Discrete spatiotemporal control of cAMP-regulated platelet functions by A-kinase anchoring proteins

Lead Research Organisation: University of Hull
Department Name: Hull York Medical School

Abstract

Platelets are small cells that play a key role in blood clotting that prevents the loss of excessive blood from the circulation when we cut ourselves. Upon activation, platelets become sticky and adhere to the walls of the blood vessels to form a clot. People who suffer from heart disease have platelets that are stickier than normal although these reasons for this are unclear. The inappropriate activation of blood platelets inside our arteries plays a key role in both heart attacks and strokes, and is a principal cause of mortality and morbidity in the UK. To protect ourselves from the increased stickiness of platelets, endothelial cells that line blood vessels release a chemical messenger called prostacyclin (PGI2), which inhibits platelet activity, making them less 'sticky' and reducing thrombosis. Subjects with heart disease, diabetes and obesity have platelets that do not respond very well to PGI2, making them more vulnerable to thrombosis. In this project we aim to investigate the molecular mechanisms that regulate platelet sensitivity to PGI2, which are currently undefined. We will use blood from human volunteers to isolate platelets and study the proteins that are activated by PGI2. By understanding how PGI2 controls platelets in normal individuals we can begin to compare this with people with disease to understand how the pathway breaks down. The study will increase our understanding of how PGI2 protects against heart disease and may lead to the development of new antithrombotic strategies.

Technical Summary

The inappropriate activation of blood platelets is associated with arterial thrombosis that underlines clinical events such as myocardial infarction. Elevation in cAMP leading to activation of protein kinase A (PKA) represents a major inhibitory pathway for blood platelet function and arterial thrombosis. Subjects with cardiovascular disease have reduced sensitivity to inhibition by cAMP and PKA, which is thought to contribute to their elevated risk of arterial thrombosis. However, the molecular basis of their resistance to the inhibitory effects of cAMP/PKA is unknown. Platelets possess two isoforms of PKA (PKA-I and II), although the specific functions of these isoforms in regulating platelet activity are unknown. In many cells PKA signalling is isoform specific and directed by A-kinase anchoring proteins (AKAPs). In preliminary experiments, inhibition of PKA-I/AKAP interactions reduced platelet inhibition by prostacyclin and PKA-mediated signalling events, suggesting that a type I AKAP may regulate PKA signalling in platelets. In the present application we will (a) examine the role of PKA isoforms in platelet signalling and function, (b) determine how this is affected by coupling with AKAPs, and (c) determine which AKAPs are present in platelets. We will use pharmacological inhibitors of PKA in platelets and gene silencing of PKA isoforms in megakaryocytes to dissect the potential non-redundant roles of PKA isozymes in these cells. This will be complemented by the use of highly selective peptides that uncouple AKAP-PKA interactions in an isoform specific manner, to determine the role of AKAPs in PKA platelet signalling and function. Furthermore, chemical proteomics methodology will be used to identify platelet AKAPs. These studies will produce highly novel data on the PKA isoform specific events and their regulation by AKAPs, and provide a platform for future studies examining how specific PKA isozyme-AKAP coupling modulate distinct aspects of platelet function.

Planned Impact

The knowledge gained from the proposed studies will provide novel information of the regulation of blood platelet activity. Since these cells participate in normal haemostasis, preventing blood loss upon injury, and in pathophysiological thrombosis, knowledge of their function provides important information on human biology. We believe our work will be of particular interest to clinicians, biomedical and clinical scientists and to the pharmaceutical industry to aid in the development of new therapeutic regimes to combat thrombotic disease. The inhibition platelet function with drugs such as aspirin and ADP receptor antagonists is supported by extensive experimental data. However, clinically, current approaches lack efficacy in many patients, and are associated with common and serious side effects, which include bleeding. The increasing rates of incidence of arterial thrombosis and underlying obesity-related metabolic disorders, will only add to this burden and therefore more refined, efficacious and safer approaches are required. cAMP activated intracellular signalling pathways are the most potent endogenous mechanisms for platelet inhibition. In contrast to clinically used platelet inhibitors (e.g. Aspirin), which only block certain functions of platelets while leaving others untouched, cAMP causes broad inhibition of all aspects of platelet function and is therefore critical to 'global' modulation of platelet activity. Therefore a detailed understanding of cAMP regulatory mechanisms in platelets is essential to (i) aid clinical understanding of why the cAMP pathway is compromised in disease states and therefore how it contributes to the pathogenesis of arterial thrombosis, (ii) understand the molecular mechanisms that lead to a comprehensive inhibition of platelet activation providing a broader frame work for clinical and biomedical scientists to design new strategies to combat excessive platelet function, and (iii) to provide new therapeutic targets for drug development by the pharmaceutical industry.
 
Description The inappropriate activation of blood platelets inside blood vessels leads to thrombosis and heart attacks. The synthesis and release of prostacyclin (PGI2) from endothelial cells that line blood vessels inhibits platelet activity and reduces thrombosis. The inhibition of platelet function is achieved through the activation of a complex protein communication network inside the cells called the cAMP signaling system. The experiments we are performing are designed to understand how this protein network is organised. This may allow us to determine why the communication network stops working correctly in disease states leading to heart attacks and strokes.
During our research we have developed a new methodology that allows us to examine how cAMP regulates platelets in whole blood, without having to isolate the cells. This more accurately reflects blood function in vivo and means that we can better understand how cAMP regulates platelet function inside the body.
In addition, we have found two new proteins that help control cAMP signaling networks in blood platelets. One of these studies is now published where we identified the first functional AKAP in platelets, while the other is currently under review
Exploitation Route The protein signalling/communication network we are studying (cAMP) is ubiquitously expressed in all human and animal cells. Consequently it has been implicated a range of biological processes including immune function, cell metabolism, blood vessel development. While our work is still on going we are hoping that the methodology we developed can help others evaluate how cAMP signaling networks influence cell function. The method offers a high throughput platform for other researchers to evaluate both cell signaling and reagents that could potentially affect these signaling networks.
The immediate beneficiaries of work will be investigators in the field of haemostasis, thrombosis and vascular biology. The identification of specific protein signalling complexes that drive platelet activation is important in order to increase the understanding of platelet function in health and improve therapeutic strategies for controlling the activity in disease. At a broader level the data may help others understand the role of the cAMP signalling network in cellular metabolism. This may be critical in the development of new drugs and treatments to control metabolic disorders such as diabetes and obesity.
Sectors Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

URL http://onlinelibrary.wiley.com/doi/10.1111/jth.13042/full
 
Description This grant is now complete. We have presented our findings at a number of conferences, both national and international, which we believe has created the platform for future impact. The methodology developed during the work has now been published and provided to collaborators for evaluation in different disciplines, mainly for clinical/patients samples. The other major focus of the work, examining new mechanisms by which cAMP signalling regulates blood platelets, is still ongoing. However, the development of new international collaborations in Germany, Norway and the USA will increase the potential impact.
First Year Of Impact 2013
Sector Education,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Title Fluorescent Barcoding Phosphoflow cytometry 
Description We have published a new method that allows biochemical signalling events in blood platelets to be evaluated in whole human blood without the need isolated and manipulate the cells. While this method has been established by other groups, we have made the critical step of applying the technology to primary human cell samples, developing the methodology to be compatible with whole human blood and devised a storage method that allows samples to be evaluated at a later date or transported to another institution. 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact None yet - collaborative work still ongoing. 
 
Description AKAP & studies 
Organisation Helmholtz Association of German Research Centres
Department The Max Delbrück Center for Molecular Medicine (MDC)
Country Germany 
Sector Academic/University 
PI Contribution WE performed experiments with reagents made available by our collaborator in MDC; these reagents were not commercially available.
Collaborator Contribution Our collaborator provided specialist reagents developed at the MDC that were used in our project.
Impact Work has been submitted for publication
Start Year 2013
 
Description Examination of A-kinase anchoring protein 9 (AKAP9) in blood platelets 
Organisation University of Houston
Country United States 
Sector Academic/University 
PI Contribution Our group has provided new biological samples in order to test the role of AKAP9 in blood platelets.
Collaborator Contribution Dr Desseur has provided specific antibodies generated by her laboratory for our group to test. These antibodies are not commercially available.
Impact Still ongoing.
Start Year 2013
 
Description Examination of A-kinase anchoring protein in blood platelets 
Organisation Helmholtz Association of German Research Centres
Department The Max Delbrück Center for Molecular Medicine (MDC)
Country Germany 
Sector Academic/University 
PI Contribution We have generated normal experimental (biological) samples for analysis.
Collaborator Contribution Our partner has generated specific antibodies for the AKAP-7 protein, which are not commercially available. These have now been supplied to us by Dr Klussmann for testing on our blood samples.
Impact Still on going
Start Year 2014
 
Description Gordon Research Conference - Cell Biology of Platelets and Megakaryocytes 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Prestigious international conference where the work was discussed at length leading to new concepts on the nature of the work.
Year(s) Of Engagement Activity 2015
 
Description UK Platelet Society Meeting (University of Leicester) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact The presentation led to broad discussion around new concepts of biochemical signalling events in blood platelets
Year(s) Of Engagement Activity 2015