[show abstract][hide abstract] ABSTRACT: Drugs are often used in combination and, for pharmacologists, the manner of their interactions can cast light on drug mechanisms and biological processes. Here we provide simplified descriptions of commonly used analytical methods for analysing drug combinations and describe a new and practical experimental solution to address the mechanistic question: 'Do two channel-blocking drugs bind at the same site?' We define two simple mathematical models that describe the effects of two channel blockers acting simultaneously at either the same (Syntopic Model) or different (Allotopic Model) binding sites within a channel pore. We find that the optimum concentrations of two drugs for distinguishing between the two models are related to the mathematical Golden Ratio.
Trends in Pharmacological Sciences 08/2013; · 9.25 Impact Factor
[show abstract][hide abstract] ABSTRACT: Recently, the ability of polymeric collagen-like peptides to regulate cell behavior has generated great interest. A triple-helical peptide known as collagen-related peptide (CRP) contains the sequence (Gly-Pro-Hyp)(10). With Gly-Pro-Cys triplets appended to both of its termini, designated CRP(cys), chemical cross-linking using heterobifunctional reagents generates CRP(cys)-XL, a potent, widely used, polymeric agonist for platelet Glycoprotein VI, whereas non-cross-linked, monomeric CRP(cys) antagonizes Glycoprotein VI. Here, we describe how cysteine in these triplets may also undergo random air-induced oxidation, especially upon prolonged storage or repeated freeze-thawing, to form disulphide bonds, resulting in a lesser degree of polymerization than with chemical cross-linking. We investigated the monomeric and polymeric states of these and other cysteine-containing collagen-derived peptides, using gel filtration and dynamic light scattering, allowing the size of a CRP-XL aggregate to be estimated. The effect of cysteine thiols upon peptide adsorption to surfaces and subsequent platelet responses was investigated. This demonstrated that cysteine is required for strong binding to glass coverslips and to plastic plates used in ELISA assays.
[show abstract][hide abstract] ABSTRACT: This study was designed to assess the effect of Factor Xa antagonists on thrombus formation at various axial positions on a tissue factor rich surface under arterial blood flow conditions. Non-anticoagulated, flowing human blood, drawn directly from an antecubital vein, was perfused over a tissue factor coated cover slip in a parallel-plate perfusion chamber. Thrombus surface coverage, thrombus mean height and fibrin surface coverage were measured at six different axial positions by confocal microscopy. Both thrombus surface coverage and mean height decreased along the cover slip axis whereas the fibrin surface coverage increased. Pre-chamber treatment of blood with the direct Factor Xa inhibitors Razaxaban and 813893 resulted in significantly reduced thrombus and fibrin formation at all axial positions investigated (P < 0.05). Thrombus and fibrin deposition in a laminar flow chamber changed with axial position with surface coverage measurements being more reproducible than thrombus mean height. Data were more reproducible towards the centre of the flow chamber than at the extremities. Razaxaban and 813893 inhibited thrombus and fibrin formation at the highest concentrations tested. No difference in drug effect was apparent at different axial positions. In conclusion, axial position influences the degree of thrombus and fibrin deposition with measurements being less reproducible at the extremities of the flow chamber. This technique may prove useful for analysing anti-thrombotic drug effects before progression to clinical trials.
Journal of Thrombosis and Thrombolysis 11/2011; 33(1):6-15. · 1.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: Collagen-induced platelet activation is a key step in the development of arterial thrombosis via its interaction with the receptors glycoprotein (GP)VI and integrin α(2) β(1) . Adhesion and degranulation-promoting adapter protein (ADAP) regulates α(IIb) β(3) in platelets and α(L) β(2) in T cells, and is phosphorylated in GPVI-deficient platelets activated by collagen.
To determine whether ADAP plays a role in collagen-induced platelet activation and in the regulation and function of α(2) β(1).
Using ADAP(-/-) mice and synthetic collagen peptides, we investigated the role of ADAP in platelet aggregation, adhesion, spreading, thromboxane synthesis, and tyrosine phosphorylation.
Platelet aggregation and phosphorylation of phospholipase Cγ2 induced by collagen were attenuated in ADAP(-/-) platelets. However, aggregation and signaling induced by collagen-related peptide (CRP), a GPVI-selective agonist, were largely unaffected. Platelet adhesion to CRP was also unaffected by ADAP deficiency. Adhesion to the α(2) β(1) -selective ligand GFOGER and to a peptide (III-04), which supports adhesion that is dependent on both GPVI and α(2) β(1), was reduced in ADAP(-/-) platelets. An impedance-based label-free detection technique, which measures adhesion and spreading of platelets, indicated that, in the absence of ADAP, spreading on GFOGER was also reduced. This was confirmed with non-fluorescent differential-interference contrast microscopy, which revealed reduced filpodia formation in ADAP(-/-) platelets adherent to GFOGER. This indicates that ADAP plays a role in mediating platelet activation via the collagen-binding integrin α(2) β(1). In addition, we found that ADAP(-/-) mice, which are mildly thrombocytopenic, have enlarged spleens as compared with wild-type animals. This may reflect increased removal of platelets from the circulation.
Journal of Thrombosis and Haemostasis 11/2011; 10(2):268-77. · 6.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Neutrophils are activated by immunoglobulin G (IgG)-containing immune complexes through receptors that recognize the Fc portion of IgG (FcγRs). Here, we used genetic and pharmacological approaches to define a selective role for the β isoform of phosphoinositide 3-kinase (PI3Kβ) in FcγR-dependent activation of mouse neutrophils by immune complexes of IgG and antigen immobilized on a plate surface. At low concentrations of immune complexes, loss of PI3Kβ alone substantially inhibited the production of reactive oxygen species (ROS) by neutrophils, whereas at higher doses, similar suppression of ROS production was achieved only by targeting both PI3Kβ and PI3Kδ, suggesting that this pathway displays stimulus strength-dependent redundancy. Activation of PI3Kβ by immune complexes involved cooperation between FcγRs and BLT1, the receptor for the endogenous proinflammatory lipid leukotriene B₄. Coincident activation by a tyrosine kinase-coupled receptor (FcγR) and a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (BLT1) may provide a rationale for the preferential activation of the β isoform of PI3K. PI3Kβ-deficient mice were highly protected in an FcγR-dependent model of autoantibody-induced skin blistering and were partially protected in an FcγR-dependent model of inflammatory arthritis, whereas combined deficiency of PI3Kβ and PI3Kδ resulted in near-complete protection in the latter case. These results define PI3Kβ as a potential therapeutic target in inflammatory disease.
[show abstract][hide abstract] ABSTRACT: Pea encodes eukaryotic translation initiation factor eIF4E (eIF4E(S)), which supports the multiplication of Pea seed-borne mosaic virus (PSbMV). In common with hosts for other potyviruses, some pea lines contain a recessive allele (sbm1) encoding a mutant eIF4E (eIF4E(R)) that fails to interact functionally with the PSbMV avirulence protein, VPg, giving genetic resistance to infection.
To study structure-function relationships between pea eIF4E and PSbMV VPg, we obtained an X-ray structure for eIF4E(S) bound to m(7)GTP. The crystallographic asymmetric unit contained eight independent copies of the protein, providing insights into the structurally conserved and flexible regions of eIF4E. To assess indirectly the importance of key residues in binding to VPg and/or m(7)GTP, an extensive range of point mutants in eIF4E was tested for their ability to complement PSbMV multiplication in resistant pea tissues and for complementation of protein translation, and hence growth, in an eIF4E-defective yeast strain conditionally dependent upon ectopic expression of eIF4E. The mutants also dissected individual contributions from polymorphisms present in eIF4E(R) and compared the impact of individual residues altered in orthologous resistance alleles from other crop species. The data showed that essential resistance determinants in eIF4E differed for different viruses although the critical region involved (possibly in VPg-binding) was conserved and partially overlapped with the m(7)GTP-binding region. This overlap resulted in coupled inhibition of virus multiplication and translation in the majority of cases, although the existence of a few mutants that uncoupled the two processes supported the view that the specific role of eIF4E in potyvirus infection may not be restricted to translation.
The work describes the most extensive structural analysis of eIF4E in relation to potyvirus resistance. In addition to defining functional domains within the eIF4E structure, we identified eIF4E alleles with the potential to convey novel virus resistance phenotypes.
PLoS ONE 01/2011; 6(1):e15873. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Extracts from the Ginkgo biloba tree are widely used as herbal medicines, and include bilobalide (BB) and ginkgolides A and B (GA and GB). Here we examine their effects on human 5-HT(3)A and 5-HT(3)AB receptors, and compare these to the effects of the structurally related compounds picrotin (PTN) and picrotoxinin (PXN), the two components of picrotoxin (PTX), a known channel blocker of 5-HT(3), nACh and GABA(A) receptors. The compounds inhibited 5-HT-induced responses of 5-HT(3) receptors expressed in Xenopus oocytes, with IC(50) values of 470 μM (BB), 730 μM (GB), 470 μM (PTN), 11 μM (PXN) and >1mM (GA) in 5-HT(3)A receptors, and 3.1mM (BB), 3.9 mM (GB), 2.7 mM (PTN), 62 μM (PXN) and >1mM (GA) in 5-HT(3)AB receptors. Radioligand binding on receptors expressed in HEK 293 cells showed none of the compounds displaced the specific 5-HT(3) receptor antagonist [(3)H]granisetron, confirming that they do not act at the agonist binding site. Inhibition by GB at 5-HT(3)A receptors is weakly use-dependent, and recovery is activity dependent, indicating channel block. To further probe their site of action at 5-HT(3)A receptors, BB and GB were applied alone or in combination with PXN, and the results fitted to a mathematical model; the data revealed partially overlapping sites of action. We conclude that BB and GB block the channel of the 5-HT(3)A receptor. Thus these compounds have comparable, although less potent, behaviour than at some other Cys-loop receptors, demonstrating their actions are conserved across the family.
[show abstract][hide abstract] ABSTRACT: Phagocytosis and activation of the NADPH oxidase are important mechanisms by which neutrophils and macrophages engulf and kill microbial pathogens. We investigated the role of PI3K signaling pathways in the regulation of the oxidase during phagocytosis of Staphylococcus aureus and Escherichia coli by mouse and human neutrophils, a mouse macrophage-like cell line and a human myeloid-like cell line. Phagocytosis of these bacteria was promoted by serum, independent of serum-derived antibodies, and effectively abolished in mouse neutrophils lacking the beta(2)-integrin common chain, CD18. A combination of PI3K isoform-selective inhibitors, mouse knock-outs, and RNA-interference indicated CD18-dependent activation of the oxidase was independent of class I and II PI3Ks, but substantially dependent on the single class III isoform (Vps34). Class III PI3K was responsible for the synthesis of PtdIns(3)P on phagosomes containing either bacteria. The use of mouse neutrophils carrying an appropriate knock-in mutation indicated that PtdIns(3)P binding to the PX domain of their p40(phox) oxidase subunit is important for oxidase activation in response to both S aureus and E coli. This interaction does not, however, account for all the PI3K sensitivity of these responses, particularly the oxidase response to E coli, suggesting that additional mechanisms for PtdIns(3)P-regulation of the oxidase must exist.
[show abstract][hide abstract] ABSTRACT: We have analyzed the adhesion of human and murine platelets, and of recombinant human and murine GpVI ectodomains, to synthetic triple-helical collagen-like peptides. These included 57 peptides derived from the sequence of human type III collagen and 9 peptides derived from the cyanogen bromide fragment of bovine type III collagen, alpha1(III)CB4. We have identified several peptides that interact with GpVI, in particular a peptide designated III-30 with the sequence GAOGLRGGAGPOGPEGGKGAAGPOGPO. Both human and murine platelets bound to peptide III-30 in a GpVI-dependent manner. III-30 also supported binding of recombinant GpVI ectodomains. Cross-linked III-30 induced aggregation of human and murine platelets, although with a lower potency than collagen-related peptide. Modifications of the peptide sequence indicated that the hydroxyproline residues play a significant role in supporting its GpVI reactivity. However, many peptides containing OGP/GPO motifs did not support adhesion to GpVI. These data indicate that the ability of a triple-helical peptide to bind GpVI is not solely determined by the presence or spatial arrangement of these OGP/GPO motifs within the peptides.
[show abstract][hide abstract] ABSTRACT: Fibrillar collagens provide the most fundamental platform in the vertebrate organism for the attachment of cells and matrix molecules. We have identified specific sites in collagens to which cells can attach, either directly or through protein intermediaries. Using Toolkits of triple-helical peptides, each peptide comprising 27 residues of collagen primary sequence and overlapping with its neighbours by nine amino acids, we have mapped the binding of receptors and other proteins on to collagens II or III. Integrin alpha2beta1 binds to several GXX'GER motifs within the collagens, the affinities of which differ sufficiently to control cell adhesion and migration independently of the cellular regulation of the integrin. The platelet receptor, Gp (glycoprotein) VI binds well to GPO (where O is hydroxyproline)-containing model peptides, but to very few Toolkit peptides, suggesting that sequence in addition to GPO triplets is important in defining GpVI binding. The Toolkits have been applied to the plasma protein vWF (von Willebrand factor), which binds to only a single sequence, identified by truncation and amino acid substitution within Toolkit peptides, as GXRGQOGVMGFO in collagens II and III. Intriguingly, the receptor tyrosine kinase, DDR2 (discoidin domain receptor 2) recognizes three sites in collagen II, including its vWF-binding site, although the amino acids that support the interaction differ slightly within this motif. Furthermore, the secreted protein BM-40 (basement membrane protein 40) also binds well to this same region. Thus the availability of extracellular collagen-binding proteins may be important in regulating and facilitating direct collagen-receptor interaction.
Biochemical Society Transactions 05/2008; 36(Pt 2):241-50. · 2.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: Platelet glycoprotein (GP) Ib-IX-V supports platelet adhesion on damaged vascular walls by binding to von Willebrand factor (VWF). For several decades it has been recognized that the alpha-subunit of GP (GPIbalpha) also binds thrombin but the physiological relevance, if any, of this interaction was unknown. Previous studies have shown that a sulfated tyrosine 276 (Tyr276) is essential for thrombin binding to GPIbalpha.
This study investigated the in vivo relevance of GPIbalpha residue Tyr276 in hemostasis and thrombosis.Methods: Transgenic mouse colonies expressing the normal human GPIbalpha subunit or a mutant human GPIbalpha containing a Phe substitution for Tyr276 (hTg(Y276F)) were generated. Both colonies were bred to mice devoid of murine GPIbalpha.
Surface-expressed GPIbalpha levels and platelet counts were similar in both colonies. hTg(Y276F) platelets were significantly impaired in binding alpha-thrombin but displayed normal binding to type I fibrillar collagen and human VWF in the presence of ristocetin. In vivo thrombus formation as a result of chemical damage (FeCl(3)) demonstrated that hTg(Y276F) mice have a delayed time to occlusion followed by unstable blood flow indicative of embolization. In models of laser-induced injury, thrombi developing in hTg(Y276F) animals were also less stable.
The results demonstrate that GPIbalpha residue Tyr276 is physiologically important, supporting stable thrombus formation in vivo.
Journal of Thrombosis and Haemostasis 01/2008; 6(4):684-691. · 6.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Comprehensive mapping of protein-binding sites within human collagen III has allowed the recognition motifs for integrin alpha(2)beta(1) and VWF A3 domain to be identified. Glycoprotein VI-binding sites are understood, although less well defined. This information, together with recent developments in understanding collagen fiber architecture, and crystal structures of the receptor collagen-binding domains, allows a coherent model for the interaction of collagen with the platelet surface to be developed. This complements our understanding of the orchestration of receptor presentation by membrane microdomains, such that the polyvalent collagen surface may stabilize signaling complexes within the heterogeneous receptor composition of the lipid raft. The ensuing interactions lead to the convergence of signals from each of the adhesive receptors, mediated by FcR gamma-chain and/or FcgammaRIIa, leading to concerted and co-operative platelet activation. Each receptor has a shear-dependent role, VWF/GpIb essential at high shear, and alpha(2)beta(1) at low and intermediate shear, whilst GpVI provides core signals that contribute to enhanced integrin affinity, tighter binding to collagen and consequent platelet activation.
Journal of Thrombosis and Haemostasis 08/2007; 5 Suppl 1:220-9. · 6.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Collagen-related peptide is a selective agonist for the platelet collagen receptor Glycoprotein VI. The triple helical peptide contains ten GPO triplets/strand (single letter amino acid nomenclature, where O is hydroxyproline) and so over-represents GPO compared with native collagen sequence. To investigate the ability of Glycoprotein VI to recognize GPO triplets in a setting more representative of the collagens, we synthesized a set of triple helical peptides containing fewer GPO triplets, varying their number and spacing within an inert (GPP)n backbone. The adhesion of recombinant human Glycoprotein VI ectodo-main, like that of human platelets, to these peptides increased with their GPO content, and platelet adhesion was abolished by the specific anti-Glycoprotein VI-blocking antibody, 10B12. Platelet aggregation and protein tyrosine phosphorylation were induced only by cross-linked peptides and only those that contained two or more GPO triplets. Such peptides were less potent than cross-linked collagen-related peptide. Our data suggest that both the sequences GPOGPO and GPO.........GPO represent functional Glycoprotein VI recognition motifs within collagen. Furthermore, we propose that the (GPO)4 motif can support simultaneous binding of two glycoprotein VI molecules, in either a parallel or anti-parallel stacking arrangement, which could play an important role in activation of signaling.
Journal of Biological Chemistry 02/2007; 282(2):1296-304. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: A set of 57 synthetic peptides encompassing the entire triplehelical domain of human collagen III was used to locate binding sites for the collagen-binding integrin alpha(2)beta(1). The capacity of the peptides to support Mg(2+)-dependent binding of several integrin preparations was examined. Wild-type integrins (recombinant alpha(2) I-domain, alpha(2)beta(1) purified from platelet membranes, and recombinant soluble alpha(2)beta(1) expressed as an alpha(2)-Fos/beta(1)-Jun heterodimer) bound well to only three peptides, two containing GXX'GER motifs (GROGER and GMOGER, where O is hydroxyproline) and one containing two adjacent GXX'GEN motifs (GLKGEN and GLOGEN). Two mutant alpha(2) I-domains were tested: the inactive T221A mutant, which recognized no peptides, and the constitutively active E318W mutant, which bound a larger subset of peptides. Adhesion of activated human platelets to GER-containing peptides was greater than that of resting platelets, and HT1080 cells bound well to more of the peptides compared with platelets. Binding of cells and recombinant proteins was abolished by anti-alpha(2) monoclonal antibody 6F1 and by chelation of Mg(2+). We describe two novel high affinity integrin-binding motifs in human collagen III (GROGER and GLOGEN) and a third motif (GLKGEN) that displays intermediate activity. Each motif was verified using shorter synthetic peptides.
Journal of Biological Chemistry 03/2006; 281(7):3821-31. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Recent studies implicate the collagen receptor, glycoprotein VI (GPVI) in activation of platelet 12-lipoxygenase (p12-LOX). Herein, we show that GPVI-stimulated 12-hydro(peroxy)eicosatetraenoic acid (H(P)ETE) synthesis is inhibited by palmityl trifluromethyl ketone or oleyloxyethylphosphocholine , but not bromoenol lactone, implicating secretory and cytosolic, but not calcium-independent phospholipase A2 (PLA2) isoforms. Also, following GPVI activation, 12-LOX co-immunoprecipitates with both cytosolic and secretory PLA2 (sPLA2). Finally, venoms containing sPLA2 acutely activate p12-LOX in a dose-dependent manner. This study shows that platelet 12-H(P)ETE generation utilizes arachidonate substrate from both c- and sPLA2 and that 12-LOX functionally associates with both PLA2 isoforms.
[show abstract][hide abstract] ABSTRACT: Lipoxygenases (LOX) contribute to vascular disease and inflammation through generation of bioactive lipids, including 12-hydro(pero)xyeicosatetraenoic acid (12-H(P)ETE). The physiological mechanisms that acutely control LOX product generation in mammalian cells are uncharacterized. Human platelets that contain a 12-LOX isoform (p12-LOX) were used to define pathways that activate H(P)ETE synthesis in the vasculature. Collagen and collagen-related peptide (CRP) (1 to 10 microg/mL) acutely induced platelet 12-H(P)ETE synthesis. This implicated the collagen receptor glycoprotein VI (GPVI), which signals via the immunoreceptor-based activatory motif (ITAM)-containing FcRgamma chain. Conversely, thrombin only activated at high concentrations (> 0.2 U/mL), whereas U46619 and ADP alone were ineffective. Collagen or CRP-stimulated 12-H(P)ETE generation was inhibited by staurosporine, PP2, wortmannin, BAPTA/AM, EGTA, and L-655238, implicating src-tyrosine kinases, PI3-kinase, Ca2+ mobilization, and p12-LOX translocation. In contrast, protein kinase C (PKC) inhibition potentiated 12-H(P)ETE generation. Finally, activation of the immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing platelet endothelial cell adhesion molecule (PECAM-1) inhibited p12-LOX product generation. This study characterizes a receptor-dependent pathway for 12-H(P)ETE synthesis via the collagen receptor GPVI, which is negatively regulated by PECAM-1 and PKC, and demonstrates a novel link between immune receptor signaling and lipid mediator generation in the vasculature.
Circulation Research 07/2004; 94(12):1598-605. · 11.86 Impact Factor
[show abstract][hide abstract] ABSTRACT: Platelet aggregation and its measurement using the turbidimetric method were discussed in the previous chapter. Many of the
same considerations relating to aggregometry that were raised there are equally valid in the context of this chapter and it
is recommended that readers acquaint themselves with Chapter 5, vol. 1 prior to commencing this one.
Methods in molecular biology (Clifton, N.J.) 02/2004; 272:77-87.