Christiane A. Helm

Close Approximation of Two Platelet Factor 4 Tetramers by Charge Neutralization Forms the Antigens Recognized by HIT Antibodies

Objective—Heparin-induced thrombocytopenia (HIT) is a prothrombotic drug reaction caused by antibodies that recognize positively charged platelet factor 4 (PF4), bound to the polyanion, heparin. The resulting immune complexes activate platelets. Unfractionated heparin (UFH) causes HIT more frequently than low-molecular-weight heparin (LMWH), whereas the smallest heparin-like molecule (the pentasaccharide, fondaparinux), induces anti-PF4/heparin antibodies as frequently as LMWH, but without exhibiting cross-reactivity with these antibodies. To better understand these findings, we analyzed the molecular structure of the complexes formed between PF4 and UFH, LMWH, or fondaparinux. Methods and Results—By atomic force microscopy and photon correlation spectroscopy, we show that with any of the 3 polyanions, but in the order, UFH>LMWH≫fondaparinux—PF4 forms clusters in which PF4 tetramers become closely apposed, and to which anti-PF4/heparin antibodies bind. By immunoassay, HIT antibodies bind strongly to PF4/H/PF4 complexes, but only weakly to single PF4/heparin molecules. Conclusion—HIT antigens are formed when charge neutralization by polyanion allows positively charged PF4 tetramers to undergo close approximation. Whereas such a model could explain why all 3 polyanions form antibodies with similar specificities, the striking differences in the relative size and amount of complexes formed likely correspond to the observed differences in immunogenicity (UFH>LMWH≈fondaparinux) and clinically relevant cross-reactivity (UFH>LMWH≫fondaparinux).

Phases of phosphatidyl ethanolamine monolayers studied by synchrotron x-ray scattering

For the first time, phospholid monolayers at the air/water interface have been studied by x-ray diffraction and reflection all along the isotherm from the laterally isotropic fluid (the so-called LE phase) to the ordered phases. The model used to analyze the data, and the accuracy of the parameters deduced, were tested by comparing the results obtained with two lipids having the same head group but different chain lengths. Compression of the fluid phase leads predominantly to a change of thickness of the hydrophobic moiety, much less of its density, with the head group extension remaining constant. The main transition involves a considerable increase (approximately 10%) of the electron density in the hydrophobic region, a dehydration of the head group and a positional ordering of the aliphatic tails, albeit with low coherence lengths (approximately 10 spacings). On further compression of the film, the ordered phase undergoes a continuous transition. This is characterized by an increase in positional ordering, a discontinuous decrease in lateral compressibility, a decrease in chain tilt angle with respect to the surface normal towards zero and probably also a head group dehydration and ordering.

Complex formation with nucleic acids and aptamers alters the antigenic properties of platelet factor 4

The tight electrostatic binding of the chemokine platelet factor 4 (PF4) to polyanions induces heparin-induced thrombocytopenia, a prothrombotic adverse drug reaction caused by immunoglobulin G directed against PF4/polyanion complexes. This study demonstrates that nucleic acids, including aptamers, also bind to PF4 and enhance PF4 binding to platelets. Systematic assessment of RNA and DNA constructs, as well as 4 aptamers of different lengths and secondary structures, revealed that increasing length and double-stranded segments of nucleic acids augment complex formation with PF4, while single nucleotides or single-stranded polyA or polyC constructs do not. Aptamers were shown by circular dichroism spectroscopy to induce structural changes in PF4 that resemble those induced by heparin. Moreover, heparin-induced anti-human-PF4/heparin antibodies cross-reacted with human PF4/nucleic acid and PF4/aptamer complexes, as shown by an enzyme immunoassay and a functional platelet activation assay. Finally, administration of PF4/44mer-DNA protein C aptamer complexes in mice induced anti-PF4/aptamer antibodies, which cross-reacted with murine PF4/heparin complexes. These data indicate that the formation of anti-PF4/heparin antibodies in postoperative patients may be augmented by PF4/nucleic acid complexes. Moreover, administration of therapeutic aptamers has the potential to induce anti-PF4/polyanion antibodies and a prothrombotic diathesis.

Effect of 3D-scaffold formation on differentiation and survival in human neural progenitor cells

Background3D-scaffolds have been shown to direct cell growth and differentiation in many different cell types, with the formation and functionalisation of the 3D-microenvironment being important in determining the fate of the embedded cells. Here we used a hydrogel-based scaffold to investigate the influences of matrix concentration and functionalisation with laminin on the formation of the scaffolds, and the effect of these scaffolds on human neural progenitor cells cultured within them.MethodsIn this study we used different concentrations of the hydrogel-based matrix PuraMatrix. In some experiments we functionalised the matrix with laminin I. The impact of concentration and treatment with laminin on the formation of the scaffold was examined with atomic force microscopy. Cells from a human fetal neural progenitor cell line were cultured in the different matrices, as well as in a 2D culture system, and were subsequently analysed with antibody stainings against neuronal markers. In parallel, the survival rate of the cells was determined by a live/dead assay.ResultsAtomic force microscopy measurements demonstrated that the matrices are formed by networks of isolated PuraMatrix fibres and aggregates of fibres. An increase of the hydrogel concentration led to a decrease in the mesh size of the scaffolds and functionalisation with laminin promoted aggregation of the fibres (bundle formation), which further reduces the density of isolated fibres. We showed that laminin-functionalisation is essential for human neural progenitor cells to build up 3D-growth patterns, and that proliferation of the cells is also affected by the concentration of matrix. In addition we found that 3D-cultures enhanced neuronal differentiation and the survival rate of the cells compared to 2D-cultures.ConclusionsTaken together, we have demonstrated a direct influence of the 3D-scaffold formation on the survival and neuronal differentiation of human neural progenitor cells. These findings emphasize the importance of optimizing 3D-scaffolds protocols prior to in vivo engraftment of stem and progenitor cells in the context of regenerative medicine.

Conformation of Poly(styrene sulfonate) Layers Physisorbed from High Salt Solution Studied by Force Measurements on Two Different Length Scales

The conformation of poly(styrene sulfonate) (PSS) layers physisorbed from 1 M NaCl is determined by force measurements and imaging on two length scales. With colloidal probe technique steric forces as predicted for neutral grafted brushes are observed. On decrease and increase of the NaCl concentration, the grafting density remains constant, yet the brush thickness swells and shrinks reversibly with the salt concentration with an exponent of -0.3. At low salt conditions, the brush length amounts to 30% of the contour length, a behavior known for polyelectrolyte brushes and attributed to the entropy of the counterions trapped in the brush. Between a PSS layer and a pure colloidal silica sphere, the same steric forces are observed, and additionally at large separations (beyond the range of the steric repulsion) an electrostatic force is found. A negatively charged AFM tip penetrates the brush--a repulsive electrostatic force between the tip and surface is found, and single chains can be imaged. Thus, with the nanometer-sized AFM tip, the flatly adsorbed fraction of the PSS chains is seen, whereas the micrometer-sized colloidal probe interacts with the fraction of the chains penetrating into solution.

Physical properties of homogeneous TiO2 films prepared by high power impulse magnetron sputtering as a function of crystallographic phase and nanostructure

Optical, photo-electrochemical, crystallographic and morphological properties of TiO2 thin films prepared by high power impulse magnetron sputtering at low substrate temperatures (<65 °C) without post-deposition thermal annealing are studied. The film composition—anatase, rutile or amorphous TiO2—is adjusted by the pressure (p ~ 0.75–15 Pa) in the deposition chamber. The different crystallographic phases were determined with grazing incidence x-ray diffractometry. The surface morphology and size of TiO2 grains/clusters were imaged with atomic force microscopy. Basic plasma parameters were determined by means of the time-resolved Langmuir probe technique. The power density influx on the substrate was estimated from calorimetric probe measurement. The data from calorimetric probe measurements and time-resolved Langmuir probe served as input parameters for the calculation of influx contributions of particular species. The band-gap energy Eg depends on the film composition and crystallographic phase. Optical parameters (refractive index n + ik, transmittance T, reflectance R and absorbance A) are measured as functions of photon energy in the UV–Vis range by spectroscopic ellipsometry. For the rutile and anatase films agreement with the respective bulk phase is found. Incident photon-current conversion efficiency determined by photo-electrochemical measurements reached the highest values (0.312) for the anatase film.

The solid-state architecture of a metallosupramolecular polyelectrolyte

Self-assembly of Fe(II) and the ditopic ligand 1,4-bis(2,2′:6′,2″-terpyridine-4′-yl)benzene results in equilibrium structures in solutions, so-called metallosupramolecular coordination polyelectrolytes (MEPEs). It is exceedingly difficult to characterize such macromolecular assemblies, because of the dynamic nature. Therefore, hardly any structural information is available for this type of material. Here, we show that from dilute solutions, where small aggregates predominate, it is possible to grow nanoscopic crystals at an interface. A near atomic resolution structure of MEPE is obtained by investigating the nanoscopic crystals with electron diffraction in combination with molecular modeling. The analysis reveals a primitive monoclinic unit cell (P21/c space group, a = 10.4 Å, b = 10.7 Å, c = 34.0 Å, α = γ = 90°, β = 95°, ρ = 1.26 g/cm3, and Z = 4). The MEPE forms linear rods, which are organized into sheets. Four sheets intersect the unit cell, while adjacent sheets are rotated by 90° with respect to each other. The pseudooctahedral coordination geometry of the Fe(II) centers is confirmed by Mössbauer spectroscopy. The combination of diffraction and molecular modeling presented here may be of general utility to address problems in structural materials science.

Binding of anti-platelet factor 4/heparin antibodies depends on the thermodynamics of conformational changes in platelet factor 4.

The chemokine platelet factor 4 (PF4) undergoes conformational changes when complexing with polyanions. This can induce the antibody-mediated adverse drug effect of heparin-induced thrombocytopenia (HIT). Understanding why the endogenous protein PF4 becomes immunogenic when complexing with heparin is important for the development of other negatively charged drugs and may also hint toward more general mechanisms underlying the induction of autoantibodies to other proteins. By circular dichroism spectroscopy, atomic force microscopy, and isothermal titration calorimetry we characterized the interaction of PF4 with unfractionated heparin (UFH), its 16-, 8-, and 6-mer subfractions, low-molecular-weight heparin (LMWH), and the pentasaccharide fondaparinux. To bind anti-PF4/heparin antibodies, PF4/heparin complexes require (1) an increase in PF4 antiparallel β-sheets exceeding ∼30% (achieved by UFH, LMWH, 16-, 8-, 6-mer), (2) formation of multimolecular complexes (UFH, 16-, 8-mer), and (3) energy (needed for a conformational change), which is released by binding of ≥11-mer heparins to PF4, but not by smaller heparins. These findings may help to synthesize safer heparins. Beyond PF4 and HIT, the methods applied in the current study may be relevant to unravel mechanisms making other endogenous proteins more vulnerable to undergo conformational changes with little energy requirement (eg, point mutations and post-translational modifications) and thereby predisposing them to become immunogenic.

A novel contact model for AFM indentation experiments on soft spherical cell-like particles

The use of the simple Hertz model for the analysis of Atomic Force Microscopy (AFM) force-distance curves measured on soft spherical cell-like particles leads to significant underestimations of the objects Youngs modulus E. To correct this error, a mixed double contact model (based on the simple Hertz model and the Johnson-Kendall-Roberts (JKR) model) was derived. The model considers two independent particle deformation sites: (i) the upper part of the particle is deformed by the AFM indenter, (ii) the bottom part is deformed by the substrate, which is usually unnoticed. It becomes apparent that for soft particles even small forces between substrate and particle can influence the resulting force-distance curves. For instance we show, that a gravity-induced compression on the particle bottom side can have significant influence on the measurements. To highlight these observations, the deviation of the particle Youngs modulus E between the simple Hertz model and our model is calculated. This error strongly depends on the ratio of the three involved radii: (i) the radius of the AFM indenter, (ii) the radius of the particle and (iii) the radius of the substrate as well as on the acting gravity force. Overall, the analysis suggests that for nanoscopic indenters the deviation is negligible, whereas the use of microscopic indenters results in significant errors that can be corrected via the presented model. This is important especially for very soft particles, since larger indenters can achieve higher signal to noise ratios. Furthermore, the applicability of the model was confirmed by indentation experiments on hydrogel microbeads. The mixed double contact model is applicable to a large range of indenter geometries and can be adapted for other contact models.

The adhesion and spreading of thrombocyte vesicles on electrode surfaces

The interaction of thrombocyte vesicles with the surface of metal electrodes, i.e., mercury, gold and gold electrodes modified with self assembled monolayers (SAM), was studied with the help of chronoamperometry, atomic force microscopy, and quartz crystal microbalance measurements. The experimental results show that the interaction of the thrombocyte vesicles with the surface of the electrodes depends on the hydrophobicity of the latter: whereas on very hydrophobic surfaces (mercury and gold functionalized with SAM) the thrombocyte vesicles disintegrate and form a monolayer of lipids, on the less hydrophobic gold surface a bilayer is formed. The chronoamperometric measurements indicate the possibility of future applications to probe membrane properties of thrombocytes.