Study of the role of secreted platelet thiol isomerases in the regulation of platelet function, haemostasis & thrombosis

Platelets are tiny blood cells whose role is to trigger the blood to clot when blood vessels become ruptured during injury. While this is clearly essential to prevent excessive and life-threatening bleeding, abnormal stimulation of blood clotting can be catastrophic, particularly if this leads to the clotting of blood within an artery or vein. This is a condition know as thrombosis, and when this happens in arteries that supply the heart muscle with blood it can cause a heart attack. Thrombosis also commonly occurs in the blood vessels that supply the brain with blood, and in this case it causes strokes. Both of these conditions are very common in the UK and are frequently fatal. The cause of thrombosis in these situations is often underlying diseases such as the formation of fatty lesions in the wall of blood vessels that are liable to rupture, but platelets are ultimately responsible for triggering a dangerous clot to form. The use of drugs to dampen down the responses of platelets has been successful in many ‘at risk’ patients in the prevention of thrombosis, although many patients gain no benefit and may even suffer side effects such as bleeding. In order to develop better drugs that target platelets to prevent thrombosis, scientists need to know more about how platelets recognise injury, and then stimulate the blood to clot. In this research project we will investigate some proteins that we have found to be released by platelets when they encounter tissue injury. We know that once released some of these molecules attach themselves to the platelet surface and somehow enhance the platelet clotting activity. The purpose of this study is to work out which of the proteins that are released are important to control platelets and how they work. We will also determine whether these molecules contribute to thrombosis using new experimental procedures. It is anticipated that this project will equip us with a new understanding of how platelets regulate their functions and the identification of the molecules involved. This knowledge may lead, in future studies, to the development of more effective drugs to successfully prevent thrombosis.

Platelets perform a vital role in haemostasis, although inappropriate platelet activation, for example at the site of a ruptured atherosclerotic lesion, causes arterial thrombosis leading to heart attacks and strokes. While substantial progress has been made in recent years in the treatment and prevention of thrombosis, there remains a substantial unmet clinical demand. Understanding of the complex mechanisms through which the function of platelets is regulated is important for the development of more effective strategies to prevent arterial thrombosis. We have reported previously that during platelet activation, which leads to aggregation and thrombus formation, platelets release ERp5, a member of the thiol isomerase family of enzymes. This protein interacts with the platelet surface, whereupon its enzymic activity enhances platelet function. While the molecular mechanisms have yet to be defined, protein disulphide isomerase (PDI), the best characterised thiol isomerase, is also secreted from platelets and influences their reactivity. Extra-cellular PDI has also been recently implicated in the regulation of tissue factor, which triggers coagulation following exposure and de-encryption at sites of injury. In preliminary studies we have identified additional platelet thiol isomerases, some of which are also secreted during activation.
The aims of this study are to characterise systematically the range thiol iomerases that are released from platelets during activation; to establish the potential roles of individual secreted thiol isomerases in the regulation of platelet function in vitro; to determine the identity of platelet surface substrates for these enzymes which will begin to explain their individual or collective mechanisms of action; and to establish the abilities of each in the regulation of thrombus formation in vivo using mouse models of thrombosis. This study will involve the generation of a panel of novel antibody-based reagents that will enable the specific inhibition of secreted thiol isomerases, and will employ a wide range of cell biology and biochemical approaches to study the function of platelets in vitro and in vivo.
Data from this project will provide a detailed and systematic understanding of the individual and collective roles of secreted thiol iomerases at the platelet surface, and the substrates through which they exert their actions. Understanding of this new paradigm for the regulation of thrombus formation may lead to the identification of new targets for the pharmacological suppression of platelets, which may include selected thiol isomerases themselves, that may result through continued studies in the generation of more effective anti-thrombotic therapies.