Animal research project summary

Key Words:

Platelets, thrombosis, heart attack, stroke, clotting

Purpose:

• Basic research
• Translational and applied research
• Maintenance of colonies of genetically altered animals

Objectives:

Through our research we aim to understand how the blood cells know as platelets that cause the blood to clot are controlled. This is important because they need to be able to sense when injury has occurred in order to prevent excessive bleeding. The function of platelets is also be triggered by diseases of the heart and blood system, which cause thrombosis, or blood clots in the circulation. Thrombosis is the trigger for heart attacks and strokes that are major causes of death and illness.

By understanding the molecules and processes that platelets use when they encounter healthy, injured or diseased blood vessels will enable us to develop new medicines that interfere with these processes, make platelets less active (in a controlled way) and therefore reduce the risk of heart attack or stroke. So our questions include what are the proteins on the surface of platelets that function as probes to detect injury or disease, what do they detect, and how to they then control the proteins present within platelets that turn blood clotting on or off.

Benefits:

The goal of this research is to determine how we could design new drugs to prevent thrombosis. Current drugs that target platelets and suppress their activity to prevent thrombosis are effective in many patients, but a large number of patients gain no benefit and go on to have a heart attack or stroke. Worryingly, most current ‘anti-platelet’ medicines also cause lots of side effects. Of particular importance are bleeding side effects, which can be life threatening and these occur frequently.

Understanding the precise molecules in platelets that control each aspect of their function will enable us to identify mechanisms that may be more effective and safer (i.e. fewer side effects) targets for new anti-thrombotic medicines. Platelets are also involved in other diseases such as cancer, and any drugs that control their function are likely to be beneficial in the treatment of these additional conditions.

Animals used:

We will breed mice in this project and use these to ask specific scientific question. We expect to breed up to 2000 mice per year, or 10000 over 5 years. Most mice that will be used in these studies will be genetically modified, which means that we need to breed these ourselves.

Adverse effects:

In most cases the breeding of genetically modified mice will result in mice that may have mildly defective blood clotting, but are essentially healthy.

On occasion it is possible that a genetically modified mouse may suffer from unhealthy bleeding or thrombosis, although in our experience this is very rare. Should this occur and exceed acceptable limits of severity (mild or moderate), mice will be killed humanely as soon as possible. The experiments that we do with the genetically modified mice to test bleeding and thrombosis will be performed under general anaesthesia without recovery, so the mice do not feel anything and do not wake up after the experiment.

These protocols therefore have a non recovery severity limited. Some mice will receive drugs that affect platelet function or platelet number prior to experiments, or on rare occasions will be allowed to recover following analysis of bleeding. In these cases the health of mice will be studied very closely, and should they fail to thrive or appear distressed or unwell, they will be killed humanely as soon as possible.

Replacement:

Platelets do a specialised job that occurs specifically in blood flowing in blood vessels. For this reason if we need to study their ability to trigger blood clotting this needs to be performed in a living animal. All of our early fundamental research is performed using human platelets in the laboratory including the study of platelet clot formation in flowing blood, but in order to study the implications of specific processes in these cells on thrombosis and bleeding, the use of animals, and particularly mice, is essential because these processes are only triggered in conditions that are present in the body.

In addition, platelets lack a nucleus so they are unable to divide in culture. This also prevents us from using molecular biology to alter genes in human platelets in the laboratory. Transgenic mice are therefore used for this purpose, where the mouse itself produces genetically modified platelets for our experiments.

The mouse is considered a good model for studying thrombosis in a way that is similar to thrombosis in humans. Well-established standardised tests allow our data to be compared with a large scientific literature.

Reduction:

We will use statistical power calculations to ensure that we use the minimum number of mice in order to answer each of our scientific questions. This will be based on our experience of the tests that we perform and how variable the readouts from these are, the size of change that is physiologically meaningful, and statistically how reliable the data are (i.e. to rule out things that might happen by chance).

Mouse usage will also be minimised in the following ways:

• Use of efficient statistical designs to increase precision, e.g. use of litter mates as controls
• Use of efficient breeding protocols that minimise the production of unwanted animals (numbers or genotype)
• Utilisation of surplus mice that do not have the correct genetic makeup (for experimental use) for breeding. This avoids the need for additional mice for this purpose
• Use of pilot studies where new types of mice are used
• Use of modern efficient laboratory tests that allow fewer animals to be used

When necessary, or appropriate, a statistician will be consulted to ensure an experimental design is optimal and minimises the number of animals required

Refinement:

Mice are established models for human haemostasis and thrombosis. While there are subtle differences between mouse and human platelets, in most cases mice model normal and diseased processes in humans well. Mice also allow us to overcome the limitations that are caused by an inability perform molecular biology on cultured platelets, due to the resource of available genetically modified mouse strains, to produce platelets that are genetically altered (removing or adding genes). Combined together the advantages of using mice rather than other species have allowed mice to become the preferred pre-clinical model for studies of platelet function.

In recent years we have been involved in studies that have resulted in the development of more refined measurements of thrombosis in mice that allow more precise and sensitive analysis in which variability is minimised. The ability to analyse multiple parameters simultaneously in a single mouse, in a single thrombus, represents an important development since this improves the depth and quality of information than is obtained while also reducing the numbers of animals used.

The breeding of new genetically modified mice may result in unexpected levels of suffering. All mice will therefore be very carefully studied for signs of ill health and killed humanely if necessary. Based on our experience, these occurrences are rare due to having first performed relevant experiments using human platelets. Measures are therefore taken to avoid making transgenic mice in which substantial health issues are likely to be encountered.