Adsorption Kinetics Of Fibrinogen Research Articles

ChemInform Abstract: Mechanisms of Fibrinogen Adsorption at Solid Substrates

AbstractReview: 100 refs.

Fibrinogen adsorption mechanisms at the gold substrate revealed by QCM-D measurements and RSA modeling

Adsorption kinetics of fibrinogen at a gold substrate at various pHs was thoroughly studied using the QCM-D method. The experimental were interpreted in terms of theoretical calculations performed according to the random sequential adsorption model (RSA). In this way, the hydration functions and water factors of fibrinogen monolayers were quantitatively evaluated at various pHs. It was revealed that for the lower range of fibrinogen coverage the hydration function were considerably lower than previously obtained for the silica sensor [33]. The lower hydration of fibrinogen monolayers on the gold sensor was attributed to its higher roughness. However, for higher fibrinogen coverage the hydration functions for both sensors became identical exhibiting an universal behavior. By using the hydration functions, the fibrinogen adsorption/desorption runs derived from QCM-D measurements were converted to the Γd vs. the time relationships. This allowed to precisely determine the maximum coverage that varied between 1.6mgm−2 at pH 3.5 and 4.5mgm−2 at pH 7.4 (for ionic strength of 0.15M). These results agree with theoretical eRSA modeling and previous experimental data derived by using ellipsometry, OWLS and TIRF. Various fibrinogen adsorption mechanisms were revealed by exploiting the maximum coverage data. These results allow one to develop a method for preparing fibrinogen monolayers of well-controlled coverage and molecule orientation.

Mechanisms of fibrinogen adsorption at the silica substrate determined by QCM-D measurements

Adsorption kinetics of fibrinogen at a silica substrate was thoroughly studied in situ using the QCM-D method. Because of low dissipation, the Sauerbrey’s equation was used for calculating the wet mass per unit area (wet coverage of the protein). Measurements were done for various bulk suspension concentrations, flow rates and pHs. These experimental data were compared with the theoretical dry coverage data derived from the solution of the mass transfer equation. In this way, the hydration functions and water factors of fibrinogen monolayers were quantitatively evaluated for various pHs. In the case of pH 7.4 and ionic strength of 0.15M, the hydration function changed from 0.75 to 0.6 for the dry coverage Γd equal to 0 and 4mgm−2, respectively. Interestingly, for pH 7.4 and 4.5 (ionic strength of 10−2M) a minimum of the hydration function appeared at Γd ca. 2mgm−2. Analytical polynomial expressions were formulated for the interpolation of the experimental results. By using the hydration functions, the fibrinogen adsorption/desorption runs derived from QCM-D measurements were converted to the Γd vs. the time relationships. This allowed to precisely determine the maximum coverage that varied between 1.2mgm−2 at pH 3.5 and 4.2mgm−2 at pH 7.4 for ionic strength of 0.15M. These results agree with theoretical modeling and previous experimental data derived by using ellipsometry, OWLS and TIRF. Various fibrinogen adsorption mechanisms were revealed by exploiting the maximum coverage data whose validity was also confirmed by the dissipation vs. the dry mass relationships. Beside significance to basic science, these results enable to develop a robust technique, based on the QCM-D measurements, suitable for precisely determining the dry mass of protein monolayers adsorbed under various physicochemical conditions.

Influence of Surface Charge/Potential of a Gold Electrode on the Adsorptive/Desorptive Behaviour of Fibrinogen

The effect of gold substrate surface charge (potential) on adsorptive/desorptive behaviour of fibrinogen (FG) was studied by employing differential capacitance (DC) and polarization modulated infrared reflection absorption spectroscopy (PM-IRRAS), in terms of FG adsorption thermodynamics, kinetics, and desorption kinetics. The gold substrate surface charge was modulated in-situ within the electrochemical double-layer region by means of electrochemical potentiostatic polarization in a FG-containing electrolyte, thus avoiding the interference of other physico-chemical properties of the gold surface on FG’s interfacial behaviour. The FG adsorption equilibrium was modeled using the Langmuir isotherm. Highly negative values of apparent Gibbs free energy of adsorption (ranging from from −52.1±0.4 to −55.8±0.8kJmol−1, depending on the FG adsorption potential) indicated a highly spontaneous and strong adsorption of FG onto the gold surface. The apparent Gibbs free energy of adsorption was found to be independent of surface charge when the surface was negatively charged. However, when the gold surface was positively charged, the apparent Gibbs free energy of adsorption exhibited a pronounced linear relationship with the surface charge, shifting to more negative values with an increase in positive electrode potential. The adsorption kinetics of FG was also found to be dependent on gold surface charge in a similar manner to the apparent Gibbs free energy of adsorption.It was suggested that the driving force for the adsorption of FG on a negatively charged surface represents a positive gain in the entropy of the system, whereas the adsorption on a positively charged gold surface was found to be controlled by electrostatic forces.FG desorption measurements revealed that when the gold surface is polarized within the electrochemical double-layer region during the desorption process, the protein desorption kinetics is rather slow. However, within the regions of hydrogen and oxygen evolution, the FG desorption kinetics accelerates significantly, due to the physical removal of the adsorbed protein layer by gas bubbles evolving from the substrate surface, which enables a complete removal of the pre-adsorbed FG layer. The latter could potentially be employed for electrochemical cleaning of electrically-conducting surfaces fouled by adsorbed protein layers (heat exchangers, filtration membranes, etc.).

Mechanisms of Fibrinogen Adsorption at Solid Substrates

The aim of this work was to critically review recent results pertinent to fibrinogen adsorption at solid/electrolyte interfaces with the emphasis focused on a quantitative analysis of these processes in terms of the electrostatic interactions. Accordingly, in the first part, the primary chemical structure of fibrinogen is analyzed. Physicochemical data pertinent to the bulk properties derived from hydrodynamic, dynamic light scattering and micro-electrophoretic measurements aided by theoretical modeling are discussed. Possible conformations and the effective charge distribution over the fibrinogen molecule for various pH an ionic strength are defined, especially the semi-collapsed conformation prevailing at physiological conditions. Adsorption kinetics of fibrinogen at hydrophilic and hydrophobic (polymer modified) substrates determined by various techniques is described. Adsorption at polymeric carrier particles, pertinent to immunological assays, studied in terms of electrokinetic and concentration depletion methods, are also considered. The reversibility of adsorption, fibrinogen molecule orientations and maximum coverages are thoroughly discussed. The stability of fibrinogen monolayers formed at these carrier particles in respect to pH and ionic strength cyclic changes is also discussed. In the final section interactions and deposition of model colloid particles on fibrinogen monolayers are analyzed which allows one to derive valuable information about molecule orientations. Based on the physicochemical data, adsorption kinetics and colloid particle deposition measurements, probable adsorption mechanisms of fibrinogen on solid/electrolyte interfaces are defined.

Fibrinogen Adsorption on Mica Studied by AFM and in Situ Streaming Potential Measurements

Adsorption of fibrinogen from aqueous solutions on mica was studied using AFM and in situ streaming potential measurements. In the first stage, bulk physicochemical properties of fibrinogen and the mica substrate were characterized for various ionic strength and pH. The zeta potential and number of uncompensated (electrokinetic) charges on the protein surfaces were determined from microelectrophoretic measurements. Analogously, using streaming potential measurements, the electrokinetic charge density of mica was determined for pH range 3-10 and the NaCl background electrolyte concentration of 10(-3) and 10(-2) M. Next, the kinetics of fibrinogen adsorption at pH 3.5 and 7.4 in the diffusion cell was studied using a direct AFM determination of the number of molecules per unit area of the mica substrate. Then, streaming potential measurements were performed to determine the apparent zeta potential of fibrinogen-covered mica for different pH and ionic strength in terms of its surface concentration. A quantitative interpretation of these streaming potential measurements was achieved in terms of the theoretical model postulating a side-on adsorption of fibrinogen molecules as discrete particles. On the basis of these results, the maximum coverage of fibrinogen Θ close to 0.29 was predicted, in accordance with previous theoretical predictions. It was also suggested that anomalous adsorption for pH 7.4, where fibrinogen and the mica substrate were both negatively charged, can be explained in terms of a heterogeneous charge distribution on fibrinogen molecules. It was estimated that the positive charge was 12 e (for NaCl concentration of 10(-2) M and pH 7.4) compared with the net charge of fibrinogen at this pH, equal to -21 e. Results obtained in this work proved that the coverage of fibrinogen can be quantitatively determined using the streaming potential method, especially for Θ < 0.2, where other experimental methods become less accurate.

Kinetics of Fibrinogen Adsorption on Hydrophilic Substrates

Irreversible side-on adsorption of fibrinogen, modeled as a linear chain of touching beads of various size, was studied theoretically using the random sequential adsorption (RSA) model. Numerical simulation of the Monte Carlo type enabled one to determine the dependence of the surface blocking function (available surface function) on the protein coverage. These numerical results were interpolated using analytical functions based on a polynomial expansion. The dependence of the jamming coverage on the size of the simulation area was also determined. By an extrapolation of these results to the infinite area size, the maximum surface concentration of fibrinogen for the side-on adsorption was determined to be 2.26 x 10(3) microm(-2). This corresponds to a jamming coverage theta(infinity) of 0.29. It was shown that the blocking function can well be approximated in the limit of high coverage by the dependence C(theta(infinity) - theta)(4). Using this interpolating expression, the kinetics of fibrinogen adsorption under convection and diffusion transport conditions were evaluated for various bulk concentrations of the protein. These kinetic curves were derived by numerically solving the mass transport equation in the bulk with the blocking function used as a nonlinear boundary condition at the interface. It was shown that our theoretical results are in agreement with experimental kinetic data obtained by AFM, ellipsometry, and other techniques for hydrophilic surfaces in the limit of low bulk fibrinogen concentration.

Conformational behavior of fibrinogen on topographically modified polymer surfaces

The influence of topographical surface features at the submicron scale on the structural changes in the surface-adsorbed fibrinogen was investigated on poly(lactic-co-glycolic-acid) (PLGA) films. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) was employed in this study for the induced conformational change of fibrinogen over various adsorption times, while the adsorption kinetics of fibrinogen was quantified by the enzyme linked immunosorbent assay (ELISA). When a PLGA surface is modified topographically, the adsorbed fibrinogen undergoes less conformational change when compared to adsorption on the pristine PLGA surface. The extent of conformational change is related to platelet adhesion. Reduced thrombogenicity was demonstrated by the higher ratios of alpha-helix to beta-turn and beta-sheet to beta-turn structures on the topographic PLGA film, which suggests that topographical manipulation of surfaces is a viable approach to influence the thrombogenicity of surfaces.

Adsorption of fibrinogen on a biomedical-grade stainless steel 316LVM surface: a PM-IRRAS study of the adsorption thermodynamics, kinetics and secondary structure changes

Polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS) was employed to investigate the interaction of serum protein fibrinogen with a biomedical-grade 316LVM stainless steel surface, in terms of the adsorption thermodynamics, kinetics and secondary structure changes of the protein. Apparent Gibbs energy of adsorption values indicated a highly spontaneous and strong adsorption of fibrinogen onto the surface. The kinetics of fibrinogen adsorption were successfully modeled using a pseudo first-order kinetic model. Deconvolution of the amide I bands indicated that the adsorption of fibrinogen on 316LVM results in significant changes in the protein's secondary structure that occur predominantly within the first minute of adsorption. Among the investigated structures, the alpha-helix structure undergoes the smallest changes, while the beta-sheet and beta-turns structures undergo significant changes. It was shown that lateral interactions between the adsorbed molecules do not play a role in controlling the secondary structure changes. An increase in temperature induced changes in the secondary structure of the protein, characterized by a loss of the alpha-helical content and its transformation into the beta-turns structure.

Monitoring fibrinogen adsorption kinetics by interfacial TIRF rheometry.

The adsorption kinetics of purified fibrinogen to unmodified and aminopropylsilane-modified quartz glass surfaces were studied under pseudo-first order (binding-unit excess) conditions by the total internal reflection fluorescence (TIRF) method. Fluorescence in the adsorbed protein layer (350 nm) was excited by the evanescent wave at 285-290 nm. In order to reduce and possibly eliminate the influence of mass transfer on the kinetics of fibrinogen adsorption, a novel protein adsorption chamber containing a cone-and-plate rheometer with total internal reflection technology was employed. The aim of the study was to obtain critical shear rates, at which the adsorption rate of fibrinogen became independent of diffusion. Therefore, shear rates were varied between 0 and 7200 s-1 at initial fibrinogen concentrations of c9 = 4.7 and 17.7 micrograms mL-1. The adsorption rate of fibrinogen increased 5-17-fold, depending on the surface, as the critical shear rate was approached. Above the critical shear rates the kinetic data of fibrinogen adsorption could be fitted at c9 = 4.7 micrograms mL-1 to a single exponential function, indicating the predominance of a single binding step with a half-life of ca 20 s. At the higher initial concentration of c9 = 17.7 micrograms/mL-, however, a significant deviation from the single exponential behavior was observed in the first 10 s of the adsorption reaction, indicating a very fast initial event with a half-life of ca 5 s in addition to a slower binding reaction with a half-life of ca 35 s. Thus the novel TIRF rheometer can resolve kinetics down to half-lives of 5 s and possibly even lower.

Kinetics of fibrinogen adsorption from solutions flowing through polymer hollow fibers

Kinetics of fibrinogen adsorption from solutions flowing through polymer hollow fibers

Effect of surface hydrophobicity on the conformational changes of adsorbed fibrinogen

The extent of conformational changes of fibrinogen adsorbed on germanium, poly(hydroxyethyl methacrylate) (poly(HEMA), Biomer, and polystyrene surfaces was studied using Fourier transform infrared spectroscopy (FTIR) coupled with attenuated total reflectance (ATR) optics. The contact angles of water on the solid surfaces were 19.3°, 35.5°, 37.6°, and 90.5°, respectively. The adsorption kinetics of fibrinogen from 1 mg/ml solution showed that the amount of adsorbed fibrinogen increased as the surface became more hydrophobic. The spectra of fibrinogen in the bulk solution and of fibrinogen tightly adsorbed on germanium, poly(HEMA), Biomer, and polystyrene surfaces were treated using Fourier self-deconvolution and the synthetic single-peak fitting techniques to resolve the overlapped peaks in the amide I and II regions. It was found that some α-helical structures were changed into the unordered structures and the content of β-turns was increased upon the protein adsorption. A weighted-peak shift method was used to examine the extent of the protein conformational changes upon adsorption on hydrophobic and hydrophilic surfaces. The results indicated that the adsorbed fibrinogen underwent a larger degree of conformational changes as the surface hydrophobicity increased.

The kinetics of baboon fibrinogen adsorption to polymers: in vitro and in vivo studies.

Fibrinogen adsorption on polymers from blood may mediate or potentiate thrombosis because of its involvement in both the intrinsic clotting system and the formation of platelet aggregates. While the kinetics of fibrinogen adsorption from plasma in vitro have previously been found to be very different on polar and nonpolar surfaces [T. A Horbett, "The kinetics of adsorption of plasma proteins to a series of hydrophilic-hydrophobic copolymers," ACS Org. Coat. Plas. Chem. 40, 642-646 (1979)] the significance of this difference with respect to thrombogenesis in vivo has not been clarified. In this study, the kinetics of deposition of baboon 125I fibrinogen from plasma in vitro or from blood in vivo on a series of polymers was measured. The polymers chosen for this study had previously been found to have a large range in surface polarity and reactivity in the in vivo baboon shunt model. The kinetics of fibrinogen adsorption in vitro were observed to be of three types, depending on the polymer: high initial adsorption decreasing to a lower steady state value; constant throughout the time course; low initial adsorption rising steadily to a plateau value. In vivo, fibrinogen deposition kinetics were of two types: low, constant deposition throughout the time course, independent of heparinization; low deposition initially followed by a second phase of greatly increased deposition (probably as fibrin) which was prevented or greatly decreased by heparinizing the animals. Polymers for which fibrinogen adsorption increased to a plateau in vitro were found to have a heparin inhibitable second phase of enhanced in vivo fibrinogen deposition. These polymers also have been found in previous studies to enhance the rate of platelet destruction when used as in vivo shunts on baboons. Conversely, most polymers with high initial in vitro fibrinogen adsorption followed by a decrease had low fibrinogen deposition behavior in vivo and were also minimally destructive of platelets. The adsorption kinetics of fibrinogen to polymers from blood in vivo and in vitro and the consumption of platelets in vivo induced by the polymers all vary with polymer polarity. More polar polymers had in vitro fibrinogen kinetics characterized by a rise to a plateau, in vivo fibrinogen deposition characterized by a second stage of great increase inhibitable by heparin, and enhanced platelet consumption. The correlation of three separate indicators of surface thrombogenicity with surface polarity suggests that more polar materials may be more thrombogenic because of an influence on the way in which fibrinogen interacts with these surfaces.