The identification and functions of regulatory proteins recruited to the cell surface during platelet activation

Lead Research Organisation: University of Reading
Department Name: Sch of Biological Sciences

Abstract

Platelets perform a central role in blood clotting although they are also responsible for triggering thrombosis, the formation of blood clots in the circulation, which leads to heart attacks and strokes. A detailed understanding of the normal processes that regulate the function of platelets is essential in developing new strategies and medication for the prevention of thrombosis. Platelets respond to a range of factors exposed, generated or released at sites of injury. In particular platelets adhere to proteins exposed in the damaged blood vessel wall. This leads to platelet activation: spreading to cover the injury site, and the secretion of a range of chemicals that act together to cause platelets to stick together to form a thrombus to plug the site of damage. The activation of platelet function is associated with the recruitment to the cell surface of a several of proteins involved in thrombus formation. The dynamic nature of proteins on the platelet surface is an integral component of the mechanisms that control platelet function. Given the potential importance of this in blood clotting and thrombosis, in this study we propose to characterise in detail the complement of proteins that are recruited to the platelet surface during activation, and also proteins whose levels are altered following stimulation. The validity of results will be determined using a range of cell biology techniques. Where suitable reagents are available, we will begin to assess the ability of these proteins to interact with other platelet surface proteins such as receptors, and determine whether they are involved in the regulation of platelet function. This project will lead to more focused future studies to determine the roles of specific proteins on the platelet surface. Through increased understanding of the complex molecular mechanisms that regulate the function of platelets this study will contribute to the development of new strategies to prevent thrombosis and may identify new processes or molecules that form the basis for the development of more effective anti-thrombotic drugs.

Technical Summary

Platelets perform a pivotal role in the regulation of haemostasis, a physiologic response to injury that prevents excessive bleeding. They also play a central role in thrombosis, the inappropriate formation of blood clots in the circulation, that lead to heart attacks and strokes. Understanding of the complex interactions of platelets with factors exposed, secreted or generated at sites of tissue injury, and the cell signalling mechanisms that stimulate and inhibit the functions of these cells, is essential in the development of new strategies and drugs for the prevention and treatment of thrombosis. Platelets adhere to sub-endothelial collagens that are exposed upon tissue injury and become activated. This leads to spreading, the secretion of a range of pro-thrombotic factors, and platelet aggregation to form a thrombus. Activation is associated with the recruitment to the cell surface of a range of proteins that are involved in the regulation of platelet function. These in include cell adhesion receptors, receptors for soluble agonists and enzymes. The recruitment of functionally important proteins to the cell surface represents an additional regulatory mechanism for the control of platelet function and thrombus formation. A comprehensive study of surface-recruited proteins has not, however, been performed. In this study we propose to characterise in detail the complement of proteins that are recruited to the platelet surface during activation with collagen and thrombin, and also to examine changes in protein surface exposure following stimulation of the inhibitory platelet receptor PECAM-1. A range of complementary proteomics technologies will be used in order to gain a comprehensive profile. The recruitment of proteins identified using mass-spectrometry will be confirmed using flow cytometry and immunoblotting techniques. Where suitable reagents such as antibodies or selective inhibitors are available, the potential involvement of selected proteins in the regulation of platelet adhesion, secretion and aggregation will begin to be examined. This, together with literature and database surveys will be used to determine which proteins are likely to be functionally important for future detailed examination.
This study will be the first stage of a larger programme that will focus in on specific proteins to determine in detail the functional significance of recruitment to the cell surface. This project will contribute to increased knowledge of the control of platelet function in health and disease, and may identify new processes or molecules that may be targeted in the development of anti-thrombotic drugs.

Publications

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Barrett NE (2008) Future innovations in anti-platelet therapies. in British journal of pharmacology

 
Description Houston, USA 
Organisation Baylor College of Medicine
Country United States 
Sector Hospitals 
PI Contribution We identified the protein of interest in our proteomics study. The postdoc performing this work and I visited our collaborators in Houston and together performed the necessary experiments.
Collaborator Contribution Our proteomics work through this study identified a new platelet protein that we believed may be important for platelet function (HIP55). Our collaborators in Houston provided access to a knockout mouse for this molecule and relevant laboratory facilities during two visits. This mouse was generated in the Tan laboratory and is exceptionally difficult to breed. This enabled us to confirm that importance of this protein in the regulation of integrin siganalling in platelets. This work will continue to establing the relavance of this in the modulation of platelet function in thrombotic disease. This work has also been in important as it demonstrates the power of targeted functional porteomics study - leading to greater understanding of cell regulation.
Impact A key publication: Proteomics, 9, 4340-4354. (2009)
Start Year 2007
 
Description Public lecture 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact The talk results in extensive discussion afterwards, and follow up inquiries. A number of the audience offered to be involved in our work.

Extensive contact from members of the public, radio and printed media coverage
Year(s) Of Engagement Activity 2009
 
Description Research lab open day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Hosted talks and visits to research laboratories to demonstrate how our research is done and how it impacts on increasing the health of the nation

Continued contact with several visitors and further visit planned next year
Year(s) Of Engagement Activity Pre-2006,2006,2010
 
Description School presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Lecture on various aspects and approaches to cardiovascular research within my lab

Students were encouraged to contact me should they have any questions. This has resulted in email-based assistance with school projects.
Year(s) Of Engagement Activity 2009