Visualization of human plasma fibrinogen adsorbed on highly oriented pyrolytic graphite by scanning probe microscopy

doi:10.1016/j.susc.2005.12.058

Surface Science

Volume 600, Issue 8, 15 April 2006, Pages 1674-1678

https://doi.org/10.1016/j.susc.2005.12.058 Get rights and content

Abstract

Human plasma fibrinogen (HPF) was observed by atomic force microscopy (AFM) and Kelvin probe force microscopy (KPFM) conducted in non-contact mode. The HPF was adsorbed on a highly oriented pyrolytic graphite (HOPG) substrate as single molecules, as aggregated bundles, and as aggregated fibers. Topographic and phase images confirmed structural changes in the HPF after exposure to air, while topographic and KPFM images confirmed fibers with the width of a single HPF molecule. Additionally, KPFM confirmed the surface potential difference between the HPF and the HOPG, and periodical potential drop reflecting the E and D domains in the fiber.

Introduction

The control of blood coagulation on biomaterial surfaces is an urgent issue in the medical field. The main process occurring in blood coagulation is the polymerization of fibrinogen into erythrocyte-trapping fibrin [1]. Other plasma proteins are also involved in initiating this process. The molecular structure of these proteins plays a great role in coagulation. Many researchers have attempted to analyze and visualize the molecular structure of fibrinogen and fibrin through various approaches including electron microscopy, amino acid sequence analysis, X-ray diffraction [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13]. Fig. 1 shows the simplified structure of fibrinogen [1], [2], [10], [11]. Fibrinogen is a 340 kDa protein consisting of polypeptide chains of Aα, Bβ, and γ. These chains are coiled as an α helix and are terminated as domains referred to as αC, D and E. Two chain units consisting of Aα, Bβ and γ chains are connected in the E domain to form a dimer. Although the molecular structure of fibrinogen as a free molecule has been investigated in detail, its adsorption state on a biomaterial surface has yet to be elucidated. Recently, the adsorption state of fibrinogen on various substrates has been the subject of intensive investigation employing atomic force microscopy (AFM) [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27]. Such use of AFM has been crucial in the study of molecular biology on surfaces. In this article, we report new information on the adsorption state of fibrinogen obtained by combining the results of topographic and phase images. Additionally, we report on surface potential images of fibrinogen obtained by Kelvin probe force microscopy (KPFM).

Section snippets

Fibrinogen adsorption on the substrate surface

Human plasma fibrinogen (HPF; Aldrich, Type I: protein 63%, containing approx. 15% sodium citrate and approx. 25% sodium chloride) was absorbed on a highly oriented pyrolite graphite (HOPG; Advanced Ceramics, STM-1) surface by immersing the HOPG substrate into a HPF solution (concentration: 0.01 μg/ml) for 5 min at 25 °C. Dulbecco’s phosphate-buffered saline (PBS; pH 7.4) without Ca and Mg was used as the solvent for the HPF solution. After immersion, the sample was rinsed once with PBS, and then

Results and discussion

Fig. 2 shows the topography of a 1 μm × 1 μm region of the HOPG surface scanned within 1 h after the adsorption experiment. The HPF was dispersed on the substrate in various configurations: (i) as single molecules, (ii) as aggregated bundles, and (iii) as aggregated fibers. The HPF’s adsorption on the HOPG surface in the PBS solution may have been due to hydrophobic interaction. Likewise, the HPF observed in this AFM image may have remained immobilized during the rinsings by PBS solution and pure

Conclusions

The adsorption state of fibrinogen was investigated based on the topographic, phase, and surface potential images obtained by non-contact SPM. HPF was adsorbed on a HOPG substrate surface as single molecules, or as aggregates of bundles or fibers. Unfolding in the HPF after exposure to air was confirmed by topographic and phase images obtained for single HPF molecules. Fibers with the width of a single HPF molecule were also confirmed from topographic images. Periodical potential drop, which

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Periodic surface potential dependence corresponding to the different polypeptide surface potentials were observed. This improved methods to control blood coagulation, which is an important issue in the medical field [75]. Similar studies were performed for the distribution of surface potentials in amyloid fibrils.
Electrical properties, such as charge propagation, dielectrics, surface potentials, conductivity, and piezoelectricity, play crucial roles in biomolecules, biomembranes, cells, tissues, and other biological samples. However, characterizing these electrical properties in delicate biosamples is challenging. Atomic Force Microscopy (AFM), the so called “Lab on a Tip” is a powerful and multifunctional approach to quantitatively study the electrical properties of biological samples at the nanometer level. Herein, the principles, theories, and achievements of various modes of AFM in this area have been reviewed and summarized.
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The single amino acid resolution level of denatured in formic acid cytochrome c and bovine serum albumin molecules was achieved with scanning tunneling microscopy under high vacuum: these molecules have demonstrated either unfolded or 2D-refolded state [15]. Different AFM studies have reported on the morphological changes of the adsorbed proteins [16–19] that may reflect surface induced denaturation, which is a well-known effect [2,20]. Recently using AFM operated in ambient environment we have visualized HOPG induced denaturation of several proteins of blood plasma [21].
Fibrinogen denaturation is an important phenomenon in biology and medicine. It has been previously investigated with bulk methods and characterized by parameters, which refer to big protein ensembles. Here we provide a new insight into fibrinogen denaturation with a high-resolution single-molecule atomic force microscopy (AFM). The ultrastructure of individual fibrinogen molecules was studied after heating or extended contact with the highly oriented pyrolytic graphite surface (HOPG) modified with oligoglycine-hydrocarbon graphite modifier (GM). Fibrinogen heating to 65 °C and 90 °C resulted in the formation of various shapes containing fibrillar and globular structures, which were attributed to the monomers and small aggregates of fibrinogen. Fibrinogen unfolded by the extended (10 min) incubation on GM-HOPG surface in water revealed a different morphology. It contained fibrillar structures only, and their organization reflected the initial native structure of fibrinogen: typically, six polypeptide chains connected by multiple disulfide bonds were seen. A combination of two morphologies – globular aggregates with dense fibrillar networks – has been revealed for thermally denatured protein adsorbed on a GM-HOPG surface with extended (10 min) rinsing with water. The obtained results provide better understanding of fibrinogen unfolding induced by different factors and are important for improvement of biomedical applications, such as fibrinogen-based protein matrixes and carbon-based biomaterials.
AFM visualization at a single-molecule level of denaturated states of proteins on graphite
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This general conclusion is based on a lot of results obtained by applications of different experimental approaches, however, the reported results of microscopic observations at the level of single molecules are often inconsistent. There are plenty of studies of the conformational changes of the proteins adsorbed on other surfaces (like silica, metal, polymer surfaces etc.); however, little attention has been paid to the convincing high resolution microscopic study of conformational behavior of protein adsorbed on hydrophobic HOPG, whilst the reported results are frequently contradictory [26⿿30]. Recent studies indicate that the probable reason of non-reproducibility is extremely high sensitivity of HOPG surface to impurities from water media and air [31⿿33].
Different graphitic materials are either already used or believed to be advantageous in biomedical and biotechnological applications, e.g., as biomaterials or substrates for sensors. Most of these applications or associated important issues, such as biocompatibility, address the problem of adsorption of protein molecules and, in particular the conformational state of the adsorbed protein molecule on graphite. High-resolution AFM demonstrates highly oriented pyrolytic graphite (HOPG) induced denaturation of four proteins of blood plasma, such as ferritin, fibrinogen, human serum albumin (HSA) and immunoglobulin G (IgG), at a single molecule level. Protein denaturation is accompanied by the decrease of the heights of protein globules and spreading of the denatured protein fraction on the surface. In contrast, the modification of HOPG with the amphiphilic oligoglycine-hydrocarbon derivative monolayer preserves the native-like conformation and provides even more mild conditions for the protein adsorption than typically used mica. Protein unfolding on HOPG may have universal character for ⿿soft⿿ globular proteins.
Influence of Surface Charge/Potential of a Gold Electrode on the Adsorptive/Desorptive Behaviour of Fibrinogen
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This, in turn, increases the probability for intermolecular interactions between the αC domains of the neighbouring FG molecules, which results in a more thermodynamically stable state for the θ1 form of FG. Consequently, the driving force for the second adsorption step, transformation of θ1 to θ2, decreases [31,32]. Next, the modeled (simulated) kinetic data were deconvoluted into the two contributions (θ1 and θ2), and presented in Fig. 6 (solid lines).
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 ± 0.8 kJ mol⁻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.).
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The knowledge of fibrinogen adsorption is of great importance because its layers formed at the liquid–solid interfaces trigger inflammatory responses that may lead to the acceptance or rejection of implants. Due to these reasons fibrinogen has been intensively studied for the last few decades [6–9]. The geometrical shape and dimensions of fibrinogen were first acquired by the electron microscopy studies of Hall and Slayter [10].
Adsorption of fibrinogen on polystyrene latex particles was studied using the micro-electrophoretic and the concentration depletion methods. Measurements were carried out for pH 3.5, 7.4 and ionic strength of 0.15 M, NaCl. Electrophoretic mobility of latex was determined as a function of the amount of adsorbed fibrinogen, expressed as a surface concentration. A monotonic increase in the electrophoretic mobility (zeta potential) of the latex was observed, indicating a significant adsorption of fibrinogen on latex for pH equal to 3.5 and 7.4, respectively. The anomalous adsorption in the latter case was explained in terms of the heterogeneous charge distribution on the fibrinogen molecule. The stability of fibrinogen monolayers formed on latex was also determined in pH cycling between 3.5 and 9.7. These measurements revealed that fibrinogen adsorption was irreversible, governed by the two main adsorption mechanisms: (i) the unoriented (random) mechanism prevailing for pH = 3.5 where adsorbing molecules significantly penetrate the latex particle core and (ii) the side-on adsorption mechanism prevailing for pH equal to 7.4. In both cases, variations in the zeta potential with the fibrinogen coverage were adequately described in terms of the electrokinetic model, previously formulated for particle adsorption on planar substrates. Based on these experimental data, an efficient procedure of preparing fibrinogen monolayers on latex particles of controlled conformations and coverage was envisaged.
Evaluation of fibrinogen self-assembly: Role of its αC region
2010, Journal of Thrombosis and Haemostasis
Background:Exposure of cryptic, functional sites on fibrinogen upon its adsorption to hydrophobic surfaces of biomaterials has been linked to an inflammatory response and fibrosis. Such adsorption also induces ordered fibrinogen aggregation which is poorly understood. Objective:To investigate hydrophobic surface‐induced fibrinogen aggregation. Methods:Contact and lateral force scanning probe microscopy, yielding topography, image dimensions and fiber elastic modulus measurements were used along with transmission and scanning electron microscopy. Fibrinogen aggregation was induced under non‐enzymatic conditions by adsorption on a trioctyl‐surface monolayer (trioctylmethylamine) grafted onto silica clay plates. Results:A more than one molecule thick coating was generated by adsorption on the plate from 100 to 200 μg mL⁻¹ fibrinogen solutions, and three‐dimensional networks formed from 4 mg mL⁻¹ fibrinogen incubated with uncoated or fibrinogen‐coated plates. Fibrils appeared laterally assembled into branching and overlapping fibers whose heights from the surface ranged from approximately 3 to 740 nm. The elastic modulus of fibrinogen fibers was 1.55 MPa. No fibrils formed when fibrinogen lacking αC‐domains was used as a coating or was incubated with intact fibrinogen‐coated plates, or when the latter plates were sequentially incubated with anti‐Aα529–539 mAb and intact fibrinogen. When an anti‐Aα241–476 mAb was used instead, fine, long fibers formed. Similarly, sequential incubations of fibrinogen‐coated plates with recombinant αC‐domain (Aα392–610 fragment) or αC‐connector (Aα221–372 fragment) and fibrinogen resulted in distinctly fine fiber networks. Conclusions:Adsorption‐induced fibrinogen self‐assembly is initiated by a more than one molecule‐thick surface layer and eventuates in three‐dimensional networks whose formation requires fibrinogen with intact αC‐domains.

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Visualization of human plasma fibrinogen adsorbed on highly oriented pyrolytic graphite by scanning probe microscopy

Abstract

Introduction

Section snippets

Fibrinogen adsorption on the substrate surface

Results and discussion

Conclusions

J. Vasc. Interv. Radiol.

J. Mol. Biol.

Surf. Sci.

Ultramicroscopy

FEBS

Surf. Sci.

J. Collod. Inter. Sci.

Blood

J. Biophys. Biochem. Cytol.

J. Biol. Chem.

Annal. N.Y. Acad. Sci.

Biochem.

Eur. J. Biochem.

Biopolym.