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 ß-catenin expression. ß-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.

Assaying the efficacy of dual-antiplatelet therapy: use of a controlled-shear-rate microfluidic device with a well-defined collagen surface to track dynamic platelet adhesion.

We report the development and demonstration of an assay that distinguishes the pharmacological effects of two widely used antiplatelet therapies, aspirin (COX-1 inhibitor) and clopidogrel (P2Y12 inhibitor). Whole blood is perfused through a low-volume microfluidic device in contact with a well-characterized (ellipsometry, atomic force microscopy) acid-soluble type I collagen surface. Whole human blood treated in vitro with a P2Y12 inhibitor 2-methylthioadenosine 5'-monophosphate triethylammonium salt (2-MeSAMP) extended the time to the start of platelet recruitment, i.e., platelet binding to the collagen surface. Treatment with 2-MeSAMP also slowed the rate of aggregate buildup, with an overall reduced average platelet aggregate area after 8 min of constant blood flow. A far smaller effect was observed for in vitro treatment with aspirin, for which the rate of change of surface coverage is indistinguishable from controls. In whole blood obtained from patients under treatment with dual-antiplatelet therapy (aspirin and clopidogrel), a significant extension of time to platelet recruitment was observed along with a slowed rate of aggregate buildup and an average aggregate size approximately half that of control measurements. Differentiation of the pharmacological effects of these two well-targeted antiplatelet pathways suggests a role for this assay in determining the antiplatelet effects of these and related new therapeutics in clinical settings.

Inhibition of platelet adhesion by peptidomimetics mimicking the interactive ß-hairpin of glycoprotein Ibα.

ß-Hairpin peptidomimetics mimicking the interaction sites of the platelet receptor glycoprotein (GP)Ibα with von Willebrand factor (vWF) were synthesised and evaluated for their ability to increase platelet velocity under high shear conditions and to inhibit shear-induced platelet aggregation. A cyclic and bridged dodecapeptide 2e containing a heterochiral diproline motif was identified as a lead compound for the generation of a novel class of potential antiplatelet agents.

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.

Microfluidic device to study arterial shear-mediated platelet-surface interactions in whole blood: reduced sample volumes and well-characterised protein surfaces.

We report a novel device to analyze cell-surface interactions under controlled fluid-shear conditions on well-characterised protein surfaces. Its performance is demonstrated by studying platelets interacting with immobilised von Willebrand Factor at arterial vascular shear rates using just 200 µL of whole human blood per assay. The device's parallel-plate flow chamber, with 0.1 mm² cross sectional area and height-to-width ratio of 1:40, provides uniform, well-defined shear rates along the chip surface with negligible vertical wall effects on the fluid flow profile while minimizing sample volumetric flow. A coating process was demonstrated by ellipsometry, atomic force microscopy, and fluorescent immunostaining to provide reproducible, homogeneous, uniform protein layers over the 0.7 cm² cell-surface interaction area. Customized image processing quantifies dynamic cellular surface coverage vs. time throughout the whole-blood-flow assay for a given drug treatment or disease state. This device can track the dose response of anti-platelet drugs, is suitable for point-of-care diagnostics, and is designed for adaptation to mass manufacture.

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 Fc gammaRIIa 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.

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 (alpha(IIb)beta3), 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.

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.

Data on the regulation of moesin and merlin by the urokinase receptor (uPAR): Model explaining distal activation of integrins by uPAR.

The data presented herein are connected to our research article (doi: 10.1016/j.biocel.2017.04.012) [1], in which we investigated the functional connections between the urokinase receptor (uPAR), and the ezrin/radixin/moesin (ERM) proteins, moesin and merlin [1]. Firstly, a model of action is proposed that enlightens how uPAR regulates distal integrins. In addition, data show the effects of expressing wild-type moesin or permanently active T558D mutant of moesin on angiogenesis and morphology of human aortic endothelial cells (HAEC). Additional data compare the effects of urokinase (uPA, the main ligand of uPAR) on the same cells. Lastly, we provide technical data demonstrating the effects of specific siRNA for moesin and merlin on moesin and merlin expression, respectively.

Moesin and merlin regulate urokinase receptor-dependent endothelial cell migration, adhesion and angiogenesis.

The glycosyl-phosphatidyl-inositol (GPI)-anchored urokinase receptor (uPAR) has no intracellular domain, but nevertheless initiates signalling through proximal interactions with other membrane receptors including integrins. The relationships between uPAR and ezrin/radixin/moesin (ERM) proteins, moesin and merlin have never been explored. Moesin and merlin are versatile membrane-actin links and regulators of receptors signalling, respectively. We show that uPAR controls moesin and merlin, which propagate uPAR-initiated signals and modulate integrin functions, thereby regulating uPAR activity. uPAR rapidly de-phosphorylates moesin and phosphorylates merlin inactivating both proteins, and enhancing cell migration and angiogenesis. Moesin behaves as a molecular switch turning either on or off uPAR signalling through cycles of de-activation/activation, or sustained activation, respectively. Furthermore, moesin is at the crossroads of uPAR-initiated outside-in and inside-out signalling promoting integrin-dependent cell adhesion suggesting that uPAR also activates integrins distally through moesin. Knocking down merlin expression enhanced cell migration and adhesion through different regulation of fibronectin- and vitronectin-binding integrins.

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 αIIbß (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 device's thrombogenic surface, does not activate platelets, and does not interfere with the action of agonists added to deliberately activate platelets.

Shear-mediated platelet adhesion analysis in less than 100 µl of blood: toward a POC platelet diagnostic.

We report a microfluidic chip-based hydrodynamic focusing approach that minimizes sample volume for the analysis of cell-surface interactions under controlled fluid-shear conditions. Assays of statistically meaningful numbers of translocating platelets interacting with immobilized von Willebrand factor at arterial shear rates (∼1500 s(-1)) are demonstrated. By controlling spatial disposition and relative flow rates of two contacting fluid streams, e.g., sample (blood) and aqueous buffer, on-chip hydrodynamic focusing guides the cell-containing stream across the protein surface as a thin fluid layer, consuming ∼50 µL of undiluted whole blood for a 2-min platelet assay. Control of wall shear stress is independent of sample consumption for a given flow time. The device design implements a mass-manufacturable fabrication approach. Fluorescent labeling of cells enables readout using standard microscopy tools. Customized image-analysis software rapidly quantifies cellular surface coverage and aggregate size distributions as a function of time during blood-flow analyses, facilitating assessment of drug treatment efficacy or diagnosis of disease state.

New trends in bioanalytical microdevices to assess platelet function.

Cardiovascular disease is the major cause of mortality globally. The role of platelets and antiplatelet drugs in the treatment of cardiovascular disease is widely appreciated. Platelets have a less well-known role in cancer and inflammation and as the role of platelets in cancer and inflammation is increasingly understood, there is a compelling need to develop diagnostic assays of platelet function to guide clinical management. Most current platelet-function tests are focused on platelet aggregation, are cumbersome, require dedicated laboratory personnel, measure a single response to a single agonist and artificially separate platelets from blood, thus, these assays do not reflect the pathophysiolgical environment of complex disease states. New technology platforms are emerging that address the physiological adhesive function of platelets to vascular-specific matrices using small volumes of whole blood, giving rapid results. These technologies will guide therapy in the prevention of cardiovascular disease and probably in risk management in both cancer and atherosclerosis.

Bioinformatic discovery of novel bioactive peptides.

Short synthetic oligopeptides based on regions of human proteins that encompass functional motifs are versatile reagents for understanding protein signaling and interactions. They can either mimic or inhibit the parent protein's activity and have been used in drug development. Peptide studies typically either derive peptides from a single identified protein or (at the other extreme) screen random combinatorial peptides, often without knowledge of the signaling pathways targeted. Our objective was to determine whether rational bioinformatic design of oligopeptides specifically targeted to potentially signaling-rich juxtamembrane regions could identify modulators of human platelet function. High-throughput in vitro platelet function assays of palmitylated cell-permeable oligopeptides corresponding to these regions identified many agonists and antagonists of platelet function. Many bioactive peptides were from adhesion molecules, including a specific CD226-derived inhibitor of inside-out platelet signaling. Systematic screens of this nature are highly efficient tools for discovering short signaling motifs in molecular signaling pathways.

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.

Shear stress modulates the interaction of platelet-secreted matrix proteins with tumor cells through the integrin alphavbeta3.

Interaction of tumor cells with the vascular wall is required for metastasis from the bloodstream. The precise interaction among metastatic cells, circulating platelets, the vessel wall, and physiological flow conditions remains to be determined. In this study, we investigated the interaction of shear on metastatic cell lines adherent to lipopolysaccharide (LPS)-treated endothelium. Tumor cells were perfused over LPS-treated human umbilical vein endothelial cells (HUVECs) at incremental venous shear rates from 50 to 800 s(-1). At a venous shear rate of 400 s(-1), 3% of adherent tumor cells formed pseudopodia under shear, a process we termed shear-induced activation. Because platelets promote tumor dissemination, we then investigated the effect of pretreating tumor cells with platelet releasate collected from activated platelet concentrate. We found that in the presence of platelet releasate, the number of tumor cells adhering to HUVECs increased and tumor "activation" occurred at a significantly lower shear rate of 50 s(-1). This was inhibited with acetylsalicylic acid. Depletion of fibronectin or vitronectin from the platelet releasate resulted in significantly less adhesion at higher venous shear rates of 600 and 800 s(-1). The integrin alphavbeta3 has been shown to mediate cell adhesion primarily through vitronectin and fibronectin proteins. Inhibition of alphavbeta3, followed by the addition of platelet releasate to the tumor cells, resulted in significantly less adhesion at higher venous shear rates of 600 and 800 s(-1). Collectively, our data suggest that alphavbeta3 promotes the metastatic phenotype of tumor cells through interactions with the secreted platelet proteins vitronectin and fibronectin under venous shear conditions.