trends in the development of antithrombotic agents

Trends in the Development of Antithrombotic Agents

JOHN HAMPTON, MA, BM, DPhil (Oxon), MRCP

Nottingham, England

In venous thrombosis and pulmonary embolism, blood solidifies to form a clot, which is a homogenous structure containing fibrin, red cells, white cells and platelets. Heparin and oral anticoagulant agents are effective in prophylaxis and treatment. In the future, fibrinolytic therapy (urokinase) and Arvin may prove more useful.

An arterial thrombus consists of platelet masses separated by fibrin strands; any agent that either prevents platelets from sticking to each other or prevents the formation of fibrin might theoretically be effective in preventing thrombosis. However, anticoagulant drugs are at best only marginally useful; although there are other ways of preventing fibrin formation, it now seems reasonable to investigate clinically agents that affect platelet behavior. Some of the methods of studying platelet behavior are described and their limitations are emphasized. Some of the many compounds known to influence platelets are discussed, and those which seem most worthy of clinical trials are indicated.

Sudden occlusion of an artery frequently results in the necrosis of distal tissue, and in the heart and brain this is a common cause of death. In most cases the occlusion is the result of thrombosis,lv2 and although our knowledge of the mechanism of this process is still far from complete it is possible to discern trends that may ul- timately lead to the development of an effective antithrombotic agent.

From the Department of Medicine, General Hospital, Nottingham, England. Manuscript received March 31, 1970, accepted May 26, 1970.

Address for reprints: John R. Hampton, MRCP, Department of Medicine, General Hospital, Nottingham, NGl-6HA, England.

It is essential that we bear in mind the differences between a thrombus and a clot, for they are sufficiently dissimilar for us to suspect that agents that inhibit the formation of one may not influence the other. A typical clot results from the solidification of blood in the test tube : It is soft, dark red and homogeneous ; mi- croscopic examination reveals it to be composed of a fine fibrin mesh enclosing red cells, white cells and platelets, each in the pro- portions present in whole blood. A thrombus, on the other hand, is pale and friable and is not homogeneous; it consists of masses of platelets surrounded by polymorphonuclear leucocytes and fibrin strands, and red cells are conspicuously absent. Occlusion of an artery usually results from the formation of a thrombus at the site of the block ; less often the artery is occluded by embohzation of a platelet-fibrin thrombus from a proximal part of the artery, or by a clot-like structure that formed in the heart. Occlusion of a vein may begin with the formation of a platelet-fibrin mass, but a clot forms rapidly in the stagnant blood stream. Most of the structure that forms a pulmonary embolus is clot, not thrombus. Thrombi, then, are characteristic of arteries, when blood flow is rapid ; when flow is slow, clots tend to form. The terms “white thrombus” and “red thrombus,” although descriptive, are misleading and should be abandoned.

VOLUME 27, JUNE 1971 659

HAMPTON

In this review, I shall be concerned primarily with the prevention of thrombus formation, and not so much with the prevention of clotting. Since a thrombus is composed mainly of fibrin and platelet masses we can consider possible antithrombotic agents under two main headings: agents that attack fibrin, and agents that attack the binding of platelets to one another. Since platelet masses form the major part of an early thrombus, and since the appearance of platelet aggregates is the earliest stage in experimentally induced thrombosis, it seems probable that agents that prevent platelets sticking to each other and to the vessel waI1 will be more effective in preventing thrombosis than agents preventing fibrin formation. I shall thus be concerned more with platelets t.han with fibrin; the role of the third component of thrombus, the white blood cell, is not yet sufficiently understood for there to be any grounds for considering as antithrombotic agents compounds that affect white blood cell behavior.

Compounds Affecting Fibrin Formation

Anticoagulant Agents

Anticoagulant agents may act directly or indirectly on the sequence of reactions that results in fibrin formation. Heparin acts directly and inter- feres with the conversion of prothrombin and with the thrombin-fibrinogen reaction. The orally effective anticoagulant agents, the coumarin and in- danedione derivatives, act indirectly by inhibiting the synthesis by the liver of factors VII, IX and X.

Although there is no doubt as to the efficiency of these compounds in preventing venous “thrombosis” and pulmonary embolism” (that is, conditions caused by clot-like structures), their effect on arterial thrombosis is still disputed. The role of anticoagulant agents in the treatment of myocardial infarction has recently been reviewed in depth by Mitchell.4 It seems that the administration of these agents over a prolonged period does benefit the survivors of myocardial infarction, but the advantage of this treatment is small and is frequently outweighed by the danger of hemorrhage. It is clear that we should look elsewhere for agents of real therapeutic value.

Fibrinolytic Agents

The direct and indirect anticoagulant agents prevent fibrin formation, but have little effect on fibrin once it has been formed. The physiologic mechanism for removing fibrin is the fibrinolytic system, which has been the subject of several recent reviews.h,” An inactive globulin called plasminogen is converted to an active substance, plasmin, by an enzyme that has been given the name “activator.” Activator is present in all body tissues, but mainly in the endothelial cells of blood vessels ;

it is excreted in urine, and the activator prepared from urine is called urokinase. Activator causes the destruction of an arginine-valine bond in plasminogen, and the resulting plasmin is an enzyme that attacks fibrin and degrades it to soluble frag- ments. When fibrin forms in vivo plasminogen is precipitated with it, and thus is available for the local destruction of the fibrin. Although activator is released by the administration of nicotinic acid, epinephrine and acetylcholine, and under conditions of “stress,” attempts to make use of it to en- hance the lysis of clots and thrombi have not been successful. The direct administration of plasmin is hazardous, since the preparations available have attacked proteins other than fibrin.

Streptokinase: The first powerful agent to be discovered which stimulated the fibrinolytic system was streptokinase’; this is an activator prepared from hemolytic streptococci, and intravenous infusion causes a marked increase in fibrinolysis. It has been claimed to be of value for the lysis of venous obstruction$ and puImonary emboli,!’ and also for the treatment of emboli and thrombosis in peripheral arterjes.1” ‘I However, there are no properly controlled studies that show it to have any definite advantage over heparin therapy for pulmonary embolism or surgery for acute peripheral arterial occlusion.

One large-scale trial of the effects of streptokinase on patients with acute myocardial infarction has been reported.*’ Of 558 patients with infarctions that had occurred less than 12 hours pre- viously, 297 were treated with streptokinase and 261 with heparin and coumadin. The mortality in the first group was 14 nercent and in the second 22 percent, a difference that was statistically significant. Unfortunately, the design of the study was not entireIy satisfactory; the patients were not allocated randomly to the 2 groups, and there was a marked preponderance of patients with infarcts of less than 3 hours’ duration in the group given streptokinase. Because of this, the treated group may have contained a higher proportion of patients without true infarctions, and since the phy- sicians knew which treatment was being used in each case, there may have been bias in the eventual decision as to which patients should be withdrawn from the trial. Having had 20 years of controversy about the value of anticoagulant agents in acute myocardial infarction, we should for the moment keep an open mind about the use of streptokinase.

Urokinase : Streptokinase has the disadvan- tage of being antigenic and frequently pyrogenic. In addition, all patients have a certain antibody titer to streptokinase because of previous strepto- coccal infections ; the amount of streptokinase needed to neutralize these antibodies and then to cause fibrinolysis therefore has to be carefully established in each patient. Urokinase can now be prepared from human urine and appears to

660 The American Journal of CARDIOLOGY

ANTITHROMBOTIC AGENTS

have none of these disadvantages. It can induce a predictable degree of fibrinolysis and can lyse artificially-produced clots in human veins.16 En- couraging accounts of its effects on pulmonary emboli have been reported, 17-lg but in view of the efficacy of heparin in this condition20 and the known rapidity of resolution of pulmonary emboli,” we must await the trial now being conducted under the auspices of the National Heart and Lung Institute, Bethesda, Md., before we can be certain of its value.

There are good reasons for optimism about the use of urokinase in the treatment of pulmonary emboli but, as I have already emphasized, a pulmonary embolus resembles a clot and fibrin alone is responsible for its structural integrity. The embolus is in an area reasonably well perfused with blood and, at least in the early stages, it is not attached to the vessel wall. Activator can, therefore, diffuse into the embolus from the proximal end and the sides. None of these factors apply to thrombi in coronary arteries: The thrombus is only partly fibrin, it is attached firmly to the vessel wall, and when there has been an infarction the vessel is usually completely occluded1 so that only a small area can be exposed to circulating activator. Although studies of the effect of urokinase in acute myocardial infarction are clearly necessary, the prospect that this agent will have a dramatic effect seems less good in treatment of this lesion than in the treatment of pulmonary emboli.

Combined phenformin and ethylestrenol: Fearnley and his colleagu~esz2-“* have suggested an entirely different approach to the use of fibrinolysis in coronary artery disease. The fibrinolytic activity of the blood can be increased over a prolonged period by the oral administration of a combination of phenformin and ethylestrenol; this might prevent fibrin formation in the bloodstream even if there is insufficient fibrinolytic activity to have much effect on a clot or thrombus large enough to cause symptoms. This regimen also has effects on platelets, as will be discussed later.

Arvin

The most recently developed method of inhibiting fibrin deposition depends on the conversion of fibrinogen to a fibrin-like substance which does not form a solid clot but which is rapidly broken down in vivo.2s,2G Bites by the Malayan pit viper (Agkis- trodon rhodostoma) render the blood incoagulable without inducing a bleeding tendency; the purified component responsible for this has now been separated and given the name Arvin and initial clinical studies27.2u have shown that it is nontoxic. Control of therapy has so far been achieved by measurement of plasma fibrinogen, but a simple bedside observation of the quality of the clot formed in a test tube may eventually prove to be adequate. Al- though Arvin acts on fibrinogen rather than

formed fibrin, venous occlusions appear to have been lysed in some patients. However, there is as yet no evidence that Arvin will cause the lysis of an arterial thrombus.

Compounds Affecting Platelet Behavior

When thrombosis and clotting were thought to represent the same process, it seemed obvious that to prevent thrombosis a drug should delay the clotting of blood in the test tube; the effect of the anticlotting drug was simple to measure since tests of blood clotting were well established. Thus, the effect of heparin can be determined by measurement of the blood clotting time, and the effect of the indirect anticoagulant agents by measurement of the prothrombin time. However, if we believe that to prevent thrombosis a drug should affect the functions of the platelets, we are presented with a dilemma. We do not know which aspect of platelet function we should be studying. In other words, there is no simple test of platelet function that will indicate with certainty whether a drug is having, or is likely to have, a beneficial effect on thrombosis.

There are many possible ways that platelet behavior can be studied; here I am concerned only with those that seem to be assessing the way platelets stick together to form the clumps seen in thrombi, and I shall not discuss the role played by platelet factors in blood clotting.

The first and greatest problem in the use of platelet function tests is that in vivo experiments are of necessity limited to animals. Platelets from different species behave differently in vitro,28 and most laboratory animals do not normally suffer from thrombosis. Any information obtained from animal studies is therefore not necessarily relevant to man. Human platelets can readilv be studied in vitro, but all such situations are highly artificial and the results of these studies may not be relevant to thrombosis. Therefore, in the assessment of a potential antithrombotic agent the only solution is to determine its effect on an in vivo test of thrombosis in animals, and on one or more in vitro tests of human platelet behavior. I shall describe 5 types of platelet function tests, 2 in the first category and 3 in the second, which are simple to set up and which, with various modifications, are in wide- spread use.

Experimental Thrombosis in Animals

In 1851 Wharton Jones first noted that mild injury to a vessel in a frog’s web caused “the ag- glomeration of colourless corpuscles” ; these were subsequently shown to be platelets, and it was found that injury to the arteries or veins of many species led to the formation of platelet-fibrin thrombi at the injury site. Convenient arteries for such experiments are those of the exposed rabbit


HAMPTON

cerebral cortex.:“)J1 If a small artery is pinched with fine forceps sufficiently hard to make it bleed transiently, a “white body” (a platelet-fibrin thrombus) will grow at the injury site until the vessel is blocked; it will then embolize distally and the embolus will gradually break up. A new “white body” will appear at the injury site, and the process of growth and embolization will be repeated regularly for several hours. The rate of formation of emboli can be timed and, hence, a quantitative assessment can be made of the activity of the thrombotic process and the effect of a drug on this process can be measured.

Thrombus Formation in Artificial Vessels

In 1927 Rowntree and Shionoya”“J:’ showed that thrombosis could be observed in a collodion tube inserted between a carotid artery and jugular vein of a rabbit. First, platelet masses could be seen sticking to the collodion and then a “white thrombus” formed; finally, the tube became blocked by clot. With the development of siliconized plastic tubing, the basic technique was considerably developed by Mustard, and Downie and their co- workers.“-‘-“” They confirmed the observations of Rowntree and Shionoya and showed that the deposition of thrombus in an extracorporeal shunt in a pig depended to a large extent on flow patterns. They also showed that the rate of thrombus formation in a shunt inserted into a rabbit can be measured quantitatively by weighing the platelet mass at different time intervals.

Platelet Adhesiveness

The term “platelet adhesiveness” is used to describe the sticking of platelets to a foreign surface such as glass. WrighV7 first demonstrated that when blood was rotated in a glass flask the platelet count fell, and that this fall in count resulted from the adhesion of platelets to the glass. In 1960 Hellem3x showed that when whole blood was passed through a glass bead column the platelet count also fell; this did not occur unless red cells were present, and it was subsequently show+ that the adhesion of platelets to glass depends on the release of adenosine diphosphate (ADP) from red blood cells. Many modifications of the glass bead column technique are now in use, differing mainly in the means by which blood is passed through the column-by motor-driven syringe,38 by suction40 or by free bleed from a vein.41

Platelet adhesiveness is increased in patients with coronary artery disease,42 but a similar increase also occurs in a wide variety of other diseases.37,43-4’ We therefore cannot say that increased adhesiveness is certainly bad, or that a reduction in adhesiveness is certainly desirable. However, the various techniques measuring adhesiveness can be used to assess the effect of an anti-

thrombotic agent administered to man or added to blood in vitro. The Hellem (glass bead) and Wright (rotating flask) techniques appear to give similar results,4H but the Salzman technique evi- dently measures a slightly different property of platelets since it is the only one with which abnormal platelet behavior (decreased adhesiveness) can be detected in von Willebrand’s disease.40

Platelet aggregation: “Platelet aggregation” is the term used to describe the sticking of platelets to each other to form clumps or aggregates. The presence of red cells is not necessary, and the process can be observed in platelet-rich plasma which is prepared by low-speed centrifugation of whole blood. Aggregation is induced by a variety of agents, of which adenosine diphosphate and tri- phosphate (ADP and ATP), epinephrine and norepinephrine, collagen and thrombin are the most important.“!’ 51 These “aggregating agents” are added in precise quantities. Therefore, platelet aggregation is a simpler experimental situation than platelet adhesiveness, where the degree of adhesiveness will depend on the way red cells release ADP as much as on abnormalities in the platelets themselves. However, because platelet aggregation is not measured in whole blood it represents a step further away from the in vivo situation.

Under certain experimental conditions the platelet aggregation that is induced by ADP can be shown to occur in 2 phases”“: the second phase is accompanied by the release of ADP and other substances from within the platelet. This (‘release reaction”“:’ is also concerned in the aggregation induced by epinephrines4 and collagen.5: It seems probable that the “second phase” of ADP-induced aggregation has a mechanism similar to that of epinephrine-induced and collagen-induced aggregation, whereas the mechanism of the first phase of ADP-induced aggregation is different.

Platelet aggregation is most conveniently measured by recording the optical density of a tube of platelet-rich plasma5”Ji : When platelet aggregates form, more light can pass through the tube and the optical density falls; when the aggregates dis- perse, the optical density rises again. This technique is simple, requires small quantities of plasma, and readily lends itself to automation. Un- fortunately, the degree of aggregation and the rate of disaggregation vary considerably in any individual from day to day, and only a few of the factors causing this variability are understood.5R This means that although measurements of platelet aggregation can be used to compare groups of subjects,5”,60 they are valueless for the study of indi- viduals or for determining the effects of drues administered in vivo unless large groups of subjects are used. However, the technique is ideal for the rapid screening of potential antithrombotic agents in vitro since several different agents can be tested against a single plasma sample.



Platelet Electrophoresis

Platelets are negatively chargedsl and therefore tend to repel each other. The charge on the platelet is directly related to its electrophoretic mobility, and this can be measured quite simply with the appropriate apparatus.62 Measurements of mobility are made on individual platelets in diluted platelet-rich plasma: Some of the complexities involved in the formation of platelet aggregates are thus avoided, but the experimental system may be further removed from the in vivo situation.

Agents that cause platelet aggregation induce changes in platelet mobility ; concentrations suffi- cient to cause aggregation induce a decrease in mobility (no aggregation actually occurs because the platelet-rich plasma is diluted and the system is not stirred), and smaller concentrations induce an increase in mobility.B”m6” The changes in mobility are similar when the platelets are challenged with ADP, ATP, norepinephrine, thrombin and collagen, but differences in the co-factors required for these changes and in the effects of various inhjbi- tors on the changes suggest that the mechanism involved in the response to ADP and ATP may differ from that involved in the response to norepinephrine, collagen and thrombin.66

Whatever the agent used to change platelet mobility, there is a maximal mobility that can be at- tained. By determining the concentrations of ADP and norepinephrine required to induce this maximal mobility, it has been possible to delineate 3 basic electrophoretic response patterns. In normal subjects, the same concentrations of ADP and norepinephrine induce maximal mobility.“3 In patients with any sort of acute illness there is an increase of platelet sensitivity to both ADP and noradrena1ine.67 In patients w&h arterial disease”* and in women taking oral contraceptives,6”v70 there is a selective increase of platelet sensitivity to ADP alone, and this increase in sensitivity is ap- parently related to a subtle change in plasma lipids.

Changes in platelet electrophoretic behavior thus provide another index against which the effects of drugs can be assessed; however, there are several possible ways that a drug could modify the electrophoretic response pattern. First, it might affect the response to ADP or norepinephrine, or both ; second, it might affect the increase or the decrease in mobility induced by either of these agents; and third, it might affect the change in platelet sensitivity to ADP or norepinephrine without influencing the biphasic mobility change. The response of the platelets of any individual subject remains reasonably constant provided his health is unchanged ; the electrophoretic technique is therefore suitable for studying the effects of drugs that have been either administered in viva or added to plasma in vitro. However, the main

disadvantages of the technique are that it is tedi- ous and time-consuming, and relatively large vol- umes (about 50 ml) of blood are required.

Selectjon of an Antithrombotic Agent

I have shown that several widely different SYS- terns can be used to assess drugs that might influence thrombosis through an effect on platelets. Neither heparin nor the coumadin derivatives affect any of these systems in therapeutic concentrations. However, many agents do affect one or more of them, and 1 shall describe some of those that might justifiably be tried as antithrombotic agents. Such a wide variety of drugs affect platelet behavior that it is difficult to classify them; however, the 2 most important groups of compounds appear to be the vasoactive and the antiinflammatory drugs.

Vasoactive Compounds

Persantin: Adenosine inhibits the formation of white bodies in the injured arteries of the rabbit cortex,71 but it is toxic when given intravenously and, moreover, it is rapidly removed from the blood. Dipyridamole (Persantin@, Boehringer) retards the destruction of adenosine,70 and it seemed possible that it could be used to potentiate the effects of small amounts of adenasine in the experimental situation. However, Emmons et a1.73,74 found that dipyridamole alone is a powerful inhibitor of white body formation in the rabbit and that, when given orally or intravenously to man, it reduces the “spontaneous” aggregation that occurs when platelet-rich plasma is stirred ; when added to platelet-rich plasma in vitro it reduces the first phase of the aggregation response to ADP. Oral administration also reduces platelet adhesiveness75 and abolishes the increase in platelet electrophoretic mobility induced by norepinephrine, although it has no effect on the electropho- retie response to ARP.7”

Dipyridamole analogs : The mechanism of action of dipyridamole is not clear. A study (unpub- lished) of the effects of a group of analogs of dipyridamole showed that there was no correlation between the inhibition of adenosine deaminase and any effect on platelets ; some of these analogs were found to be more effective than dipyridamole in inhibiting white body formation in the rabbit cortex and yet completely ineffective in the in vitro tests, whereas others (particularly RA 433, which was studied in some detaiV7) were highly effective in vitro but not in vivo. It is possible that there is a spectrum of activity among the dipyridamole- like substances, with some affecting experimental thrombosis in vivo and therefore perhaps acting on the vessel wall, and others having most effect in the in vitro tests and therefore acting against the platelets.


HAMPTON

Vasodilating agents : It seems probable that all vasoactive agents have some effect on platelets, although it is not possible to predict which aspect of platelet behavior will be influenced. Hampton et al.76 studied a variety of vasoactive drugs used in the treatment of cardiovascular disease and found that Intensaina (Abbott) and papaverine inhibited both ADP and norepinephrine-induced platelet aggregation ; glyceryl trinitrate inhibited aggregation induced by ADP but not that induced by norepinephrine ; and adrenergic blocking agents (phentolamine, tolazoline, propranolol, isoxuprine and iproveratril) all inhibited norepinephrine- induced aggregation, but only phentolamine and propranolol also affected the response to ADP. In- tensain, glyceryl trinitrate, tolazoline and propranolol all inhibited the electrophoretic mobility changes induced bv both ADP and norepinephrine.

No comprehensive theory explains the action of all vasodilator agents on smooth muscle, and there is none to account for their effects on platelets. However, the common effect of such agents re- emphasizes the similarities between platelets and smooth muscle cells: Both contain a contractile protein (thrombasthenin and actomyosin, respec- tively) and both contain a calcium-dependent adenosine triphosphatase.

Antiinflammatory Agents

Aspirin : Aspirin prolongs the bleeding time; Weiss and Alecort7x showed that although it did not affect (first phase) ADP-induced aggregation, it did diminish the response to collagen and the ac- companying release from the platelets of ADP. Zucker and Peterson7!’ showed that the second phase of the response to ADP was inhibited. The effects of aspirin on platelet aggregation therefore appear to be limited to situations in which aggregation depends on the release of ADP and other substances by the platelet. Platelet adhesiveness (measured by several different techniques) is not affected bv aspirin.iH.8f’ Changes in platelet aggregation car; be detected after a single dose of as little as 150 mg of acetyl salicylic acid (sodium sali- cylate is far less effective), and the effect of a single dose of aspirin may persist for several days.fi* Aspirin has also been shown to have an effect on platelet behavior in vivo; administration to rabbits causes a decrease in the rate of deposition of platelets in an extracorporeal shunt.*l

Pyrazole compounds (phenylbutazone) : Plate- let behavior is also affected by the pyrazole compounds, of which the most important clinically is phenylbutazone. Smythe et al.R2 showed that sul- finpyrazone (Anturan) increased the life of platelets in man and caused a decrease in their adhesiveness. Mustard et a1.8” showed that this agent caused a slight reduction in the rate of formation of a thrombus in an artificial arteriovenous shunt,

Fantlxs showed that sulfinpyrazole, phenylbutazone, antipyrine and Tanderil all inhibited ADP- induced platelet aggregation in vitro.

Other antiinflammatory agents: O’Brien85 studied the effects of a wide variety of other antiinflammatory agents on the platelet aggregation induced by epinephrine and collagen. He found that many agents had an inhibitory effect and that indomethacin was as powerful as aspirin; amido- pyrine and paracetamol were, however, less active and phenacetin and codeine were ineffective.

Dextran

The intravenous administration of dextran has been claimed to be of value in a variety of thrombotic conditions. x~i Bygdeman et al.A7 showed that intravenously administered dextran reduced the platelet adhesiveness to glass beads induced by ADP, and Bennett et al.xx showed that platelet adhesiveness in whole blood and platelet aggregation induced by ADP, were also diminished. However, platelet aggregation induced by collagen is not affected.Y” Dextran 70 (molecular weight 70,000) appears to be more effective than Dextran 40 (molecular weight 40,000) ; the maximal effect on platelets occurs about 3 hours after the infusion, per- sists for a further 4 hours or so, and has disappeared after 24 hours.8’,“n The infusion of dextran has no immediate effects on experimental thrombosis in rabbit arterioles, but 4 hours after an infusion the formation of thrombi is inhibited.“l

Polyvinylpyrrolidone : In vitro the dextrans have little effect on platelet behavior except when present in very high concentrations.“” This, together with their delayed effect after intravenous administration, suggests that their action may be on some plasma component rather than on the platelets themselves. Another substance that can be used as a plasma-volume expander, polyvinylpyrrolidone, also inhibits platelet adhesiveness.g2 This substance is chemically unrelated to the dextrans, but both appear to have an effect on plasma lipids and may possibly affect the platelets by this means, since changes in plasma lipids have been implicated in some aspects of abnormal platelet behavior.“3

Chlorphenoxyisobutyrate (Atromid)

The administration of Atromid@ reduces plasma cholesterol and triglycerides and it is claimed to “correct other thrombogenic tendencies.” Carson et al.“’ and Symons et al.!‘” showed that a dose of 2 a day for 1 to 2 months caused a significant reduction in platelet adhesiveness, and Robinson and LeBea@ showed that ADP-induced aggregation was also diminished. Chakrabarti et a1.g7 found that Atromid reduced platelet adhesiveness after 2 months, but that its effect had largely disappeared after it had been administered for a further 4

The American Journal of CARDIOLOGY


months. However, O’Brien and Heywoodg8 found that Atromid had no effect on platelet adhesiveness, and Hampton and MitchellO* concluded that it had no effect on abnormal platelet electrophoretic behavior.

It seems that Atromid does have an effect on platelets, but whether it can be said to correct this aspect of the “thrombogenic tendency of the blood” is by no means clear. Atromid probably does not affect the platelets directly, for its time course of action is slow and in vitro it increases platelet aggregation”” ; whether the change in platelet behavior is related to alterations in the plasma lipids is not known.

Other Compounds

Antihistaminic agents: A wide variety of other unrelated drugs affect various aspects of platelet behavior. O’BrienlnO showed that when antihistaminic, antimalarial and local anesthetic agents were added to blood or platelet-rich plasma in high concentrations in vitro, platelet adhesiveness and aggregation were inhibited. Further studies with antihistaminic preparations showed that there was no correlation between the efficacy of a drug as an antihistamine and its effect on platelets.lOl

Phenothiazine derivatives: Many drugs used in the treatment of psychiatric disorders influence platelets. Mills and Robert@ showed that if nor- tryptiline, amitryptiline, chlorpromazine and pro- methazine were added to platelet-rich plasma in vitro, the second phase of ADP-induced platelet aggregation was diminished. Like aspirin, therefore, these compounds probably act by preventing the release of endogenous ADP by the platelet. The monoamine oxidase inhibitor nialamide has also been shown to reduce platelet adhesiveness.lOZi

Glyceryl guaiacolate : This compound, a component of some preparations used for the treatment of bronchial asthma, reduces the degree to which platelets stick to polystyrene containersloZ ; when added to platelet-rich plasma in vitro in relatively high concentrations, it also inhibits ADP-induced platelet aggregation.lO”

Combined phenformin and ethylestrenol: The administration of a combination of phenformin and ethylestrenol (of interest because of its effect of stimulating fibrinolysis) has been shown to reduce platelet adhesiveness.24 Plasma cholesterol is also reduced by this regimen, but this is not related to the change in platelet behavior.g7

Prostaglandin E, : A large group of unrelated compounds known to affect platelet behavior are toxic and cannot at present be considered as potential antithrombotic agents. Nevertheless, some are worth mentioning briefly since they could form the starting point for profitable lines of research. The naturally occurring compound Prostaglandin E, is probably the most powerful inhibitor of platelet behavior yet described. It reduces the adhesive-

ness, aggregation and electrophoretic response of human platelets in vitro, and it inhibits experimental thrombosis in animals.106J07 When administered intravenously to man, platelet behavior is again affected, but the side effects are sometimes unpleasant and sufficiently severe to prevent its use as a therapeutic agent.lo8 Platelet aggregation and adhesiveness are depressed in patients with uremia, and an important abnormality in the blood appears to be guadinosuccinic acid.lOoJ1o Platelet aggregation is also inhibited by the group of substituted aminoacids typified by arginine methyl esterlll ; it is also affected by various enzyme poi- sons, and particularly by agents that block sulphy- dry1 groups.l12

Diet : Finally, mention must be made of the possibility of altering platelet behavior by dietary manipulation. Nord6y11n showed that a diet rich in saturated fat increased platelet adhesiveness in rats, whereas a diet containing linseed oil reduced adhesiveness. A report that the platelet adhesiveness of atherosclerotic patients could be reduced by adding linseed oil to the diet114 was not confirmed by a later studyll”; nevertheless, the possible interrelationship between platelets and diet should not be forgotten.

Clinical Prospects for Antithrombotic Agents that Attack Platelets

It is evident that there are a large number of compounds that might prevent thrombosis because of their action on platelets. However, none of these compounds affect all aspects of platelet behavior and our problem is to decide which particular “platelet function test” is most relevant to thrombosis in man.ll” Would a drug that is highly active against experimental thrombosis in the rabbit but not very active against platelets in vitro (for example, Persantin) be likely to be of more therapeutic use than one which is active in the in vitro tests but inactive in vivo in rabbits (for example, RA 433) ? Is a drug that is active against the second phase of ADP-induced platelet aggregation (aspirin) likely to be of more use than one that affects the first phase of the response (glyceryl trinitrate) ? More important, can we be sure that by reducing the degree to which platelets stick to glass we reduce the likelihood of clinical thrombosis? Although there are many compounds that inhibit some aspect of the response to ADP, none has yet been found that makes the platelets of a patient with arterial disease respond normally to ADP in the electrophoretic system. Is it this that we need to achieve?

None of these questions can yet be answered. We have the means to study only limited aspects of the effects of drugs on platelet behavior and, until we know which of the platelet function tests is the most relevant to thrombosis in man, only clinical


HAMPTON

TABLE I

Action of Persantin and Aspirin Against Platelets

Persantin Aspirin

In vivo (animals) +* + In vitro (human platelets)

Platelet adhesiveness + - Platelet aggregation with ADP

First phase + - Second phase - +

* + = possible effect on platelets.

trials will tell us whether an “antiplatelet” agent is a useful antithrombotic drug. Which, then, of the many compounds that affect platelets should we select for clinical trials?

Dextran is undoubtedly useful for the prevention of venous thrombosis,l17 and it may be of use for the prevention of arterial thrombosis when the latter is predictable (such as after endarterec- tomy and arterial catheterization). However, the need to give it intravenously and its relatively short-lived effect make Dextran unsuitable for large scale trials in a population at risk of coronary thrombosis.

Atromid is clearly an important and interesting compound. It has no effect on the mortality of survivors of acute myocardial infarction, but it is not, known whether it is effective in preventing infarction in subjects initially without overt arterial disease. Since its effect on platelets only lasts a few months the justification for undertaking a large clinical trial with Atromid rests on its action on plasma lipids rather than on its action on platelets.

Aspirin vs. Persantin: Of the antiinflammatory agents, aspirin is clearly the most interesting since it is cheap and, in the doses required to influence platelets, it is nontoxic. Of the vasoactive agents dipyridamole (Persantinm) is the most promising; this is the first drug to have received a

trial as an antithrombotic agent on the basis of platelet function tests. Although Persantin has been shown to have no effect on mortality when given for a month after an acute myocardial infarction,llx it does reduce the frequency of thromboembolic events in patients with artificial heart valves.T4.11S’ The actions of Persantin and aspirin against platelets are very different (Table I).

Although there is still much that we do not know about the actions of Persantin and aspirin on platelets, no further laboratory experiments will tell us which (if either) of these is likely to be a useful antithrombotic agent. A large-scale and long-term clinical trial is needed.

Conclusions

Clotting and thrombosis are 2 different pro- cesses that result in the solidification of blood. In venous “thrombosis” and pulmonary embolism the structure responsible for the clinical state is mainly clot; heparin and the indirect anticoagulant drugs are valuable for the prophylaxis and treatment of these conditions, and in the future fibrinolytic therapy and Arvin may prove to be even more effective. There is, however, no medical regimen of proved efficacy for the prophylaxis and treatment of arterial thrombosis.

Rather than attempting further to affect the fibrin component of the thrombus, it now seems more promising to attempt to modify the behavior of the blood platelets so as to prevent their sticking to the artery wall and to each other. Although we have several methods of assessing platelet behavior in vivo (in animals) and in vitro, and although many compounds are known to influence some aspects of platelet behavior, we do not know which “platelet function test” is most relevant to thrombosis in man. The compounds that affect platelets have been identified, but there is no substitute for carefully controlled clinical studies.

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