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, UFHϾLMWHϾ Ͼfondaparinux-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 (UFHϾLMWHϷfondaparinux) and clinically relevant cross-reactivity (UFHϾLMWHϾ Ͼfondaparinux

With X-ray and neutron reflectivity, the structure and composition of polyelectrolyte multilayers from poly(allyl amine) (PAH) and poly(styrene sulfonate) (PSS) are studied as function of preparation conditions (salt concentration and solution temperature, T). The onset of a temperature effect occurs at 0.05 M NaCl (Debye length approximately 1 nm). At 1 M salt, the film thickness increases by a factor of 3 on heating the deposition solution from 5 to 60 degrees C. The PAH/PSS bilayer thickness is independent of the kind of salt (NaCl or KCl), yet its composition is different (more bound water for NaCl). At low T, the internal roughness is 33% of the bilayer thickness; it increases to 60% at high T. The roughening is accompanied by a total loss of bound water. At which temperature the roughening starts is a function of the kind of salt (50 degrees C for NaCl and 35 degrees C for KCl). The strong temperature dependence and the eventual loss of bound water molecules may be attributed to the hydrophobic force; however, there is an isotope effect, since the loss of bound water is less pronounced in the deuterated layers.

Polyelectrolyte multilayers are prepared by consecutively adsorbing poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (PSS) from aqueous KCl solution (1 mol/L) at different temperatures, approaching the precipitation temperature at 55 °C. The films are characterized by X-ray and neutron reflectometry, using selectively deuterated PSS layers. When the preparation solution is heated, the film thickness increases up to 75%; the film/air and the internal roughness increase by up to a factor of 3. Below 35 °C preparation temperature, the internal roughness is about 40% of the thickness of a polycation/polyanion layer pair, the same result as obtained when the bilayer thickness is controlled by varying the salt content in the deposition solution. At higher preparation temperatures, the interpenetration is more pronounced, a feature which may be attributed to the theoretically predicted changes of the polymer conformation in solution from extended configuration to pearl-necklace or even globular structures.

Bundle formation of the vertically oriented polyelectrolytes within polyelectrolyte brushes is studied with x-ray reflectivity and grazing-incidence diffraction as a function of grafting density and ion concentration. At 0.8 Molar monomer concentration and without added salt, a bundle consists of two chains and is 50 A long. On the addition of up to 1M CsCl, the aggregation number increases up to 15 whereas the bundle length approaches a limiting value, 20 A. We suggest that the bundle formation is determined by a balance between long-ranged electrostatic repulsion, whose range and amplitude is decreased on salt addition, and short-ranged attraction.

Polystyrene sulfonate (PSS 77 kDa) adsorbed onto oppositely charged dioctadecyldimethylammonium bromide (DODA) monolayers at the air/water interface is investigated with X-ray reflectivity and grazing incidence diffraction. The alkyl tails of DODA in the condensed phase form an oblique lattice with large tilts and intermediate azimuth angle. On PSS adsorption, the alkyl tail structure is maintained; only the tilt angle changes. Bragg peaks caused by flatly adsorbed, aligned PSS chains are observed, when DODA is in the fluid and also when it is in the condensed phase. The two-dimensional lamellar phase is only found at intermediate PSS bulk concentrations (0.001-1 mmol/L). In this phase, the PSS coverage can be varied by a factor of 3, depending on DODA molecular area and polymer bulk concentration. Charge compensation in the lamellar phase is almost achieved at 1 mmol/L. At larger bulk concentrations, PSS adsorbs flatly yet without chain alignment. Presumably, a necessary condition for a two-dimensional lamellar phase is a pronounced electrostatic force which causes a large persistence length as well as repulsion between the aligned chains.