243 results on '"Richard W. Farndale"'
Search Results
2. The effects of inhibition and siRNA knockdown of collagen-binding integrins on human umbilical vein endothelial cell migration and tube formation
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Emma J. Hunter, Samir W. Hamaia, Peter S.-K. Kim, Jean-Daniel M. Malcor, and Richard W. Farndale
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Medicine ,Science - Abstract
Abstract Blood vessels in the body are lined with endothelial cells which have vital roles in numerous physiological and pathological processes. Collagens are major constituents of the extracellular matrix, and many adherent cells express several collagen-binding adhesion receptors. Here, we study the endothelium–collagen interactions mediated by the collagen-binding integrins, α1β1, α2β1, α10β1 and α11β1 expressed in human umbilical vein endothelial cells (HUVECs). Using qPCR, we found expression of the α10 transcript of the chondrocyte integrin, α10β1, along with the more abundant α2, and low-level expression of α1. The α11 transcript was not detected. Inhibition or siRNA knockdown of the α2-subunit resulted in impaired HUVEC adhesion, spreading and migration on collagen-coated surfaces, whereas inhibition or siRNA knockdown of α1 had no effect on these processes. In tube formation assays, inhibition of either α1 or α2 subunits impaired the network complexity, whereas siRNA knockdown of these integrins had no such effect. Knockdown of α10 had no effect on cell spreading, migration or tube formation in these conditions. Overall, our results indicate that the collagen-binding integrins, α1β1 and α2β1 play a central role in endothelial cell motility and self-organisation.
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- 2022
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3. Local Net Charge State of Collagen Triple Helix Is a Determinant of FKBP22 Binding to Collagen III
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Yoshihiro Ishikawa, Arkadiusz Bonna, Douglas B. Gould, and Richard W. Farndale
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endoplasmic reticulum ,extracellular matrix ,Ehlers–Danlos Syndrome ,collagen Toolkit ,triple-helical peptides ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Mutations in the FKBP14 gene encoding the endoplasmic reticulum resident collagen-related proline isomerase FK506 binding protein 22 kDa (FKBP22) result in kyphoscoliotic Ehlers–Danlos Syndrome (EDS), which is characterized by a broad phenotypic outcome. A plausible explanation for this outcome is that FKBP22 participates in the biosynthesis of subsets of collagen types: FKBP22 selectively binds to collagens III, IV, VI, and X, but not to collagens I, II, V, and XI. However, these binding mechanisms have never been explored, and they may underpin EDS subtype heterogeneity. Here, we used collagen Toolkit peptide libraries to investigate binding specificity. We observed that FKBP22 binding was distributed along the collagen helix. Further, it (1) was higher on collagen III than collagen II peptides and it (2) was correlated with a positive peptide charge. These findings begin to elucidate the mechanism by which FKBP22 interacts with collagen.
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- 2023
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4. Mild hyperlipidemia in mice aggravates platelet responsiveness in thrombus formation and exploration of platelet proteome and lipidome
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Johanna P. van Geffen, Frauke Swieringa, Kim van Kuijk, Bibian M. E. Tullemans, Fiorella A. Solari, Bing Peng, Kenneth J. Clemetson, Richard W. Farndale, Ludwig J. Dubois, Albert Sickmann, René P. Zahedi, Robert Ahrends, Erik A. L. Biessen, Judith C. Sluimer, Johan W. M. Heemskerk, and Marijke J. E. Kuijpers
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Medicine ,Science - Abstract
Abstract Hyperlipidemia is a well-established risk factor for cardiovascular diseases. Millions of people worldwide display mildly elevated levels of plasma lipids and cholesterol linked to diet and life-style. While the prothrombotic risk of severe hyperlipidemia has been established, the effects of moderate hyperlipidemia are less clear. Here, we studied platelet activation and arterial thrombus formation in Apoe −/− and Ldlr −/− mice fed a normal chow diet, resulting in mildly increased plasma cholesterol. In blood from both knockout mice, collagen-dependent thrombus and fibrin formation under flow were enhanced. These effects did not increase in severe hyperlipidemic blood from aged mice and upon feeding a high-fat diet (Apoe −/− mice). Bone marrow from wild-type or Ldlr −/− mice was transplanted into irradiated Ldlr −/− recipients. Markedly, thrombus formation was enhanced in blood from chimeric mice, suggesting that the hyperlipidemic environment altered the wild-type platelets, rather than the genetic modification. The platelet proteome revealed high similarity between the three genotypes, without clear indication for a common protein-based gain-of-function. The platelet lipidome revealed an altered lipid profile in mildly hyperlipidemic mice. In conclusion, in Apoe −/− and Ldlr −/− mice, modest elevation in plasma and platelet cholesterol increased platelet responsiveness in thrombus formation and ensuing fibrin formation, resulting in a prothrombotic phenotype.
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- 2020
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5. Factor XIII is a newly identified binding partner for platelet collagen receptor GPVI‐dimer—An interaction that may modulate fibrin crosslinking
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Masaaki Moroi, Isuru Induruwa, Richard W. Farndale, and Stephanie M. Jung
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crosslinking ,factor XIII ,fibrin ,fibrin clot ,GPVI ,GPVI‐dimer ,Diseases of the blood and blood-forming organs ,RC633-647.5 - Abstract
Abstract Background In the fibrin‐forming process, thrombin cleaves fibrinogen to fibrin, which form fibrils and then fibers, producing a gel‐like clot. Thrombin also activates coagulation factor XIII (FXIII), which crosslinks fibrin γ‐chains and α‐chains, stabilizing the clot. Many proteins bind to fibrin, including FXIII, an established regulation of clot structure, and platelet glycoprotein VI (GPVI), whose contribution to clot function is largely unknown. FXIII is present in plasma, but the abundant FXIII in platelet cytosol becomes exposed to the surface of strongly activated platelets. Objectives We determined if GPVI interacts with FXIII and how this might modulate clot formation. Methods We measured interactions between recombinant proteins of the GPVI extracellular domain: GPVI‐dimer (GPVI‐Fc2) or monomer (GPVIex) and FXIII proteins (nonactivated and thrombin‐activated FXIII, FXIII subunits A and B) by ELISA. Binding to fibrin clots and fibrin γ‐chain crosslinking were analyzed by immunoblotting. Results GPVI‐dimer, but not GPVI‐monomer, bound to FXIII. GPVI‐dimer selectively bound to the FXIII A‐subunit, but not to the B‐subunit, an interaction that was decreased or abrogated by the GPVI‐dimer–specific antibody mFab‐F. The GPVI‐dimer–FXIII interaction decreased the extent of γ‐chain crosslinking, indicating a role in the regulation of clot formation. Conclusions This is the first report of the specific interaction between GPVI‐dimer and the A‐subunit of FXIII, as determined in an in vitro system with defined components. GPVI‐dimer–FXIII binding was inhibitory toward FXIII‐catalyzed crosslinking of fibrin γ‐chains in fibrin clots. This raises the possibility that GPVI‐dimer may negatively modulate fibrin crosslinking induced by FXIII, lessening clot stability.
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- 2022
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6. Activation‐induced changes in platelet surface receptor expression and the contribution of the large‐platelet subpopulation to activation
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Masaaki Moroi, Richard W. Farndale, and Stephanie M. Jung
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activation ,GPIb ,GPVI ,IIbIIIa ,large platelets ,receptors ,Diseases of the blood and blood-forming organs ,RC633-647.5 - Abstract
Abstract Objective Platelet surface receptors are also present subcellularly in organelle membranes and can be expressed on the surface upon platelet activation. However, some receptors were reported to be decreased after activation. We analyzed the mechanism of activation‐dependent expression for different receptors. Methods Flow cytometry using platelet‐rich plasma or washed platelets was used to analyze receptor‐expression changes after platelet activation by glycoprotein (GP) VI–specific agonists, crosslinked collagen‐related peptide (CRP‐XL) and convulxin (Cvx), and thrombin. Platelets prelabeled with fluorescent antibody specific for a receptor were allowed to adhere on immobilized collagen or fibrinogen and post‐stained with antibody against the same receptor labeled with another fluorophore, allowing us to differentiate preexisting receptors from newly expressed receptors. Results Surface expression of αIIbβ3 increased in CRP‐XL–, Cvx‐, or thrombin‐stimulated platelets, but GPIb decreased due to shedding and internalization. Both total and dimeric GPVI increased in thrombin‐induced platelets, but decreased in platelets stimulated by Cvx, as a result of internalization. The larger platelets showed a greater increase in surface receptor (α2β1, αIIbβ3, GPVI, GPIb) expression upon activation compared to the smaller ones. Pre‐ and postlabeling with antibody specific for the same receptor, but conjugated with different fluorophores, allowed us to differentiate the receptors expressed on the surface of resting platelets from receptors newly exposed to the surface upon platelet activation. Conclusions Increased receptor expressions after activation are mainly manifested in the larger platelets. On platelets adhered on fibrinogen, the newly expressed receptors, especially GPVI, are localized in the lamellipodia of the spread platelets.
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- 2020
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7. Proline provides site-specific flexibility for in vivo collagen
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Wing Ying Chow, Chris J. Forman, Dominique Bihan, Anna M. Puszkarska, Rakesh Rajan, David G. Reid, David A. Slatter, Lucy J. Colwell, David J. Wales, Richard W. Farndale, and Melinda J. Duer
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13Cγ Chemical Shifts ,Proline Ring ,Collagen Model Peptides ,Collagen Triple Helix ,Backbone Dihedrals ,Medicine ,Science - Abstract
Abstract Fibrillar collagens have mechanical and biological roles, providing tissues with both tensile strength and cell binding sites which allow molecular interactions with cell-surface receptors such as integrins. A key question is: how do collagens allow tissue flexibility whilst maintaining well-defined ligand binding sites? Here we show that proline residues in collagen glycine-proline-hydroxyproline (Gly-Pro-Hyp) triplets provide local conformational flexibility, which in turn confers well-defined, low energy molecular compression-extension and bending, by employing two-dimensional 13C-13C correlation NMR spectroscopy on 13C-labelled intact ex vivo bone and in vitro osteoblast extracellular matrix. We also find that the positions of Gly-Pro-Hyp triplets are highly conserved between animal species, and are spatially clustered in the currently-accepted model of molecular ordering in collagen type I fibrils. We propose that the Gly-Pro-Hyp triplets in fibrillar collagens provide fibril “expansion joints” to maintain molecular ordering within the fibril, thereby preserving the structural integrity of ligand binding sites.
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- 2018
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8. Identification of HSP47 Binding Site on Native Collagen and Its Implications for the Development of HSP47 Inhibitors
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Haiyan Cai, Parvathy Sasikumar, Gemma Little, Dominique Bihan, Samir W. Hamaia, Aiwu Zhou, Jonathan M. Gibbins, and Richard W. Farndale
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HSP47 inhibitor ,collagen ,fibrosis ,molecular docking ,structural analysis ,Microbiology ,QR1-502 - Abstract
HSP47 (heat shock protein 47) is a collagen-specific molecular chaperone that is essential for procollagen folding and function. Previous studies have shown that HSP47 binding requires a critical Arg residue at the Y position of the (Gly-Xaa-Yaa) repeats of collagen; however, the exact binding sites of HSP47 on native collagens are not fully defined. To address this, we mapped the HSP47 binding sites on collagens through an ELISA binding assay using collagen toolkits, synthetic collagen peptides covering the entire amino acid sequences of collagen types II and III assembled in triple-helical conformation. Our results showed that HSP47 binds to only a few of the GXR motifs in collagen, with most of the HSP47 binding sites identified located near the N-terminal part of the triple-helical region. Molecular modelling and binding energy calculation indicated that residues flanking the key Arg in the collagen sequence also play an important role in defining the high-affinity HSP47 binding site of collagen. Based on this binding mode of HSP47 to collagen, virtual screening targeting both the Arg binding site and its neighboring area on the HSP47 surface, and a subsequent bioassay, we identified two novel compounds with blocking activity towards HSP47 binding of collagen. Overall, our study revealed the native HSP47 binding sites on collagen and provided novel information for the design of small-molecule inhibitors of HSP47.
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- 2021
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9. Data on hyper-activation of GPVI signalling in obese patients: Towards the identification of novel antiplatelet targets in obesity
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María N. Barrachina, Irene Izquierdo, Lidia Hermida-Nogueira, Aurelio M. Sueiro, Esteban Guitián, Felipe F. Casanueva, Richard W. Farndale, Masaaki Moroi, Stephanie M. Jung, María Pardo, and Ángel García
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Computer applications to medicine. Medical informatics ,R858-859.7 ,Science (General) ,Q1-390 - Abstract
This data article is associated with the manuscript “GPVI surface expression and signalling pathway activation are increased in platelets from obese patients: elucidating potential anti-atherothrombotic targets in obesity” [1]. The study refers to a combination of different approaches in order to identify platelet-derived biomarkers in obesity. A total of 34 obese patients and their lean-matched controls were included in the study. We carried out a proteomic and functional (aggregation assays) analysis to find alterations in platelet-derived signalling pathways. After that, biochemical and mechanistic (flow cytometry assays) approaches were done in order to confirm a hyperactivation of the GPVI-related signalling pathway.
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- 2019
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10. Role of Platelet Glycoprotein VI and Tyrosine Kinase Syk in Thrombus Formation on Collagen-Like Surfaces
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Natalie J. Jooss, Ilaria De Simone, Isabella Provenzale, Delia I. Fernández, Sanne L.N. Brouns, Richard W. Farndale, Yvonne M.C. Henskens, Marijke J.E. Kuijpers, Hugo ten Cate, Paola E.J. van der Meijden, Rachel Cavill, and Johan W.M. Heemskerk
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calcium ,collagen ,glycoprotein VI ,platelet activation ,protein tyrosine kinase ,thrombus ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Platelet interaction with collagens, via von Willebrand factor, is a potent trigger of shear-dependent thrombus formation mediated by subsequent engagement of the signaling collagen receptor glycoprotein (GP)VI, enforced by integrin α2β1. Protein tyrosine kinase Syk is central in the GPVI-induced signaling pathway, leading to elevated cytosolic Ca2+. We aimed to determine the Syk-mediated thrombogenic activity of several collagen peptides and (fibrillar) type I and III collagens. High-shear perfusion of blood over microspots of these substances resulted in thrombus formation, which was assessed by eight parameters and was indicative of platelet adhesion, activation, aggregation, and contraction, which were affected by the Syk inhibitor PRT-060318. In platelet suspensions, only collagen peptides containing the consensus GPVI-activating sequence (GPO)n and Horm-type collagen evoked Syk-dependent Ca2+ rises. In whole blood under flow, Syk inhibition suppressed platelet activation and aggregation parameters for the collagen peptides with or without a (GPO)n sequence and for all of the collagens. Prediction models based on a regression analysis indicated a mixed role of GPVI in thrombus formation on fibrillar collagens, which was abolished by Syk inhibition. Together, these findings indicate that GPVI-dependent signaling through Syk supports platelet activation in thrombus formation on collagen-like structures regardless of the presence of a (GPO)n sequence.
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- 2019
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11. Measurement of the Interaction Between Recombinant I-domain from Integrin alpha 2 beta 1 and a Triple Helical Collagen Peptide with the GFOGER Binding Motif Using Molecular Force Spectroscopy
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Mark E. Welland, Richard W. Farndale, Dominique Bihan, Debdulal Roy, Simon J. Attwood, Anna M. C. Simpson, and Samir W. Hamaia
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integrin ,alpha 2 beta 1 ,collagen ,atomic force microscopy ,single moleculeforce spectroscopy ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
The role of the collagen-platelet interaction is of crucial importance to the haemostatic response during both injury and pathogenesis of the blood vessel wall. Of particular interest is the high affinity interaction of the platelet transmembrane receptor, alpha 2 beta 1, responsible for firm attachment of platelets to collagen at and around injury sites. We employ single molecule force spectroscopy (SMFS) using the atomic force microscope (AFM) to study the interaction of the I-domain from integrin alpha 2 beta 1 with a synthetic collagen related triple-helical peptide containing the high-affinity integrin-binding GFOGER motif, and a control peptide lacking this sequence, referred to as GPP. By utilising synthetic peptides in this manner we are able to study at the molecular level subtleties that would otherwise be lost when considering cell-to-collagen matrix interactions using ensemble techniques. We demonstrate for the first time the complexity of this interaction as illustrated by the complex multi-peaked force spectra and confirm specificity using control blocking experiments. In addition we observe specific interaction of the GPP peptide sequence with the I-domain. We propose a model to explain these observations.
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- 2013
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12. A Simple Bioconjugate Attachment Protocol for Use in Single Molecule Force Spectroscopy Experiments Based on Mixed Self-Assembled Monolayers
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Myriam Ouberai, Richard W. Farndale, Debdulal Roy, Samir W. Hamaia, Rachael Stone, Anna M. C. Simpson, Simon J. Attwood, and Mark E.Welland
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atomic force microscopy ,single molecule force spectroscopy ,biotin-avidin interaction ,mixed self-assembled monolayers ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Single molecule force spectroscopy is a technique that can be used to probe the interaction force between individual biomolecular species. We focus our attention on the tip and sample coupling chemistry, which is crucial to these experiments. We utilised a novel approach of mixed self-assembled monolayers of alkanethiols in conjunction with a heterobifunctional crosslinker. The effectiveness of the protocol is demonstrated by probing the biotin-avidin interaction. We measured unbinding forces comparable to previously reported values measured at similar loading rates. Specificity tests also demonstrated a significant decrease in recognition after blocking with free avidin.
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- 2012
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13. Platelet adhesion to decorin but not collagen I correlates with the integrin α2 dimorphism E534K, the basis of the human platelet alloantigen (HPA)-5 system
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Thomas J. Kunicki, Shirley A. Williams, Daniel Diaz, Richard W. Farndale, and Diane J. Nugent
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Diseases of the blood and blood-forming organs ,RC633-647.5 - Abstract
A single nucleotide polymorphism in the integrin α2 gene ITGA2 (rs1801106; G1600A) creates the non-conservative amino acid substitution E534K, the basis of the human platelet alloantigen system HPA-5. Yet HPA-5 alleles do not influence binding of α2β1 to its primary ligand collagen I, and the effect of HPA-5 on platelet function has not been determined. We used a direct platelet adhesion assay to evaluate whether differential inheritance of HPA-5 alleles influences platelet adhesion to collagen I or an alternative ligand, decorin. Platelets from donors bearing one or more minor allele HPA-5b showed attenuated adhesion to purified decorin but not collagen I. Adhesion to decorin was significantly inhibited by human alloantibodies specific for HPA-5a but not by the collagen I sequence GFOGER or α2-specific inhibitory monoclonal antibodies. The minor allele 534K attenuates platelet adhesion to decorin but not collagen I, providing the first evidence of a functional effect of HPA-5 alleles.
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- 2012
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14. Pharmacological Inhibition of Glycoprotein VI- and Integrin α2β1-Induced Thrombus Formation Modulated by the Collagen Type
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Natalie J. Jooss, Yvonne M.C. Henskens, Steve P. Watson, Richard W. Farndale, Meinrad P. Gawaz, Martine Jandrot-Perrus, Natalie S. Poulter, Johan W. M. Heemskerk, Biochemie, RS: Carim - B03 Cell biochemistry of thrombosis and haemostasis, MUMC+: DA CDL Algemeen (9), RS: Carim - B04 Clinical thrombosis and Haemostasis, and Central Diagnostic Lab
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collagen ,ROLES ,IDENTIFICATION ,SURFACE ,FLOW ,protein tyrosine kinase ,Hematology ,AGGREGATION ,glycoprotein VI ,ACTIVATION ,PLATELET-ADHESION ,thrombus ,GPVI ,BINDING ,KINASE ,integrin alpha 2 beta 1 - Abstract
Background In secondary cardiovascular disease prevention, treatments blocking platelet-derived secondary mediators pose a risk of bleeding. Pharmacological interference of the interaction of platelets with exposed vascular collagens is an attractive alternative, with clinical trials ongoing. Antagonists of the collagen receptors, glycoprotein VI (GPVI), and integrin α2β1, include recombinant GPVI-Fc dimer construct Revacept, 9O12 mAb based on the GPVI-blocking reagent Glenzocimab, Syk tyrosine-kinase inhibitor PRT-060318, and anti-α2β1 mAb 6F1. No direct comparison has been made of the antithrombic potential of these drugs. Methods Using a multiparameter whole-blood microfluidic assay, we compared the effects of Revacept, 9O12-Fab, PRT-060318, or 6F1 mAb intervention with vascular collagens and collagen-related substrates with varying dependencies on GPVI and α2β1. To inform on Revacept binding to collagen, we used fluorescent-labelled anti-GPVI nanobody-28. Results and Conclusion In this first comparison of four inhibitors of platelet–collagen interactions with antithrombotic potential, we find that at arterial shear rate: (1) the thrombus-inhibiting effect of Revacept was restricted to highly GPVI-activating surfaces; (2) 9O12-Fab consistently but partly inhibited thrombus size on all surfaces; (3) effects of GPVI-directed interventions were surpassed by Syk inhibition; and (4) α2β1-directed intervention with 6F1 mAb was strongest for collagens where Revacept and 9O12-Fab were limitedly effective. Our data hence reveal a distinct pharmacological profile for GPVI-binding competition (Revacept), GPVI receptor blockage (9O12-Fab), GPVI signaling (PRT-060318), and α2β1 blockage (6F1 mAb) in flow-dependent thrombus formation, depending on the platelet-activating potential of the collagen substrate. This work thus points to additive antithrombotic action mechanisms of the investigated drugs.
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- 2023
15. Structural insights into collagen binding by platelet receptor glycoprotein VI
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Louris J. Feitsma, Harma C. Brondijk, Gavin E. Jarvis, Dominique Hagemans, Dominique Bihan, Natasia Jerah, Marian Versteeg, Richard W. Farndale, and Eric G. Huizinga
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Blood Platelets ,Binding Sites ,Immunology ,Animals ,Thrombosis ,Collagen ,Platelet Membrane Glycoproteins ,Cell Biology ,Hematology ,Peptides ,Platelet Activation ,Biochemistry ,Protein Binding - Abstract
Glycoprotein VI (GPVI) mediates collagen-induced platelet activation after vascular damage and is an important contributor to the onset of thrombosis, heart attack, and stroke. Animal models of thrombosis have identified GPVI as a promising target for antithrombotic therapy. Although for many years the crystal structure of GPVI has been known, the essential details of its interaction with collagen have remained elusive. Here, we present crystal structures of the GPVI ectodomain bound to triple-helical collagen peptides, which reveal a collagen-binding site across the β-sheet of the D1 domain. Mutagenesis and binding studies confirm the observed binding site and identify Trp76, Arg38, and Glu40 as essential residues for binding to fibrillar collagens and collagen-related peptides (CRPs). GPVI binds a site on collagen comprising two collagen chains with the core formed by the sequence motif OGPOGP. Potent GPVI-binding peptides from Toolkit-III all contain OGPOGP; weaker binding peptides frequently contain a partial motif varying at either terminus. Alanine-scanning of peptide III-30 also identified two AGPOGP motifs that contribute to GPVI binding, but steric hindrance between GPVI molecules restricts the maximum binding capacity. We further show that no cooperative interactions could occur between two GPVI monomers binding to a stretch of (GPO)5 and that binding of ≥2 GPVI molecules to a fibril-embedded helix requires non-overlapping OGPOGP motifs. Our structure confirms the previously suggested similarity in collagen binding between GPVI and leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1) but also indicates significant differences that may be exploited for the development of receptor-specific therapeutics.
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- 2022
16. Integrins in GtoPdb v.2023.1
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Gavin E. Jarvis and Richard W. Farndale
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General Medicine ,General Chemistry - Abstract
Integrins are unusual signalling proteins that function to signal both from the extracellular environment into the cell, but also from the cytoplasm to the external of the cell. The intracellular signalling cascades associated with integrin activation focus on protein kinase activities, such as focal adhesion kinase and Src. Based on this association between extracellular signals and intracellular protein kinase activity, we have chosen to include integrins in the 'Catalytic receptors' section of the database until more stringent criteria from NC-IUPHAR allows precise definition of their classification.Integrins are heterodimeric entities, composed of α and β subunits, each 1TM proteins, which bind components of the extracellular matrix or counter-receptors expressed on other cells. One class of integrin contains an inserted domain (I) in its α subunit, and if present (in α1, α2, α10, α11, αD, αE, αL, αM and αX), this I domain contains the ligand binding site. All β subunits possess a similar I-like domain, which has the capacity to bind ligand, often recognising the RGD motif. The presence of an α subunit I domain precludes ligand binding through the β subunit. Integrins provide a link between ligand and the actin cytoskeleton (through typically short intracellular domains). Integrins bind several divalent cations, including a Mg2+ ion in the I or I-like domain that is essential for ligand binding. Other cation binding sites may regulate integrin activity or stabilise the 3D structure. Integrins regulate the activity of particular protein kinases, including focal adhesion kinase and integrin-linked kinase. Cellular activation regulates integrin ligand affinity via inside-out signalling and ligand binding to integrins can regulate cellular activity via outside-in signalling.Several drugs that target integrins are in clinical use including: (1) abciximab (αIIbβ3) for short term prevention of coronary thrombosis, (2) vedolizumab (α4β7) to reduce gastrointestinal inflammation, and (3) natalizumab (α4β1) in some cases of severe multiple sclerosis.
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- 2023
17. Tyrosine-sulfated dermatopontin shares multiple binding sites and recognition determinants on triple-helical collagens with proteins implicated in cell adhesion and collagen folding, fibrillogenesis, cross-linking, and degradation
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Morten M. Jensen, Arkadiusz Bonna, Sigurd J. Frederiksen, Samir W. Hamaia, Peter Højrup, Richard W. Farndale, and Henrik Karring
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Binding Sites ,Swine ,Fibrillar Collagens ,Biophysics ,Collagen-binding ,Chaperone ,Tyrosine sulfation ,Arginine ,Biochemistry ,complex mixtures ,Collagen Type I ,Analytical Chemistry ,Cornea ,Cell Adhesion ,otorhinolaryngologic diseases ,Animals ,Tyrosine ,Dermatopontin ,Collagen ,Connective tissue disorders ,Peptides ,Molecular Biology - Abstract
Dermatopontin (DPT), a small extracellular matrix protein that stimulates collagen fibrillogenesis, contains sulfotyrosine residues but neither its level of sulfation nor its binding sites on fibrillar collagens are known. Here, we discovered that DPT is present in a relatively high mass concentration (~ 0.02%) in porcine corneal stroma, from which we purified five DPT charge variants (A-E) containing up to six sulfations. The major variant (C), containing four sulfotyrosine residues, was used to locate binding sites for DPT on triple-helical collagens II and III using the Collagen Toolkits. DPT-binding loci included the triple helix crosslinking sites and collagenase cleavage site. We find that strong DPT-binding sites on triple-helical collagen comprise an arginine-rich, positively-charged sequence that also contains hydrophobic residues. This collagen-binding signature of DPT is similar to that of the chaperone HSP47. Thus, we propose that DPT assumes the role of HSP47 as a collagen chaperone during and after the secretion. Peptide II-44, harbouring the conserved collagenase cleavage site, shows the strongest DPT-binding of the Collagen Toolkit II peptides. Substituting any of the three arginine residues (R) with alanine in the sequence GLAGQRGIVGLOGQRGER of II-44 resulted in almost complete loss of DPT binding. Since osteogenesis imperfecta, spondyloepiphyseal dysplasia, and spondyloepimetaphyseal dysplasia congenita are associated with missense mutations that substitute the corresponding arginine residues in collagens alpha-1(I) and alpha-1(II), we suggest that disrupted DPT binding to fibrillar collagens may contribute to these connective tissue disorders. In conclusion, the present work provides a cornerstone for further elucidation of the role of DPT.
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- 2022
18. Multimerin 1 supports platelet function in vivo and binds to specific GPAGPOGPX motifs in fibrillar collagens that enhance platelet adhesion
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Heyu Ni, Bradley W. Doble, Samir W. Hamaia, Peter L. Gross, Subia Tasneem, Catherine P.M. Hayward, Dominique Bihan, D'Andra Parker, Arkadiusz Bonna, Richard W. Farndale, Alexander Leatherdale, David Lillicrap, and Yiming Wang
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Blood Platelets ,Impaired platelet adhesion ,Fibrillar Collagens ,Multimerin 1 ,030204 cardiovascular system & hematology ,von Willebrand factor ,Fibrinogen ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Von Willebrand factor ,Platelet adhesiveness ,medicine ,Animals ,Platelet ,multimerin ,platelet adhesiveness ,biology ,Chemistry ,Hematology ,Adhesion ,Original Articles ,Blood Proteins ,PLATELETS ,Cell biology ,biology.protein ,Original Article ,Ex vivo ,medicine.drug - Abstract
Background Multimerin 1 (human: MMRN1, mouse: Mmrn1) is a homopolymeric, adhesive, platelet and endothelial protein that binds to von Willebrand factor and enhances platelet adhesion to fibrillar collagen ex vivo. Objectives To examine the impact of Mmrn1 deficiency on platelet adhesive function, and the molecular motifs in fibrillar collagen that bind MMRN1 to enhance platelet adhesion. Methods Mmrn1‐deficient mice were generated and assessed for altered platelet adhesive function. Collagen Toolkit peptides, and other triple‐helical collagen peptides, were used to identify multimerin 1 binding motifs and their contribution to platelet adhesion. Results MMRN1 bound to conserved GPAGPOGPX sequences in collagens I, II, and III (including GPAGPOGPI, GPAGPOGPV, and GPAGPOGPQ) that enhanced activated human platelet adhesion to collagen synergistically with other triple‐helical collagen peptides (P
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- 2020
19. Versican binds collagen via its G3 domain and regulates the organization and mechanics of collagenous matrices
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Dongning Chen, Yu Du, Jessica Llewellyn, Arkadiusz Bonna, Biao Zuo, Paul A. Janmey, Richard W. Farndale, and Rebecca G. Wells
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carbohydrates (lipids) - Abstract
Type I collagen is the most abundant structural protein in the body and, with other fibrillar collagens, forms the fibrous network of the extracellular matrix (ECM). Another group of ECM polymers, the glycosaminoglycans (GAGs) and GAG-modified proteoglycans, play important roles in regulating collagen behaviors and contribute to the compositional, structural and mechanical complexity of the ECM. While the binding between collagen and small leucine-rich proteoglycans (SLRPs) has been studied in detail, the interactions between collagen and the large bottlebrush proteoglycan versican are not well understood. Here, we report that versican binds collagen directly and regulates collagen structure and mechanics. Versican colocalizes with collagen fibers in vivo and binds to collagen via its C-terminal G3 domain (a non-GAG-modified domain present in all known versican isoforms) in vitro; it promotes the deposition of a highly-aligned collagen-rich matrix by fibroblasts. Versican also shows an unexpected effect on the rheology of collagen gels in vitro, causing decreased stiffness and attenuated shear strain stiffening, and the cleavage of versican in liver results in reduced tissue compression stiffening. Thus, versican is an important collagen binding partner, playing a role in modulating collagen organization and mechanics.
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- 2022
20. Cleavage by MMP‐13 renders VWF unable to bind to collagen but increases its platelet reactivity
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Joanna-Marie Howes, Richard W. Farndale, Vera Knäuper, and Jean-Daniel Malcor
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Blood Platelets ,congenital, hereditary, and neonatal diseases and abnormalities ,Endothelium ,von Willebrand factor ,030204 cardiovascular system & hematology ,Matrix metalloproteinase ,03 medical and health sciences ,Platelet Adhesiveness ,0302 clinical medicine ,matrix metalloproteinase‐13 ,Von Willebrand factor ,hemic and lymphatic diseases ,Matrix Metalloproteinase 13 ,medicine ,Humans ,Platelet ,Secretion ,Thrombus ,thrombosis ,Whole blood ,biology ,Chemistry ,Endothelial Cells ,Original Articles ,Hematology ,medicine.disease ,PLATELETS ,ADAMTS13 ,Cell biology ,medicine.anatomical_structure ,glycoprotein Ib alpha ,cardiovascular system ,biology.protein ,Original Article ,Collagen ,circulatory and respiratory physiology - Abstract
Background Atherosclerotic plaque rupture and subsequent thrombosis underpin thrombotic syndromes. Under inflammatory conditions in the unstable plaque, perturbed endothelial cells secrete von Willebrand Factor (VWF) which, via its interaction with GpIbα, enables platelet rolling across and adherence to the damaged endothelium. Following plaque rupture, VWF and platelets are exposed to subendothelial collagen, which supports stable platelet adhesion, activation, and aggregation. Plaque‐derived matrix metalloproteinase (MMP)‐13 is also released into the surrounding lumen where it may interact with VWF, collagen, and platelets. Objectives We sought to discover whether MMP‐13 can cleave VWF and whether this might regulate its interaction with both collagen and platelets. Methods We have used platelet adhesion assays and whole blood flow experiments to assess the effects of VWF cleavage by MMP‐13 on platelet adhesion and thrombus formation. Results Unlike the shear‐dependent cleavage of VWF by a disintegrin and metalloprotease with thrombospondin motif member 13 (ADAMTS13), MMP‐13 is able to cleave VWF under static conditions. Following cleavage by MMP‐13, immobilized VWF cannot bind to collagen but interacts more strongly with platelets, supporting slower platelet rolling in whole blood under shear. Compared with intact VWF, the interaction of cleaved VWF with platelets results in greater GpIbα upregulation and P‐selectin expression, and the thrombi formed on cleaved VWF–collagen co‐coatings are larger and more contractile than platelet aggregates on intact VWF‐collagen co‐coatings or on collagen alone. Conclusions Our data suggest a VWF‐mediated role for MMP‐13 in the recruitment of platelets to the site of vascular injury and may provide new insights into the association of MMP‐13 in atherothrombotic and stroke pathologies.
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- 2020
21. Activation‐induced changes in platelet surface receptor expression and the contribution of the large‐platelet subpopulation to activation
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Stephanie M. Jung, Masaaki Moroi, and Richard W. Farndale
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media_common.quotation_subject ,Receptor expression ,receptors ,Thrombin ,Original Articles: Hemostasis ,GPVI ,medicine ,GPIb ,Platelet ,Platelet activation ,Internalization ,Receptor ,media_common ,IIbIIIa ,Chemistry ,lcsh:RC633-647.5 ,large platelets ,Convulxin ,αIIbβ3 ,Hematology ,lcsh:Diseases of the blood and blood-forming organs ,Cell biology ,Original Article ,activation ,medicine.drug - Abstract
Objective Platelet surface receptors are also present subcellularly in organelle membranes and can be expressed on the surface upon platelet activation. However, some receptors were reported to be decreased after activation. We analyzed the mechanism of activation‐dependent expression for different receptors. Methods Flow cytometry using platelet‐rich plasma or washed platelets was used to analyze receptor‐expression changes after platelet activation by glycoprotein (GP) VI–specific agonists, crosslinked collagen‐related peptide (CRP‐XL) and convulxin (Cvx), and thrombin. Platelets prelabeled with fluorescent antibody specific for a receptor were allowed to adhere on immobilized collagen or fibrinogen and post‐stained with antibody against the same receptor labeled with another fluorophore, allowing us to differentiate preexisting receptors from newly expressed receptors. Results Surface expression of αIIbβ3 increased in CRP‐XL–, Cvx‐, or thrombin‐stimulated platelets, but GPIb decreased due to shedding and internalization. Both total and dimeric GPVI increased in thrombin‐induced platelets, but decreased in platelets stimulated by Cvx, as a result of internalization. The larger platelets showed a greater increase in surface receptor (α2β1, αIIbβ3, GPVI, GPIb) expression upon activation compared to the smaller ones. Pre‐ and postlabeling with antibody specific for the same receptor, but conjugated with different fluorophores, allowed us to differentiate the receptors expressed on the surface of resting platelets from receptors newly exposed to the surface upon platelet activation. Conclusions Increased receptor expressions after activation are mainly manifested in the larger platelets. On platelets adhered on fibrinogen, the newly expressed receptors, especially GPVI, are localized in the lamellipodia of the spread platelets.
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- 2020
22. Chain Alignment of Collagen I Deciphered using Computationally Designed Heterotrimers
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Katherine Stott, Samir W. Hamaia, Birgit Leitinger, Douglas R. Walker, Jeffrey D. Hartgerink, Douglas Sammon, Paul Brear, Richard W. Farndale, Abhishek A. Jalan, and Emma Hunter
- Subjects
STRUCTURAL BASIS ,Models, Molecular ,Biochemistry & Molecular Biology ,Protein family ,VON-WILLEBRAND-FACTOR ,Protein Conformation ,DISCOIDIN DOMAIN RECEPTORS ,0601 Biochemistry and Cell Biology ,Article ,Collagen Type I ,03 medical and health sciences ,Protein structure ,Recognition sequence ,A3 DOMAIN ,Heterotrimeric G protein ,DDR2 ,Humans ,Amino Acid Sequence ,Amino Acids ,Molecular Biology ,Peptide sequence ,030304 developmental biology ,0303 health sciences ,Science & Technology ,TYROSINE KINASES ,IDENTIFICATION ,0304 Medicinal and Biomolecular Chemistry ,Chemistry ,030302 biochemistry & molecular biology ,RECOGNITION ,Computational Biology ,Cell Biology ,Cell biology ,AFFINITY BINDING ,Collagen ,Peptides ,Life Sciences & Biomedicine ,Discoidin domain ,Type I collagen ,Triple helix - Abstract
The most abundant member of the collagen protein family, collagen I (also known as type I collagen; COL1), is composed of one unique (chain B) and two similar (chain A) polypeptides that self-assemble with one amino acid offset into a heterotrimeric triple helix. Given the offset, chain B can occupy either the leading (BAA), middle (ABA) or trailing (AAB) position of the triple helix, yielding three isomeric biomacromolecules with different protein recognition properties. Despite five decades of intensive research, there is no consensus on the position of chain B in COL1. Here, three triple-helical heterotrimers that each contain a putative von Willebrand factor (VWF) and discoidin domain receptor (DDR) recognition sequence from COL1 were designed with chain B permutated in all three positions. AAB demonstrated a strong preference for both VWF and DDR, and also induced higher levels of cellular DDR phosphorylation. Thus, we resolve this long-standing mystery and show that COL1 adopts an AAB register.
- Published
- 2020
23. Impact of UV- and carbodiimide-based crosslinking on the integrin-binding properties of collagen-based materials
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Samir W. Hamaia, Richard W. Farndale, Natalia Davidenko, Daniel V. Bax, Ruth E. Cameron, Serena M. Best, Bax, Daniel [0000-0002-1162-5319], Farndale, Richard [0000-0001-6130-8808], Best, Serena [0000-0001-7866-8607], Cameron, Ruth [0000-0003-1573-4923], and Apollo - University of Cambridge Repository
- Subjects
Ultraviolet Rays ,Integrin binding ,0206 medical engineering ,Integrin ,Biomedical Engineering ,Biocompatible Materials ,macromolecular substances ,02 engineering and technology ,Plasma protein binding ,Biochemistry ,Biomaterials ,chemistry.chemical_compound ,Platelet Adhesiveness ,Protein Domains ,Tissue engineering ,Cell Movement ,Cell Line, Tumor ,Animals ,Humans ,UV crosslinking ,Cell adhesion ,Molecular Biology ,Cell Proliferation ,Carbodiimide ,biology ,Chemistry ,Cell growth ,technology, industry, and agriculture ,General Medicine ,021001 nanoscience & nanotechnology ,020601 biomedical engineering ,In vitro ,3. Good health ,Carbodiimides ,Cross-Linking Reagents ,biology.protein ,Biophysics ,Cattle ,Collagen ,Integrin alpha2beta1 ,0210 nano-technology ,Protein Binding ,Biotechnology - Abstract
Collagen constructs are widely used for tissue engineering. These are frequently chemically crosslinked, using EDC, to improve their stability and tailor their physical properties. Although generally biocompatible, chemical crosslinking can modify crucial amino acid side chains, such as glutamic acid, that are involved in integrin-mediated cell adhesion. Instead UV crosslinking modifies aromatic side chains. Here we elucidate the impact that EDC, in combination with UV, exerts on the activity of integrin-binding motifs. By employing a model cell line that exclusively utilises integrin α2β1, we found that whilst EDC crosslinking modulated cell binding, from cation-dependent to cation-independent, UV-mediated crosslinking preserved native-like cell binding, proliferation and surface colonisation. Similar results were observed using a purified recombinant I-domain from integrin α1. Conversely, binding of the I-domain from integrin α2 was sensitive to UV, particularly at low EDC concentrations. Therefore, from this in vitro study, it appears that UV can be used to augment EDC whist retaining a specific subset of integrin-binding motifs in the native collagen molecule. These findings, delineating the EDC- and UV-susceptibility of cell-binding motifs, permit controlled cell adhesion to collagen-based materials through specific integrin ligation in vitro. However, in vivo, further consideration of the potential response to UV wavelength and dose is required in the light of literature reports that UV initiated collagen scission may lead to an adverse inflammatory response. STATEMENT OF SIGNIFICANCE: Recently, there has been rapid growth in the use of extracellular matrix-derived molecules, and in particular collagen, to fabricate biomaterials that replicate the cellular micro-environment. Often chemical or physical crosslinkers are required to enhance the biophysical properties of these materials. Despite extensive use of these crosslinkers, the cell-biological consequences have not been ascertained. To address this, we have investigated the integrin-binding properties of collagen after chemically crosslinking with EDC and physically crosslinking with UV-irradiation. We have established that whilst EDC crosslinking abates all of the integrin binding sites in collagen, UV selectively inhibits interaction with integrin-α2 but not -α1. By providing a mechanistic model for this behaviour, we have, for the first time, defined a series of crosslinking parameters to systematically control the interaction of collagen-based materials with defined cellular receptors.
- Published
- 2019
24. THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: Catalytic receptors
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Tom P. Monie, Simon D. Harding, Csaba Szabó, Lincoln R. Potter, Stephen P.H. Alexander, Peter Brouckaert, Jamie A. Davies, Harald H.H.W. Schmidt, Elena Faccenda, Christopher Southan, Eamonn Kelly, John Garthwaite, Scott A. Waldman, Jane F. Armstrong, Andreas Papapetropoulos, Michaela Kuhn, Adam J. Pawson, John A. Peters, David J. MacEwan, John C. Burnett, Clare E. Bryant, Annie Beuve, Richard W. Farndale, Andreas Friebe, Adrian J. Hobbs, Emma L. Veale, Gavin E. Jarvis, Alistair Mathie, Doriano Fabbro, RS: MHeNs - R3 - Neuroscience, Pharmacology and Personalised Medicine, Alexander, Stephen Ph [0000-0003-4417-497X], Fabbro, Doriano [0000-0002-9400-4517], Mathie, Alistair [0000-0001-6094-2890], Peters, John A [0000-0002-4277-4245], Veale, Emma L [0000-0002-6778-9929], Armstrong, Jane F [0000-0002-0524-0260], Faccenda, Elena [0000-0001-9855-7103], Harding, Simon D [0000-0002-9262-8318], Pawson, Adam J [0000-0003-2280-845X], Southan, Christopher [0000-0001-9580-0446], Davies, Jamie A [0000-0001-6660-4032], Bryant, Clare [0000-0002-2924-0038], Farndale, Richard W [0000-0001-6130-8808], Jarvis, Gavin E [0000-0003-4362-1133], MacEwan, David [0000-0002-2879-0935], Monie, Tom P [0000-0003-4097-1680], Papapetropoulos, Andreas [0000-0002-4253-5930], and Apollo - University of Cambridge Repository
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Pharmacology ,Clinical pharmacology ,Computer science ,Databases, Pharmaceutical ,Biology and Life Sciences ,Receptors, Cytoplasmic and Nuclear ,Ligands ,Ion Channels ,law.invention ,Receptors, G-Protein-Coupled ,Summary information ,law ,Medicine and Health Sciences ,Humans ,Catalytic receptors - Abstract
The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15541. Catalytic receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.
- Published
- 2021
25. Unravelling Form and Function: Improved function of engineered cardiac tissue through extra-cellular anisotropy
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Sanjay Sinha, Richard W. Farndale, Vera Graup, Jamie A. Cyr, Ruth E. Cameron, Semih Bayraktar, Maria Colzani, and Serena M. Best
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Cell physiology ,Contractility ,Scaffold ,Tissue engineering ,Chemistry ,Signal transduction ,Embryonic stem cell ,Intracellular ,Function (biology) ,Cell biology - Abstract
Cardiac tissue engineering is a promising therapeutic option for myocardial repair after injury, however, so far engineered heart patches have shown limited translational utility due to poor electrical integration and tissue contractility. Emerging research suggests that scaffolds that recapitulate the three-dimensional structure of the native myocardium improve physiological function. Complex scaffold fabrication remains a technical challenge and the isolated impact of scaffold architecture on tissue function and cellular physiology is poorly understood. Here, we provided a direct comparison between isotropic and aligned collagen scaffold morphologies where all confounding physio-mechanical features, such as strut wall thickness and surface roughness are conserved. This enabled the independent and systematic assessment of the effects of pore macro-architecture on global tissue function and cellular maturation. We seeded our scaffolds with embryonic stem cell derived cardiomyocytes (hESC-CM) and measured tissue function through calcium signal transduction and dynamic contractile strain. The aligned tissue constructs facilitated improved signalling synchronicity and directional contractility. We further examined the influence of scaffold macrostructure on intercellular organization and intracellular development. Cells on aligned constructs conformed to the orientation of the scaffold macro-structure and were found to have phenotypic and genetic markers of increased maturity. Our results isolate the influence of scaffold macro-structure on engineered tissue function at multiple length scales. These findings inform the design of optimized cardiac tissue and expand the potential for engineered tissue in regenerative and model medical systems by reducing the gaps in tissue functionality that limit their utility.
- Published
- 2021
26. The voltage-gated K
- Author
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Joy R, Wright, Sarah, Jones, Sasikumar, Parvathy, Leonard K, Kaczmarek, Ian, Forsythe, Richard W, Farndale, Jonathan M, Gibbins, and Martyn P, Mahaut-Smith
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Blood Platelets ,Platelet Adhesiveness ,Platelet Aggregation ,Potassium Channels, Voltage-Gated ,Humans ,Collagen ,Integrin alpha2beta1 - Abstract
Kv1.3 is a voltage-gated K
- Published
- 2021
27. Identification of HSP47 Binding Site on Native Collagen and Its Implications for the Development of HSP47 Inhibitors
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Samir W. Hamaia, Dominique Bihan, Gemma Little, Haiyan Cai, Parvathy Sasikumar, Jonathan M. Gibbins, Richard W. Farndale, Aiwu Zhou, Zhou, Aiwu [0000-0002-2555-5091], Gibbins, Jonathan M. [0000-0002-0372-5352], Farndale, Richard W. [0000-0001-6130-8808], Apollo - University of Cambridge Repository, Little, Gemma [0000-0001-9810-4358], Gibbins, Jonathan M [0000-0002-0372-5352], and Farndale, Richard W [0000-0001-6130-8808]
- Subjects
0301 basic medicine ,collagen ,animal structures ,Binding energy ,Microbiology ,Biochemistry ,Article ,03 medical and health sciences ,Residue (chemistry) ,0302 clinical medicine ,Humans ,structural analysis ,Binding site ,Molecular Biology ,HSP47 Heat-Shock Proteins ,Heat shock protein 47 ,chemistry.chemical_classification ,Binding Sites ,integumentary system ,biology ,Ligand binding assay ,fibrosis ,molecular docking ,HSP47 inhibitor ,QR1-502 ,Amino acid ,Molecular Docking Simulation ,Procollagen peptidase ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,embryonic structures ,biology.protein ,Function (biology) - Abstract
HSP47 (heat shock protein 47) is a collagen-specific molecular chaperone that is essential for procollagen folding and function. Previous studies have shown that HSP47 binding requires a critical Arg residue at the Y position of the (Gly-Xaa-Yaa) repeats of collagen, however, the exact binding sites of HSP47 on native collagens are not fully defined. To address this, we mapped the HSP47 binding sites on collagens through an ELISA binding assay using collagen toolkits, synthetic collagen peptides covering the entire amino acid sequences of collagen types II and III assembled in triple-helical conformation. Our results showed that HSP47 binds to only a few of the GXR motifs in collagen, with most of the HSP47 binding sites identified located near the N-terminal part of the triple-helical region. Molecular modelling and binding energy calculation indicated that residues flanking the key Arg in the collagen sequence also play an important role in defining the high-affinity HSP47 binding site of collagen. Based on this binding mode of HSP47 to collagen, virtual screening targeting both the Arg binding site and its neighboring area on the HSP47 surface, and a subsequent bioassay, we identified two novel compounds with blocking activity towards HSP47 binding of collagen. Overall, our study revealed the native HSP47 binding sites on collagen and provided novel information for the design of small-molecule inhibitors of HSP47.
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- 2021
28. Covalent Capture of a Heterotrimeric Collagen Helix
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Richard W. Farndale, Douglas R. Walker, Jeffrey D. Hartgerink, I-Che Li, Abhishek A. Jalan, and Sarah A. H. Hulgan
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Models, Molecular ,Protein Conformation, alpha-Helical ,Chemistry ,Collagen helix ,Organic Chemistry ,Supramolecular chemistry ,Biochemistry ,Folding (chemistry) ,Crystallography ,Protein structure ,Covalent bond ,Helix ,Peptide bond ,Amino Acid Sequence ,Collagen ,Protein Multimerization ,Physical and Theoretical Chemistry ,Triple helix - Abstract
Stabilizing the three-dimensional structure of supramolecular materials can be accomplished through covalent capture of the assembled system. The lysine-aspartate charge pairs designed to direct the self-assembly of a collagen triple helix were subsequently used to covalently capture the helix through proximity-directed amide bond formation using EDC/HOBT activation. The triple helix thus stabilized maintains its folded structure and can now be used for applications previously inaccessible due to problematic folding equilibria.
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- 2019
29. A Comprehensive UHPLC Ion Mobility Quadrupole Time-of-Flight Method for Profiling and Quantification of Eicosanoids, Other Oxylipins, and Fatty Acids
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Stephanie M. Jung, Gabriele Mocciaro, Sonia Liggi, Isuru Induruwa, Clare E. Bryant, Valerie B. O'Donnell, John C. Fjeldsted, Christine Hinz, Richard W. Farndale, Milton Pereira, Julian L. Griffin, Sven W. Meckelmann, Liggi, Sonia [0000-0003-1802-357X], Griffin, Julian L [0000-0003-1336-7744], and Apollo - University of Cambridge Repository
- Subjects
Chemie ,0904 Chemical Engineering ,010402 general chemistry ,Tandem mass spectrometry ,Mass spectrometry ,01 natural sciences ,LIPIDOMICS ,Analytical Chemistry ,Ion ,INFLAMMATION ,Tandem Mass Spectrometry ,ACTIVATED HUMAN PLATELETS ,Liquid chromatography–mass spectrometry ,Ion Mobility Spectrometry ,0399 Other Chemical Sciences ,Lipidomics ,Structural isomer ,Animals ,Humans ,ESTERIFIED EICOSANOIDS ,Oxylipins ,TANDEM MASS-SPECTROMETRY ,Cells, Cultured ,Chromatography, High Pressure Liquid ,chemistry.chemical_classification ,Science & Technology ,Chromatography ,Chemistry ,Fatty Acids ,Chemistry, Analytical ,010401 analytical chemistry ,Fatty acid ,Oxylipin ,DOCOSAHEXAENOIC-ACID ,0104 chemical sciences ,Mice, Inbred C57BL ,Physical Sciences ,Eicosanoids ,LIQUID-CHROMATOGRAPHY ,0301 Analytical Chemistry - Abstract
Analysis of oxylipins by liquid chromatography mass spectrometry (LC/MS) is challenging because of the small mass range occupied by this diverse lipid class, the presence of numerous structural isomers, and their low abundance in biological samples. Although highly sensitive LC/MS/MS methods are commonly used, further separation is achievable by using drift tube ion mobility coupled with high-resolution mass spectrometry (DTIM-MS). Herein, we present a combined analytical and computational method for the identification of oxylipins and fatty acids. We use a reversed-phase LC/DTIM-MS workflow able to profile and quantify (based on chromatographic peak area) the oxylipin and fatty acid content of biological samples while simultaneously acquiring full scan and product ion spectra. The information regarding accurate mass, collision-cross-section values in nitrogen (DTCCSN2), and retention times of the species found are compared to an internal library of lipid standards as well as the LIPID MAPS Structure Database by using specifically developed processing tools. Features detected within the DTCCSN2 and m/ z ranges of the analyzed standards are flagged as oxylipin-like species, which can be further characterized using drift-time alignment of product and precursor ions distinctive of DTIM-MS. This not only helps identification by reducing the number of annotations from LIPID MAPS but also guides discovery studies of potentially novel species. Testing the methodology on Salmonella enterica serovar Typhimurium-infected murine bone-marrow-derived macrophages and thrombin activated human platelets yields results in agreement with literature. This workflow has also annotated features as potentially novel oxylipins, confirming its ability in providing further insights into lipid analysis of biological samples.
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- 2019
30. Tailoring the biofunctionality of collagen biomaterials via tropoelastin incorporation and EDC-crosslinking
- Author
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Malavika Nair, Richard W. Farndale, Anthony S. Weiss, Daniel V. Bax, Serena M. Best, and Ruth E. Cameron
- Subjects
Biomedical Engineering ,Biocompatible Materials ,Cell morphology ,Biochemistry ,Biomaterials ,Extracellular matrix ,Cell surface receptor ,Tropoelastin ,Cell Adhesion ,Animals ,Cell adhesion ,Molecular Biology ,integumentary system ,biology ,Cell growth ,Chemistry ,Biomaterial ,Cellular response ,General Medicine ,Cell biology ,Elastin ,Rats ,biology.protein ,HT1080 ,Collagen ,Biotechnology - Abstract
Recreating the cell niche of virtually all tissues requires composite materials fabricated from multiple extracellular matrix (ECM) macromolecules. Due to their wide tissue distribution, physical attributes and purity, collagen, and more recently, tropoelastin, represent two appealing ECM components for biomaterials development. Here we blend tropoelastin and collagen, harnessing the cell-modulatory properties of each biomolecule. Tropoelastin was stably co-blended into collagen biomaterials and was retained after EDC-crosslinking. We found that human dermal fibroblasts (HDF), rat glial cells (Rugli) and HT1080 fibrosarcoma cells ligate to tropoelastin via EDTA-sensitive and EDTA-insensitive receptors or do not ligate with tropoelastin, respectively. These differing elastin-binding properties allowed us to probe the cellular response to the tropoelastin-collagen composites assigning specific bioactivity to the collagen and tropoelastin component of the composite material. Tropoelastin addition to collagen increased total Rugli cell adhesion, spreading and proliferation. This persisted with EDC-crosslinking of the tropoelastin-collagen composite. Tropoelastin addition did not affect total HDF and HT1080 cell adhesion; however, it increased the contribution of cation-independent adhesion, without affecting the cell morphology or, for HT1080 cells, proliferation. Instead, EDC-crosslinking dictated the HDF and HT1080 cellular response. These data show that a tropoelastin component dominates the response of cells that possess non-integrin based tropoelastin receptors. EDC modification of the collagen component directs cell function when non-integrin tropoelastin receptors are not crucial for cell activity. Using this approach, we have assigned the biological contribution of each component of tropoelastin-collagen composites, allowing informed biomaterial design for directed cell function via more physiologically relevant mechanisms. Statement of significance Biomaterials fabricated from multiple extracellular matrix (ECM) macromolecules are required to fully recreate the native tissue niche where each ECM macromolecule engages with a specific repertoire of cell-surface receptors. Here we investigate combining tropoelastin with collagen as they interact with cells via different receptors. We identified specific cell lines, which associate with tropoelastin via distinct classes of cell-surface receptor. These showed that tropoelastin, when combined with collagen, altered the cell behaviour in a receptor-usage dependent manner. Integrin-mediated tropoelastin interactions influenced cell proliferation and non-integrin receptors influenced cell spreading and proliferation. These data shed light on the interplay between biomaterial macromolecular composition, cell surface receptors and cell behaviour, advancing bespoke materials design and providing functionality to specific cell populations.
- Published
- 2021
31. Nonredundant Roles of Platelet Glycoprotein VI and Integrin αIIbβ3 in Fibrin-Mediated Microthrombus Formation
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Paola E. J. van der Meijden, Gina Perrella, Jingnan Huang, Mark Roest, Floor C J I Heubel-Moenen, Isabella Provenzale, Richard W. Farndale, Robert A. S. Ariëns, Steve P. Watson, Frauke Swieringa, Martine Jandrot-Perrus, Mark R Thomas, Johan W. M. Heemskerk, Biochemie, RS: Carim - B03 Cell biochemistry of thrombosis and haemostasis, MUMC+: MA Hematologie (9), Interne Geneeskunde, RS: Carim - B04 Clinical thrombosis and Haemostasis, and RS: Carim - B01 Blood proteins & engineering
- Subjects
Male ,0301 basic medicine ,ADHESION ,030204 cardiovascular system & hematology ,0302 clinical medicine ,fibrin ,blood platelet ,biology ,Chemistry ,Microfluidic Analytical Techniques ,Ligand (biochemistry) ,thrombin ,3. Good health ,Cell biology ,platelet aggregation ,Female ,GPVI ,Cardiology and Cardiovascular Medicine ,Thrombasthenia ,KEY ROLE ,medicine.drug ,Blood Platelets ,VON-WILLEBRAND-FACTOR ,Integrin ,microfluidics ,Platelet Glycoprotein GPIIb-IIIa Complex ,Platelet Membrane Glycoproteins ,Fibrin ,03 medical and health sciences ,Tissue factor ,Platelet Adhesiveness ,PROCOAGULANT ACTIVITY ,Thrombin ,Von Willebrand factor ,KINASE ,medicine ,Humans ,Syk Kinase ,Calcium Signaling ,Thrombus ,Blood Coagulation ,RECEPTOR ,Thrombosis ,medicine.disease ,COLLAGEN ,THROMBUS FORMATION ,030104 developmental biology ,TISSUE FACTOR ,Case-Control Studies ,biology.protein - Abstract
Objective: Fibrin is considered to strengthen thrombus formation via integrin αIIbβ3, but recent findings indicate that fibrin can also act as ligand for platelet glycoprotein VI. Approach and Results: To investigate the thrombus-forming potential of fibrin and the roles of platelet receptors herein, we generated a range of immobilized fibrin surfaces, some of which were cross-linked with factor XIIIa and contained VWF-BP (von Willebrand factor-binding peptide). Multicolor microfluidics assays with whole-blood flowed at high shear rate (1000 s −1 ) indicated that the fibrin surfaces, regardless of the presence of factor XIIIa or VWF-BP, supported platelet adhesion and activation (P-selectin expression), but only microthrombi were formed consisting of bilayers of platelets. Fibrinogen surfaces produced similar microthrombi. Markedly, tiggering of coagulation with tissue factor or blocking of thrombin no more than moderately affected the fibrin-induced microthrombus formation. Absence of αIIbβ3 in Glanzmann thrombasthenia annulled platelet adhesion. Blocking of glycoprotein VI with Fab 9O12 substantially, but incompletely reduced platelet secretion, Ca 2+ signaling and aggregation, while inhibition of Syk further reduced these responses. In platelet suspension, glycoprotein VI blockage or Syk inhibition prevented fibrin-induced platelet aggregation. Microthrombi on fibrin surfaces triggered only minimal thrombin generation, in spite of thrombin binding to the fibrin fibers. Conclusions: Together, these results indicate that fibrin fibers, regardless of their way of formation, act as a consolidating surface in microthrombus formation via nonredundant roles of platelet glycoprotein VI and integrin αIIbβ3 through signaling via Syk and low-level Ca 2+ rises.
- Published
- 2021
32. Selectivity of the collagen-binding integrin inhibitors, TC-I-15 and obtustatin
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Richard W. Farndale, Emma Hunter, Samir W. Hamaia, Donald Gullberg, Jean-Daniel Malcor, Farndale, Richard [0000-0001-6130-8808], and Apollo - University of Cambridge Repository
- Subjects
Angiogenesis ,Integrin ,Angiogenesis Inhibitors ,Viper Venoms ,Toxicology ,Article ,Cell Line ,Obtustatin ,Mice ,TC-I-15 ,Disintegrin ,Animals ,Cell adhesion ,Pharmacology ,biology ,Dose-Response Relationship, Drug ,Chemistry ,Transfection ,Adhesion ,Small molecule ,Cell biology ,biology.protein ,Collagen peptides ,C2C12 ,Collagen ,Integrin alpha2beta1 ,Protein Binding - Abstract
Integrins are a family of 24 adhesion receptors which are both widely-expressed and important in many pathophysiological cellular processes, from embryonic development to cancer metastasis. Hence, integrin inhibitors are valuable research tools which may have promising therapeutic uses. Here, we focus on the four collagen-binding integrins α1β1, α2β1, α10β1 and α11β1. TC-I-15 is a small molecule inhibitor of α2β1 that inhibits platelet adhesion to collagen and thrombus deposition, and obtustatin is an α1β1-specific disintegrin that inhibits angiogenesis. Both inhibitors were applied in cellular adhesion studies, using synthetic collagen peptide coatings with selective affinity for the different collagen-binding integrins and testing the adhesion of C2C12 cells transfected with each. Obtustatin was found to be specific for α1β1, as described, whereas TC-I-15 is shown to be non-specific, since it inhibits both α1β1 and α11β1 as well as α2β1. TC-I-15 was 100-fold more potent against α2β1 binding to a lower-affinity collagen peptide, suggestive of a competitive mechanism. These results caution against the use of integrin inhibitors in a therapeutic or research setting without testing for cross-reactivity., Graphical abstract Unlabelled Image, Highlights • TC-I-15 inhibits the collagen-binding integrin, α2β1, with IC50
- Published
- 2021
33. Modulating hESC-derived cardiomyocyte and endothelial cell function with triple-helical peptides for heart tissue engineering
- Author
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Serena M. Best, Emma Hunter, Jean-Daniel Malcor, Thomas Krieg, Sanjay Sinha, Richard W. Farndale, Semih Bayraktar, Murray Polkinghorne, Ruth E. Cameron, Maria Colzani, Krieg, Thomas [0000-0002-5192-580X], Cameron, Ruth [0000-0003-1573-4923], Best, Serena [0000-0001-7866-8607], Farndale, Richard [0000-0001-6130-8808], Sinha, Sanjay [0000-0001-5900-1209], and Apollo - University of Cambridge Repository
- Subjects
Cell type ,Human Embryonic Stem Cells ,Biophysics ,Bioengineering ,02 engineering and technology ,Cardiac tissue engineering ,Regenerative medicine ,Article ,Biomaterials ,03 medical and health sciences ,Tissue engineering ,Pluripotent stem cells ,Humans ,Myocytes, Cardiac ,Induced pluripotent stem cell ,030304 developmental biology ,0303 health sciences ,Tissue Engineering ,Chemistry ,Endothelial Cells ,Biomaterial ,Cell Differentiation ,021001 nanoscience & nanotechnology ,Embryonic stem cell ,In vitro ,Cell biology ,Endothelial stem cell ,Collagen biomaterials ,Mechanics of Materials ,Triple-helical peptides ,Ceramics and Composites ,Peptides ,0210 nano-technology - Abstract
In this study, we investigated the role of cardiomyocyte (CM) and endothelial cell (EC) specific interactions with collagen in the assembly of an operational myocardium in vitro. Engineered cardiac patches represent valuable tools for myocardial repair following infarction and are generally constituted of a suitable biomaterial populated by CMs and supportive cell types. Among those, ECs are required for tissue vascularization and positively modulate CM function. To direct the function of human embryonic stem cell (hESC)-derived CM and EC seeded on biomaterials, we replicated cell-collagen interactions, which regulate cellular behaviour in the native myocardium, using triple-helical peptides (THPs) that are ligands for collagen-binding proteins. THPs enhanced proliferation and activity of CMs and ECs separately and in co-culture, drove CM maturation and enabled coordinated cellular contraction on collagen films. These results highlight the importance of collagen interactions on cellular response and establish THP-functionalized biomaterials as novel tools to produce engineered cardiac tissues., Graphical abstract Image 1
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- 2020
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34. Platelet-primed interactions of coagulation and anticoagulation pathways in flow-dependent thrombus formation
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Sanne L. N. Brouns, Elisabetta Castoldi, Isabella Provenzale, Remco Verdoold, Henri M. H. Spronk, Johan W. M. Heemskerk, Paolo Simioni, Rachel Cavill, Marijke J E Kuijpers, Luca Spiezia, Frauke Swieringa, Paola E. J. van der Meijden, Elena Campello, Rene van Oerle, Richard W. Farndale, Johanna P. van Geffen, Kenneth J. Clemetson, Apollo - University of Cambridge Repository, Biochemie, RS: Carim - B03 Cell biochemistry of thrombosis and haemostasis, Promovendi CD, Interne Geneeskunde, RS: Carim - B04 Clinical thrombosis and Haemostasis, DKE Scientific staff, RS: FSE DACS, RS: FSE DACS Mathematics Centre Maastricht, MUMC+: HVC Pieken Trombose (9), RS: Carim - B01 Blood proteins & engineering, and Farndale, Richard [0000-0001-6130-8808]
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Male ,0301 basic medicine ,Thrombomodulin ,lcsh:Medicine ,030204 cardiovascular system & hematology ,692/4019/592/1339 ,ACTIVATION ,0302 clinical medicine ,Platelet ,lcsh:Science ,610 Medicine & health ,BLOOD-COAGULATION ,Multidisciplinary ,biology ,PLASMA ,Chemistry ,article ,Middle Aged ,Phenotype ,Coagulation ,PROTEIN-C ,Female ,Platelets ,Adult ,Blood Platelets ,631/250/2500 ,Fibrin ,Thromboplastin ,03 medical and health sciences ,Tissue factor ,medicine ,Humans ,HEMOSTASIS ,Platelet activation ,Thrombus ,Blood Coagulation ,lcsh:R ,Anticoagulants ,Thrombosis ,medicine.disease ,Coagulation system ,COLLAGEN ,FIBRIN ,CONCENTRATE ,Kinetics ,030104 developmental biology ,Case-Control Studies ,Hemostasis ,Hemorheology ,biology.protein ,Cancer research ,lcsh:Q ,RESISTANCE ,Protein C ,GENERATION - Abstract
In haemostasis and thrombosis, platelet, coagulation and anticoagulation pathways act together to produce fibrin-containing thrombi. We developed a microspot-based technique, in which we assessed platelet adhesion, platelet activation, thrombus structure and fibrin clot formation in real time using flowing whole blood. Microspots were made from distinct platelet-adhesive surfaces in the absence or presence of tissue factor, thrombomodulin or activated protein C. Kinetics of platelet activation, thrombus structure and fibrin formation were assessed by fluorescence microscopy. This work revealed: (1) a priming role of platelet adhesion in thrombus contraction and subsequent fibrin formation; (2) a surface-independent role of tissue factor, independent of the shear rate; (3) a mechanism of tissue factor-enhanced activation of the intrinsic coagulation pathway; (4) a local, suppressive role of the anticoagulant thrombomodulin/protein C pathway under flow. Multiparameter analysis using blood samples from patients with (anti)coagulation disorders indicated characteristic defects in thrombus formation, in cases of factor V, XI or XII deficiency; and in contrast, thrombogenic effects in patients with factor V-Leiden. Taken together, this integrative phenotyping approach of platelet–fibrin thrombus formation has revealed interaction mechanisms of platelet-primed key haemostatic pathways with alterations in patients with (anti)coagulation defects. It can help as an important functional add-on whole-blood phenotyping.
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- 2020
35. Collagen scaffolds functionalized with triple-helical peptides support 3D HUVEC culture
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Serena M. Best, Natalia Davidenko, Jean-Daniel Malcor, Richard W. Farndale, Sanjay Sinha, Daniel V. Bax, Emma Hunter, Ruth E. Cameron, Bax, Daniel [0000-0002-1162-5319], Cameron, Ruth [0000-0003-1573-4923], Best, Serena [0000-0001-7866-8607], Sinha, Sanjay [0000-0001-5900-1209], Farndale, Richard [0000-0001-6130-8808], and Apollo - University of Cambridge Repository
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Integrin ,02 engineering and technology ,Regenerative medicine ,Umbilical vein ,Biomaterials ,03 medical and health sciences ,collagen biomaterial ,Tissue engineering ,Receptor ,Research Articles ,030304 developmental biology ,collagen-mimetic peptides ,0303 health sciences ,biology ,Chemistry ,Biological activity ,Adhesion ,021001 nanoscience & nanotechnology ,endothelial cells ,Cell biology ,biology.protein ,AcademicSubjects/SCI01410 ,AcademicSubjects/MED00010 ,0210 nano-technology ,Discoidin domain ,3D cell scaffold - Abstract
Porous biomaterials which provide a structural and biological support for cells have immense potential in tissue engineering and cell-based therapies for tissue repair. Collagen biomaterials that can host endothelial cells represent promising tools for the vascularization of engineered tissues. Three-dimensional collagen scaffolds possessing controlled architecture and mechanical stiffness are obtained through freeze–drying of collagen suspensions, followed by chemical cross-linking which maintains their stability. However, cross-linking scaffolds renders their biological activity suboptimal for many cell types, including human umbilical vein endothelial cells (HUVECs), by inhibiting cell–collagen interactions. Here, we have improved crucial HUVEC interactions with such cross-linked collagen biomaterials by covalently coupling combinations of triple-helical peptides (THPs). These are ligands for collagen-binding cell-surface receptors (integrins or discoidin domain receptors) or secreted proteins (SPARC and von Willebrand factor). THPs enhanced HUVEC adhesion, spreading and proliferation on 2D collagen films. THPs grafted to 3D-cross-linked collagen scaffolds promoted cell survival over seven days. This study demonstrates that THP-functionalized collagen scaffolds are promising candidates for hosting endothelial cells with potential for the production of vascularized engineered tissues in regenerative medicine applications.
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- 2020
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36. Structural studies of the MMP-3 interaction with triple-helical collagen introduce new roles for the enzyme in tissue remodelling
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Szymon W. Manka, Richard W. Farndale, Dominique Bihan, and Apollo - University of Cambridge Repository
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Models, Molecular ,631/45 ,Protein Folding ,lcsh:Medicine ,Plasma protein binding ,631/1647/2258/1267 ,Matrix metalloproteinase ,Biochemistry ,Extracellular matrix ,03 medical and health sciences ,Zymogen ,Humans ,lcsh:Science ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,Chemistry ,lcsh:R ,030302 biochemistry & molecular biology ,article ,Cooperative binding ,Hemopexin ,Cell biology ,lcsh:Q ,Matrix Metalloproteinase 3 ,Molecular modelling ,Collagen ,Matrix Metalloproteinase 1 ,Wound healing ,Structural biology ,631/535 ,Triple helix ,Protein Binding - Abstract
Matrix metalloproteinase-3 (MMP-3) participates in normal extracellular matrix turnover during embryonic development, organ morphogenesis and wound healing, and in tissue-destruction associated with aneurysm, cancer, arthritis and heart failure. Despite its inability to cleave triple-helical collagens, MMP-3 can still bind to them, but the mechanism, location and role of binding are not known. We used the Collagen Toolkits, libraries of triple-helical peptides that embrace the entire helical domains of collagens II and III, to map MMP-3 interaction sites. The enzyme recognises five sites on collagen II and three sites on collagen III. They share a glycine-phenylalanine-hydroxyproline/alanine (GFO/A) motif that is recognised by the enzyme in a context-dependent manner. Neither MMP-3 zymogen (proMMP-3) nor the individual catalytic (Cat) and hemopexin (Hpx) domains of MMP-3 interact with the peptides, revealing cooperative binding of both domains to the triple helix. The Toolkit binding data combined with molecular modelling enabled us to deduce the putative collagen-binding mode of MMP-3, where all three collagen chains make contacts with the enzyme in the valley running across both Cat and Hpx domains. The observed binding pattern casts light on how MMP-3 could regulate collagen turnover and compete with various collagen-binding proteins regulating cell adhesion and proliferation.
- Published
- 2019
37. Corrigendum to ‘Anti-thrombotic efficacy of S007-867: Pre-clinical evaluation in experimental models of thrombosis in vivo and in vitro’ [Biochem. Pharmacol. 148 (2018) 288–297]
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Prem Prakash, Ankita Misra, Dominique Bihan, Manoj Kumar Barthwal, Hobby Aggarwal, Nicholas Pugh, Chandra Prakash Pandey, Dinesh K. Dikshit, Madhu Dikshit, Richard W. Farndale, Sachin Kumar, and Priyanka Dhankani
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Pharmacology ,Text mining ,In vivo ,business.industry ,medicine ,medicine.disease ,business ,Biochemistry ,Thrombosis ,Clinical evaluation ,In vitro - Published
- 2018
38. Integrins (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database
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Richard W. Farndale and Gavin E. Jarvis
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biology ,Computer science ,Integrin ,biology.protein ,Computational biology - Abstract
Integrins are unusual signalling proteins that function to signal both from the extracellular environment into the cell, but also from the cytoplasm to the external of the cell. The intracellular signalling cascades associated with integrin activation focus on protein kinase activities, such as focal adhesion kinase and Src. Based on this association between extracellular signals and intracellular protein kinase activity, we have chosen to include integrins in the 'Catalytic receptors' section of the database until more stringent criteria from NC-IUPHAR allows precise definition of their classification.Integrins are heterodimeric entities, composed of α and β subunits, each 1TM proteins, which bind components of the extracellular matrix or counter-receptors expressed on other cells. One class of integrin contains an inserted domain (I) in its α subunit, and if present (in α1, α2, α10, α11, αD, αE, αL, αM and αX), this I domain contains the ligand binding site. All β subunits possess a similar I-like domain, which has the capacity to bind ligand, often recognising the RGD motif. The presence of an α subunit I domain precludes ligand binding through the β subunit. Integrins provide a link between ligand and the actin cytoskeleton (through typically short intracellular domains). Integrins bind several divalent cations, including a Mg2+ ion in the I or I-like domain that is essential for ligand binding. Other cation binding sites may regulate integrin activity or stabilise the 3D structure. Integrins regulate the activity of particular protein kinases, including focal adhesion kinase and integrin-linked kinase. Cellular activation regulates integrin ligand affinity via inside-out signalling and ligand binding to integrins can regulate cellular activity via outside-in signalling.Several drugs that target integrins are in clinical use including: (1) abciximab (αIIbβ3) for short term prevention of coronary thrombosis, (2) vedolizumab (α4β7) to reduce gastrointestinal inflammation, and (3) natalizumab (α4β1) in some cases of severe multiple sclerosis.
- Published
- 2019
39. Structural studies of MMP-3 interaction with triple-helical collagen introduce the enzyme’s new roles in tissue remodelling
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Richard W. Farndale, Szymon W. Manka, and Dominique Bihan
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0303 health sciences ,Chemistry ,030302 biochemistry & molecular biology ,Cooperative binding ,Hemopexin ,Matrix metalloproteinase ,Stromelysin 1 ,Cell biology ,Extracellular matrix ,03 medical and health sciences ,Zymogen ,Wound healing ,030304 developmental biology ,Triple helix - Abstract
Matrix metalloproteinase-3 (MMP-3 or stromelysin 1) participates in normal extracellular matrix (ECM) turnover during embryonic development, organ morphogenesis and wound healing, and in tissue-destructive diseases, such as aneurysm, cancer, arthritis and heart failure. Despite its ability to hydrolyse numerous proteins in the ECM, MMP-3 fails to cleave the triple helix of interstitial fibrillar collagens. Nonetheless, it can still bind to these collagens although the mechanism, location and role of binding are not known. We used the Collagen Toolkits, libraries of triple-helical peptides that embrace the entire helical domains of collagens II and III, to map MMP-3 interaction sites. The enzyme recognises five sites on collagen II and three sites on collagen III. They share a glycine-phenylalanine-hydroxyproline/alanine (GFO/A) motif that is recognised by the enzyme in a context-dependent manner. Neither MMP-3 zymogen (proMMP-3) nor the individual catalytic (Cat) and hemopexin (Hpx) domains of MMP-3 interact with the peptides, revealing cooperative binding of both domains to the triple helix. The Toolkit binding data combined with molecular modelling enabled us to deduce the putative collagen-binding mode of MMP-3, where all three collagen chains make contacts with the enzyme in the valley running across both Cat and Hpx domains. The observed binding pattern casts light on how MMP-3 could regulate collagen turnover and compete with various collagen-binding proteins regulating cell adhesion and proliferation.
- Published
- 2019
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40. Role of Platelet Glycoprotein VI and Tyrosine Kinase Syk in Thrombus Formation on Collagen-Like Surfaces
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Marijke J.E. Kuijpers, Johan W. M. Heemskerk, Rachel Cavill, Delia I. Fernandez, Ilaria De Simone, Hugo ten Cate, Natalie J Jooss, Richard W. Farndale, Isabella Provenzale, Paola E. J. van der Meijden, Yvonne M. C. Henskens, Sanne L. N. Brouns, De Simone, Ilaria [0000-0003-1037-8456], Farndale, Richard W [0000-0001-6130-8808], Cavill, Rachel [0000-0002-3796-1687], Apollo - University of Cambridge Repository, RS: CARIM - R1 - Thrombosis and haemostasis, RS: Carim - B03 Cell biochemistry of thrombosis and haemostasis, Promovendi CD, Biochemie, Faculteit FHML Centraal, RS: CARIM - R1.04 - Clinical thrombosis and haemostasis, MUMC+: DA CDL Algemeen (9), Med Microbiol, Infect Dis & Infect Prev, MUMC+: HVC Pieken Trombose (9), RS: CARIM - R1.03 - Cell biochemistry of thrombosis and haemostasis, MUMC+: DA CDL Analytisch cluster 1K (9), Interne Geneeskunde, MUMC+: MA Alg Interne Geneeskunde (9), RS: Carim - B04 Clinical thrombosis and Haemostasis, MUMC+: HVC Trombosezorg (8), DKE Scientific staff, and RS: FSE DACS
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0301 basic medicine ,collagen ,Platelet Aggregation ,Syk ,030204 cardiovascular system & hematology ,ADHESION ,Collagen receptor ,lcsh:Chemistry ,ACTIVATION ,0302 clinical medicine ,BINDING ,Platelet ,lcsh:QH301-705.5 ,Spectroscopy ,Cells, Cultured ,Cyclohexylamines ,biology ,Chemistry ,General Medicine ,3. Good health ,Computer Science Applications ,Cell biology ,thrombus ,GPVI ,Tyrosine kinase ,Proto-oncogene tyrosine-protein kinase Src ,circulatory and respiratory physiology ,SRC ,Blood Platelets ,Integrin ,Platelet Membrane Glycoproteins ,Catalysis ,Article ,Inorganic Chemistry ,glycoprotein VI ,03 medical and health sciences ,platelet activation ,Humans ,Syk Kinase ,Platelet activation ,Physical and Theoretical Chemistry ,Molecular Biology ,Protein Kinase Inhibitors ,calcium ,IDENTIFICATION ,RECEPTOR ,Organic Chemistry ,protein tyrosine kinase ,Thrombosis ,RECOGNIZES ,Peptide Fragments ,FIBRIN ,030104 developmental biology ,Pyrimidines ,lcsh:Biology (General) ,lcsh:QD1-999 ,biology.protein - Abstract
Platelet interaction with collagens, via von Willebrand factor, is a potent trigger of shear-dependent thrombus formation mediated by subsequent engagement of the signaling collagen receptor glycoprotein (GP)VI, enforced by integrin &alpha, 2&beta, 1. Protein tyrosine kinase Syk is central in the GPVI-induced signaling pathway, leading to elevated cytosolic Ca2+. We aimed to determine the Syk-mediated thrombogenic activity of several collagen peptides and (fibrillar) type I and III collagens. High-shear perfusion of blood over microspots of these substances resulted in thrombus formation, which was assessed by eight parameters and was indicative of platelet adhesion, activation, aggregation, and contraction, which were affected by the Syk inhibitor PRT-060318. In platelet suspensions, only collagen peptides containing the consensus GPVI-activating sequence (GPO)n and Horm-type collagen evoked Syk-dependent Ca2+ rises. In whole blood under flow, Syk inhibition suppressed platelet activation and aggregation parameters for the collagen peptides with or without a (GPO)n sequence and for all of the collagens. Prediction models based on a regression analysis indicated a mixed role of GPVI in thrombus formation on fibrillar collagens, which was abolished by Syk inhibition. Together, these findings indicate that GPVI-dependent signaling through Syk supports platelet activation in thrombus formation on collagen-like structures regardless of the presence of a (GPO)n sequence.
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- 2019
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41. Intrinsic local destabilization of the C-terminus predisposes integrin α1 I domain to a conformational switch induced by collagen binding
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Jean Baum, Richard W. Farndale, Conceição A.S.A. Minetti, Jie Zhu, Ana Monica Nunes, Jacqueline Jezioro, David P. Remeta, and Samir W. Hamaia
- Subjects
0301 basic medicine ,030102 biochemistry & molecular biology ,biology ,Chemistry ,C-terminus ,Allosteric regulation ,Protein domain ,Integrin ,Mutant ,Wild type ,Isothermal titration calorimetry ,Biochemistry ,03 medical and health sciences ,030104 developmental biology ,Biophysics ,biology.protein ,Hydrogen–deuterium exchange ,Molecular Biology - Abstract
Integrin-collagen interactions play a critical role in a myriad of cellular functions that include immune response, and cell development and differentiation, yet their mechanism of binding is poorly understood. There is increasing evidence that conformational flexibility assumes a central role in the molecular mechanisms of protein-protein interactions and here we employ NMR hydrogen-deuterium exchange (HDX) experiments to explore the impact of slower timescale dynamic events. To gain insight into the mechanisms underlying collagen-induced conformational switches, we have undertaken a comparative study between the wild type integrin α1 I and a gain-of-function E317A mutant. NMR HDX results suggest a relationship between regions exhibiting a reduced local stability in the unbound I domain and those that undergo significant conformational changes upon binding. Specifically, the αC and α7 helices within the C-terminus are at the center of such major perturbations and present reduced local stabilities in the unbound state relative to other structural elements. Complementary isothermal titration calorimetry experiments have been performed to derive complete thermodynamic binding profiles for association of the collagen-like triple-helical peptide with wild type α1 I and E317A mutant. The differential energetics observed for E317A are consistent with the HDX experiments and support a model in which intrinsically destabilized regions predispose conformational rearrangement in the integrin I domain. This study highlights the importance of exploring different timescales to delineate allosteric and binding events.
- Published
- 2016
42. Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase
- Author
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Dominique Bihan, Sebastian Kalamajski, Arkadiusz Bonna, Richard W. Farndale, Kristofer Rubin, Kalamajski, Sebastian [0000-0002-6600-9302], and Apollo - University of Cambridge Repository
- Subjects
collagen ,0301 basic medicine ,fibromodulin ,extracellular matrix ,Molecular Sequence Data ,Glycobiology and Extracellular Matrices ,Lysyl oxidase ,macromolecular substances ,Matrix (biology) ,Fibril ,Biochemistry ,Cell Line ,Protein-Lysine 6-Oxidase ,Extracellular matrix ,Mice ,03 medical and health sciences ,Enzyme activator ,Animals ,Humans ,Amino Acid Sequence ,Protein Interaction Maps ,Binding site ,Molecular Biology ,Peptide sequence ,Mice, Knockout ,Extracellular Matrix Proteins ,small leucine-rich proteoglycan (SLRP) ,Binding Sites ,Chemistry ,Cell Biology ,Cell biology ,Enzyme Activation ,Collagen, type I, alpha 1 ,030104 developmental biology ,Proteoglycans ,lysyl oxidase ,Gene Deletion ,cross-linking - Abstract
The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.
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- 2016
43. Structural basis for collagen recognition by the immune receptor OSCAR
- Author
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Alexander D. Barrow, Dimitri Y. Chirgadze, Michal Blaszczyk, Andrew B. Herr, Deborah G. Conrady, Richard W. Farndale, Long Zhou, Jeanette L.C. Miller, Jennifer M. Hinerman, and Dominique Bihan
- Subjects
0301 basic medicine ,Immunology ,Cell Biology ,Hematology ,Immune receptor ,Plasma protein binding ,Biology ,Platelet membrane glycoprotein ,Biochemistry ,Molecular biology ,Collagen receptor ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Ectodomain ,030220 oncology & carcinogenesis ,GPVI ,Binding site ,Receptor - Abstract
The osteoclast-associated receptor (OSCAR) is a collagen-binding immune receptor with important roles in dendritic cell maturation and activation of inflammatory monocytes as well as in osteoclastogenesis. The crystal structure of the OSCAR ectodomain is presented, both free and in complex with a consensus triple-helical peptide (THP). The structures revealed a collagen-binding site in each immunoglobulin-like domain (D1 and D2). The THP binds near a predicted collagen-binding groove in D1, but a more extensive interaction with D2 is facilitated by the unusually wide D1-D2 interdomain angle in OSCAR. Direct binding assays, combined with site-directed mutagenesis, confirm that the primary collagen-binding site in OSCAR resides in D2, in marked contrast to the related collagen receptors, glycoprotein VI (GPVI) and leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). Monomeric OSCAR D1D2 binds to the consensus THP with a KD of 28 µM measured in solution, but shows a higher affinity (KD 1.5 μM) when binding to a solid-phase THP, most likely due to an avidity effect. These data suggest a 2-stage model for the interaction of OSCAR with a collagen fibril, with transient, low-affinity interactions initiated by the membrane-distal D1, followed by firm adhesion to the primary binding site in D2.
- Published
- 2016
44. NMR spectroscopy of native and in vitro tissues implicates polyADP ribose in biomineralization
- Author
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Maggie Green, Richard W. Farndale, Karin H. Müller, W. Ying Chow, Catherine M. Shanahan, Dominique Bihan, David G. Reid, Jeremy N. Skepper, David A. Slatter, Wai Ching Wong, Rakesh Rajan, Roger A. Brooks, Melinda J. Duer, Rajan, Rakesh [0000-0002-3647-6233], Mueller, Karin [0000-0003-4693-8558], Reid, David [0000-0003-2464-5295], Farndale, Richard [0000-0001-6130-8808], Duer, Melinda [0000-0002-9196-5072], and Apollo - University of Cambridge Repository
- Subjects
Carbon Isotopes ,Poly Adenosine Diphosphate Ribose ,Multidisciplinary ,Bone Development ,Sheep ,Nitrogen Isotopes ,Adenosine diphosphate ribose ,Chemical shift ,Nuclear magnetic resonance spectroscopy ,Models, Biological ,In vitro ,Extracellular Matrix ,NMR spectra database ,chemistry.chemical_compound ,Mice ,Calcification, Physiologic ,chemistry ,Solid-state nuclear magnetic resonance ,Biochemistry ,Ribose ,Animals ,Growth Plate ,Nuclear Magnetic Resonance, Biomolecular ,Biomineralization - Abstract
Fundamentals of Bone Formation In vitro models can help guide research for tissue engineering or drug delivery, but the extent to which results from in vitro experiments may mimic in vivo ones will depend on the robustness of the model. For complex tissues like the extracellular matrix or bone, this means matching the chemical organization of the tissue at both the atomic scale and the structural level. Chow et al. (p. 742 ) used nuclear magnetic resonance (NMR) spectroscopy to analyze a sample on both these length scales. First an isotope-enriched mouse was produced to enhance the NMR signal. Samples from these mice were then used to study the extracellular matrix of developing bone and the calcification front during fetal bone growth.
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- 2018
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45. Cellular response to collagen-elastin composite materials
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Daniel V, Bax, Helen E, Smalley, Richard W, Farndale, Serena M, Best, and Ruth E, Cameron
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Solubility ,Cell Movement ,Cell Line, Tumor ,Cell Adhesion ,Animals ,Humans ,Biocompatible Materials ,Cattle ,Cell Count ,Collagen ,Stress, Mechanical ,Cell Proliferation ,Elastin - Abstract
Collagen is used extensively in tissue engineering due to its biocompatibility, near-universal tissue distribution, low cost and purity. However, native tissues are composites that include diverse extracellular matrix components, which influence strongly their mechanical and biological properties. Here, we provide important new findings on the differential regulation, by collagen and elastin, of the bio-response to the composite material. Soluble and insoluble elastin had differing effects on the stiffness and failure strength of the composite films. We established that Rugli cells bind elastin via EDTA-sensitive receptors, whilst HT1080 cells do not. These cells allowed us to probe the contribution of collagen alone (HT1080) and collagen plus elastin (Rugli) to the cellular response. In the presence of elastin, Rugli cell attachment, spreading and proliferation increased, presumably through elastin-binding receptors. By comparison, the attachment and spreading of HT1080 cells was modified by elastin inclusion, but without affecting their proliferation, indicating indirect modulation by elastin of the response of cells to collagen. These new insights highlight that access to elastin dominates the cellular response when elastin-binding receptors are present. In the absence of these receptors, modification of the collagen component and/or physical properties dictate the cellular response. Therefore, we can attribute the contribution of each constituent on the ultimate bioactivity of heterogeneous collagen-composite materials, permitting informed, systematic biomaterials design. STATEMENT OF SIGNIFICANCE: In recent years there has been a desire to replicate the complex extracellular matrix composition of tissues more closely, necessitating the need for composite protein-based materials. In this case both the physical and biochemical properties are altered with the addition of each component, with potential consequences on the cell. To date, the different contributions of each component have not been deconvolved, and instead the cell response to the scaffold as a whole has been observed. Instead, here, we have used specific cell lines, that are sensitive to specific components of an elastin-collagen composite, to resolve the bio-activity of each protein. This has shown that elastin-induced alteration of the collagen component can modulate early stage cell behaviour. By comparison the elastin component directly alters the cell response over the short and long term, but only where appropriate receptors are present on the cell. Due to the widespread use of collagen and elastin, we feel that this data permits, for the first time, the ability to systematically design collagen-composite materials to promote desired cell behaviour with associated advantages for biomaterials fabrication.
- Published
- 2018
46. Thrombospondin-1 promotes matrix homeostasis by interacting with collagen and lysyl oxidase precursors and collagen cross-linking sites
- Author
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Nicholas Pugh, Josephine C. Adams, Richard W. Farndale, Arkadiusz Bonna, Silvia Rosini, and David J.S. Hulmes
- Subjects
0301 basic medicine ,Male ,Sequence Homology ,Lysyl oxidase ,Matrix metalloproteinase ,Matrix (biology) ,Biochemistry ,Collagen Type I ,Extracellular matrix ,Protein-Lysine 6-Oxidase ,Thrombospondin 1 ,03 medical and health sciences ,Mice ,0302 clinical medicine ,medicine ,Extracellular ,Animals ,Homeostasis ,Humans ,Amino Acid Sequence ,Fibroblast ,Thrombospondins ,Molecular Biology ,Cells, Cultured ,Skin ,Mice, Knockout ,Extracellular Matrix Proteins ,Chemistry ,Cell Biology ,Fibroblasts ,Cell biology ,Extracellular Matrix ,Mice, Inbred C57BL ,030104 developmental biology ,medicine.anatomical_structure ,Cross-Linking Reagents ,030220 oncology & carcinogenesis ,Intracellular - Abstract
Fibrillar collagens of the extracellular matrix are critical for tissue structure and physiology; however, excessive or abnormal deposition of collagens is a defining feature of fibrosis. Regulatory mechanisms that act on collagen fibril assembly potentially offer new targets for antifibrotic treatments. Tissue weakening, altered collagen fibril morphologies, or both, are shared phenotypes of mice lacking matricellular thrombospondins. Thrombospondin-1 (TSP1) plays an indirect role in collagen homeostasis through interactions with matrix metalloproteinases and transforming growth factor–1 (TGF-1). We found that TSP1 also affects collagen fibril formation directly. Compared to skin from wild-type mice, skin from Thbs1-/- mice had reduced collagen cross-linking and reduced prolysyl oxidase (proLOX) abundance with increased conversion to catalytically active LOX. In vitro, TSP1 bound to both the C-propeptide domain of collagen I and the highly conserved KGHR sequences of the collagen triple-helical domain that participate in cross-linking. TSP1 also bound to proLOX and inhibited proLOX processing by bone morphogenetic protein-1. In human dermal fibroblasts (HDFs), TSP1 and collagen I colocalized in intracellular vesicles and on extracellular collagen fibrils, whereas TSP1 and proLOX colocalized only in intracellular vesicles. Inhibition of LOX-mediated collagen cross-linking did not prevent the extracellular association between collagen and TSP1; however, treatment of HDFs with KGHR-containing, TSP1-binding, triple-helical peptides disrupted the collagen-TSP1 association, perturbed the collagen extracellular matrix, and increased myofibroblastic differentiation in a manner that depended on TGF- receptor 1. Thus, the extracellular KGHR-dependent interaction of TSP1 with fibrillar collagens contributes to fibroblast homeostasis.
- Published
- 2018
47. MMP-13 binds to platelet receptors αIIbβ3 and GPVI and impairs aggregation and thrombus formation
- Author
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Richard W. Farndale, Samir W. Hamaia, Joanna-Marie Howes, Stephanie M. Jung, Vera Knäuper, Nicholas Pugh, Jean-Daniel Malcor, Jung, Stephanie M [0000-0002-7409-9715], and Apollo - University of Cambridge Repository
- Subjects
0301 basic medicine ,GPVI collagen receptor ,Matrix metalloproteinase ,Platelet membrane glycoprotein ,03 medical and health sciences ,0302 clinical medicine ,matrix metalloproteinase‐13 ,medicine ,Platelet ,Platelet activation ,cardiovascular diseases ,Thrombus ,Receptor ,thrombosis ,Original Articles: Haemostasis ,Chemistry ,Hematology ,medicine.disease ,Thrombosis ,030104 developmental biology ,platelets ,Cancer research ,Original Article ,Integrin alphaIIbbeta3 (αIIbβ3) ,GPVI ,030217 neurology & neurosurgery - Abstract
Essentials MMP‐13 has the potential to influence platelet function and thrombus formation directly.We sought to elucidate whether MMP‐13 is able to bind to specific platelet receptors.MMP‐13 is able to bind to platelet alphaIIbbeta3 (αIIbβ3) and glycoprotein (GP)VI.These interactions are sufficient to inhibit platelet aggregation and thrombus formation. Background Acute thrombotic syndromes lead to atherosclerotic plaque rupture with subsequent thrombus formation, myocardial infarction and stroke. Following rupture, flowing blood is exposed to plaque components, including collagen, which triggers platelet activation and aggregation. However, plaque rupture releases other components into the surrounding vessel which have the potential to influence platelet function and thrombus formation. Objectives Here we sought to elucidate whether matrix metalloproteinase‐13 (MMP‐13), a collagenolytic metalloproteinase up‐regulated in atherothrombotic and inflammatory conditions, affects platelet aggregation and thrombus formation. Results We demonstrate that MMP‐13 is able to bind to platelet receptors alphaIIbbeta3 (αIIbβ3) and platelet glycoprotein (GP)VI. The interactions between MMP‐13, GPVI and αIIbβ3 are sufficient to significantly inhibit washed platelet aggregation and decrease thrombus formation on fibrillar collagen. Conclusions Our data demonstrate a role for MMP‐13 in the inhibition of both platelet aggregation and thrombus formation in whole flowing blood, and may provide new avenues of research into the mechanisms underlying the subtle role of MMP‐13 in atherothrombotic pathologies.
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- 2018
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48. Correction to: Evaluation of cell binding to collagen and gelatin: a study of the effect of 2D and 3D architecture and surface chemistry
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Natalia Davidenko, Carlos F. Schuster, Serena M. Best, Samir W. Hamaia, Daniel V. Bax, Ruth E. Cameron, and Richard W. Farndale
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0301 basic medicine ,food.ingredient ,Materials science ,Polymer science ,Chemistry ,Biomedical Engineering ,Biophysics ,Bioengineering ,02 engineering and technology ,Biomaterials Synthesis and Characterization ,021001 nanoscience & nanotechnology ,Gelatin ,Biomaterials ,03 medical and health sciences ,Cell binding ,030104 developmental biology ,food ,0210 nano-technology - Abstract
Studies of cell attachment to collagen-based materials often ignore details of the binding mechanisms—be they integrin-mediated or non-specific. In this work, we have used collagen and gelatin-based substrates with different dimensional characteristics (monolayers, thin films and porous scaffolds) in order to establish the influence of composition, crosslinking (using carbodiimide) treatment and 2D or 3D architecture on integrin-mediated cell adhesion. By varying receptor expression, using cells with collagen-binding integrins (HT1080 and C2C12 L3 cell lines, expressing α2β1, and Rugli expressing α1β1) and a parent cell line C2C12 with gelatin-binding receptors (αvβ3 and α5β1), the nature of integrin binding sites was studied in order to explain the bioactivity of different protein formulations. We have shown that alteration of the chemical identity, conformation and availability of free binding motifs (GxOGER and RGD), resulting from addition of gelatin to collagen and crosslinking, have a profound effect on the ability of cells to adhere to these formulations. Carbodiimide crosslinking ablates integrin-dependent cell activity on both two-dimensional and three-dimensional architectures while the three-dimensional scaffold structure also leads to a high level of non-specific interactions remaining on three-dimensional samples even after a rigorous washing regime. This phenomenon, promoted by crosslinking, and attributed to cell entrapment, should be considered in any assessment of the biological activity of three-dimensional substrates. Spreading data confirm the importance of integrin-mediated cell engagement for further cell activity on collagen-based compositions. In this work, we provide a simple, but effective, means of deconvoluting the effects of chemistry and dimensional characteristics of a substrate, on the cell activity of protein-derived materials, which should assist in tailoring their biological properties for specific tissue engineering applications. Graphical Abstract
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- 2018
49. Anti-thrombotic efficacy of S007-867: pre-clinical evaluation in experimental models of thrombosis in vivo and in vitro
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Manoj Kumar Barthwal, Nicholas Pugh, Dominique Bihan, Prem Prakash, Dinesh K. Dikshit, Ankita Misra, Sachin Kumar, Chandra Prakash Pandey, Madhu Dikshit, Richard W. Farndale, Hobby Aggarwal, Priyanka Dhankani, Farndale, Richard [0000-0001-6130-8808], and Apollo - University of Cambridge Repository
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0301 basic medicine ,Male ,Pyrrolidines ,Platelet Aggregation ,Blood flow velocity ,030204 cardiovascular system & hematology ,Pharmacology ,Biochemistry ,Ferric Compounds ,Rats, Sprague-Dawley ,03 medical and health sciences ,Thromboxane A2 ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,Platelet Adhesiveness ,Chlorides ,Fibrinolytic Agents ,Piperidines ,Bleeding time ,Platelet adhesiveness ,Antithrombotic ,medicine ,Animals ,Platelet ,Platelet activation ,Blood Coagulation ,Hemostasis ,medicine.diagnostic_test ,Aspirin ,Dose-Response Relationship, Drug ,business.industry ,Thrombosis ,Clopidogrel ,Rats ,030104 developmental biology ,chemistry ,Collagen ,business ,medicine.drug - Abstract
Pharmacological inhibition of platelet collagen interaction is a promising therapeutic strategy to treat intra-vascular thrombosis. S007-867 is a novel synthetic inhibitor of collagen-induced platelet aggregation. It has shown better antithrombotic protection than aspirin and clopidogrel with minimal bleeding tendency in mice. The present study is aimed to systematically investigate the antithrombotic efficacy of S007-867 in comparison to aspirin and clopidogrel in vivo and to delineate its mechanism of action in vitro. Aspirin, clopidogrel, and S007-867 significantly reduced thrombus weight in arterio-venous (AV) shunt model in rats. In mice, following ferric chloride induced thrombosis in either carotid or mesenteric artery; S007-867 significantly prolonged the vessel occlusion time (1.2-fold) and maintained a sustained blood flow velocity for >30 min. Comparatively, clopidogrel showed significant prolongation in TTO (1.3-fold) while aspirin remained ineffective. Both S007-867 and aspirin did not alter bleeding time in either kidney or spleen injury models, and thus maintained hemostasis, while clopidogrel showed significant increase in spleen bleeding time (1.7-fold). The coagulation parameters namely thrombin time, prothrombin time or activated partial thromboplastin time remained unaffected even at high concentration of S007-867 (300 µM), thus implying its antithrombotic effect to be primarily platelet mediated. S007-867 significantly inhibited collagen-mediated platelet adhesion and aggregation in mice ex-vivo. Moreover, when blood was perfused over a highly thrombogenic combination of collagen mimicking peptides like CRP-GFOGER-VWF-III, S007-867 significantly reduced total thrombus volume or ZV50 (53.4 ± 5.7%). Mechanistically, S007-867 (10-300 μM) inhibited collagen-induced ATP release, thromboxane A2 (TxA2) generation, intra-platelet [Ca+2] flux and global tyrosine phosphorylation including PLCγ2. Collectively the present study highlights that S007-867 is a novel synthetic inhibitor of collagen induced platelet activation, that effectively maintains blood flow velocity and delays vascular occlusion. It inhibits thrombogenesis without compromising hemostasis. Therefore, S007-867 may be further developed for the treatment of thrombotic disorders in clinical settings.
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- 2018
50. Collagen Gly missense mutations: Effect of residue identity on collagen structure and integrin binding
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Richard W. Farndale, Yu-Shan Lin, Barbara Brodsky, Yimin Qiu, Samir W. Hamaia, Sezin Yigit, Hongtao Yu, David L. Kaplan, and Arya Mekkat
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0301 basic medicine ,Protein Folding ,animal structures ,Protein Conformation ,Glycine ,Mutation, Missense ,medicine.disease_cause ,Collagen Type I ,Protein Structure, Secondary ,Article ,law.invention ,03 medical and health sciences ,Residue (chemistry) ,Structural Biology ,law ,medicine ,Missense mutation ,Humans ,Amino Acid Sequence ,Integrin binding ,Mutation ,030102 biochemistry & molecular biology ,integumentary system ,Chemistry ,Circular Dichroism ,Hydrogen Bonding ,Osteogenesis Imperfecta ,Trypsin ,Molecular biology ,030104 developmental biology ,Amino Acid Substitution ,embryonic structures ,Recombinant DNA ,Type I collagen ,Triple helix ,medicine.drug ,Protein Binding - Abstract
Gly missense mutations in type I collagen, which replace a conserved Gly in the repeating (Gly-Xaa-Yaa)(n) sequence with a larger residue, are known to cause Osteogenesis Imperfecta (OI). The clinical consequences of such mutations range from mild to lethal, with more serious clinical severity associated with larger Gly replacement residues. Here, we investigate the influence of the identity of the residue replacing Gly within and adjacent to the integrin binding (502)GFPGER(507) sequence on triple-helix structure, stability and integrin binding using a recombinant bacterial collagen system. Recombinant collagens were constructed with Gly substituted by Ala, Ser or Val at four positions within the integrin binding region. All constructs formed a stable triple-helix structure with a small decrease in melting temperature. Trypsin was used to probe local disruption of the triple helix, and Gly to Val replacements made the triple helix trypsin sensitive at three of the four sites. Any mutation at Gly505, eliminated integrin binding, while decreased integrin binding affinity was observed in the replacement of Gly residues at Gly502 following the order Val > Ser > Ala. Molecular dynamics simulations indicated that all Gly replacements led to transient disruption of triple-helix interchain hydrogen bonds in the region of the Gly replacement. These computational and experimental results lend insight into the complex molecular basis of the varying clinical severity of OI.
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- 2018
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