Kinlough-Rathbone Rl

Synergism between platelet aggregating agents: the role of the arachidonate pathway.

Abstract Aggregation of platelets by low concentrations of ADP is augmented by non-aggregating concentrations of collagen, thrombin, arachidonate or the divalent cation ionophore A23,187. Release-inducing agents act synergistically with ADP and with each other. Both collagen and thrombin cause aggregation by releasing ADP and by freeing platelet arachidonate to form prostaglandin endoperoxides which give rise to thromboxane A2. In these experiments the role of the arachidonate pathway in the synergism between pairs of aggregating and release-inducing agents was examined. Indomethacin was used to inhibit conversion of arachidonate to prostaglandin endoperoxides and thromboxane A2 and creatine phosphate/creatine phosphokinase (CP/CPK) was used in some experiments to convert released ADP to ATP. Synergism of collagen with ADP, arachidonate or thrombin was inhibited by indomethacin indicating that the arachidonate pathway plays a major role in the synergistic effects to which collagen contributes. Synergism of thrombin with collagen or arachidonate was inhibited by indomethacin but synergism of thrombin with ADP was only slightly affected. Indomethacin had little influence on the combined effects of these two agents on platelet aggregation. Thus it appears that the conversion of platelet arachidonate to prostaglandin endoperoxides and thromboxane A2 plays a minor part in the synergistic effects in which thrombin or A23,187 are involved. Thus, the non-steroidal anti-inflammatory drugs may have only limited use in inhibiting the contribution of thrombin and ADP to the formation of platelet thrombi at sites of vessel injury.

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.

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.

Platelet adherence to the vessel wall and to collagen-coated surfaces.

A rotating probe system has been developed to quantitate the adherence of 51Cr-labeled platelets to collagen-coated glass rods or to the subendothelium of the rabbit aorta. The platelets were suspended in a medium containing physiological concentrations of calcium and magnesium, albumin, and apyrase to prevent aggregation. Chelation of divalent cations in the medium by addition of sodium citrate, EDTA, or EGTA markedly diminished platelet adherence. These observations indicate that the interpretation of platelet adherence studies performed in the presence of these agents is questionable. Increasing albumin concentrations in the medium decreased the number of adherent platelets, whereas increasing the hematocrit increased platelet adherence. Thus the factors influencing platelet adherence were studied in the presence of 4% albumin and 40% hematocrit.

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.

Effects of reserpine and serotonin on adenine nucleotide metabolism and glucose oxidation of washed rabbit platelets

Abstract Reserpine inhibits the transfer of ATP from the metabolically active pool into the releasable pool (amine storage organelles) of washed rabbit platelets. Since reserpine is known to interfere with oxidative phosphorylation of isolated mitochondria, possible effects of reserpine on the energy metabolism of intact platelets were examined with the aim of determining whether or not such effects could explain the reduced transfer of metabolic ATP into the releasable pool of ATP. Reserpine caused a small increase in [ 14 C]hypoxanthine and [ 14 C]inosine accumulation in a suspension of washed rabbit platelets prelabeled with [ 14 C]adenosine or [ 14 C]adenine. Reserpine also caused increased oxidation of [ 14 C]glucose. Serotonin mimicked these effects of reserpine, and imipramine inhibited the serotonin-induced as well as the reserpine-induced increases in [ 14 C]hypoxanthine and [ 14 C]inosine formation and glucose oxidation. However, imipramine did not prevent the reserpine-induced inhibition of [ 14 C]ATP transfer from the metabolically active pool into the releasable pool. It is concluded, that the effects of reserpine on energy metabolism are at least partially attributable to the increased demand for energy for the uptake of serotonin, liberated from the platelets during incubation with reserpine, and not to uncoupling of oxidative phosphorylation. Furthermore, the small decrease of the metabolically active ATP pool upon incubation of platelets with reserpine cannot explain the reduced transfer of ATP from the metabolic pool into the amine storage granule pool.

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.