LICAMM Modulation of hypofibrinolysis in diabetes.

Hypofibrinolysis is associated with increased risk of thrombotic disease. Diabetes is characterized by hypofibrinolysis and elevated levels of plasminogen-activator inhibitor (PAI)-1 is one of the mechanisms implicated. We hypothesise that inhibition of PAI-1 activity using a novel methodology improves hypofibrinolysis in high risk conditions, including diabetes, and consequently decreases the risk of thrombosis.

<p>We developed a phage display library of a scaffold protein that constrains two variable conformational 9AA peptides (>3x1010 random peptides), mimicking the configuration of antibodies, termed Affimers. These Affimers have a low molecular weight and are able to interact with proteins. We will use the library of high diversity random library to screen for PAI-1 binders employing a phage display system. The project will have three main objectives:</p> <p>Isolate high affinity PAI-1 binding Affimers and characterise their role in modulating fibrinolysis.</p> <p>Study binding affinities/specificity of Affimers of interests and pinpoint interaction sites.</p> <p>Conduct preliminary in vivo studies to establish the role of Affimers in bleeding and thrombosis.</p> <h3>Objective 1.</h3> <p>Isolate high affinity PAI-1 binding Affimers and characterise their role in modulating fibrinolysis.</p> <p>Screening for PAI-1 binding Affimers. Screening the library of Affimers against immobilised PAI-1 will be undertaken and high affinity binders enriched. A total of 250 clones will be picked and sequenced. Affimers with distinct sequences will be subcloned for large scale protein production.</p> <p>Functional analysis of Affimers. This will be undertaken using turbidimetric and thromboelastometric assays employing plasma and whole blood systems. Plasma systems will investigate the role of Affimers in modulating fibrinolysis in the presence of other plasma proteins, whereas whole blood will study the potential effects of cells. Initially, pooled plasma samples and the validated high-throughput turbidimetric assay will be employed to identify PAI-1 binding Affimers the modulate clot lysis. Subsequently, individual samples from healthy control and various clinical conditions (ischaemic heart disease, type 1 and type 2 diabetes plasma; n=40 in each group), will be tested to establish inter-individual variability in response in various conditions. Turbidimetric experiments will be complemented by confocal and electron microscopy to visualise the clot and ensure the structure remains physiological.</p> <p>Having narrowed down the Affimers of interest, clot formation/lysis experiments on whole blood using rotational elastometry will be conducted on individual Affimers as well as a combination of Affimers as appropriate.</p> <p>Testing combination of Affimers. In the unlikely event that none of the high affinity binding Affimers shows an effect on fibrinolysis, a number of Affimers will be used in combination to establish whether more than one Affimer is required to effectively block PAI-1 activity.</p> <h3>Objective 2.</h3> <p>Study binding affinities/specificity of Affimers of interests and pinpoint interaction sites</p> <p>Binding assays. These will be conducted using surface plasmon resonance to establish the binding affinity of Affimer to PAI-1 and study association/dissociation rates. PAI-1 will be coupled to a CM5 sensor chip using amine-coupling chemistry and Affimers will be passed over the surface of PAI-1 with binding constants calculated using BIAeval software. Experiments will be repeated by immobilizing Affimers of interests and flow PAI-1 over the protein.</p> <p>Pull down assays. This will be performed to investigate interaction of Affimers of interest with PAI-1 in solution and to ensure that this interaction is specific and does not involve other plasma proteins. Optimisation will be carried out using purified proteins before using plasma samples.</p> <p>Microarray analysis. Peptide chips of overlapping 15 AA peptides will be created for PAI-1. Affimers of interest will be added to these peptide chips and after extensive washing binding will be detected with biotinylated anti-Affimer antibody.</p> <p>Crystallography. this will be used to study Affimers-PAI-1 complexes. Data generated will help to understand molecular interactions that underlie interactions sites of Affiemrs of interests with PAI-1.</p> <p>Objective 3. Conduct preliminary in vivo studies to establish the role of Affimers in bleeding and thrombosis.</p> <p>Our work so far has shown that >70% of Affimers that modulate coagulation protein activity, other than PAI-1, have a similar effects in human and mouse plasma. Affimers of interest with equal effect in human and mouse plasma will further undergo in vivo testing. Mice will be anaesthetised and cannulae placed in the jugular vein followed by injection of selected Affimers. Simultaneously, femoral vessels will be dissected and thrombosis induced by application of filter paper soaked in ferric chloride for 3 minutes with serial images taken to quantify thrombus size. In a separate set of experiments, tail bleeding time will be measured in anaesthetised animals after injecting Affimers of interest with scaffold only protein used as control in all the in vivo experiments.</p> <h3>Outcome:</h3> <p>The risk of atherothrombotic events in diabetes subjects is increased and the prognosis following vascular ischaemia remains poor despite advances in treatment. Hypofibrinolysis is one of the mechanisms implicated in increased thrombosis risk in diabetes, which is partially mediated by raised PAI-1 levels. Using both human and animal studies, the proposed work comprehensively investigates the role of inhibiting PAI-1, using a novel methodology, in modulating hypofibrinolysis, which may have future therapeutic implications in high vascular risk conditions.</p> <h3>References:</h3> <p>Yarmolinsky J, Bordin Barbieri N, Weinmann T, Ziegelmann PK, Duncan BB, Inês Schmidt M. Plasminogen activator inhibitor-1 and type 2 diabetes: a systematic review and meta-analysis of observational studies. Sci Rep. 2016 Jan 27;6:17714.</p> <p>Kearney K, Tomlinson D, Smith K, Ajjan R. Hypofibrinolysis in diabetes: a therapeutic target for the reduction of cardiovascular risk. Cardiovasc Diabetol. 2017 Mar 9;16(1):34.</p> <p>Tiede et al. Affimer proteins are versatile and renewable affinity reagents. Elife. 2017, 27;6, pii: e24903.</p>

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