Papers by Richard Farndale
Thrombosis and Haemostasis, Jun 1, 2007
The adhesion of leukocytes to immobilised platelets may contribute to inflammatory and thrombotic... more The adhesion of leukocytes to immobilised platelets may contribute to inflammatory and thrombotic responses in damaged tissue. To investigate the conditions under which platelets and leukocytes might be deposited together in vessels, we perfused fluorescently-labelled whole blood through glass capillaries coated with various collagen preparations. Video-microscopic observations of the surface showed that platelets formed numerous, individual, rolling and stationary attachments to surfaces coated with acid-soluble, monomeric collagen. However, leukocyte interactions with the deposited platelets were rare. If the blood was washed out, the adherent platelets became more activated, and many rolling adherent leukocytes were observed if a second bolus of blood was perfused over them. This suggested that platelet activation had initially been inadequate to support leukocyte capture. Next, fibrillar collagen was adsorbed to the capillaries to present an ordered array of peptide motifs to platelet receptor glycoprotein (Gp)VI and transduce an activating signal. In this case, platelets were deposited in discrete, stable aggregates and the bound platelets captured many flowing leukocytes. Alternatively, acid-soluble collagen was seeded with collagen-related peptide (CRP) known to contain a GpVI-binding motif. Again, platelet adhesion became stable, and numerous flowing leukocytes were captured. Addition of antibody against GpVI or against P-selectin greatly reduced leukocyte adhesion to the platelets. Thus, in whole blood, platelets binding to exposed collagen need to be activated through GpVI in order to expose sufficient P-selectin to allow efficient capture of flowing leukocytes to take place.
Protein science : a publication of the Protein Society, Jan 24, 2016
Integrin-collagen interactions play a critical role in a myriad of cellular functions that includ... more Integrin-collagen interactions play a critical role in a myriad of cellular functions that include immune response, cell development and differentiation, and 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 lo...
Endocrinology, 1991
Leukotriene B4 is one of a number of agents which stimulate bone resorption by acting on osteobla... more Leukotriene B4 is one of a number of agents which stimulate bone resorption by acting on osteoblasts. Some agonists, such as PTH or prostaglandins, are known to activate adenylate cyclase in osteoblasts, whereas others, such as vitamin D3, have no effect on adenylate cyclase. Recent evidence suggests that both classes of agonist may raise the intracellular calcium concentration, although the relative importance for bone resorption of calcium mobilization and adenylate cyclase activity in the osteoblast is not clear. Here it is shown 1) that leukotriene B4 does not activate but may be inhibitory toward adenylate cyclase in intact osteoblasts or membrane preparations, 2) that leukotriene B4 causes an elevation of intracellular calcium levels in osteoblast monolayers, 3) leukotriene B4 rapidly activates phosphatidylinositol bisphosphate breakdown in osteoblast membranes and intact osteoblasts, and 4) that leukotriene B4 stimulates phosphatidylinositol kinase activity concurrently with phosphoinositidase C in intact osteoblasts over a similar timescale. These results suggest that leukotriene B4 may increase the concentration of intracellular calcium in osteoblasts by stimulating phosphoinositide turnover, and support the proposal that calcium signaling rather than activation of adenylate cyclase in osteoblasts may be of overriding importance in the regulation of bone resorption.
J Mol Biol, 2004
We have determined the 1.8 Å crystal structure of a triple helical integrin-binding collagen pept... more We have determined the 1.8 Å crystal structure of a triple helical integrin-binding collagen peptide (IBP) with sequence (Gly-Pro-Hyp)2-Gly-Phe-Hyp-Gly-Glu-Arg-(Gly-Pro-Hyp)3. The central GFOGER hexapeptide is recognised specifically by the integrins α2β1, α1β1, α10β1 and α11β1. These integrin/collagen interactions are implicated in a number of key physiological processes including cell adhesion, cell growth and differentiation, and pathological states such as thrombosis and tumour metastasis. Comparison of the IBP structure with the previously determined structure of an identical collagen peptide in complex with the integrin α2-I domain (IBPc) allows the first detailed examination of collagen in a bound and an unbound state. The IBP structure shows a direct and a water-mediated electrostatic interaction between Glu and Arg side-chains from adjacent strands, but no intra-strand interactions. The interactions between IBP Glu and Arg side-chains are disrupted upon integrin binding. A comparison of IBP and IBPc main-chain conformation reveals the flexible nature of the triple helix backbone in the imino-poor GFOGER region. This flexibility could be important to the integrin–collagen interaction and provides a possible explanation for the unique orientation of the three GFOGER strands observed in the integrin-IBPc complex crystal structure.
Cell, 2000
We have determined the crystal structure of a complex between the I domain of integrin α2β1 and a... more We have determined the crystal structure of a complex between the I domain of integrin α2β1 and a triple helical collagen peptide containing a critical GFOGER motif. Three loops on the upper surface of the I domain that coordinate a metal ion also engage the collagen, with a collagen glutamate completing the coordination sphere of the metal. Comparison with the unliganded I domain reveals a change in metal coordination linked to a reorganization of the upper surface that together create a complementary surface for binding collagen. Conformational changes propagate from the upper surface to the opposite pole of the domain, suggesting both a basis for affinity regulation and a pathway for signal transduction. The structural features observed here may represent a general mechanism for integrin–ligand recognition.
Studies have suggested a pivotal role for free sulfhydryls in platelet integrin function, and enz... more Studies have suggested a pivotal role for free sulfhydryls in platelet integrin function, and enzyme-mediated reduction of disulfide bonds on platelets has been implicated. The platelet fibrinogen receptor alpha(IIb)beta(3) is the best-studied platelet integrin and serves as a model system for studying the structure-function relation in this family of adhesion receptors. The demonstration of free sulfhydryls on the exofacial domain of purified alpha(IIb)beta(3), specifically in its activated conformation, prompted us to explore the potential for activation-dependent, enzymatically catalyzed thiol expression on intact platelets and the possible role of surface-associated protein disulfide isomerase (PDI) in alpha(IIb)beta(3) ligation. Using the membrane-impermeant sulfhydryl blocker para-chloromercuriphenyl sulfonate, the inhibitor of disulfide exchange bacitracin, and the monoclonal anti-PDI antibody RL90, we examined fibrinogen binding to alpha(IIb)beta(3) as well as ligation-induced allosteric changes in the conformation of alpha(IIb)beta(3). We sought to distinguish the possible involvement of disulfide exchange in agonist-induced platelet stimulation from its role in integrin ligation. Analysis of the role of free thiols in platelet aggregation suggested a thiol-independent initial ligation followed by a thiol-dependent stabilization of binding. Flow cytometric analysis showed that sustained binding of fibrinogen, as well as expression of ligand-induced binding site epitopes and ligand-bound conformation, depended on free thiols and disulfide exchange. Expression of P-selectin was minimally affected, even with complete inhibition of alpha(IIb)beta(3) function. These data indicate that although agonist-induced platelet stimulation is independent of ecto-sulfhydryls, engagement of integrin alpha(IIb)beta(3) on the intact platelet depends totally on their enzymatically catalyzed surface expression.
Blood 2012, 2012
Inhibition of Ca(2+) mobilization by cyclic nucleotides is central to the mechanism whereby endot... more Inhibition of Ca(2+) mobilization by cyclic nucleotides is central to the mechanism whereby endothelial-derived prostacyclin and nitric oxide limit platelet activation in the intact circulation. However, we show that ∼ 50% of the Ca(2+) response after stimulation of glycoprotein VI (GPVI) by collagen, or of Toll-like 2/1 receptors by Pam(3)Cys-Ser-(Lys)(4) (Pam(3)CSK(4)), is resistant to prostacyclin. At low agonist concentrations, the prostacyclin-resistant Ca(2+) response was predominantly because of P2X1 receptors activated by ATP release via a phospholipase-C-coupled secretory pathway requiring both protein kinase C and cytosolic Ca(2+) elevation. At higher agonist concentrations, an additional pathway was observed because of intracellular Ca(2+) release that also depended on activation of phospholipase C and, for TLR 2/1, PI3-kinase. Secondary activation of P2X1-dependent Ca(2+) influx also persisted in the presence of nitric oxide, delivered from spermine NONOate, or increased ectonucleotidase levels (apyrase). Surprisingly, apyrase was more effective than prostacyclin and NO at limiting secondary P2X1 activation. Dilution of platelets reduced the average extracellular ATP level without affecting the percentage contribution of P2X1 receptors to collagen-evoked Ca(2+) responses, indicating a highly efficient activation mechanism by local ATP. In conclusion, platelets possess inhibitor-resistant Ca(2+) mobilization pathways, including P2X1 receptors, that may be particularly important during early thrombotic or immune-dependent platelet activation.
The pathophysiology of microthrombocy- topenia in the Wiskott-Aldrich syndrome (WAS) and its mild... more The pathophysiology of microthrombocy- topenia in the Wiskott-Aldrich syndrome (WAS) and its milder form, X-linked throm- bocytopenia (XLT), is unclear. Although quantitative defects are correctable by splenectomy, residual platelet abnormali- ties are suggestive of intrinsic distur- bances of production. In contrast to hu- man patients, murine models of WASp deficiency exhibit only mild thrombocyto- penia, and platelets are of normal
Thrombosis and Haemostasis
In the platelet, ATP-gated P2X1 receptors have been reported to amplify functional responses to c... more In the platelet, ATP-gated P2X1 receptors have been reported to amplify functional responses to collagen, however the relative importance of early CCa2+ mobilisation events is unknown. We now report that selective desensitisation of P2X1 receptor activity leads to a major reduction in the initial intracellular Ca2+ responses to a wide range of collagen concentrations (0.25-2 microg ml(-1)). Peak [Ca2+](i) increases were reduced to 8.5 and 55% of control, and the maximum rate of rise was reduced to 12 and 33% of control, at low and high collagen concentrations, respectively. This P2X1-dependent acceleration and enhancement of collagen-stimulated Ca2+ responses was not observed in the absence of extracellular Ca2+. These results demonstrate a major role for ATP-gated Ca2+ influx in the early collagen-evoked Ca2+ signals and can at least partly explain the important contribution of P2X1 receptors to arterial thrombosis.
Neuropharmacology, Jan 28, 2015
There are no commercially available, small, receptor-specific P2X1 ligands. There are several syn... more There are no commercially available, small, receptor-specific P2X1 ligands. There are several synthetic derivatives of the natural agonist ATP and some structurally-complex antagonists including compounds such as PPADS, NTP-ATP, suramin and its derivatives (e.g. NF279, NF449). NF449 is the most potent and selective ligand, but potencies of many others are not particularly high and they can also act at other P2X, P2Y and non-purinergic receptors. While there is clearly scope for further work on P2X1 receptor pharmacology, screening can be difficult owing to rapid receptor desensitisation. To reduce desensitisation substitutions can be made within the N-terminus of the P2X1 receptor, but these could also affect ligand properties. An alternative is the use of fluorescent voltage-sensitive dyes that respond to membrane potential changes resulting from channel opening. Here we utilised this approach in conjunction with fragment-based drug-discovery. Using a single concentration (300 μM) ...
The Journal of biological chemistry, Jan 29, 2014
Remodeling of collagen by matrix metalloproteinases (MMPs) is crucial to tissue homeostasis and r... more Remodeling of collagen by matrix metalloproteinases (MMPs) is crucial to tissue homeostasis and repair. MMP-13 is a collagenase with a substrate preference for collagen II over collagens I and III. It recognizes a specific, well-known site in the tropocollagen molecule where its binding locally perturbs the triple helix, allowing the catalytic domain of the active enzyme to cleave the collagen α chains sequentially, at Gly(775)-Leu(776) in collagen II. However, the specific residues upon which collagen recognition depends within and surrounding this locus have not been systematically mapped. Using our triple-helical peptide Collagen Toolkit libraries in solid-phase binding assays, we found that MMP-13 shows little affinity for Collagen Toolkit III, but binds selectively to two triple-helical peptides of Toolkit II. We have identified the residues required for the adhesion of both proMMP-13 and MMP-13 to one of these, Toolkit peptide II-44, which contains the canonical collagenase cl...
Thrombosis and haemostasis, 2005
In the platelet, ATP-gated P2X1 receptors have been reported to amplify functional responses to c... more In the platelet, ATP-gated P2X1 receptors have been reported to amplify functional responses to collagen, however the relative importance of early CCa2+ mobilisation events is unknown. We now report that selective desensitisation of P2X1 receptor activity leads to a major reduction in the initial intracellular Ca2+ responses to a wide range of collagen concentrations (0.25-2 microg ml(-1)). Peak [Ca2+](i) increases were reduced to 8.5 and 55% of control, and the maximum rate of rise was reduced to 12 and 33% of control, at low and high collagen concentrations, respectively. This P2X1-dependent acceleration and enhancement of collagen-stimulated Ca2+ responses was not observed in the absence of extracellular Ca2+. These results demonstrate a major role for ATP-gated Ca2+ influx in the early collagen-evoked Ca2+ signals and can at least partly explain the important contribution of P2X1 receptors to arterial thrombosis.
Journal of molecular biology, Jan 23, 2004
We have determined the 1.8A crystal structure of a triple helical integrin-binding collagen pepti... more We have determined the 1.8A crystal structure of a triple helical integrin-binding collagen peptide (IBP) with sequence (Gly-Pro-Hyp)(2)-Gly-Phe-Hyp-Gly-Glu-Arg-(Gly-Pro-Hyp)(3). The central GFOGER hexapeptide is recognised specifically by the integrins alpha2beta1, alpha1beta1, alpha10beta1 and alpha11beta1. These integrin/collagen interactions are implicated in a number of key physiological processes including cell adhesion, cell growth and differentiation, and pathological states such as thrombosis and tumour metastasis. Comparison of the IBP structure with the previously determined structure of an identical collagen peptide in complex with the integrin alpha2-I domain (IBP(c)) allows the first detailed examination of collagen in a bound and an unbound state. The IBP structure shows a direct and a water-mediated electrostatic interaction between Glu and Arg side-chains from adjacent strands, but no intra-strand interactions. The interactions between IBP Glu and Arg side-chains ar...
Blood, Jan 2, 2014
Mitochondrial DNA (mtDNA) is a highly potent inflammatory trigger and is reportedly found outside... more Mitochondrial DNA (mtDNA) is a highly potent inflammatory trigger and is reportedly found outside the cells in blood in various pathologies. Platelets are abundant in blood where they promote hemostasis. Although lacking a nucleus, platelets contain functional mitochondria. On activation, platelets produce extracellular vesicles known as microparticles. We hypothesized that activated platelets could also release their mitochondria. We show that activated platelets release respiratory-competent mitochondria, both within membrane-encapsulated microparticles and as free organelles. Extracellular mitochondria are found in platelet concentrates used for transfusion and are present at higher levels in those that induced acute reactions (febrile nonhemolytic reactions, skin manifestations, and cardiovascular events) in transfused patients. We establish that the mitochondrion is an endogenous substrate of secreted phospholipase A2 IIA (sPLA2-IIA), a phospholipase otherwise specific for bact...
Thrombosis and Haemostasis, 2007
The adhesion of leukocytes to immobilised platelets may contribute to inflammatory and thrombotic... more The adhesion of leukocytes to immobilised platelets may contribute to inflammatory and thrombotic responses in damaged tissue. To investigate the conditions under which platelets and leukocytes might be deposited together in vessels, we perfused fluorescently-labelled whole blood through glass capillaries coated with various collagen preparations. Video-microscopic observations of the surface showed that platelets formed numerous, individual, rolling and stationary attachments to surfaces coated with acid-soluble, monomeric collagen. However, leukocyte interactions with the deposited platelets were rare. If the blood was washed out, the adherent platelets became more activated, and many rolling adherent leukocytes were observed if a second bolus of blood was perfused over them. This suggested that platelet activation had initially been inadequate to support leukocyte capture. Next, fibrillar collagen was adsorbed to the capillaries to present an ordered array of peptide motifs to platelet receptor glycoprotein (Gp)VI and transduce an activating signal. In this case, platelets were deposited in discrete, stable aggregates and the bound platelets captured many flowing leukocytes. Alternatively, acid-soluble collagen was seeded with collagen-related peptide (CRP) known to contain a GpVI-binding motif. Again, platelet adhesion became stable, and numerous flowing leukocytes were captured. Addition of antibody against GpVI or against P-selectin greatly reduced leukocyte adhesion to the platelets. Thus, in whole blood, platelets binding to exposed collagen need to be activated through GpVI in order to expose sufficient P-selectin to allow efficient capture of flowing leukocytes to take place.
Journal of Thrombosis and Haemostasis, 2007
To cite this article: Farndale RW, Slatter DA, Siljander PRM, Jarvis GE. Platelet receptor recogn... more To cite this article: Farndale RW, Slatter DA, Siljander PRM, Jarvis GE. Platelet receptor recognition and cross-talk in collagen-induced activation of platelets. J Thromb Haemost 2007; 5 (Suppl. 1): 220-9.
Journal of Molecular Biology, 2004
We have determined the 1.8 Å crystal structure of a triple helical integrinbinding collagen pepti... more We have determined the 1.8 Å crystal structure of a triple helical integrinbinding collagen peptide (IBP) with sequence (Gly-Pro-Hyp) 2 -Gly-Phe-Hyp-Gly-Glu-Arg-(Gly-Pro-Hyp) 3 . The central GFOGER hexapeptide is recognised specifically by the integrins a2b1, a1b1, a10b1 and a11b1. These integrin/collagen interactions are implicated in a number of key physiological processes including cell adhesion, cell growth and differentiation, and pathological states such as thrombosis and tumour metastasis. Comparison of the IBP structure with the previously determined structure of an identical collagen peptide in complex with the integrin a2-I domain (IBP c ) allows the first detailed examination of collagen in a bound and an unbound state. The IBP structure shows a direct and a water-mediated electrostatic interaction between Glu and Arg side-chains from adjacent strands, but no intra-strand interactions. The interactions between IBP Glu and Arg side-chains are disrupted upon integrin binding. A comparison of IBP and IBP c main-chain conformation reveals the flexible nature of the triple helix backbone in the imino-poor GFOGER region. This flexibility could be important to the integrin -collagen interaction and provides a possible explanation for the unique orientation of the three GFOGER strands observed in the integrin-IBP c complex crystal structure.
Journal of Biological Chemistry, 1996
Signal transduction pathways that mediate C5a and fMet-Leu-Phe (fMLP)-induced pertussis toxin (PT... more Signal transduction pathways that mediate C5a and fMet-Leu-Phe (fMLP)-induced pertussis toxin (PTx)-sensitive activation of phospholipase C (PLC) have been investigated using a cotransfection assay system in COS-7 cells. The abilities of the receptors for C5a and fMLP to activate PLC beta2 and PLC beta3 through the Gbetagamma subunits of endogenous Gi proteins in COS-7 cells were tested because both PLC beta2 and PLC beta3 were shown to be activated by the betagamma subunits of G proteins in in vitro reconstitution assays. Neither of the receptors can activate endogenous PLC beta3 or recombinant PLC beta3 in transfected COS-7 cells. However, both receptors can clearly activate PLC beta2 in a PTx-sensitive manner, suggesting that the receptors may interact with endogenous PTx-sensitive G proteins and activate PLC beta2 probably through the Gbetagamma subunits. These findings were further corroborated by the results that PLC beta3 could only be slightly activated by Gbeta1gamma1 or Gbeta1gamma5 in the cotransfection assay, whereas the Gbetagamma subunits strongly activated PLC beta2 under the same conditions. PLC beta3 can be activated by Galphaq, Galpha11, and Galpha16 in the cotransfection assay. In addition, the Ggamma2 and Ggamma3 mutants with substitution of the C-terminal Cys residue by a Ser residue, which can inhibit wild type Gbetagamma-mediated activation of PLC beta2, were able to inhibit C5a or fMLP-mediated activation of PLC beta2. These Ggamma mutants, however, showed little effect on m1-muscarinic receptor-mediated PLC activation, which is mediated by the Gq class of G proteins. These results all confirm that the Gbetagamma subunits are involved in PLC beta2 activation by the two chemoattractant receptors and suggest that in COS-7 cells activation of PLC beta3 by Gbetagamma may not be the primary pathway for the receptors.
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Papers by Richard Farndale