Localised inhibition of thrombin at endothelial surfaces - a translational strategy to inhibit immune responses to localised antigen

Innate immune mechanisms mediate multiple human inflammatory diseases and shape the way that adaptive immune responses (i.e. T and B cell-dependent responses) develop. Although therapeutics against innate effectors are becoming available, for instance agents targeting complement or specific cytokines, there is still a dearth of therapies that target inflammation per se, and corticosteroids, with multiple troublesome side effects, remain the mainstay of therapy for most inflammatory diseases.
Movement of leukocytes along chemical gradients into underlying tissues underpins all inflammation. The chemicals involved are called 'chemokines'. The supervisors of this project have shown that an enzyme involved in blood clotting, called thrombin, is absolutely required to generate these chemokine gradients to initiate recruitment of cells at the beginning of inflammation. In this case, thrombin is acting through receptors expressed on the blood vessels at the site where inflammation is about to develop, rather than causing a blood clot to form.
There are lots of drugs currently available to inhibit clotting, some of which inhibit the actions of thrombin. These drugs, called anticoagulants, are very effective at inhibiting inflammation in small and large animals. However, when tested in humans, they have been shown to cause significant problems related to bleeding, sometimes into the brain (causing strokes). Therefore, they are not routinely used to inhibit inflammation. The supervisors have invented a completely novel type of drug called Thrombalexin, that tethers to the cells lining a blood vessel and inhibits thrombin, but only at that site. It is therefore a good way to prevent clotting at specific sites, such as inside a transplanted organ. Based on promising preliminary data, Thrombalexin is undergoing rigorous testing and development that is required before it can be used as a drug. In the first clinical trial, which is planned for late 2017/early 2018, it will be perfused into a transplanted organ before being transplanted into recipients who are at high risk of clotting in the immediate post-transplant period.
However, based on what we know about the role of thrombin in generating chemokines, Thrombalexin might have significant impact in inflammatory situations where clotting is not a problem, and by localising to specific sites, it might turn out to be a drug that can be used safely to inhibit localised inflammation. This is what my project will investigate.
Because a transplanted organ undergoing rejection is a very good example of localised inflammation, I will predominantly (though not exclusively) use transplant models in my work, testing the ability of Thrombalexin to inhibit both aggressive and smouldering rejection, and showing that one of the main mechanisms by which it works is through inhibition of chemokine gradients and inhibition of cell recruitment into the transplant. Because my project will overlap with the phase 1 clinical trial of Thrombalexin in transplant patients, I will have an ideal opportunity to study whether Thrombalexin also inhibits chemokine secretion in humans, and ultimately to link this to changes in organ performance, cell recruitment and rejection-free survival.

Monocytes are important in local inflammation and transplantation, where the number of infiltrating monocytes during rejection closely correlates with the severity of rejection, and the degree of functional impairment that results, indicating that strategies to limit monocyte infiltration might have significant impact on survival. The chemokines involved in monocyte recruitment are well-known. Although chemokine receptor antagonists have shown promise in models of inflammation, clinical development has been limited by toxicity. Underpinning this project is the discovery, by the supervisors, that hearts from transgenic mice expressing an endothelial cell (EC)-tethered direct thrombin inhibitor (hirudin) were completely resistant to antibody-mediated rejection, and were not infiltrated by monocytes (or NK cells), whereas in hearts in which thrombosis was inhibited but thrombin signalling (through protease activated receptor-1) was left intact, there was a very heavy monocyte infiltrate. From this they have invented Thrombalexin to use as a localized anticoagulant: my project will be to explore its capacity to inhibit localized inflammation via inhibition of chemokine expression. I will use well characterised models of mouse acute and chronic rejection to study the impact of Thrombalexin, localised in the transplant or injected intravenously, on chemokine gradient generation, monocyte recruitment and transplant survival. I will also investigate whether signalling through other members of the protease activated receptor family, can augment the protection offered by Thrombalexin; here I will also use a well characterised model of localised skin inflammation. Finally, I will study samples from the phase 1 trial of Thrombaelxin, to establish the link, in humans between thrombin signalling and chemokine gradient generation. This project will address fundamental aspects of the biology of inflammation and inform clinical development of a novel therapeutic.

Transplantation is the "Gold standard" treatment for end stage renal disease; enhancing quality of life while being more cost effective than dialysis. One of the most challenging aspects of transplantation management is improving graft longevity. Over the last few decades, advances in surgical techniques and pre transplantation cross-match have improved short-term graft survival. Yet, disappointingly, there has been little improvement in long term graft failure rates which remain at 4% graft loss per year, causing thousands of patients across the world to return to dialysis each year. Immune mediated damage remains the single largest cause of graft loss. The overarching aim of this research is to maximise the lifespan of solid organ transplants by seeking ways to address the problem of allograft rejection and shedding new light on the molecular mechanisms that underpin it in the hope to develop new avenues for future therapeutic manipulation.

Donor organs are in high demand and short supply, Currently 1000 patients per year die waiting for solid organ transplantation. Maximising allograft lifespan reduces the need for re-transplantation and reduces the numbers returning to dialysis, which in itself provides a significant impact on morbidity. Economically, improving graft longevity improves cost-effectiveness by reducing the need for dialysis and re-transplantation and allows patients to return to work and remain economically active while also immeasurably improving their quality of life. Reducing re-transplantation in turn increases the potential donor pool for patients on the waiting list. This work will benefit not only the UK but also all other societies offering organ transplantation.

Key to graft longevity is the development of tolerance where by patients maintain stable graft function in the absence of immunosuppression. While immunosuppressive drugs have allowed us to offer this treatment to a wide audience, they are not without their side effects including infection, diabetes and malignancy, all of which place a significant burden on the NHS. The ability to induce tolerance will also service a wider society by eradicating the significant cost of funding these medications and the treatment of their complications, freeing resources for alternative areas of expenditure.

Although focused on kidney transplant recipients, this work has the potential to be translated to recipient of all other solid organs, and will have broad applicability to other diseases involving an immune response to localised antigen, such as arthritis, cancer, and atherosclerosis. Indeed, thrombin has already been implicated in autoimmunity and atherosclerosis. Gaining a deeper understanding of the role of thrombin in leucocyte migration will significantly contribute to the current knowledge of the role of coagulation in innate immune responses and has repercussions across other disciplines in innate immunity such as the complement system, autoimmunity, infection and malignancy.