1.3.1 SUMMARY
Intermittent claudication is a common and due to impaired blood supply to the lower limb, usually caused by atherosclerosis. The outlook for the affected limb is relatively good, but intermittent claudication is associated with an increased risk o f myocardial infarction and stroke. The fact that atherosclerosis is a generalised condition affecting the coronary and cerebral circulations is usually quoted as the explanation for the increased morbidity and mortality in claudicants. However, evidence from multivariate analysis suggests an effect independent of co-existing diseases, cardiovascular risk factors or the extent of coronary or cerebral atherosclerosis. Claudication itself may be an independent risk factor for these cardiovascular events.
Activation o f neutrophils, platelets and the coagulation system are intimately involved in the pathophysiology of thrombosis and infarction. Platelets and the coagulation system cause thrombosis and neutrophils are intimately involved in the response to and outcome of infarction. Intermittent claudication is a mild, repetitive form of ischaemia-reperfusion. In this situation oxygenation of previously ischaemic tissue results in the generation o f oxygen free radicals, with subsequent cellular and humoral activation, and increased tissue damage. There is evidence that neutrophil activation is fundamental to this phenomenon, and that platelet activation and thrombosis may contribute to the injury.
There is ample evidence supporting a role for neutrophils, platelets and the coagulation system in both atherogenesis and the progression and complications of atherosclerotic disease. Most of this comes from experimental and clinical work on the coronary circulation and myocardial infarction, with some work suggesting similar mechanisms at other sites. Differences in neutrophils, platelets and the coagulation system have also been reported in peripheral vascular disease, and claudication has been shown to activate neutrophils, generate arachidonic acid metabolites, activate coagulation factors and increase microvascular permeability.
1.3.2 HYPOTHESIS
Activation, or increased potential to be activated, i.e., priming, of neutrophils, platelets or the coagulation system could result in an amplified response to pathological events. In these circumstances the response to plaque rupture could be more pronounced. A fissured plaque which would otherwise have formed a thrombotic cap and gradually remodelled, may evolve into an occlusive thrombus in the presence of a “primed” neutrophils, platelets and/or coagulation system. Enhanced coagulation may stabilise a developing thrombus, preventing early lysis. Similarly, a “primed” system may impair recovery of the peri-infarct tissue due to micro thrombosis and/or increased numbers o f activated neutrophils. Such priming could turn a subclinical event into a symptomatic stroke or myocardial infarction.
W e hypothesised that if claudication were to activate and/or “prime” neutrophils, platelets and/or the coagulation system this may explain some of the increased cardiovascular risk found in claudicants.
L3.3 AIM OF THESIS
To investigate whether intermittent claudication causes activation and/or priming of blood cells, and activation of the coagulation system. In detail the work will:
1. To determine whether exercise precipitates activation and/or priming of neutrophils in claudicants, and compare this with healthy, matched controls.
We planned to measure neutrophil counts, and other leucocyte populations, before and after exercise, as this may influence other measurements. Markers of neutrophil activation will be measured, mmnly to compare baseline differences and to consider local activation. To measure neutrophil activation in response to stimulation we aim to use an established microplate technique and a more sensitive flow cytometric technique. We also want to study the duration of any activation, and will measure changes over two hours after exercise.
2. To determine platelet activation and priming in systemic blood from claudicants and controls, at rest, after a single episode of exercise and after repeated
exercise.
These studies are limited by constraints of techniques used to assess activation. Differences between the two groups, and changes following exercise, are likely to be small and subtle, particularly in stable claudicants, and sampling artefact and the basic nature of some of the measures may mask such changes. We aim to use a flow cytometric technique, as this involves minimal preparation, and is more likely to detect subtle differences. Experiments will be run in parallel with more established techniques to measure platelet activation and assess activation in response to stimuli. Flow cytometry, as a method of studying platelet reactivity, is new to our laboratory. Hence, before outlining the experimental design we will discuss flow cytometry and the necessary set up and standardisation procedures.
3. To determine whether repeated exercise causes activation and/or priming of the coagulation system in systemic blood from claudicants, and compare this with
controls.
A measure of the response of the coagulation system to stimulation would be ideal, but currently available techniques are very crude and would be unlikely to detect any small differences we might expect in claudication. Consequently, we plan to measure activation using new, highly sensitive assays of activation as an indirect measure.
4. Does aspirin influence the activation and/or priming of platelets and/or the coagulation system in claudicants at rest, after a single episode of exercise and after
repeated exercise.
Many patients with atherosclerotic disease are taking aspirin, which may have a profound effect on platelet activation, and may affect the coagulation response. We aim to look at platelet and coagulation responses in patients taking and not taking aspirin.