Date of Award
Doctor of Philosophy (PhD)
The usefulness of drugs which inhibit platelet function and may be effective antithrombotic agents can only be demonstrated in clinical studies. Unfortunately, since the stimuli which cause thrombosis in patients are likely to vary extensively and since the mode of action of these drugs on such stimuli, although not completely understood, are likely to be different, the results from clinical studies cannot be generalized either for drug or thrombotic condition. Furthermore, the initial investigations with promising drugs cannot be made clinically for economic and ethical reasons. Therefore, there exists the need for some screening test(s) with which these drugs can be evaluated in vitro. In addition, there exists a need for an experimental in vivo test in order to study in greater detail the drug effects, their modes of action and possible synergism. Three drugs have been identified as possible anti-thrombotic drugs, these are aspirin, sulfinpyrazone and dipyridamole. Both dipyridamole and sulfinpyrazone appear to correct shortened platelet survival seen in patients with prosthetic heart valves whereas aspirin does not. However, aspirin in combination with a low dose of dipyridamole, which itself has no effect, does appear useful in correcting reduced platelet survival seen in similar patients. Sulfinpyrazone also has been shown to reduce the incidence of thrombosis of A/V shunts in patients undergoing renal dialysis. Nonetheless, when these drugs are tested ex vivo, different results are observed. Dipyridamole and sulfinpyrazone appear to have little or not effect on platelet adherence, aggregation and release whereas aspirin has been shown to inhibit platelet function for the lifespan of the aspirin-exposed platelet. In spite of these in vivo-ex vivo inconsistencies, all three drugs have been shown to effectively inhibit thrombosis in various animal models.
In addition, little data is available on the relation between drug effect, dosage and plasma level. Aspirin, which appears to have a permanent effect on the platelet, has circulating life span of less than 60 minutes. No studies have previously examined the effects of dipyridamole and sulfinpyrazone in vivo and their relation to their circulating plasma concentrations determined ex vivo.
The general objective of this study was to investigate the effects of these three drugs in vivo in an attempt to answer some questions concerning the dose-response and mechanisms of action of the drugs. Specifically, this study undertook: 1) to determine some possible explanations for the discrepancies observed between the results of platelet survival studies and ex vivo tests of platelet function observed with aspirin, dipyridamole and sulfinapyrazone, and 2)having established the existence of some possible artifacts of ex vivo testing, to use an in vivo test to study i) the dose-response relationship of the drugs and ii) some aspects of the mechanism of action of aspirin and sulfinpyrazone.
The results of this study have 1) provided a better understanding of some of the problems which exist when assessing potential useful antithrombotic agents, 2) introduced a useful in vivo model with which to study the effect of these drugs in vivo, and 3) provided new data concerning the mechanism of action of aspirin and sulfinpyrazone. The following conclusions can be drawn from the results:
1) Screening of drugs in vitro and ex vivo are limited and probably not appropriate in their present form. this is because citrate masks the effect of dipyridamole. Possibly a substitute anticoagulant such as heparin may avoid this problem. Alternatively, such investigations may require the use of washed platelet suspensions in which no anticoagulants are present. But even this type of preparation has its limitations. In addition, by limiting the investigations of the effects of these drugs on platelet function ex vivo and in vitro, any effects produced by drug characteristics such as distribution, metabolism and elimination would be overlooked.
2) The use of an in vivo model such as the one described in this study appears to be a useful test for examining the in vivo effects of these and other drugs. Furthermore, various stimuli can be used in place of collagen (i.e., arachidonic acid, ADP, thrombin) in order to evaluate the effects on or interaction between the drug in question and a variety of stimuli.
3) the relationship between drug-plasma concentration and drug-effect varies with each drug studied. The effect of dipyridamole is directly related to the concentration of the drug present in the platelet poor plasma. In order to maintain a dipyridamole effect, it may be necessary to increase the frequency of dipyridamole ingestion if the same relationship exists in humans. With aspirin, the effect is influenced by the initial plasma concentration, suggesting that this drug could be taken less frequently. The relationship between sulfinpyrazone-plasma concentration and sulfinpyrazone-effect is complex. The results from this study suggest that further studies must be made before any conclusions can be made about the optimal dose regime.
4) The "synergistic effect" of aspirin/dipyridamole observed clinically may be due, in part, to the alterations of the pharmacokinetics of dipyridamole bu the salicylate moiety of aspirin present in the plasma.
5) the prolonged in vivo effect of aspirin is not due to acetylation of megakaryocyes not exchange of the acetyl group from the plasma proteins onto new platelets entering the circulation. The aspirin effect slowly disappears with time and is consistent with the hypothesis that the appearance of new platelets into the circulation can overcome the aspirin effect.
6) the inhibitory effect of sulfinpyrazone is bi-phasic. Part of the effect is due to sulfinpyrazone per se and part of the effect is due to a metabolite not previously described. If a similar metabolite exists in humans, the current thoughts about sulfinpyrazone therapy may need revision.
Buchanan, Michael R., "Assessment of antithrombotic drugs in vivo and ex vivo" (1977). Open Access Dissertations and Theses. Paper 3353.