Periodic Reporting for period 2 - TAPAS (TArgeting Platelet Adhesion receptors in thrombosiS)
Período documentado: 2020-01-01 hasta 2022-06-30
What is the problem/issue being addressed?
Targeting Platelet Adhesion Receptors in Thrombosis (TAPAS) is a Marie Sklodowska-Curie Innovative Training Network, European Joint Doctorate. The primary goal of TAPAS was to train a uniquely qualified cohort of 15 Early-Stage Researchers (ESRs) in a highly intersectoral and multi-disciplinary programme of work tackling the problem of thrombosis (blood clots) by targeting the specialised blood cell, the platelet. Platelets are involved in the prevention of bleeding; however, their inappropriate activation can lead to thrombosis and its associated cardiovascular diseases. A thrombus can be generated either in arteries or veins. Venous thrombosis commonly happens in the legs and pelvis, and thrombi can embolise to the lungs (pulmonary embolism). Arterial thrombosis causes common diseases such as stroke and heart attack. TAPAS focused on the study of platelets in both arterial and venous thrombosis.
Why is it important for society?
Cardiovascular diseases are the leading cause of morbidity and mortality worldwide. In 2010, thromboembolic conditions were estimated to account for 1 in 4 deaths worldwide and 1 in 3 deaths in the European Union (EU), with a cost of over €200 billion a year to the EU economy. Current anti-platelet treatments are effective in less than half of patients and are limited by bleeding side effects which can be life-threatening. With an ageing population in the EU, who are at a greater risk of thrombosis, new powerful therapeutics are needed that target thrombosis and preserve haemostasis (i.e. prevention of bleeding). TAPAS, aimed to tackle this by combining innovative approaches to identify novel molecular targets and test new reagents for prevention and treatment of thrombotic diseases.
What were the overall objectives?
1. To study the molecular basis of clustering of the major platelet glycoprotein signalling receptors, focusing on CLEC-2, GPIb and GPVI. These receptors are promising targets for development of novel antiplatelet drugs that will preserve haemostasis.
2. To further define the role of glycoprotein receptors in haemostasis, thrombosis and blood vessel integrity.
3. To identify new epitopes in glycoprotein receptors for therapeutic intervention and to identify novel inhibitory compounds or biologics.
4. To train the next generation of researchers that can apply the knowledge gained in TAPAS to the development of clinically effective medicines for thrombosis and other complex diseases.
Targeting Platelet Adhesion Receptors in Thrombosis (TAPAS) is a Marie Sklodowska-Curie Innovative Training Network, European Joint Doctorate. The primary goal of TAPAS was to train a uniquely qualified cohort of 15 Early-Stage Researchers (ESRs) in a highly intersectoral and multi-disciplinary programme of work tackling the problem of thrombosis (blood clots) by targeting the specialised blood cell, the platelet. Platelets are involved in the prevention of bleeding; however, their inappropriate activation can lead to thrombosis and its associated cardiovascular diseases. A thrombus can be generated either in arteries or veins. Venous thrombosis commonly happens in the legs and pelvis, and thrombi can embolise to the lungs (pulmonary embolism). Arterial thrombosis causes common diseases such as stroke and heart attack. TAPAS focused on the study of platelets in both arterial and venous thrombosis.
Why is it important for society?
Cardiovascular diseases are the leading cause of morbidity and mortality worldwide. In 2010, thromboembolic conditions were estimated to account for 1 in 4 deaths worldwide and 1 in 3 deaths in the European Union (EU), with a cost of over €200 billion a year to the EU economy. Current anti-platelet treatments are effective in less than half of patients and are limited by bleeding side effects which can be life-threatening. With an ageing population in the EU, who are at a greater risk of thrombosis, new powerful therapeutics are needed that target thrombosis and preserve haemostasis (i.e. prevention of bleeding). TAPAS, aimed to tackle this by combining innovative approaches to identify novel molecular targets and test new reagents for prevention and treatment of thrombotic diseases.
What were the overall objectives?
1. To study the molecular basis of clustering of the major platelet glycoprotein signalling receptors, focusing on CLEC-2, GPIb and GPVI. These receptors are promising targets for development of novel antiplatelet drugs that will preserve haemostasis.
2. To further define the role of glycoprotein receptors in haemostasis, thrombosis and blood vessel integrity.
3. To identify new epitopes in glycoprotein receptors for therapeutic intervention and to identify novel inhibitory compounds or biologics.
4. To train the next generation of researchers that can apply the knowledge gained in TAPAS to the development of clinically effective medicines for thrombosis and other complex diseases.
RRO1: Development and use of innovative methods to elucidate receptor clustering and signalling
We have taken advantage of recent advances in fluorescent imaging techniques to study the mechanism that led to clustering of platelet glycoprotein receptors and generation of intracellular signal. These include the application of advanced microscopy and computational methods that allow visualisation of receptors below the defraction limit of light, including expansion microscopy, stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM), DNA-paint and fluorescence correlation spectroscopy (FCS), and FRET-flow cytometry. We have also labelled novel protein-based probes with fluorescent ligands based on nanobody technology (nanbodies are small antibodies made in camelids which makes suitable for imaging) and applied these to measurement of thrombus formation.
RO2: Modelling the molecular events of receptor clustering and screening for new targets
We have used ordinary differential equations (ODE) and agent-based modelling (ABM) to model the clustering of platelet glycoprotein receptors and tested the models using advanced microscopy methods and through the development of divalent and tetravalent ligands based on crosslinking of novel nanobodies. We have screened for small molecule inhibitors of platelet glycoprotein receptors using large chemical libraries based on alpha-screen technology and virtual screening. Several lead compounds have been identified including both agonists and antagonists. Several blocking nanobodies have been identified.
RO3: Validation of approaches and verification of lead compounds
The knowledge gleamed from the increased understanding of the mechanisms of receptor activation and lead compounds have been applied to in vitro microfluidic models, including vessel-on-a-chip and in in vivo models of thrombosis using humanised (h) transgenic mice (hGPVI and hCLEC-2). These studies have shown efficacy of inhibitors and provided critical information on pharmacokinetics of lead compounds. These findings have further developed the understanding of the mode of activation of platelet glycoprotein receptors and identified novel ligands with important implications for therapeutic exploitation.
We have taken advantage of recent advances in fluorescent imaging techniques to study the mechanism that led to clustering of platelet glycoprotein receptors and generation of intracellular signal. These include the application of advanced microscopy and computational methods that allow visualisation of receptors below the defraction limit of light, including expansion microscopy, stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM), DNA-paint and fluorescence correlation spectroscopy (FCS), and FRET-flow cytometry. We have also labelled novel protein-based probes with fluorescent ligands based on nanobody technology (nanbodies are small antibodies made in camelids which makes suitable for imaging) and applied these to measurement of thrombus formation.
RO2: Modelling the molecular events of receptor clustering and screening for new targets
We have used ordinary differential equations (ODE) and agent-based modelling (ABM) to model the clustering of platelet glycoprotein receptors and tested the models using advanced microscopy methods and through the development of divalent and tetravalent ligands based on crosslinking of novel nanobodies. We have screened for small molecule inhibitors of platelet glycoprotein receptors using large chemical libraries based on alpha-screen technology and virtual screening. Several lead compounds have been identified including both agonists and antagonists. Several blocking nanobodies have been identified.
RO3: Validation of approaches and verification of lead compounds
The knowledge gleamed from the increased understanding of the mechanisms of receptor activation and lead compounds have been applied to in vitro microfluidic models, including vessel-on-a-chip and in in vivo models of thrombosis using humanised (h) transgenic mice (hGPVI and hCLEC-2). These studies have shown efficacy of inhibitors and provided critical information on pharmacokinetics of lead compounds. These findings have further developed the understanding of the mode of activation of platelet glycoprotein receptors and identified novel ligands with important implications for therapeutic exploitation.
The research performed over the last four years has given rise to a sea-change in our understanding of the organisation of glycoprotein receptors and their mechanism of activation and has identified candidate lead agents for therapeutic exploitation. The work has given rise to fundamental changes in our understanding of the organisation of glycoprotein receptors in the membrane, including the observation many exist as a mixture of monomers and dimers and do not adopt a unique conformation on dimerisation. The work has also uncovered several mechanisms of receptor dimerisation and shown how these work in synergy to drive clustering and intracellular signalling. We have shown how advanced methodology including microscopy, proteomics and lipidomics and flow-based models can progress our understanding of platelet glycoprotein receptors, as well as generating novel agents (small molecule inhibitors, biologics, fluorescent probes, mouse models) to further study their function.
TAPAS has also developed a cohort of 15 highly skilled and specialist researchers that will lead to further academic-industry interactions and collaborations. Several of the several of the ESRs have already secured prestigious postdoctoral positions in leading laboratories and in industry, while the others are at advanced stage of completion of their doctoral thesis.
A key benefit of the TAPAS programme is the synergy in expertise between academic laboratories and the private sector in terms of understanding and commercial exploitation of processes surrounding thrombosis and haemostasis, molecular target validation and assay development. By working together in this network, the ESRs have gained a unique insight into ‘team’ science, including handling of large data sets, in vivo analyses, patient studies and commercialisation.
TAPAS has fostered accelerated advancements in the development of antiplatelet agents targeted at platelet glycoprotein receptors for prevention of thrombosis with reduced morbidity relative to current antiplatelet drugs and so will be of significance beyond the lifetime of TAPAS. Nevertheless, the societal benefits are many years away as the primary focus has been on generating knowledge that provides a base for translation. TAPAS has helped to create interest in Biotech/Pharma in regard to targeting platelet glycoprotein receptors in thrombosis. Further, this has taken place in parallel with early phase clinical trials on a GPVI-blocking Fab, glenzocimab in ischaemic stroke from the Biotech Acticor. The precursor of glenzocimab, 9012, has been shown to powerful inhibit platelet activation.
TAPAS has also developed a cohort of 15 highly skilled and specialist researchers that will lead to further academic-industry interactions and collaborations. Several of the several of the ESRs have already secured prestigious postdoctoral positions in leading laboratories and in industry, while the others are at advanced stage of completion of their doctoral thesis.
A key benefit of the TAPAS programme is the synergy in expertise between academic laboratories and the private sector in terms of understanding and commercial exploitation of processes surrounding thrombosis and haemostasis, molecular target validation and assay development. By working together in this network, the ESRs have gained a unique insight into ‘team’ science, including handling of large data sets, in vivo analyses, patient studies and commercialisation.
TAPAS has fostered accelerated advancements in the development of antiplatelet agents targeted at platelet glycoprotein receptors for prevention of thrombosis with reduced morbidity relative to current antiplatelet drugs and so will be of significance beyond the lifetime of TAPAS. Nevertheless, the societal benefits are many years away as the primary focus has been on generating knowledge that provides a base for translation. TAPAS has helped to create interest in Biotech/Pharma in regard to targeting platelet glycoprotein receptors in thrombosis. Further, this has taken place in parallel with early phase clinical trials on a GPVI-blocking Fab, glenzocimab in ischaemic stroke from the Biotech Acticor. The precursor of glenzocimab, 9012, has been shown to powerful inhibit platelet activation.