Gerardene Meade

Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily.

The glutathione transferases (GSTs; also known as glutathione S-transferases) are major phase II detoxification enzymes found mainly in the cytosol. In addition to their role in catalysing the conjugation of electrophilic substrates to glutathione (GSH), these enzymes also carry out a range of other functions. They have peroxidase and isomerase activities, they can inhibit the Jun N-terminal kinase (thus protecting cells against H(2)O(2)-induced cell death), and they are able to bind non-catalytically a wide range of endogenous and exogenous ligands. Cytosolic GSTs of mammals have been particularly well characterized, and were originally classified into Alpha, Mu, Pi and Theta classes on the basis of a combination of criteria such as substrate/inhibitor specificity, primary and tertiary structure similarities and immunological identity. Non-mammalian GSTs have been much less well characterized, but have provided a disproportionately large number of three-dimensional structures, thus extending our structure-function knowledge of the superfamily as a whole. Moreover, several novel classes identified in non-mammalian species have been subsequently identified in mammals, sometimes carrying out functions not previously associated with GSTs. These studies have revealed that the GSTs comprise a widespread and highly versatile superfamily which show similarities to non-GST stress-related proteins. Independent classification systems have arisen for groups of organisms such as plants and insects. This review surveys the classification of GSTs in non-mammalian sources, such as bacteria, fungi, plants, insects and helminths, and attempts to relate them to the more mainstream classification system for mammalian enzymes. The implications of this classification with regard to the evolution of GSTs are discussed.

A serine‐rich glycoprotein of Streptococcus sanguis mediates adhesion to platelets via GPIb

Streptococcus sanguis is the most common oral bacterium causing infective endocarditis and its ability to adhere to platelets, leading to their activation and aggregation, is thought to be an important virulent factor. Previous work has shown that S. sanguis can bind directly to platelet glycoprotein (GP) Ib but the nature of the adhesin was unknown. Here, we have shown that a high molecular weight glycoprotein of S. sanguis mediates adhesion to glycocalacin. The bacterial glycoprotein was purified from cell extracts by chromatography on GPIb‐ and wheatgerm agglutinin affinity matrices and its interaction with GPIb was shown to be sialic acid‐dependent. We designated the glycoprotein serine‐rich protein A (SrpA). An insertional inactivation mutant lacking the SrpA of S. sanguis showed significantly reduced binding to glycocalacin, reduced adherence to platelets and a prolonged lag time to platelet aggregation. In addition, under flow conditions, platelets rolled and subsequently adhered on films of wild‐type S. sanguis cells at low shear (50/s) but did not bind to films of the SrpA mutant. Platelets did not bind to wild‐type bacterial cells at high shear (1500/s). These findings help to understand the mechanisms by which the organism might colonize platelet‐fibrin vegetations.

Conformation-Specific Blockade of the Integrin GPIIb/IIIa: A Novel Antiplatelet Strategy That Selectively Targets Activated Platelets

Platelet activation causes conformational changes of integrin GPIIb/IIIa (&agr;IIb&bgr;3), resulting in the exposure of its ligand-binding pocket. This provides the unique possibility to design agents that specifically block activated platelets only. We used phage display of single-chain antibody (scFv) libraries in combination with several rounds of depletion/selection to obtain human scFvs that bind specifically to the activated conformation of GPIIb/IIIa. Functional evaluation of these scFv clones revealed that fibrinogen binding to human platelets and platelet aggregation can be effectively inhibited by activation-specific scFvs. In contrast to clinically used GPIIb/IIIa blockers, which are all conformation unspecific, activation-specific GPIIb/IIIa blockers do not induce conformational changes in GPIIb/IIIa or outside-in signaling, as evaluated by ligand-induced binding-site (LIBS) exposure in flow cytometry or P-selectin expression in immunofluorescence microscopy, respectively. In contrast to the conformation-unspecific blocker abciximab, activation-specific scFvs permit cell adhesion and spreading on immobilized fibrinogen, which is mediated by nonactivated GPIIb/IIIa. Mutagenesis studies and computer modeling indicate that exclusive binding of activation-specific scFv is mediated by RXD motifs in the heavy-chain complementary-determining region (CDR) 3 of the antibodies, which in comparison with other antibodies forms an exceptionally extended loop. In vivo experiments in a ferric-chloride thrombosis model of the mouse carotid artery demonstrate similar antithrombotic potency of activation-specific scFv, when compared with the conformation-unspecific blockers tirofiban and eptifibatide. However, in contrast to tirofiban and eptifibatide, bleeding times are not prolonged with the activation-specific scFvs, suggesting lower bleeding risks. In conclusion, activation-specific GPIIb/IIIa blockade via human single-chain antibodies represents a promising novel strategy for antiplatelet therapy.

Molecular Basis for Staphylococcus aureus–Mediated Platelet Aggregate Formation Under Arterial Shear In Vitro

Objective—Staphylococcus aureus is the most frequent causative organism of infective endocarditis (IE) and is characterized by thrombus formation on a cardiac valve that can embolize to a distant site. Previously, we showed that S aureus clumping factor A (ClfA) and fibronectin-binding protein A (FnBPA) can stimulate rapid platelet aggregation. Methods and Results—In this study we investigate their relative roles in mediating aggregate formation under physiological shear conditions. Platelets failed to interact with immobilized wild-type S aureus (Newman) at shear rates <500 s−1 but rapidly formed an aggregate at shear rates >800 s−1. Inactivation of the ClfA gene eliminated aggregate formation at any shear rate. Using surrogate hosts that do not interact with platelets bacteria overexpressing ClfA supported rapid aggregate formation under high shear with a similar profile to Newman whereas bacteria overexpressing FnBPA did not. Fibrinogen binding to ClfA was found to be essential for aggregate formation although fibrinogen-coated surfaces only allowed single-platelets to adhere under all shear conditions. Blockade of the platelet immunoglobulin receptor FcγRIIa inhibited aggregate formation. Conclusions—Thus, fibrinogen and IgG binding to ClfA is essential for aggregate formation under arterial shear conditions and may explain why S aureus is the major cause of IE.

Cyclic Strain–Mediated Regulation of Vascular Endothelial Occludin and ZO-1 Influence on Intercellular Tight Junction Assembly and Function

Objective—The vascular endothelium constitutes a highly effective fluid/solute barrier through the regulated apposition of intercellular tight junction complexes. Because endothelium-mediated functions and pathology are driven by hemodynamic forces (cyclic strain and shear stress), we hypothesized a dynamic regulatory link between endothelial tight junction assembly/function and hemodynamic stimuli. We, therefore, examined the effects of cyclic strain on the expression, modification, and function of 2 pivotal endothelial tight junction components, occludin and ZO-1. Methods and Results—For these studies, bovine aortic endothelial cells were subjected to physiological levels of equibiaxial cyclic strain (5% strain, 60 cycles/min, 24 hours). In response to strain, both occludin and ZO-1 protein expression increased by 2.3±0.1-fold and 2.0±0.3-fold, respectively, concomitant with a strain-dependent increase in occludin (but not ZO-1) mRNA levels. These changes were accompanied by reduced occludin tyrosine phosphorylation (75.7±8%) and increased ZO-1 serine/threonine phosphorylation (51.7±9% and 82.7±25%, respectively), modifications that could be completely blocked with tyrosine phosphatase and protein kinase C inhibitors (dephostatin and rottlerin, respectively). In addition, there was a significant strain-dependent increase in endothelial occludin/ZO-1 association (2.0±0.1-fold) in parallel with increased localization of both occludin and ZO-1 to the cell–cell border. These events could be completely blocked by dephostatin and rottlerin, and they correlated with a strain-dependent reduction in transendothelial permeability to FITC-dextran. Conclusions—Overall, these findings indicate that cyclic strain modulates both the expression and phosphorylation state of occludin and ZO-1 in vascular endothelial cells, with putative consequences for endothelial tight junction assembly and barrier integrity.

A palmitylated peptide derived from the glycoprotein Ibβ cytoplasmic tail inhibits platelet activation

Summary.  The platelet receptor GPIb/IX/V mediates a crucial role in hemostasis, yet the signaling mechanisms involved are incompletely understood. The complex consists of four polypeptides GPIbα, GPIbβ, GPIX and GPV. We identified an amino acid sequence in the cytoplasmic tail of the GPIbβ subunit between residues R151 and A161 that is highly conserved across species and hypothesized that it has functional importance. To target this motif, we synthesized a corresponding cell‐permeable palmitylated peptide (Pal‐RRLRARARARA) and investigated its effect on platelet function. Pal‐RRLRARARARA completely inhibited low dose thrombin‐ and ristocetin‐induced aggregation in washed platelets but only partially inhibited collagen‐ and U46619‐induced aggregation. Thromboxane production in platelets stimulated with thrombin was significantly reduced by Pal‐RRLRARARARA compared with collagen. Activation of the integrin αIIbβ3 in response to thrombin was significantly reduced when platelets were preincubated with Pal‐RRLRARARARA. The adhesion of washed platelets to von Willebrand factor (VWF) under static conditions was significantly reduced by Pal‐RRLRARARARA. Under conditions of high shear, the velocity of platelets rolling on VWF was significantly increased when platelets are preincubated with Pal‐RRLRARARARA. This study defines a novel function for the RRLRARARARA motif of GPIbβ in platelet activation.

Integrated system investigating shear-mediated platelet interactions with von Willebrand factor using microliters of whole blood.

We report an integrated platelet translocation analysis system that measures complex dynamic platelet-protein surface interactions in microliter volumes of unmodified anticoagulated whole blood under controlled fluid shear conditions. The integrated system combines customized platelet-tracking image analysis with a custom-designed microfluidic parallel plate flow chamber and defined von Willebrand factor surfaces to assess platelet trajectories. Using a position-based probability function that accounts for image noise and preference for downstream movement, outputs include instantaneous and mean platelet velocities, periods of motion and stasis, and bond dissociation kinetics. Whole blood flow data from healthy donors at an arterial shear rate (1500 s(-1)) show mean platelet velocities from 8.9+/-1.0 to 12+/-4 microm s(-1). Platelets in blood treated with the antiplatelet agent c7E-Fab fragment spend more than twice as much time in motion as platelets from untreated control blood; the bond dissociation rate constant (k(off)) increases 1.3-fold, whereas mean translocation velocities do not differ. Blood from healthy unmedicated donors was used to assess flow assay reproducibility, donor variability, and the effects of antiplatelet treatment. This integrated system enables reliable, rapid populational quantification of platelet translocation under pathophysiological vascular fluid shear using as little as 150 microl of blood.

Canonical Wnt signaling in megakaryocytes regulates proplatelet formation

Wnt signaling is involved in numerous aspects of vertebrate development and homeostasis, including the formation and function of blood cells. Here, we show that canonical and noncanonical Wnt signaling pathways are present and functional in megakaryocytes (MKs), with several Wnt effectors displaying MK-restricted expression. Using the CHRF288-11 cell line as a model for human MKs, the canonical Wnt3a signal was found to induce a time and dose-dependent increase in β-catenin expression. β-catenin accumulation was inhibited by the canonical antagonist dickkopf-1 (DKK1) and by the noncanonical agonist Wnt5a. Whole genome expression analysis demonstrated that Wnt3a and Wnt5a regulated distinct patterns of gene expression in MKs, and revealed a further interplay between canonical and noncanonical Wnt pathways. Fetal liver cells derived from low-density-lipoprotein receptor-related protein 6-deficient mice (LRP6(-/-)), generated dramatically reduced numbers of MKs in culture of lower ploidy (2N and 4N) than wild-type controls, implicating LRP6-dependent Wnt signaling in MK proliferation and maturation. Finally, in wild-type mature murine fetal liver-derived MKs, Wnt3a potently induced proplatelet formation, an effect that could be completely abrogated by DKK1. These data identify novel extrinsic regulators of proplatelet formation, and reveal a profound role for Wnt signaling in platelet production.

A novel functional role for the highly conserved α-subunit KVGFFKR motif distinct from integrin αIIbβ3 activation processes

Summary.  Background: The highly conserved integrin α‐subunit membrane‐proximal motif KVGFFKR plays a decisive role in modulating the activation of integrin αIIbβ3. Previously, we have shown that a platelet permeable palmityl (pal)‐peptide with this seven amino acid sequence can directly activate αIIbβ3 leading to platelet aggregation. Objectives: To investigate further the role of the KVGFFKR motif in integrin αIIbβ3 function. Methods: We used two sequence‐specific complementary model systems, palmityl pal‐peptides in platelets, and mutant αIIbβ3‐expressing Chinese Hamster Ovary (CHO) cell lines. Results: In platelets we show that the two phenylalanine amino acids in pal‐KVGFFKR (pal‐FF) peptide are critical for stimulating platelet aggregation. Pal‐FF peptide treatment of platelets also gives rise to a tyrosine phosphorylation signal despite the presence of inhibitors of fibrinogen binding. In CHO cells, a double alanine substitution, αIIb(F992A, F993A)β3, induces constitutive integrin activation but prevents actin stress fiber formation upon adhesion to fibrinogen, suggesting that αIIbβ3‐mediated cytoskeletal reorganization is also dependent on F992 and F993. This further highlights a critical role for the two phenylalanine residues in both of these αIIbβ3‐mediated processes. Conclusion: In addition to regulating integrin αIIbβ3 activation state, the KVGFFKR motif also influences cytoskeletal reorganization. This activity is critically determined by F992 and F993 within the seven amino acid sequence.

Effective Hydrodynamic Shaping of Sample Streams in a Microfluidic Parallel-Plate Flow-Assay Device: Matching Whole Blood Dynamic Viscosity

We report the development of an aqueous buffer system tailored to the fluidic and hemodynamic requirements of our recently reported microfluidic platelet dynamic assay device, which uses hydrodynamic focusing to “shape” a blood sample into a thin flowing layer adjacent to its protein-functionalized surface. By matching the dynamic viscosity of whole blood (3.13 ± 0.08 mPa·s, from healthy donors), the selected buffer minimizes interfacial fluid mixing and better controls shear rate within the device, permitting platelet/protein-surface interaction assays with as little as 50 μL of whole blood. Buffers containing the viscosity-enhancing components bovine serum albumin (BSA), gelofusine/glycine, or histopaque (Ficoll gradient solution) were found not to activate platelets when incubated with blood at concentrations up to 50%, as assessed by flow cytometry quantitation of P-selectin expression and αIIbβ 3 activation. In contrast, glycerol-based buffer activated platelets (two-fold increase in P-selectin levels) at concentrations as low as 10% by volume. BSA- and gelofusine/glycine-based buffers were problematic in preparation and use, and therefore, were not used beyond initial characterization. The histopaque solution selected as the best choice for flow studies stabilizes sample contact with the devices thrombogenic surface, does not activate platelets, and does not interfere with the action of agonists added to deliberately activate platelets.