British Medical Bulletin (1978) VoL 34, No. 2, pp. 123-128


CONTRIBUTION OF PLATELETS TO THROMBUS FORMATION A M WHITE BSc PhD Horsham Research Centre CIBA-GEIGYPharmaceuticals Division CIBA Laboratories, Horsham, Sussex

S HEPTINSTALL BSc PhD Department of Medicine University of Nottingham Platelet behaviour a Agents that induce platelet activity b Physiology c Biochemistry Experimental systems for analysing the thrombotic process a Physical determinants b Factors affecting formation of the platelet monolayer c Factors affecting mural thrombus formation Conclusions References

Platelets adhere to subendothelium, they can be stimulated to adhere to each other so that macroscopic platelet aggregates are produced, and they can release mediators that induce other platelets to aggregate and that promote blood coagulation. It is known that through these activities platelets contribute to haemostasis and it is widely believed that these activities are relevant to thrombosis, particularly to arterial thrombosis where platelet masses form the bulk of the occlusion. Whether thrombus formation is akin to haemostasis where tissue damage initiates platelet deposition, or whether factors present in the plasma provide the initial stimulus, is unknown. In this paper we discuss platelet behaviour and the biochemistry of the processes that may contribute to thrombus formation. We then go on to describe attempts that have been made to specify the rate-determining steps in experimental systems that possess the blood-flow characteristics of arteries. Although it is only a hypothesis on which the model is based—that exposed subendothelium initiates thrombus formation—it is an approach that has been explored in some depth.

b Physiology

In general, platelets respond to such agents in the following way. After the platelets have been activated by the particular agent they are transformed from their normal disc shape into a spherical form with pseudopodia. In the case of soluble agents, the spherical structures interact one with another to form macroscopic aggregates, while insoluble agents are covered by a monolayer of adherent platelets. Aggregation requires participation of extracellular cofactors: platelets will not aggregate unless calcium ions and fibrinogen are present. Platelets can also undergo a release reaction during which prostaglandins and thromboxanes are synthesized and the contents of intracellular storage granules are secreted. Osmiophilic granules known as dense bodies contain ADP, ATP, serotonin, catecholamines and calcium ions, and so provide agents which amplify the effect of the original stimulatory agent; other granules contain platelet factor 4, fibrinogen and acid hydrolases, and so contribute to procoagulant activity. The precise way in which platelets respond depends on the

1 Platelet Behaviour Most of the studies designed to give information on the way platelets behave in the circulation are carried out under highly artificial conditions. Platelets are usually studied after they have been separated from other blood cells and whilst they are suspended in anticoagulated plasma or in denned chemical media. However, the results of such in-vitro investigations are valuable in that they allow us to speculate on the function of platelets in vivo. 123 Vol. 34 No. 2

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a Agents that Induce Platelet Activity A great number and variety of agents, including some lowmolecular-weight molecules, proteolytic enzymes, gh/coproteins and particulate matter, induce platelets to aggregate (Mustard & Packham, 1970); but perhaps those that have most relevance to thrombosis are thrombin and collagen. The presence of fibrin in the thrombus leaves us in no doubt that thrombin is formed, and the fact that thrombin is a potent platelet-aggregating agent indicates that it may be doubly important. Collagen lies beneath the endothelial cells that form the innermost layer of the wall, but it may be exposed in areas of damaged vessel such as are present in the region of atheromatous plaques. Certainly platelets are deposited and platelet thrombi form where vessels are experimentally injured (Honour & Mitchell, 1964) or where the endothelial layer has been experimentally removed (Baumgartner, 1974a). Fatty acids, bacteria, viruses and antigen-antibody complexes are also aggregating agents, so it is possible that diet, infection or immunological reactions are also determinants of thrombotic states. ADP is an aggregating agent that has received special attention, because thrombin, collagen, and many other materials act, at least in part, by releasing ADP from intracellular storage granules (Hovig, 1963; Haslam, 1964). It is now known that platelets are also induced to synthesize aggregating agents that are more potent than ADP. These are the prostaglandin endoperoxides PGG 2 and PGH 2 , and thromboxane A2. Although they are produced hi very small quantities and are extremely short lived they serve to amplify the response of the platelet to a given stimulatory agent (Samuelsson et al. 1976). Serotonin (5-hydroxytryptamine), adrenaline and noradrenaline are aggregating agents that regularly appear in plasma, are taken up by platelets, and are released from them along with ADP (Mustard & Packham, 1970). Although the plasma concentrations of these biogenic amines are usually not high enough for them to aggregate platelets directly, they may be important when they are released from platelets because they can act synergistically with ADP to enhance its effect (Ardlie et al. 1966; Mills & Roberts, 1967; Baumgartner & Born, 1968; Michal & Motamed, 1976).


A M White & S HeptinstaU

plasma fibrinogen level, but it may provide a high concentration between the platelets in the aggregate. Platelet factor 4 has heparin-neutralizing activity and so could have a role in overcoming any natural heparin-like inhibitors of coagulation that may be present.

nature of the agent, on its concentration, and on the experimental conditions. There are also species differences and differences within the same species.

c Biochemistry Although all aspects of platelet behaviour cannot yet be interpreted in biochemical terms, in the last few years significant advances have been made. Activation It is likely that the first step in the activation of platelets by most agents involves interaction between the agent and specific receptors on the platelet surface. Thus ADP does not enter platelets, and ADP-binding sites are present on platelet membranes (Nachman & Ferris, 1974). It is thought that these sites may be important in the activation of platelets because both binding and aggregation are inhibited by nucleotides that are structurally similar to ADP. Although adrenaline and serotonin are transported into platelets it is unlikely that the uptake is associated with aggregation. Inhibitors of uptake are not necessarily inhibitors of aggregation (Barthel & Markwardt, 1975), and rat platelets have been shown to have separate receptors for serotonin uptake and serotonin-induced shape change (Drummond & Gordon, 1975). Thrombin-specific receptors are present on the platelet surface (Detwiler & Feinman, 1973; Ganguly & Sonnichsen, 1976; Tollefsen & Majerus, 1976). There has been some discussion as to whether a form of thrombin-receptor complex is sufficient to activate platelets or whether subsequent proteolysis is also required (Detwiler et al. 1975). However, thrombin that is devoid of proteorytic activity, although able to bind tightly to the platelet, fails to cause secretion (Tollefsen et al. 1974), and a hydrolytic product has now been isolated (Phillips & Agin, 1977). Although fibrinogen is present in the platelet membrane (Nachman et al. 1967), it is unlikely that it is directly involved (Flengsrud et al. 1972; Tollefsen & Majerus, 1975). Perhaps least is known about the way collagen activates platelets. It has been suggested that an enzyme-acceptor complex is formed between glucosyltransferase on the platelet and galactosyl-hydroxylysine side chains on the collagen molecules (Jamieson et al. 1971), but this proposition now appears untenable for a number of reasons (Michaeli & Orloff, 1976). Collagen must be in the fibrillar form to cause platelets to aggregate (Muggli & Baumgartner, 1973; Brass & Bensusan, 1974; Gordon & Dingle, 1974; Jaffe & Deykin, 1974), and differences between collagen types with respect to their ability to cause platelets to aggregate in vitro probably reflect variations in the rate of fibril formation arising from different methods of preparation (Barnes et al. 1976). Human plasma may contain an inhibitor of fibrillogenesis (Barnes et al. 1976).

Procoagulmt Activity There are several ways in which platelets can contribute towards the coagulation process. First, the platelet surface protects active clotting factors from inactivation by their natural inhibitors (Walsh & Biggs, 1972). Second, platelets contribute through aggregation, during which platelet phospholipids (platelet factor 3) are made available (Joist et al. 1974) and stimulate activation of factor X and the conversion of prothrombin to thrombin. In the presence of ADP, the coagulation pathway may be activated through factor XII (Walsh, 1972a), and interaction of platelets with collagen can activate factor XI (Walsh, 1972b). Finally, during the release reaction, fibrinogen and platelet factor 4 are made available. The amount of fibrinogen released is small in relation to the

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Response to Different Agents Agents range from weak inducers to potent inducers of platelet activity. Perhaps the weakest of those we have mentioned is serotonin. This activates human platelets to undergo the change in shape and to aggregate (Mitchell & Sharp, 1964), but the release reaction rarely occurs. Even the aggregation is only transient; a minute or so after the platelets start to aggregate they begin to disaggregate (Baumgartner & Born, 1968). ADP can induce change in shape (Macmillan & Oliver, 1965; Born, 1970) and a transient reversible aggregation (Born, 1962; O'Brien, 1962) in the same way as serotonin, but unlike the effect of serotonin that of ADP is markedly concentration dependent. In addition, platelets from some individuals can undergo the release reaction. This can sometimes be seen as a second wave of aggregation (Macmillan, 1966; Mills et al. 1968), as the newly synthesized and released materials contribute to the aggregation process, but this wave occurs only when the extent of release is large (HeptinstaU & Mulley, 1977) or is artificially enhanced by, for instance, the presence of citrate in the suspension (Macfarlane et al. 1975; Mustard et al. 1975). The effects of adrenaline and noradrenaline on human platelets are similar to those of ADP (O'Brien, 1963; Mitchell & Sharp, 1964). They differ, however, in that they induce the second wave of aggregation more readily (Lages & Weiss, 1977) and apparently stimulate aggregation without the prior shape change (Bull & Zucker, 1965; O'Brien & Woodhouse, 1968; Seaman & Brooks, 1969). It would appear that the release reaction induced by ADP, adrenaline and noradrenaline is dependent on the platelets aggregating. When aggregation is inhibited by removing calcium ions from the suspension the release reaction does not occur. In contrast, agents like thrombin or collagen can stimulate the release reaction directly (Feinman & Detwiler, 1974; Kattlove & Gomez, 1975). Indeed, the aggregation that is induced by these agents is largely brought about by the synthesized and released materials (Izrael et al. 1974; Samuelsson et al. 1976). Thrombin and collagen are considered to be the most potent of the agents that have been mentioned. As far as we are aware they aggregate platelets from all humans and all species, although different platelet preparations may respond to different extents.

Shape Change and Aggregation

So far it has not been possible to associate any of the surface receptors with any activity that might indicate how platelets change their shape and develop an adhesive surface, but it is almost certain that a contractile process is involved (Pollard

124 Br. Med. Bull. 1978


Release Reaction Platelets have enzymic pathways for metabolizing phospholipid-bound arachidonic acid. They have phospholipase activity, which liberates the arachidonic acid from phos125 Vol. 34 No. 2

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phatidylcholine and phosphatidylinositol (Bills et al. 1976), and a cydo-oxygenase, which converts it to the prostaglandin endoperoxide intermediates PGG2 and PGH 2 (Hamberg & Samuelsson, 1974). These endoperoxides can either be converted to thromboxane B 2 via the unstable thromboxane A 2 (Hamberg et al. 1975; Needkman et al. 1976), to the prostaglandins Ej and F ^ , or to non-prostenoate structures. Much interest is centred on the intermediates of these pathways, PGG2, PGH 2 and particularly thromboxane A^ because these substances are also potent stimulators of platelet aggregation and release reaction (Hamberg et al. 1974; Willis et al. 1974; Hamberg et al. 1975). They or their metabolites are produced when platelets are aggregated by adrenaline or collagen (Smith et al. 1974; Hamberg et al. 1975), by thrombin (Malmsten et al. 1975; Ellis et al. 1976) or by arachidonic acid itself (Smith et al. 1974; Hamberg et al. 1975; Smith et al. 1976). PGG2 and PGH 2 may also be physiological substrates for prostacyclin production at the vessel wall which, in turn, may inhibit platelet deposition. This is discussed in detail by Moncada & Vane (1978). The contribution of these intermediates to platelet behaviour can be estimated by observing the effect of inhibiting the activity of the cyclo-oxygenase, so that the intermediates are not synthesized. The activity of this enzyme is inhibited by some non-steroidal anti-inflammatory agents. Aspirin is particularly useful in this respect, because it irreversibly acetylates the enzyme and destroys its activity (Roth & Majerus, 1975; Roth et al. 1975). The intermediates are not involved in aggregation itself; aspirin has no effect, for instance, on the reversible aggregation induced by ADP or by serotonin. However, neither adrenaline nor ADP can induce the release reaction or the second wave of aggregation when platelets have been treated with aspirin (O'Brien, 1968; Zucker & Peterson, 1968), so it would seem that the intermediates are required to amplify the effect of these weak inducers of platelet activity. They are less important in mediating the effects of the more potent agents, as platelets that have been exposed to aspirin will still respond to collagen or thrombin (Macmillan, 1968; O'Brien, 1968; Fukamief a/. 1976). A high intracellular level of cyclic AMP can affect the extent of release reaction as well as prevent shape change and aggregation. It inhibits the phospholipase that provides the arachidonic acid for conversion into the prostaglandin endoperoxides (Malmsten et al. 1976; Minkes et al. 1977). Unlike other secretory cells, platelets can undergo secretion in the absence of extracellular calcium ions. Nevertheless, movement of intracellular calcium between cell compartments is probably important because calcium ionophores, which facilitate the movement of calcium across lipid membranes, can induce the release reaction (Feinman & Detwfler, 1974; Massini & Luscher, 1974; White et al. 1974; Feinstein & Fraser, 1975; Mflrer et al. 1975; WSmer & Brossmer, 1975) and secretion can be blocked by calcium antagonists (Charo et al. 1976). It is possible that calcium from a source analogous to sarcoplasmic reticulum (Statland et al. 1969; Robblee et al. 1973) is moved into the cytoplasm, where it may then exert an effect on the microtubules. These normally lie beneath the circumferential edge of platelets, but during the release reaction they move towards the centre of the cell in a "contractile wave" (White, 1971). It is assumed that the contents of the storage granules are finally ejected after fusion of their membranes with a surface-connecting membrane system.

et al. 1977). About 16% of platelet protein is present as thrombosthenin (Bettex-Galland & Luscher, 1965), an actomyosin-like contractile protein, and mkrotubules are also present The relation of these structures to shape change is at present the subject of active investigation (Crawford & Taylor, 1977). Although extracellular calcium ions are not required for shape change, there is evidence that intracellular calcium is involved (Le Breton et al. 1976a; Le Breton et al. 1976b; Le Breton & Dinerstein, 1977). It may be significant that the adcnosinetriphosphatase (ATPase) activity of thrombosthenin is activated by calcium (Hanson et al. 1973; Thorens et al. 1973; Mahendran, 1974). Extracellular bivalent cations are needed for aggregation (Born & Cross, 1963; Hovig, 1964). Calcium ions are absolutely necessary, while magnesium ions only partially fulfil the bivalent cation requirement (Heptinstall, 1976). The presence of binding sites on platelets which are specific for, and have a high affinity for, calcium may be relevant (Heptinstall, 1977). Fibrinogen also needs to be present before platelets will aggregate (Harbury et al. 1972; Tollefsen & Majerus, 1975), but it is not clear how it is involved. It is unlikely that it is converted to fibrin since this would involve thrombin. There is controversial evidence that thrombin is involved when agents like ADP induce the release reaction (Han & Ardlie, 1974; Ardlic & Huzoor-Akbar, 1977; but see Macfarlane et al. 1975), but there is no evidence for its involvement in aggregation itself. Heparin, an inhibitor of thrombin production, appears to enhance aggregation rather than inhibit it (Thomson et al. 1973). At its simplest, aggregation is reversible; provided that the release reaction does not occur the platelets either disaggregate spontaneously or can be induced to disaggregate experimentally. Thus when ADP is removed from the system by enzymes that degrade h (Izrael et al. 1974) or when free ADP is converted into Ca-ADP or Mg-ADP complexes (Heptinstall, 1976), the platelets disaggregate. However, when platelets disaggregate spontaneously they do so even when ADP is still present (Packham et al. 1969). This suggests that platelets have a compensatory mechanism to neutralize the effects of the stimulatory agents. That platelets become refractory to a particular agent (Rozenberg & Holmsen, 1968) is another indication that this is the case. Reversibility of aggregation and refractoriness are both consistent with the involvement of a contractile process. Both shape change and aggregation are inhibited by agents that increase the intracellular level of cyclic AMP (Salzman, 1972; Haslam, 1975). Some investigators observe a reduction in the level of cyclic AMP during aggregation (Marquis et al. 1970; Salzman, 1972; Chiang et al. 1975), but this needs to be confirmed by the use of more sensitive assays when they become available. So far the mode of action of cyclic AMP is unclear, but it is possible that its influence is through the activation of protein kinases or lipid kinases. Platelet proteins are phosphorylated (Lyons et al. 1975; Haslam & Lynham, 1976; Chiang et al. 1977) and phospholipids rapidly turn over (Lloyd & Mustard, 1974; Kaulen & Gross, 1976) during platelet activity. These events may be associated with changes in calcium flux.

CONTRIBUTION OF PLATELETS TO THROMBUS FORMATION A M White & S Heptinstall 2 Experimental Systems for Analysing the Thrombotic Process

systems, appears to be mainly a physical one and is dramatic (Turitto & Baumgartner, 1975). Thus a 54-fold increase in initial rates of surface coverage by platelets was seen when suspensions of platelets were compared with whole blood in laminar flow systems. The rate of surface coverage is inversely proportional to the diameter of the vessel (Turitto & Baumgartner, 1974). b Factors Affecting Formation of the Platelet Monolayer Specific enzymes have been used to destroy certain components of the subendothelial structures and thus to reveal their relative capacities for platelet adhesion (Baumgartner, 1974b). Treatment of the subendothelium with collagenase, leaving elastin and non-collagen-containing microfibrillar elements, almost entirely destroyed the attractiveness of the surface for platelets. By contrast, an a-chymotrypsin-treated surface from which the endothelium had been removed retained its attractiveness for platelets. Platelet adhesion to the subendothelium parallels adhesion to collagen in that bivalent cations are required for both processes. Thus the inclusion of either EDTA (3mM final concentration) or a high concentration of sodium citrate (80 mM final concentration) reduces the adhesion of platelets to denuded endothelium in annular flow systems from 80 % to 20% surface coverage, thus showing that platelet adhesion and spreading is largely a Ca2+-dependent process (Baumgartner et al. 1976). Aspirin, which inhibits the platelet release reaction (Zucker & Peterson, 1968), slightly stimulates adhesion of platelets to subendothelium (Baumgartner et al. 1976). Thus, it appears that neither the production of prostaglandin intermediates nor the release of ADP is necessary for this process. Prostaglandin E t (1 (XM final concentration) partially inhibited platelet adhesion to subendothelium in flowing systems (Baumgartner et al. 1976), suggesting a possible role of cyclic AMP in the process. c Factors Affecting Mural Thrombus Formation Whereas platelet adhesion to exposed subendothelium is not inhibited by aspirin, this agent does inhibit the build-up of mural thrombi (Baumgartner et al. 1976), suggesting that prostaglandin intermediates and secreted materials are important. To what extent the individual products of platelet aggregation contribute to the formation of mural thrombi in these laminar flow systems remains unknown, but direct evidence of the importance of ADP as a mediator in these systems has been recently obtained (Tschopp & Baumgartner, 1976). In these experiments, rabbit aorta denuded of endothelium was exposed in the annular perfusion chamber to titrated flowing blood containing creatine phosphate and creatine kinase to convert ADP to ATP. The results were consistent with the notion that ADP secreted by platelets plays a major part in mural thrombus formation, since the thrombus component surmounting the pseudo-endothelium was significantly reduced.

a Physical Determinants The rate of coverage of a natural surface which has been denuded of endothelium by platelets and mural thrombi increases with increasing rate of blood flow (Baumgartner, 1973). Moreover, a slow rate of flow can be shown experimentally to encourage the deposition of fibrin (Baumgartner, 1977). Thus, shear rates of 500 s"1, 2000 s"1 and 4000 s"1 enabled 23%, 43% and 66 % of subendothelium, respectively, to be covered by platelets from native blood after 3 minutes, whereas 67 %, 26 % and 10 % of the surface, respectively, were covered by fibrin (Baumgartner, 1977). In that fibrin was produced at all at the highest shear rate, there must have been some thrombin production, with consequently direct action on platelets. The effect of red cells, at least in experimental

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Platelets do not adhere to damaged (Baumgartner, 1972) or undamaged (French et al. 1964; Baumgartner et al. 1967) endothelial cells either because of their capability for prostacydin production or because of apyrase (ADPase) activity in the membrane (Lieberman et al. 1977). Thus, for investigation of those factors that could determine the various stages of thrombus formation in injured vessels a reproducible method of removing endothelial cells from long stretches of vessel was required. The balloon catheter method was developed (Baumgartner, 1963) for this purpose. Essentially, a small noninflated rubber balloon at the end of a thin plastic tube (usually a Fogarty embolectomy catheter) is inserted into an artery ofan experimental animal and then inflated. Movement of the inflated balloon along the vessel then removes the endothelium, after which the balloon is deflated and withdrawn through the point of entry. For in-vitro experiments the animal is perfused with a suitable medium, and after removal of the endothelium in the manner described above the vessel is dissected out and mounted in the apparatus of choice. Much work has been done with the use of everted damaged aorta mounted in a system to give rapid laminar flow similar to that in vessels (Turitto, 1975), and although some of the problems described below have been investigated in vivo many have been investigated with the use of this system or variants of it. The most crucial part of the technique and that which lends great reliability to the results is the use of quantitative stereomorphometric analysis after the damaged vessel, either in vitro or in vivo, has been rapidlyfixedwith glutaraldehyde and processed for light and electron microscopy (Baumgartner, 1972; Baumgartner & Haudenschild, 1972). Two main components of mural thrombus formation have been quantitatively investigated by this technique: (i) the rate of deposition of the platelet monolayer and (ii) the rate of formation and removal of the platelet aggregate that surmounts the monolayer. Results show that the number of platelets in the monolayer and in the overlying aggregate reach a maximum after about 10 minutes, irrespective of whether in-vivo or in-vitro methods are employed (Baumgartner, 1973); but, whereas aggregates are removed over the enstring 30 minutes, the pseudo-endothelium of platelets remains stable and apparently unreactive to components offlowingblood. However, the platelets that become detached, originally as micro-emboli, appear more likely to reattach to subendothelium than do native platelets (Baumgartner et al. 1976).

3 Conclusions We still do not know how thrombi form in arteries and veins. We do know that thrombi contain masses of platelets in a fibrin mesh, that platelets are deposited and mural thrombi form on areas of exposed subendothelium, and that platelets display activities in vitro that suggest a causal 126 Br. MetL Bull 1978


ISeoTurpie &Hinh, pp. 183-190 of thl» Bulletin.—ED.


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of the release reaction, such as sulphinpyrazone, may also have considerable antithrombotic activity (Kaegi et al. 1975)1. likewise, one might expect that a correlation should exist between platelet hyperactivity and thrombo-embolic episodes. Although studies of groups of individuals have indicated that this may be the case, it is not yet possible to diagnose a thrombotic tendency in individuals after studies of in-vitro platelet activity (Sahud, 1976). If we had a biochemical measurement that correlated well with thrombo-embolic states, and which could be used to monitor therapy with drugs, we might be nearer to solving the problem of thrombosis.

relation. However, we do not know whether damaged endothelium is the main or the only initiator of thrombus formation or whether platelet activities in vitro have any relevance in vivo. The studies that have been discussed in this paper seem to indicate that the platelet release reaction is central to thrombosis, in that it provides amplification mechanisms for aggregation, provides procoagulant activity, and is involved when mural thrombi grow on areas of exposed subendothelium. Specific inhibitors of the release reaction, for example aspirin, should therefore be ideal antithrombotic agents. So far, however, the clinical evidence with aspirin is equivocal. Although some positive results have been obtained, for example in transient ischaemic attacks (Fields et al. 1977), agents that appear to be less potent as inhibitors

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128 Br. Med. Bull. 1978

Contribution of platelets to thrombus formation.

British Medical Bulletin (1978) VoL 34, No. 2, pp. 123-128 CONTRIBUTION OF PLATELETS TO THROMBUS FORMATION A M White & S Heptinstall CONTRIBUTION OF...
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