Reversibility in the Physical Properties of Agarose Gels following an Exchange in Solvent and Non-Solvent.

Agarose forms a homogeneous thermoreversible gel in an aqueous solvent above a critical polymer concentration. Contrary to the prevailing consensus, recent confirmations indicate that agarose gels are also stable in non-solvents like acetone and ethanol. A previous study compared gel characterisations and behaviours in water and ethanol, discussing the gelation mechanism. In the current work, the ethanol gel is exchanged with water to explore the potential reversibility of the displacement of water in agarose. Initially, the structure is characterised using 1H NMR in DMSO-d6 and D2O solvents. Subsequently, a very low yield (0.04) of methyl substitution per agarobiose unit is determined. The different gels after stabilisation are characterised using rheology, and their physical properties are compared based on the solvent used. The bound water molecules, acting as plasticizers in aqueous medium, are likely removed during the exchange process with ethanol, resulting in a stronger and more fragile gel. Next, the gel obtained after the second exchange from ethanol back to water is compared with the initial gel prepared in water. This is the first time where such gel has been characterised without undergoing a phase transition when switching from a good solvent to a non-solvent, and vice versa, thereby testing the reversibility of the solvent exchange. Reversibility of this behaviour is demonstrated through swelling and rheology experiments. This study extends the application of agarose in chromatography and electrophoresis.

Are coagulation profiles in Andean highlanders with excessive erythrocytosis favouring hypercoagulability?

Chronic mountain sickness is a maladaptive syndrome that affects individuals living permanently at high altitude and is characterized primarily by excessive erythrocytosis (EE). Recent results concerning the impact of EE in Andean highlanders on clotting and the possible promotion of hypercoagulability, which can lead to thrombosis, were contradictory. We assessed the coagulation profiles of Andeans highlanders with and without excessive erythrocytosis (EE+ and EE-). Blood samples were collected from 30 EE+ and 15 EE- in La Rinconada (Peru, 5100-5300 m a.s.l.), with special attention given to the sampling pre-analytical variables. Rotational thromboelastometry tests were performed at both native and normalized (40%) haematocrit using autologous platelet-poor plasma. Thrombin generation, dosages of clotting factors and inhibitors were measured in plasma samples. Data were compared between groups and with measurements performed at native haematocrit in 10 lowlanders (LL) at sea level. At native haematocrit, in all rotational thromboelastometry assays, EE+ exhibited hypocoagulable profiles (prolonged clotting time and weaker clot strength) compared with EE- and LL (all P < 0.01). At normalized haematocrit, clotting times were normalized in most individuals. Conversely, maximal clot firmness was normalized only in FIBTEM and not in EXTEM/INTEM assays, suggesting abnormal platelet activity. Thrombin generation, levels of plasma clotting factors and inhibitors, and standard coagulation assays were mostly normal in all groups. No highlanders reported a history of venous thromboembolism based on the dedicated survey. Collectively, these results indicate that EE+ do not present a hypercoagulable profile potentially favouring thrombosis.

Characterization of Agarose Gels in Solvent and Non-Solvent Media.

Agarose is known to form a homogeneous thermoreversible gel in an aqueous medium over a critical polymer concentration. The solid-liquid phase transitions are thermoreversible but depend on the molecular structure of the agarose sample tested. The literature has mentioned that agarose gels could remain stable in non-solvents such as acetone or ethanol. However, there has been no characterization of their behavior nor a comparison with the gels formed in a good solvent such as water. In the first step of this article, the structure was characterized using 1H and 13C NMR in both D2O and DMSO-d6 solvents. DMSO is a solvent that dissolves agarose regardless of the temperature. First, we have determined a low yield of methyl substitution on the D-galactose unit. Then, the evolution of the 1H NMR spectrum was monitored as a function of temperature during both increasing and decreasing temperature processes, ranging from 25 to 80 °C. A large thermal hysteresis was obtained and discussed, which aided in the interpretation of rheological behavior. The hysteresis of NMR signals is related to the mobility of the agarose chains, which follows the sol/gel transition depending on the chains' association with H-bonds between water and the -OH groups of agarose for tightly bound water and agarose/agarose in chain packing. In the second step of the study, the water in the agarose gel was exchanged with ethanol, which is a non-solvent for agarose. The resulting gel was stable, and its properties were characterized using rheology and compared to its behavior in aqueous media. The bound water molecules that act as plasticizers were likely removed during the exchange process, resulting in a stronger and more brittle gel in ethanol, with higher thermal stability compared to the aqueous gel. It is the first time that such gel is characterized without phase transition when passing from a good solvent to a non-solvent. This extends the domains of application of agarose.

Rheological Behavior of Pectin Gels Obtained from Araçá (Psidium cattleianum Sabine) Fruits: Influence of DM, Pectin and Calcium Concentrations.

In this work, purified pectins from Araçá fruits (Psidium cattleianum Sabine) were obtained and characterized after partial demethylation. On each prepared sample, the carboxylic yield was obtained by titration, the degree of methylation (DM) by 1H-NMR, and the molecular weight distribution by steric exclusion chromatography (SEC). Then, the gelation ability in the presence of calcium counterions was investigated and related to DM (59-0%); the pectin concentration (2-10 g L-1); and the CaCl2 concentration (0.1-1 mol L-1) used for dialysis. The critical pectin concentration for homogeneous gelation was above 2 g L-1 when formed against 1 mol L-1 CaCl2. The elastic modulus (G') increased with pectin concentration following the relationship G'~C2.8 in agreement with rigid physical gel network predictions. The purified samples APP and APP-A with DM ≥ 40% in the same conditions released heterogeneous systems formed of large aggregates. Gels formed against lower concentrations of CaCl2 down to 0.1 mol L-1 had a higher degree of swelling, indicating electrostatic repulsions between charged chains, thus, counterbalancing the Ca2+ cross-linkage. Compression/traction experiments demonstrated that an irreversible change in the gel structure occurred during small compression with an enhancement of the G' modulus.

Aggregates Dramatically Alter Fibrin Ultrastructure.

Among the many factors influencing fibrin formation and structure (concentration, temperature, composition, pH, etc.), it has been suggested that the polydispersity of fibrinogen may play an important role. We propose here a detailed investigation of the influence of this parameter on fibrin multiscale structure. Two commercial fibrinogen preparations were used, a monodisperse and a polydisperse one. First, the respective compositions of both fibrinogen preparations were thoroughly determined by measuring the fibrin-stabilizing factor; fibronectin; α, ß, and γ intact chain contents; the γ/γ' chains ratio; the N-glycosylation; and the post-translational modifications. Slight variations between the composition of the two fibrinogen preparations were found that are much smaller than the compositional variations necessary to alter significantly fibrin multiscale structure as observed in the literature. Conversely, multiangle laser light scattering-coupled size exclusion chromatography and dynamic light scattering measurements showed that the polydisperse preparation contains significant amounts of aggregates, whereas the other preparation is essentially monodisperse. The multiscale structure of the fibrins produced from those two fibrinogen preparations was determined by using x-ray scattering, spectrophotometry, and confocal microscopy. Results show that fibers made from the aggregate-free fibrinogen present a crystalline longitudinal and lateral structure and form a mikado-like network. The network produced from the aggregates containing fibrinogen looks to be partly built around bright spots that are attributed to the aggregate. The multiscale structure of mixtures between the two preparations shows a smooth evolution, demonstrating that the quantity of aggregates is a major determining factor for fibrin multiscale structure. Indeed, the effect of a few percent in the mass of aggregates is larger than any other effect because of compositional differences under the same reaction conditions. Finally, we propose a mechanistic interpretation of our results, which points at a direct role of the aggregates during polymerization, which disrupts the ideal ordering of monomers inside fibrin protofibrils and fibers.

Fibrinography: A Multiwavelength Light-Scattering Assay of Fibrin Structure.

We have previously developed a fibrin structural assay dedicated to purified fibrinogen-thrombin system. Here, we extend the pertinence of this test, called Fibrinography, to tissue factor-triggered plasma coagulation. We show that Fibrinography determines quantitatively the structure of fibrin fibers in plasma with an excellent reproducibility. We compare this assay with the commonly used single wavelength turbidity method, showing that the latter is not a proper structural assay, but determines essentially the fibrinogen content in plasma. In addition, we also show, in model plasmas, that Fibrinography is able to discriminate normal and hypocoagulant plasmas, and even between hypercoagulant plasmas. Therefore, Fibrinography, by measuring the final step of the coagulation cascade, may be used to evaluate patients' plasma in hypo- or hypercoagulant diseases.

The influence of coagulation on the drying dynamics of blood pools.

Little is currently known about the importance of clotting during the drying of blood pools. While this is of little moment for droplets where drying occurs faster than contact-phase-induced clotting, clotting may significantly influence blood pools drying as it transform a liquid into a gel. To investigate this influence, we compare the drying of citrated and unmodified blood pools at constant haematocrit, showing large morphological differences during drying, both in the surface appearance, in the colour lightness, as well as in the generation and location of cracks. Further, we designed a clotting-reactivation protocol which allowed recovering the morphological evolution of pure blood drying while using citrate-sampled blood. This result opens the way to the use of citrated blood in blood pools investigations.

Antithrombin-independent effects of heparins on fibrin clot nanostructure.

OBJECTIVE: Because of the widespread clinical use of heparins, their effects on the enzymatic cascade are very well known. In contrast, little is known about the direct effect of heparins on the nanostructure of fibrin fibers, even though this nanostructure plays a major role in the mechanical strength and lysis of clots. This lack of reliable data can be correlated with the lack of a nonintrusive, quantitative method to determine this structure. We recently developed such a method that allows the simultaneous determination of the average fiber radius and the protein content using spectrometric data. In this study, we assessed the nanostructure of fibrin in a system composed of human thrombin and fibrinogen. METHODS AND RESULTS: We provide quantitative evidence showing that both unfractionated heparin and low molecular weight heparin directly alter the nanostructure of fibrin fibers independent of their other actions on the coagulation cascade; as expected, the pentasaccharide fondaparinux has no effect. CONCLUSIONS: Our results show that in addition to the effect of heparin on the coagulation cascade, modifications of the fibrin nanostructure may also contribute to improved fibrinolysis.

Size determination by use of two-dimensional Mueller matrices backscattered by optically thick random media.

Here we are concerned with the systematic study of polarized light transport in thick, isotropic, homogeneous random media and of the associated inverse problem. An original spatial and intensity rescaling of the polarization transport allows one to account implicitly for the volume fraction. This parameter elimination permits a complete exploration, by means of Monte Carlo simulations of the dependence of polarized light transport on microscopic parameters. Analysis of the Mueller matrices obtained from the simulations show that additional correlations (with respect to scalar transport) are obtained between the microscopic parameters and the spatial distribution of specific elements of the Mueller matrix. As a consequence, using carefully chosen polarization states, one can determine an average particle size independently of the volume fraction of particles, with only the knowledge of the refractive-index ratio being required. This analysis is validated with experimental Mueller matrices obtained for emulsions of various size, concentration, and polydispersity.