Exploring bias in platelet P2Y1 receptor signalling: host defence versus haemostasis.

Blood contains cells to kill germs. In order to defend us against germs, they need to leave the bloodstream and travel through the inflamed tissue to reach the germs, engulf them and secrete materials that help kill them. The body does this via a protective mechanism called the inflammatory response. Sometimes this inflammatory response occurs inappropriately in diseases unrelated to infection, which then unfortunately causes self-inflicted harm to the body, for example in asthma, chronic obstructive pulmonary disease (COPD, mostly caused by smoking), or inflammation caused by sudden trauma. Previously, our research group have discovered that white blood cells called leukocytes require the help of yet another blood cell type, the platelets, to be able to migrate from the blood stream to sites of inflammation and infection. Platelets are best known for forming blood clots, thereby preventing us from bleeding to death every time we have an injury (this process is known as haemostasis), but because they also guide the white blood cells to reach inflamed organs as well as helping the white blood cells to fight and kill germs, they are also indispensable for our immune system. Yet, the processes of haemostasis and inflammation are distinct. The main aim of our research project here is to learn how platelets get activated in response to the protective process of inflammation, without affecting the essential process of haemostasis, because this might lead to excessive bleeding, or blood clots.
Research into how platelets become activated during inflammation is important for basic science, but also because current medical treatments are just not good enough. We require better medicines - because 5.4 million people in the United Kingdom suffer from asthma, and 1.2 million suffer from COPD. Despite better management of patients, and the introduction of new drugs, 3 people die every day in the UK as a result of an asthma attack, 80 people die every day as a result of COPD, and people who have inflammation of the lungs associated with acute trauma or pneumonia have between a 20 and 50% death rate. If we can find new ways of modulate platelet activity at the site of inflammation, without affecting normal blood clotting, we might be able to create new medicines that improve the health of millions of people.
Platelets express proteins called purinergic receptors that are important for switching platelets 'on' during blood clotting, but when certain types of purinergic receptors get switched 'on' in an inflammatory environment, they boost activity to help fight germs instead of forming blood clots. We will research how a particular purinergic receptor (P2Y1) works to control platelet activity during inflammation, compared to their function in blood clotting. To do this, we have assembled a team of scientists from within King's College London, with different areas of expertise (computer modellers, synthetic chemists, pharmacologists) to create new molecules that are designed to change P2Y1 function. Receptors change shape continuously, and depending on what shape they are, when activated, platelets will function differently based on the shape or conformation of the receptor. The new molecules will be designed to bind to P2Y1 when it is a particular shape. We hope to change selective functions of platelets necessary for inflammation, to fight infection, or for clotting of blood. Together, we will create a library of these molecules to be used as research tools (called 'biased' agonists-activation antagonists-blocking) after computer-aided modelling. We will measure the functions of platelets in the presence of these agonists and antagonists, and their effects on white blood cell activation, both in the test tube and in actual inflammation and infection. This research project will help us and other scientists understand exactly how P2Y1 on platelets works during inflammation compared to blood clotting.

The activation of platelets during inflammation affects their adhesive properties, motility, the release of soluble anti-pathogen factors and their communication with other immune cells to coordinate the innate immune response to infection, or inappropriately in inflammatory disorders, for example asthma and COPD. These functions are distinct to platelet function during haemostasis. Similar activities in leukocytes are controlled by signalling molecules of the Rho family GTPases (RhoA, Rac1), which can be activated by purinergic (P2Y) receptors. Platelets express P2Y1, the role of which in platelet aggregation and haemostasis is well established, yet we have shown that the activation of P2Y1, signalling via RhoA and Rac1 in platelets is required for platelet-dependent neutrophil recruitment, and other platelet functions pertinent to inflammation - however this signalling pathway is ultimately redundant in platelet activation during aggregation. It therefore appears that functional selectivity can occur with platelet P2Y1, dependent on the local tissue environment, to dictate platelet responses (function). The incidence of biased agonism for P2Y1 is novel, but has been described elsewhere for related GPCRS. To understand how platelet P2Y1 might act in a selective manner, we will synthesize novel agonists and antagonists as experimental tools for the orthosteric binding site at P2Y1, and evaluate their potential to influence biased signalling (whether they confer a positive, negative or neutral bias to inhibiting platelet function in the context of inflammation, host defence, and haemostasis) using particular in vitro and in vivo assays to predict platelet activity in the context of respiratory inflammation and infection compared to haemostasis (aggregation, and thromboemboli). This will determine the suitability of targeting platelet P2Y1 as a novel therapeutic area to modulate the inflammatory response.

Who might benefit from this research?
The benefits of our research are of prime impact in the field of platelet pharmacology. However, the impact stretches well beyond this into general understanding of platelet physiology and its modulation via purinergic receptors. This has the potential to stimulate the development of other tool compounds, to address fundamental questions in inflammation and immunity research. Identification of academic communities has been discussed in the 'academic beneficiaries' section.
Pharmaceutical and biotechnology companies are very active in researching pathways that modulate leukocyte recruitment and activation, to get a mechanistic insight and for the development of either anti-inflammatory drugs or conversely drugs to boost the immune system to fight infections. Similar therapeutic strategies are being pursued for combating cancer metastasis.
The concept that platelets play important roles in processes pertinent to inflammation, infection, autoimmune diseases and cancer has gathered a very marked momentum (using the research terms ['platelet' and 'inflammation'] on Pubmed, 16,000 papers have been published since 1970, with 1300 since the start of 2018), to the extent that clinical trials are currently evaluating the use of established anti-platelet drugs for orphan diseases. These clinical groups require a strong pre-clinical insight into the likely importance of the various receptors and platelet signalling pathways of relevance to inflammatory responses, rather than aggregation and haemostasis.

How will they benefit from this research?
Pharmaceutical companies will enhance their knowledge and research capacity as we publish novel pathways that uncover the existence of selective platelet signalling pathways to functions around the inflammatory response offering a strong avenue of inquiry. An element of risk is taken away from interested companies, since we would be conducting the necessary early discovery research to characterize the suitability of targeting P2Y1 and platelets to develop antagonists as novel anti-inflammatory drugs or conversely as potential targets for novel agents (agonists) to boost the immune response to fight infection. Indeed, 3-4 companies have synthetic chemistry programmes to modulate P2Y1, but these are currently placed as novel anti-platelet therapies for cardiovascular disease. They might deserve evaluation for their anti-inflammatory properties. Companies could benefit from having access to our library of novel P2Y1 agonists or antagonists, or our expertise that we will gain at evaluating biased signalling of P2Y1 (as a concept), or the production of experimental tools (as a process) to create early lead compounds to influence design iteration.
The exploitation of biased signalling pathways in disease (especially with respect to purinergic receptors) could create important novel drug target opportunities, for the rational design of compounds with more diverse properties than traditional pharmacological concepts predict. Our research will provide practical evidence as to how these strategies should move forward with regards to purinergic receptor pharmacology. We also have the environment, skills, and plans to exploit our novel pharmacology.
There are at least 2 phase II clinical trials investigating P2Y12 antagonists - for example, prasugrel, and clopidogrel as anti-inflammatory/ respiratory drugs at this present time (Clinicaltrials.gov identifiers NCT01305369, and NCT01955512, respectively). Our preliminary data and publications predict that trialling established anti-platelet drugs designed for the treatment of cardiovascular disease is a misinterpretation, or over-simplification of how platelet involvement in host defence or inflammatory diseases should be exploited for patient wellbeing. Thus, our research publications will inform clinicians and national health authorities as to the likelihood of success of their choice of trial strategies.