Identification of a major GpVI-binding locus in human type III collagen

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, alpha 1(III)CB4. We have identified several peptides that interact with GpVI, in particular a peptide designated III-30 with the sequence GAOGLRGGAGPOG-PEGGKGAAGPOGPO. 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.

Structural basis for the platelet-collagen interaction - The smallest motif within collagen that recognizes and activates platelet glycoprotein VI contains two glycine-proline-hydroxyproline triplets

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 ectodomain, 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 center dot center dot center dot center dot center dot center dot center dot center dot center dot 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.

Glycoprotein VI/Fc receptor gamma chain-independent tyrosine phosphorylation and activation of murine platelets by collagen

We have investigated the ability of collagen to induce signalling and functional responses in suspensions of murine platelets deficient in the FcRgamma (Fc receptor gamma) chain, which lack the collagen receptor GPVI (glycoprotein VI). In the absence of the FcRgamma chain, collagen induced a unique pattern of tyrosine phosphorylation which was potentiated by the thromboxane analogue U46619. Immunoprecipitation studies indicated that neither collagen alone nor the combination of collagen plus U46619 induced phosphorylation of the GPVI-regulated proteins Syk and SLP76 (Src homology 2-containing leucocyte protein of 76 kDa). A low level of tyrosine phosphorylation of phospholipase Cgamma2 was observed, which was increased in the presence of U46619, although the degree of phosphorylation remained well below that observed in wild-type platelets (similar to 10%). By contrast, collagen-induced phosphorylation of the adapter ADAP (adhesion- and degranulation-promoting adapter protein) was substantially potentiated by U46619 to levels equivalent to those observed in wild-type platelets. Collagen plus U46619 also induced significant phosphorylation of FAK (focal adhesion kinase). The functional significance of collagen-induced non-GPVI signals was highlighted by the ability of U46619 and collagen to induce the secretion of ATP in FcRgamma chain-deficient platelets, even though neither agonist was effective alone. Protein tyrosine phosphorylation and the release of ATP were abolished by the anti(alpha2 integrin) antibodies Ha1/29 and HMalpha2, but not by blockade of alphaIIbbeta3. These results illustrate a novel mechanism of platelet activation by collagen which is independent of the GPVI-FcRgamma chain complex, and is facilitated by binding of collagen to integrin alpha2beta1.

Use of synthetic peptides to locate novel integrin alpha(2)beta(1)-binding motifs in human collagen III

A set of 57 synthetic peptides encompassing the entire triple-helical 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 Mg2+-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 Mg2+. 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.

Platelet 12-lipoxygenase activation via glycoprotein VI - Involvement of multiple signaling pathways in agonist control of H(P)ETE synthesis

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 mug/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.

Endocrine disrupting effects of zearalenone, alpha- and beta-zearalenol at the level of nuclear receptor binding and steroidogenesis.

The mycotoxin zearalenone (ZEN) is a secondary metabolite of fungi which is produced by certain species of the genus Fusarium and can occur in cereals and other plant products. Reporter gene assays incorporating natural steroid receptors and the H295R steroidogenesis assay have been implemented to assess the endocrine disrupting activity of ZEN and its metabolites -zearalenol (-ZOL) and -zearalenol (-ZOL). -ZOL exhibited the strongest estrogenic potency (EC50 0.022 ± 0.001 nM), slightly less potent than 17- estradiol (EC50 0.015 ± 0.002 nM). ZEN was ~70 times less potent than -ZOL and twice as potent as -ZOL. Binding of progesterone to the progestagen receptor was shown to be synergistically increased in the presence of ZEN, -ZOL or -ZOL. ZEN, -ZOL or -ZOL increased production of progesterone, estradiol, testosterone and cortisol hormones in the H295R steroidogenesis assay, with peak productions at 10 M. At 100 M, cell viability decreased and levels of hormones were significantly reduced except for progesterone. -ZOL increased estradiol concentrations more than -ZOL or ZEN, with a maximum effect at 10 M, with -ZOL (562 ± 59 pg/ml) > -ZOL (494 ± 60 pg/ml) > ZEN (375 ± 43 pg/ml). The results indicate that ZEN and its metabolites can act as potential endocrine disruptors at the level of nuclear receptor signalling and by altering hormone production.

Examination of the Silver Colloid Binding Behavior of Disulfide-Tethered Bipyridine Ligands and Their fac-Tricarbonylrhenium(I) Complexes

The syntheses of 2,2'-bipyridin-5-ylmethyl-5-(1,2-dithiolan-3-yl)pentanoate (L1) and N-(2,2'-bipyridin-5-ylmethyl)-5-(1,2-dithiolan-3-yl)pentanamide (L2) and their neutral fac carbonylrhenium(I) complexes [Re(L1)(CO)(3)Br] and [Re(L2)(CO)(3)Br] are reported. The. electronic absorption and emission spectra of the complexes are similar to the spectrum of the reference compound [Re(bipy)(CO)(3)Br] and correlate well with the density functional theory calculations undertaken. The surface-enhanced Raman spectroscopy (SERS) spectra (excited at both 532 and 785 nm) of the ligands and complexes were examined and compared to the spectrum of ethyl 5-(1,2-dithiolan-3-yl)pentanoate (L3), revealing that there is very little contribution to the spectra of these species from the dithiolated alkyl chains. The spectra are dominated by the characteristic peaks of a metalated 2,2'-bipyridyl group,arising from the silver colloid/ion complexation, and the rhenium center. The rhenium complexes show weak SERS bands related to the CO stretches and a broad band at 510 cm(-1) assigned to Re-CO stretching. Concentration dependent studies, measured by the relative intensity of several assigned peaks, indicate that, as the surface coverage increases, the bipyridine moiety lifts off the surface In the case of L1 and L2, this gives rise to complexes with silver at low concentration, enhancing the signals observed, while for the tricarbonylbromorhenium complexes of these ligands, the presence of the disulfide tether allows an enhancement in the limits of detection of these surface-borne species of 20 times in the case of [ReL2(CO)(3)Br] over [Re(bipy)(CO)(3)Br].

Receptor binding and biologic activity of synthetic ET-1 peptides in the retinal pericyte

Purpose. The purpose of this study was to examine the effect of synthetic endothelin (ET)-1 peptides with antigenic potential for binding and biologic activity using an in vitro model of microvascular pericytes.

Binding modes of diketo-acid inhibitors of HIV-1 integrase: A comparative molecular dynamics simulation study

HIV-1 integrase (IN) has become an attractive target since drug resistance against HIV-1 reverse transcriptase (RT) and protease (PR) has appeared. Diketo acid (DKA) inhibitors are potent and selective inhibitors of HIV-1 IN: however the action mechanism is not well understood. Here, to study the inhibition mechanism of DKAs we performed 10 ns comparative molecular dynamics simulations on HIV-1 IN bound with three most representative DMA inhibitors: Shionogi inhibitor, S-1360 and two Merck inhibitors L-731,988 and L-708,906. Our simulations show that the acidic part of S-1360 formed salt bridge and cation-pi interactions with Lys159. In addition, the catalytic Glu152 in S-1360 was pushed away from the active site to form an ion-pair interaction with Arg199. The Merck inhibitors can maintain either one or both of these ion-pair interaction features. The difference in potencies of the DMA inhibitors is thus attributed to the different binding modes at the catalytic site. Such structural information at atomic level, not only demonstrates the action modes of DMA inhibitors but also provides a novel starting point for structural-based design of HIV-1 IN inhibitors.