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Fibrinogen Interaction with Complement C3: Ferrata Storti Foundation a Potential Therapeutic Target to Reduce Thrombosis Risk

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Fibrinogen Interaction with Complement C3: Ferrata Storti Foundation a Potential Therapeutic Target to Reduce Thrombosis Risk ARTICLE Coagulation & its Disorders Fibrinogen interaction with complement C3: Ferrata Storti Foundation a potential therapeutic target to reduce thrombosis risk Rhodri J. King,1* Katharina Schuett,2* Christian Tiede,3 Vera Jankowski,4 Vicky John,1 Abhi Trehan,1 Katie Simmons,5 Sreenivasan Ponnambalam,6 Robert F. Storey,7 Colin W.G. Fishwick,5 Michael J. McPherson,3 Darren C. Tomlinson3 and Ramzi A. Ajjan1 Haematologica 2021 1Leeds Institute for Cardiovascular and Metabolic Medicine, Leeds University, Leeds, Volume 106(6):1616-1623 UK; 2Department of Internal Medicine I, University Hospital Aachen, Aachen, Germany, 3Bioscreening Technology Group in the School of Molecular and Cellular Biology, University of Leeds, Leeds, UK, 4Institute for Molecular and Cardiovascular Research, Aachen University, Aachen, Germany, 5School of Chemistry, University of Leeds, Leeds, UK, 6School of Molecular & Cellular Biology, University of Leeds, Leeds, UK and 7School of Medicine, University of Sheffield, Sheffield, UK *RJK and KS contributed equally as co-first authors. ABSTRACT omplement C3 binds fibrinogen and compromises fibrin clot lysis, thereby enhancing the risk of thrombosis. We investigated the role Cof the fibrinogen-C3 interaction as a novel therapeutic target to reduce thrombosis risk by analyzing: (i) consistency in the fibrinolytic properties of C3; (ii) binding sites between fibrinogen and C3; and (iii) modulation of fibrin clot lysis by manipulating fibrinogen-C3 interactions. Purified fibrinogen and C3 from the same individuals (n=24) were used to assess inter-individual variability in the anti-fibrinolytic effects of C3. Microarray screening and molecular modeling evaluated C3 and fibrinogen interaction sites. Novel synthetic conformational proteins, termed affimers, were used to modulate the C3-fibrinogen interaction and fibri- nolysis. C3 purified from patients with type 1 diabetes showed enhanced prolongation of fibrinolysis compared with healthy control protein (195±105 and 522±166 s, respectively; P=0.04), with consistent effects but a wider range (5-51% and 5-18% lysis prolongation, respectively). Peptide Correspondence: microarray screening identified two potential C3-fibrinogen interaction RA AJJAN sites within the fibrinogen b chain (residues 424-433 and 435-445). One [email protected] fibrinogen-binding affimer that was isolated displayed sequence identity with C3 in an exposed area of the protein. This affimer abolished C3- Received: October 1, 2019. induced prolongation of fibrinolysis (728±25.1 s to 632±23.7 s; P=0.005) Accepted: April 29, 2020. and showed binding to fibrinogen in the same region that is involved in C3-fibrinogen interactions. Moreover, it shortened plasma clot lysis of Pre-published: April 30, 2020. patients with diabetes, cardiovascular disease or controls by 7-11%. C3 binds fibrinogen b-chain and disruption of the fibrinogen-C3 interaction https://doi.org/10.3324/haematol.2019.239558 using affimer proteins enhances fibrinolysis, which represents a potential novel tool to reduce thrombosis in high-risk individuals. ©2021 Ferrata Storti Foundation Material published in Haematologica is covered by copyright. Introduction All rights are reserved to the Ferrata Storti Foundation. Use of published material is allowed under the following terms and 1-3 conditions: Hypofibrinolysis is associated with increased risk of atherothrombotic events. https://creativecommons.org/licenses/by-nc/4.0/legalcode. Although most studies have only shown an association between hypofibrinolysis Copies of published material are allowed for personal or inter- and cardiovascular disease, a more recent longitudinal study in a large population of nal use. Sharing published material for non-commercial pur- patients with acute coronary syndrome demonstrated that prolonged fibrin clot lysis poses is subject to the following conditions: 4 https://creativecommons.org/licenses/by-nc/4.0/legalcode, is an independent predictor of cardiovascular mortality. Therefore, it was proposed sect. 3. Reproducing and sharing published material for com- that reducing residual thrombosis risk in patients with coronary artery disease mercial purposes is not allowed without permission in writing requires targeting the fibrinolytic system in addition to administering antiplatelet from the publisher. therapies. Indeed the combination of anticoagulant and antiplatelet therapies reduces vascular thrombotic events but at the expense of increased risk of bleeding.5 Therefore, a more targeted approach is required which improves hypofibrinolysis 1616 haematologica | 2021; 106(6) Hypofibrinolytic effects of complement C3 and its modulation and reduces the risk of vascular thrombosis without Panning and protein production increasing bleeding events. A phage display library of affimer proteins, comprising 1.3x1010 Complement C3, which is incorporated into fibrin clots, random variable clones, was screened against human fibrinogen. has been shown to modulate fibrin clot lysis,6 an effect that In order to select specific affimers capable of targeting fibrinogen– may be exaggerated in patients with higher vascular risk.7 C3 interactions, competitive elution with C3 for 20 min was C3 plasma levels have shown correlations with ex-vivo plas- applied in the fourth panning. ma clot lysis in individuals with diabetes as well as in healthy controls.7,8 Moreover, plasma levels of this protein Affimers and fibrinolysis were independent predictors of resistance to fibrinolysis in Fibrinogen was incubated with affimer A6 at room temperature 875 high-vascular-risk patients with type 2 diabetes.9 These for 30 min after which C3 and factor XIII were added and clot for- data suggest that C3 represents a credible therapeutic target mation/lysis was triggered as described previously.13 to shorten fibrin clot lysis and ultimately reduce thrombosis Plasma samples were mixed with increasing concentrations of risk. However, these functional studies were conducted affimer A6 or control scaffold and then turbidimetric experiments using pooled C3 and studies assessing consistency of the were performed, as described elsewhere.13 response in different individuals, crucial to establish the therapeutic potential of C3, are lacking. Molecular modeling We have recently demonstrated that fibrin clot lysis can In order to identify potential sites of ligand binding on C3, the be modulated, leading to stabilization of the clot, by using molecular modeling software Autoligand was employed to scan small conformational proteins, termed affimers.10,11 Affimer the whole protein.14 Molecular modeling was used to predict bind- technology was able to correct abnormal lysis of clots made ing sites of any peptide sequences identified from the microarray from plasma samples of individuals with hemophilia, indi- screening and affimer work using the webserver Pepsite2® cating a potential therapeutic role for these proteins. (http://pepsite2.russelllab.org). Images were viewed and produced However, it remains unknown whether affimers can be using Pymol (The PyMOL Molecular Graphics System, Version used to enhance clot lysis, particularly in individuals at high 2.0 Schrödinger, LLC).15 vascular risk. The aim of this work was to establish the role of C3 as a Mass spectrometry therapeutic target for enhancing fibrinolysis and reducing Purified C3 from six subjects with type 1 diabetes mellitus and thrombosis risk. Therefore, we studied: (i) consistency in six healthy controls was digested by trypsin and analyzed by the fibrinolytic properties of C3 in healthy controls and matrix-assisted laser desorption/ionization -time-of-flight mass patients with high vascular risk; (ii) potential binding sites spectrometry (MALDI-TOF/MS) as described elsewhere.16 To between fibrinogen and C3; and (iii) modulation of fibrin compare potential differences between in vivo and in vitro C3 gly- clot lysis by targeted interference of fibrinogen-C3 interac- cation, we undertook in vitro glycation of the protein as previously tions. described17 and post-translational modifications were assessed as above. Methods Statistical analysis Between-group comparisons of normally and non-normally dis- Only a brief description is provided here; full details can be tributed variables were carried out by independent Student t and found in the Online Supplement. Mann–Whitney U tests, respectively. Pearson and Spearman coef- ficients were applied to assess correlations between normally and Recruitment of patients non-normally distributed variables, respectively. Based on previ- We recruited 12 patients with type 1 diabetes (T1DM) and 12 ous data7 and assuming a common standard deviation of the vari- age- and sex-matched controls to purify fibrinogen and C3. We able studied of 225 seconds (s) we calculated that the number of additionally tested 24 stored plasma samples from individuals with samples analyzed (n=24 in total) would be enough to detect a dif- high vascular risk. Ethical approval for the study was provided by ference of 270 s in C3-induced prolongation of clot lysis time the Leeds East, National Research Ethics Service committee and (power 80% at P<0.05). informed consent was obtained from participants in accordance with the Declaration of Helsinki. Results Protein purification and turbidimetric analysis Fibrinogen and complement C3 were purified as described else- Patients’ characteristics where7 and clots were formed from purified fibrinogen in the pres- Twelve patients with type 1 diabetes and 12 healthy ence or absence of corresponding
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