Jf Mustard

Platelets, Thrombosis and Drugs

SummaryThe development of thrombosis involves 4 main factors: the vessel wall, the formed elements of the blood, blood coagulation, and blood flow. In venous thrombosis the dominant mechanism is blood coagulation. In arterial thrombosis, however, the major part in both the initiation and growth of thrombi is played by the platelets.In selecting drugs which inhibit platelet function it is helpful to know which of the platelet reactions that contribute to thrombus formation can be inhibited by various agents. Platelets adhere to the damaged vessel wall, collagen being probably the most important constituent involved. They are then stimulated to release the contents of their storage granules. Release-inducing agents promote the discharge of adenosine diphosphate (ADP) which causes platelets in the vicinity to swell to a more spherical shape, extend pseudopods and adhere to each other. Platelet aggregation is reversible, and a number of drugs have been shown to be capable of inhibiting platelet function at various stages, both in vitro and in vivo. Adrenaline, noradrenaline, oestrogens and nicotine enhance aggregation. Drugs which inhibit platelet function include the non-steroidal anti-inflammatory drugs, the pyrimido-pyrimidines (e.g. dipyridamole), hydroxychloroquine, clofibrate, and dextran.In this review the effects of drugs which inhibit platelet function are outlined and the extent to which they can be used to influence the course of thromboembolic disease in man is discussed. It is suggested that combinations of anti-platelet drugs with anticoagulants could prove clinically useful.

Platelets, Thrombosis and Atherosclerosis

The interaction of platelets with the vessel wall can contribute to the early stages in the development of atherosclerosis through effects on smooth muscle cell proliferation, endothelial permeability, and possibly by causing vessel wall injury. Platelets are involved in the development of thrombi in response to vessel injury, and the repeated formation of platelet emboli and platelet-fibrin emboli from the mural thrombi may be one of the factors that cause clinical complications of atherosclerosis. Drugs which inhibit platelet function, particularly those that prolong shortened platelet survival (sulfinpyrazone and dipyridamole) may prove to be important in inhibiting the response of blood to vessel injury and thereby modifying the extent of atherosclerosis and its complications.

The effect of aspirin inhibition of PGI2 production on platelet adherence to normal and damaged rabbit aortae

Abstract These experiments were designed to investigate whether or not PGI2 production by vessel walls is responsible for their non-thrombogenic property. Adherence of rabbit platelets to the endothelium and sub-endothelium of the rabbit aorta was studied in vitro and in vivo when PGI2 production by the vessel wall was inhibited by aspirin. For in vitro studies, everted segments of rabbit aorta were mounted on a probe and rotated in a suspension of washed 51Cr-labeled platelets; the number of adherent platelets was calculated from the radioactivity associated with the platelets adherent to the segments. (This method measures the adherence of individual platelets, not platelet thrombi.) When adherence to the subendothelium was to be measured, the endothelium was removed by passage of a balloon catheter. The platelets and/or the vessel wall were pretreated with aspirin (1 mM and 2 mM respectively), washed and resuspended in fresh medium before adherence was measured. For in vivo studies, 51Cr-labeled platelets were injected into rabbits and the aortae were perfused and fixed in situ 20 min after administration of 25 or 100 mg of aspirin per kg. Platelet accumulation on both undamaged and de-endothelialized aortae was examined. PGI2-like activity produced by the vessel walls was measured by a bioassay; it was demonstrated that the aspirin treatment abolished the ability of the vessels to form PGI2, even when sodium arachidonate was supplied as a precursor. Both in vitro and in vivo prevention of PGI2 production did not affect the number of platelets that adhered to the endothelium or the subendothelium under these experimental conditions. Thus it is unlikely that the production of PGI2 by the vessel wall prevents platelets from adhering to the endothelium or subendothelium, although PGI2 may limit subsequent thrombus formation at an injury site.

Effects of the cell adhesion peptide, Arg-Gly-Asp-Ser, on responses of washed platelets from humans, rabbits, and rats

Fibrinogen is a cofactor in the aggregation of human platelets, and is required for ADP-induced aggregation of washed platelets; however, exogenous fibrinogen is not required for ADP-induced aggregation of washed platelets from rabbits or rats. Because with human platelets the cell adhesion peptide, Arg-Gly-Asp-Ser (RGDS), inhibits aggregation and the binding of 125I-fibrinogen to ADP-stimulated platelets, its effects on rabbit and rat platelets were studied to investigate the differences in the fibrinogen requirements of platelets from the three species. RGDS (50 mumol/L) caused greater than 80% inhibition of thrombin-induced or (ADP + fibrinogen)-induced aggregation of human platelets, but only 3% to 9% inhibition of the aggregation of rabbit or rat platelets, regardless of whether fibrinogen was added. RGDS inhibited the binding of 125I-fibrinogen to ADP-stimulated human platelets by 80% to 90%, but by only 15% to 27% in the case of rabbit or rat platelets. The differences were due to the species of platelets, since human and rabbit fibrinogens gave similar results. In addition, RGDS failed to displace fibrinogen from the surface of rabbit platelets that had been stimulated with ADP. Thus, there are species differences in the ability of the cell adhesion peptide, RGDS, to block the platelet fibrinogen receptor, even within the mammalian species.

Platelets and Thrombosis in the Development of Atherosclerosis and Its Complications

Platelets, blood flow, and endothelial injury are among the factors that determine the sites at which atherosclerosis develops. Smooth muscle cells in the vessel wall proliferate in response to endothelial injury;and in addition, endothelial injury plays a role in the development of mural and occlusive thrombi in major arteries and thereby contributes to the development of clinical complications of atherosclerosis. Factors that cause endothelial injury therefore appear to be involved both in the development of atherosclerosis and in its thromboembolic complications. Platelets appear to have a key role in both processes. In animal experiments, platelets that adhere to the subendothelium release a factor which stimulates smooth muscle cell proliferation. In addition, platelets that interact with the damaged vessel wall release factors that result in further accumulation of platelets at an injury site, and also accelerate the coagulation process. Recently it has been shown that repeated endothelial injury produces a surface on the vessel wall that is more thrombogenic than that produced by a single injury, and also results in the accumulation of proteoglycans in the vessel wall. Proteoglycans may trap lipoproteins and thus promote their accumulation at sites of endothelial injury. Injury itself leads to increased permeability of the vessel wall to plasma proteins.

Effect of ticlopidine on platelet aggregation, adherence to damaged vessels, thrombus formation and platelet survival

Ticlopidine (100 mg/kg/day or 400 mg/kg/day) was administered to rats and rabbits for 48 hr before and during the experiments. Aggregation studies of twice-washed platelets resuspended in Tyrode solution containing apyrase and 0.35% albumin showed that inhibition by ticlopidine of aggregation induced by ADP, collagen, sodium arachidonate or thrombin persisted after resuspension, as did inhibition of the release of 14C-serotonin from prelabeled platelets. Thus the inhibitory effect of ticlopidine or its metabolite is not readily reversed. In both species, ticlopidine prolonged platelet survival when it had been shortened by the insertion of an indwelling aortic catheter, although only the higher dose was effective in rabbits. In this species, this dose also prolonged platelet survival in sham-operated animals. Ticlopidine did not have a significant effect on the clearance of rabbit platelets when their survival had been shortened by pretreatment with neuraminidase. Ticlopidine did not affect the number of 51Cr-labeled platelets that accumulated on the injured vessel wall in rats with indwelling aortic catheters or the amount of thrombus that formed around the catheters in the aortas of the rabbits. It also did not affect the accumulation of platelets in vivo on rabbit aortas de-endothelialized with a balloon catheter. Thus, although ticlopidine inhibited platelet aggregation and release and prolonged shortened platelet survival, it did not inhibit platelet adherence to the damaged wall or thrombosis caused by chronic arterial injury. It is evident that effects on platelet survival and thrombosis do not correlate. The reason for the prolongation of platelet survival is unknown.

Investigation of possible mechanisms of pyridoxal 5′-phosphate inhibition of platelet reactions

Abstract Pyridoxal 5′-phosphate (PLP) inhibits ADP-induced platelet shape change and aggregation. PLP also causes rapid deaggregation of platelets aggregated by ADP. Effects on platelet aggregation and coagulation are demonstrable following administration to man. Several suggestions concerning the possible mechanism of inhibition have been investigated. PLP did not inhibit the nucleoside diphosphokinase activity of intact rabbit platelets nor of isolated platelet membranes. PLP had no measurable effect on rabbit platelet cyclic AMP either alone or in the presence of prostaglandin E 1 and caffeine. ADP-induced aggregation and 125 I-fibrinogen binding of rabbit platelets were inhibited concomitantly; however, fibrinogen-induced aggregation of chymotrypsin-treated platelets was only slightly inhibited by PLP. 125 I-fibrinogen binding to chymotrypsintreated platelets was partially inhibited by PLP; when PLP was added to these platelets, bound fibrinogen was displaced. With chymotrypsin-treated platelets, PLP inhibited the ADP component of the synergistic aggregating effect of the combination of fibrinogen and ADP. It seems unlikely that the inhibitory effects of PLP are due to interference with nucleoside diphosphokinase, increasing platelet cyclic AMP or inhibition of fibrinogen binding. The mechanism of inhibition remains to be established.

Drug effects on platelet adherence to collagen and damaged vessel walls.

The interaction of platelets with damaged vessel walls leads to the formation of platelet-fibrin thrombi and may also contribute to the development of atherosclerotic lesions because platelets adherent to exposed collagen release a mitogen that stimulates smooth muscle cell proliferation. The first step in thrombus formation, platelet adherence to an injured vessel wall, can be studied quantitatively by the use of platelets labeled with 51chromium. In these investigations, rabbit aortas were damaged by passage of a balloon catheter and segments of the aortas were everted on probes that were rotated in platelet suspensions. Collagen-coated glass cylinders were also used. Adherence was measured in a medium containing approximately physiologic concentrations of calcium, magnesium, protein and red blood cells. Conditions of testing influence the effect of non-steroidal anti-inflammatory drugs, sulfinpyrazone, and dipyridamole on platelet adherence. Aspirin and sulfinpyrazone were not inhibitory when tested in a medium with a 40% hematocrit; this indicates that products formed by platelets from arachidonate probably do not play a major part in the adherence of the first layer of platelets to the surface, although they may be involved in thrombus formation. Indomethacin, dipyridamole, prostaglandin E1, methylprednisolone and penicillin G and related antibiotics did inhibit platelet adherence although the concentrations required were higher than would likely be achieved in vivo upon administration to human patients. None of the non-steroidal anti-inflammatory drugs inhibited the release of granule contents from adherent platelets. Pretreatment of the damaged vessel wall with aspirin increased platelet adherence, presumably because it prevented the formation of PGI2 by the vessel wall. Platelet adherence to undamaged or damaged vessel walls was enhanced by prior exposure of the wall to thrombin. Platelet reactions with aggregating agents and platelet survival can be modified by changes in dietary lipids but there is very little evidence concerning the effects of lipids on platelet adherence. If some forms of dietary fat damage the endothelium, platelet interaction with the damaged area and release of the mitogen for smooth muscle cells would contribute to the development of atherosclerotic lesions.

Platelet Aggregation: Relevance to Thrombotic Tendencies

Platelets change shape, aggregate, and release their granule contents in response to a number of agents that they may encounter in vivo. Some understanding has been gained concerning the mechanisms involved in these platelet reactions; released ADP, products formed from platelet arachidonic acid when platelet phospholipase A2 is activated, and at least one other mechanism contribute to aggregation responses. Many investigators have reported that platelets are more responsive to aggregating agents in vitro or aggregate spontaneously in some conditions associated with cardiovascular disease. Tests that are thought to detect circulating platelet aggregates have also been developed. However, the increased sensitivity demonstrated by all these tests may be a result of vascular disease rather than its cause.

Effect of Drugs on Platelets and Complications of Vascular Disease

Platelets play a fundamental role in the formation of arterial thrombi. Thrombosis is involved in the development of vascular disease and its complications in at least four ways: 1) Platelets can take part in causing vessel injury. They can increase vessel permeability and they can influence the vessel wall, particularly the smooth muscle cells. 2) Some mural thrombi can become organized and incorporated into the vessel wall. 3) The mural thrombi which form in diseased atherosclerotic vessels may cause clinical complications because of their embolization into the circulation distal to the site of their formation. 4) Thrombi which occlude major arteries in vital organs such as the heart and the brain, cause ischemia, necrosis of tissue, and sometimes death.