were counted. 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 and valuable discussion. ** The autor is indebted to all three members of this successful group of chemists, for supply of the described drugs and invaluable scientific advice.

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taining increasing concentrations of the tested drug indicated the percentage of platelet adhesiveness prevention. A second set of test tubes was run as described, but adenosine diphosphate (ADP) was added at a concentration of 0.5 ~g/ml. This induced increased platelet adhesiveness, and a further drop in the number of remaining platelets. Here again, the difference between test tubes containing ~iiDP alone, 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 elements of agglutinated blood. By this method, the pressure required to force blood at a constant rate through a screen with multiple 20 micron-square pores is measured. When the blood elements are separated they pass through .the pores without any appreciable resistance. An increase in pressure indicates an increase in the number of aggregated cells in the blood. Swank demonstrated that the addition of ADP to the tested blood induced a sharp rise in the SFP, which was probably dependent on an increased platelet aggregation, although other cells may have participated in this action. In order to clarify the effect of the studied compounds on platelets in a simplified system, ADP was added to PRP (platelet-rich plasma) at a concentration of 1 ~g/ml, and then increasing doses of the tested drug were added at successive runs. A d~crease in the SFP indicated prevention of the ADP induced platelet aggregation. c) Photoelectric method The photoelectric method to determine platelet aggregation in platelet-rich plasma (PRP) has been described by Born (6). Continuously stirred PRP is placed in a transilluminated test tube, and the amount of light transmitted is measured with a photocell. Platelet aggregation, induced by ADP 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 the determination of the minimum effective concentration of the tested drug that prevented platelet aggregation.

Fig. 2. Simultaneous recording of the membrane capacitance aggregometer (upper tracing) and Borns photoelectric method (lower tracing). A. Aggregation induced by collagen, note good response in both tracings. B. Lack of response to collagen after pretreatment of PRP with ~BLR-743<< (100 t~g/ml). For explanation see text.

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d) ~,Membrane Capacitance(~ aggregometer This method allows for the determination of platelet aggregation in whole blood, without the somewhat cumbersome technical procedures involved in SFP readings. It is based on the assumption that the changes in platelet membrane function and ion fluxes to and from these cells that occur during aggregation, as will be described later in this paper, should create detectable changes in blood 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 oxalated blood were placed between two sensitive plates facing each other on both sides of a 5 mi siliconized test tube. The blood was continuously stirred and its temperature maintained at 37 o C in the same way as is usually done in Borns method (6, 7). The output of the capacitance meter was recorded on a model 5 Grass polygraph. Platelet aggregation was induced by adding either ADP, 0.2 [tg/ml, or collagen to the tested blood. The minimum effective dose of the evaluated drugs that prevented aggregation was determined using the same procedure as in method In preliminary series of experiments, methods ?~c** and ~d~ were used in parallel, from the same PRP, and the simultaneous records compared (Fig. 2). In vivo platelet experiments In vivo experiments were performed for all four platelet procedures a, b, c, and d on Nembutal anesthetized cats and dogs, a~er arterial, venular and tracheal cannulation. Blood samples were obtained from the artery while all solutions were injected into the vein. Polyethylene cannulae were used. 2. Testing of anti-red cell aggregation properties of chemical compounds e) Erythrocyte sedimentation rate (ESR) Thorsen and Hint method. Quantitative in vitro studies This procedure, first published by Thorsen and Hint (47) provides for a simple quantitative estimation of the erythrocyte aggregation properties of colloids or plasma. It has been adapted for the determination of the anti-aggregation properties of drugs, as a screening method that lends itself to the numerical comparison of the relative strength of action of the different compounds. The principle 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 of dilutions of plasma or artificial colloids. One part of washed human red cells was suspended in two parts 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 concentrations in the respective tubes. The points formed straight lines with nearly the same slopes. The points where lines cut the abscissae gave the concentration of colloid which caused a sedimentation rate of 1 mm per hour. This parameter, called the critical point by Thorsen and Hint, was dependent on the colloid concentration in individual tubes and could, therefore, be considered as a quantitative measure for their erythrocyte aggregation power. When the colloid concentration was lower than critical, the erythrocyte aggregation disappeared; when the concentration wa~ raised, aggregation and sedimentation rate rose very rapidly. For the determination of the anti-aggregation properties of substance ~86~ and related compounds, a fixed aggregation force was chosen, giving a sedimentation rate of 40 ram/hour (in the present 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 as represented by the decrease in the sedimentation rate, was measured as percentages of the control. D Vital microscopy o~ the peripheral circulation. In vivo experiments Microscopic observations of the microcirculation were performed on the transilluminared omenturn of the cat. Using a Leitz dissection microscope, the observations were made at magnification

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of 180 and never continued for more than 10 minutes at a time. Subsequent observations were made occasionally, but not more than twice in the same animal. Intravascular red cell aggregation was induced by injection of high molecular weight dextran intravenously at a concentration of 1 g/kg. The flow improving effect of the anti-adhesive drugs was followed a~er their intravenous administration. The effect of dextran infusion and antiadhesive drugs on the erythrocyte sedimentation rates were measured using a standard Westergren technique.

B. Potassium flux at the platelet membrane during aggregation


All glassware with which platelets had contact was coated with silicone. Rabbit blood was obtained 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 ml portions of PRP were sedimented and washed twice with, and finally resuspended in 1.8 ml of tris buffered 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. The final concentration of platelets was approximately 250,000 per mm3. The washed platelets were incubated without agitation at 37 o C under various experimental conditions. After incubation, the platelets to be analyzed for potassium were washed once with tris buffered saline containing EDTA but no potassium or glucose. This final washing procedure was performed at 4 o C and required approximately 10 minutes. The platelets were then sedimented by centrifugation at 22,000 g for 5 minutes and lysed ~ 3 ml of a 3 % solution of trichloracetic acid. Potassium in the lysates was measured by use of a flame photometer employing an internal lithium standard. Radioactive potassium (K42) was measured by use of a Welb scintillation detector.

C. Electron microscopic studies


The effects of auti-adhesive drugs on the ultrastructure of rabbit platelets were studied by incubating aliquotes of platelet-rich plasma (PRP) with each of these agents at different concentrations in 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 .

Results
Prevention of platelet aggregation and adhesiveness

Substance ,~86,~, substance ,,86B,,, and r~BLR-743,, were active in preventing platelet aggregation and adhesiveness in all four tests performed in vitro (methods a, b, c, and d). These results are summarized in Table 1. Using substance ~86,,-as reference unit of activity, it can be concluded that substance ,~86 B,, was five times more active than substance ,~ 86,, in preventing platelet aggregation in all tests, and ,>BLR-743,~ twice as active in preventing ADP induced platelet aggregation and ten times more active in preventing collagen induced platelet aggregation and platelet glass adhesiveness (Rolling Tube Test).

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Table 1. Relative potency of action of the anti-adhesive drugs -- in vitro experiments. Figures represent mean effective doses preventing blood ceil aggregation in lzg/mI. DRUG ESR SFP 2,000 400 -(ADP) 2,000 400 1,000 BORN (collagen) 1,000 100 MCA 2,000 400 1,000 ROLLING TUBE 2,000 400 100

250 ~86~ 25 ~86B~ ~BLR-743 ~ 100

ESR = Erythrocyte sedimentation r~te-modified Thorsen and Hint Method. = Screen Filtration pressure method. SFP 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 adhesiveness were 200 mg/kg for substance ,,86~ and 30 mg/kg for substance ,,86B(<. >,BLR-743~< prevented collagen induced aggregation and platelet adhesiveness at a dose of 30 mg/kg, i.v.
Prevention of red cell aggregation

The 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<< was ten times more active than substance ,, 86 ~ on this test, while >>BLR-743,, was only two to three times more active. Another fact to be considered is that only one fourth to one tenth as much of these drugs was required to prevent red cell aggregation than to prevent platelet aggregation. In vivo, all three substances were tested at the same doses as for platelet aggregation (see above). Af[er the intravenous injection of the drugs, ;he erythrocyte sedimentation rates of the studied animals were inhibited for a period of four to six hours. Microscopic observations of the microcirculation aPcer infusion of high molecular weight dextran revealed a slow circulation of blood. The red cells were aggregated into large masses (sludge) with spaces of cell free plasma separating them (plasma skimming). The sludge formation was especially heavy in venules, but was present also in capillaries and arterioles. In many places, red cell impaction of small post capillary venules could be discerned (Fig. 3A). Aiier the intravenous administration of anti-adhesive drugs the circulation became faster, red cell aggregates separated, and the plasma skimming disappeared, especially in the arterioles. Venular sludge remained in most cases. This effect lasted for one hour, alter which time observations were discontinued. The most striking improvement occurred during the first 15 minutes after injection (Fig. 3B).

Anti-Adhesive Drugs in Thrombosis

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

A. Effect of ADP and anti-adhesive drugs on potassium release from platelets


1. I~latelets incubated in plasma

The normal content of potassium in rabbit platelet~ was found to be 93 micro moles per l011 platelets. There were no significant changes in the potassium content of platelets incubated in plasma for periods of up to two hours. This experiment was reproduced 5 times 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 (see Fig. 4). The mean potassium content (in 10 experiments) was 68 moles per 1011 platelets. The platelets were coarsely aggregated during the incubation period, the platelet aggregates discernible with the unaided eye. The addition of anti-adhesive drugs completely prevented the loss of platelet potassium induced by ADP. The minimal concentration of these drugs required was 2.5 mg/ml. Platelet aggregation during the incubation period was also prevented. Anti-adhesive drugs incubated with platelets in plasma did not induce any potassium release.

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H. I. Bicher

....

ADP alone ADP end anti adhes =re drugs

Time of incubation(min)

Fig. 4. ADP induced loss of potassium from platelets incubated in plasma. 2. I~latelets incubated in potassium free buffer Washed platelets incubated in bui~er containing EDTA and glucose but no potassium 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 potassium release from the platelets was induced by the addition of anti-adhesive drugs, at the same concentrations as in the previous experiment (Fig. 5). This experiment was repeated t 0 times. The action of ADP on this system could not be estimated because of the erratic responses obtained. Usually, a slight increase in the rate o~ potassium release was obtained.

-- Control ptatelets ---- Platelets treated with onti adhesive drugs Time ofincubation (mini

1o io

Fig. 5. Increased loss of potassium from platelets incubated in potassium free buffer, induced by anti-adhesive drugs.

Anti-Adhesive Drugs in Thrombosis

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B. Effect of ADP and anti-adhesive drugs on the uptake of radioactive K42 by platelets

In 5 experiments, twice washed platelets from 3 ml portions of PRP were incubated at room temperature in tris buffered saline containing radioactive potassium (K42) in addition to stable potassium, EDTA and glucose. The concentration of stable potassium in the buffer differed slightly in each experiment in order to maintain the amount or radioactivity (counts per minute) sufficiently high for accurate counting. This variation did not influence the results. A~er varying periods of time the platelets were separated from the buffer, washed once, and the radioactivityfrom the total potassium of the buffer as well as lysates of platelets was measured. 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 - m moles) of the potassium in the platelets was 54 % of the .specific activity of the buffer. In 6 experiments when ADP (1, 2, or 5 ~g/ml) was added to the same mixture, the uptake of radioactive potassium was inhibited by 40 %, while the total amount of platelet 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, in the same concentrations as in the previous experiments. Both the specific activity and potassium contents of these platelets was in the same range as those of controls (Fig. 6).
~000Specific octivity in buffer -- Control

7,ooo.

6~3oo- .... ADP ..... ADP and anti adhesive drugs 5,ooo3,ooo2,000-

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

Time{rain}

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H. I. Bicher

Electron microscopic studies

Substance ~> 86~ and ~BLR-743 ~ were studied with this method, at concentrations ranging 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 shape and contained a preponderance of spherical shaped vesicles.These vesicles appeared to be part of the surface connected system which in untreated platelets shows vesicles 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 substance ~86~ was apparent, no significant amount of sphericity was induced in the surface connected vesicles of platelets so treated.

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

Disscussion A variety of methods have been developed to study the adhesive characteristics of platelets (23, 33, 48). In Wrights technique PRP is rotated in a specially designed glass bulb and the percentage change in the platelet count is determined. Moolten et al. (33) passed whole blood through a glass wool filter and estimated the ratio of the platelet to the red cell loss. Hellem (23) developed a method in which whole blood plasma was passed through a column containing a known weight of glass beads of standard size. This test provided a good system for the evaluation of the adhesive action of ADP on platelets.

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The more widely used procedure for the evaluation of the ADP induced platelet aggregation is that developed by Born (6) as described above. It is ,noteworthy that Rozenberg and Firkin (40) using this method were unable to show increased tendency to platelet aggregation in several diseases, as has been described using other procedures (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 technique, differed from those obtained when the Moolten te&nique (33) was used. Reber and Studer (38) also pointed out that under certain circumstances the Born and Wright techniques gave false positive results when drugs were tested for their anti-aggregative action, and concluded that both tests were not specific for the same property of blood platelets. Taking these facts ~nto consideration, we decided to run our platelet tests in parallel using four different procedures both in vitro and in vivo, as described above. The results obtained demonstrated the following facts: 1. All three anti-adhesive drugs tested were active in all tests for platelet adhesiveness and aggregation. 2. They acted in a similar way under the different conditions imposed by each procedure. 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 this compound, related to its specific effect on platelets, has been described by Flemming et al. (15). 3. The relative potency of action of the different compounds remained fairly stable. These results can be interpreted as an indication that these drugs interfered with a physiological process at the membrane level of the platelet, thus rendering the cell less responsive to different agglutinating stimuli, such as foreign and wettable surfaces, collagen, or adenosine diphosphate. Our modification of the Thorsen and Hint method (41) provided an accurate tool for the easy and quick quantitative evaluation of the anti-adhesive power of new chemical compounds. The results obtained, summarized in Table 1 show a good parallelism, both in respect to the type of activity, and relative potency of action of the different drugs when tested using this method, or the methods previously described for platelet agglutination. The concentrations of the drugs needed to disperse the red cell aggregates were less than those required to disperse platelets, probably because the forces holding the erythrocytes together (mainly a matter of coating or physical attraction [3, 10]) are not as strong as those linking platelet aggregates (an active chemical process requiring energy consumption [ 8, 17]). The fact that all three compounds acted in the same way in preventing both red cell and platelet aggregation indicated that this property of preventing blood cells

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H. I. Bicher

from sticking to each other was more generalized and affected not only the blood platelets. OBrien (37) described the ability of certain anti-malarials, local anesthetics, and imiprami~ne derivatives, to inhibit the ADP induced platelet aggregation. Constantine (!1) reported that histamine possessed a similar effect and that this property was also shared by some antihistamines and anti-inflammatory compounds. OBrien proposed the name of ,,anti-adhesive drugs<< to include all chemicals sharing this type of activity. Anti-adhesive drugs have been reported to induce platelet swelling also probably by inhibiting a phosphoprotein kinase (27). They also affected the active uptake of serotonin by platelets, but not the passive .histamine absorption (32). These results again suggest an interference with the functions of the platelet membrane. Zieve (49) has demonstrated that thrombin, also a platelet aggregation-inducing factor (19, 40), induced a loss of potassium from washed platelets suspended in a potassium containing medium and increased the platelet uptake of radioactive K42. .The present studies suggest that ADP has a strong action on the mechanisms that regulate the intracellular concentration of potassium in platelets. During ADP induced platelet aggregation, a net loss of potassium from the platelets has been found. At the same time, a decreased uptake of radioactive K42 by platelets has been demonstrated. Whether the diminished K+ influx into the cell may account for all of the decrease in the intracellular potassium content, or whether there is a concomitant mechanism that produces an active outflow of potassium during ADP i, nduced aggregation, can not be elucidated at the present time. According to the present theories for the resting potential of nerve and other cells, a decrease in intracellular potassium should lead to a decrease in resting potential No data are available to this effect for platelets, but a change in membrane potential could play a role in the aggregation process. Electrophoretic studies by Seaman (41) and Hampton and Mitchell (21) suggest a similar change in platelet electrophoretic mobility during aggregation. The present results support the concept of a direct membrane action for the antiadhesive drugs. They were able to reverse the effect of ADP on potassium permeability, inhibiting both the potassium loss from platelets, and the decrease in potassium uptake by platelets. These drugs seem to possess a direct action on the platelet membrane in the absence of ADP. In this case there is a liberation of potassium, but only in a potassium free medium, at high concentrations. When these concentrations are used, this could be the beginning of a lyric process, as the platelet swelling reported by Judah (27) may indicate. Our electron microscopic studies confirmed this platelet swelling which seems to occur with all the tested anti-adhesive drugs. Some of them also showed a specific effect on the tubular surface connected system of the platelet, which has

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been shown to contain Ca++ ions and to act in a similar way to the tubular systemof the muscle, in coupling contraction of thrombosthenin, the contractile protein of the platelets (30). These results are highly suggestive of a similar mechanism in platelet aggregation and muscle coupling ~nd contraction. Both systems use high energy phosphates, ionized Ca++, and induce K+ ion fluxes, however, more extensive studies will be necessary before any final ~onclussions can be drawn. These changes, both in membrane structure and ionic strength served as theoretical basis for the development of our capacitance aggregometer. The results here reporte.d proved its usefullness as a simple method to determine platelet aggregation in whole blood. Recently (1) we. have presented a concept that might explain the in vivo antithrombotic effect of the anti-adhesive drugs. This is summarized in Figure 8, and basically consists of the following steps: Phase A: Intravascular red ceil aggregation, combined with atherosclerosis or transient hypoxia. The absence of intravascular red cell aggregation during health has been confirmed in man (28). Based on microscopic observations of microthrombotic occlusions in capillary vessels, showing marked sludging of blood cells, a relation between sludge and thrombosis has been suggested. Experimental studies (9) and clinical observations (4, 22) confirmed this relationship. Since sludge is present in thrombotic disease, the obvious question is: Is it a consequence or the beginning of the thrombotic process? Gelin (18) summarized the possible pathogenetic mechanisms by which sludge could induce tissue injury. Our experiments (1, 2, 3) clearly showed that when sludge is combi,ned with atherosclerosis or a relatively short period of transient hypoxia, anoxic tissue damage is a consequence, both in the brain and heart. Phase B: Anoxic damage to vascular endotheIium. In the post capillary venules and the venous limb of the capillary, the delivery of oxygen is normally very precarious. It is in this region that hypoxic damage to vessel walls due to restricted blood flow is more easily evidenced alter vasoconstriction or intravascular agglutination, or by the combined effects of both and atherosclerosis. Knisely (28) described this damage that leads to endothelial lesion and protein leakage. Recently, Reneau et al. (39) developed a mathematical model in which the factors influencing oxygen diffusion to tissue were analyzed, and predicted the formation of minute hypoxic areas at the venous ends of brain capillaries under different pathological conditions. Phase C: Platelet to vessel wall reaction (platelet adhesiveness phase). Contact is established through a ~leaky,, endothelium between platelets and

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tissue factors (collagen, etc.) that enhance platelet adhesiveness, thus initiating a more active chain of events in the thrombotic process. Platelet adhesiveness has been correlated with collagen induced platelet aggregation. Native collagen fibrils aggregate platelets in citrated plasma. The fibrils release considerable amounts of ADP from the platelets, and the aggregation is inhibited by AMP (25). Evidence has also been obtained that collagen releases serotonin from platelets and renders platelet factor 3 available (43). The electron microscopic studies of Hovig (25) indicated that the platelet membrane remains intact during interaction with collagen while the intracellular granules disappear. The release is therefore probably either due to an increased membrane permeability to ADP and serotonin, or to an extrusion of platelet granular material without membrane rupture. Recent work by Johnson (26) has provided further details of this complicated process. The release of these factors leads to the next steps of thrombus formation. Phase D: Platelet aggregation w~th release of more endogenous platelet aggregation factors (epaf), platelet ~actor 3, minor red ceil trapping, and enhanced local
anoxia.

The importance of platelet aggregation for hemostasis and for the pathogenesis of thrombotic processes is well established. Changes in platelet reactivity of ADP, the chemical agent most frequently considered to be the natural platelet aggregation-inducer has been determined in a number of hemorrhagic and thrombotic conditions (13, 34) with the aid of different methods that lend themselves to a quantitative determination of platelet function. Platelet aggregate embolism is also considered to be part of the mechanism leading to atherosclerosis, according to Duguids theory (12). Once the process is initiated, more .endogenous ADP and other aggregation factors are liberated, and the process tends to perpetuate itself, Our experiments (1) demonstrated that red cells are trapped in the platelet aggregates, becoming a maior factor to increase the mass of the microemboli a,nd probably increase the local hypoxic condition. At this point, coagulation factors liberated from tissue (thromboplastin), platelets (factors 3, 5), and red cells probably initiate changes in plasma which lead to: Phase E: ~Cascade* or ~Thrombin Activation* process of Fibrin Formation. The well known theories (MacFarlane [311 and Seegers et al. [42]) are not discussed in this paper which mainly tends to demonstrate the manner in which the needed coagulation factors are made available by the hypoxia-aggregation process, which can, being reversible in nature, regulate the amounts and availability of these factors. Phase F: Major red cell trapping. Formation of the >>red thrombus~. French (16) has described how the actual length of a thrombus is determined by

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the 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 mesh formed.
The site of activity of the anti-adhesive drugs

According to this scheme of thrombus formation we propose that the antiadhesive drugs exert their anti-thrombotic action on phases A, B, C, D, and F Of the thrombotic sequence, (marked with X in Fig. 8). This concept establishes the rationale 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,~.
Summary

Both platelet and red cell aggregation have been related to the etiology of thrombosis. This paper describes a new category of drugs which prevent both types of .aggregation a,nd are devoid of any other pharmacological activity. These substances are called ,,anti-adhesive~ drugs. A photoelectric method, a new ,,membrane capacitance,, aggregometer, the screen filtration pressure method, and a new simple glass contact method were used to measure platelet aggregation and adhesiveness. A sedimentation rate technique and microcirculation observations determined red cell aggregation. The anti-ad-

X Phase A
X Phase B X Phase C X Phase D

Intravascular red cell aggregation combined with atherosclerosis and/or transient hypoxia Anoxic damage to vascular endothelium Platelet to vessel wal! reaction-ADP serotonin release (epaf) Platelet aggregation -- further epaf, plat,let factor 3 liberation, minor blood cell trapping Fibrin formation Major red cell trapping

1
Phase E X Phase F

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 of the anti-adhesive drugs.

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hesive drugs counteracted blood cell aggregation in vitro and in vivo, and also prevented experimental thrombosis and anoxic myocardial damage. The effect of the anti-adhesive drugs on platelet membrane mechanisms was determined using physiological and morphological techniques. It was found that K" permeability changes during ADP induced aggregation, leading to decreased intracellular potassium levels. This effect is prevented by the anti-adhesive drugs. Electron microscopic studies revealed that swelling and rounding of the surface connected system of the platelet occurs after incubation with anti-adhesive drugs. A possible mechanism for the anti-thrombotic action of these drugs is discussed.
References
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