Revealing fibrinogen monolayer conformations at different pHs: Electrokinetic and colloid deposition studies

https://doi.org/10.1016/j.jcis.2014.10.001Get rights and content

Highlights

  • Electrokinetic properties of fibrinogen monolayers on mica are characterized.

  • Orientation of fibrinogen in monolayers at different pHs is determined.

  • Presence of side-on and end-on oriented molecules at different pHs is confirmed.

Abstract

Adsorption mechanism of human fibrinogen on mica at different pHs is studied using the streaming potential and colloid deposition measurements. The fibrinogen monolayers are produced by a controlled adsorption under diffusion transport at pH of 3.5 and 7.4. Initially, the electrokinetic properties of these monolayers and their stability for various ionic strength are determined. It is shown that at pH 3.5 fibrinogen adsorbs irreversibly on mica for ionic strength range of 4 × 10−4 to 0.15 M. At pH 7.4, a partial desorption is observed for ionic strength below 10−2 M. This is attributed to the desorption of the end-on oriented molecules whereas the side-on adsorbed molecules remain irreversibly bound at all ionic strengths. The orientation of molecules and monolayer structure is evaluated by the colloid deposition measurements involving negatively charged polystyrene latex microspheres, 820 nm in diameter. An anomalous deposition of negative latex particles on substrates exhibiting a negative zeta potential is observed. At pH 3.5 measurable deposition of latex is observed even at low ionic strength where the approach distance of latex particles exceeded 70 nm. At pH 7.4 this critical distance is 23 nm. This confirms that fibrinogen monolayers formed at both pHs are characterized by the presence of the side-on and end-on oriented molecules that prevail at higher coverage range. It is also shown that positive charge is located at the end parts of the αA chains of the adsorbed fibrinogen molecules. Therefore, it is concluded that the colloid deposition method is an efficient tool for revealing protein adsorption mechanisms at solid/electrolyte interfaces.

Introduction

Fibrinogen is a multifunctional glycoprotein occurring in the blood plasma at the physiological level of varying between 1.5 and 3.5 g L−1 that can rapidly increase after tissue stress or infection [1].

Fibrinogen plays a major physiological function in the clotting cascade initiated by thrombin that removes two peptides from the fibrinogen molecule. This leads to formation of fibrin that spontaneously polymerizes forming a highly interconnected network of fibers. Fibrin fibers are the most important temporary extracellular matrix in the wound area, providing a suitable scaffold for the capture of leukocytes, endothelial cells, and another tissue cells during the healing process. In pathological situations the networks entraps a large number of erythrocytes and leucocytes forming a thrombus that may occlude a blood vessel. Therefore, fibrinogen and fibrin play a significant role in various physiological reactions including fibrinolysis, cellular and matrix interactions, wound healing and neoplasia [2].

Fibrinogen molecules also exhibit a strong tendency to adsorb on various surfaces under broad range of conditions [3] mediating cellular interactions that are key event in determining biocompatibility of these materials [4]. It is believed that adsorbed fibrinogen molecules promote leukocyte activation by stimulation of integrin receptor Mac-1 [5].

On the other hand, fibrinogen monolayers adsorbed on various synthetic materials may promote platelet adhesion that often leads to fouling of artificial organs [6], [7].

Because of its fundamental significance, the chemical structure of fibrinogen and its bulk physicochemical properties have extensively been studied [8], [9], [10], [11], [12], [13], [14], [15].

It was established [9] that the fibrinogen molecule is a symmetric dimer composed of three identical pairs of polypeptide chains, refereed to traditionally as Aα Bβ and γ chains [10]. They are coupled in the middle of the molecule through a few disulfide bridges forming a central E nodule (see Table 1). The longest Aα chain is composed of 610 amino-acids, the Bβ chain comprises 460 amino-acids and the γ chain 411 amino-acids. Accordingly, the molecular mass of the fibrinogen molecule equals to 338 kDa [10]. Moreover, from the chemical structure it can be deduced that a considerable part of the Aα chains extends from the core of the molecule forming two polar appendages (arms) having each molecular mass equal to 42,300 Da [10]. These end part of the Aα chains are called the αC domains [11].

Information about fibrinogen’s molecule shape and dimensions was derived from electron [12], [13], [14], [15] and atomic force microscopy (AFM) [16], [17], [18], [19], [20], [21] imaging of single molecules adsorbed on various substrates (most often mica). It was established that the molecule has a co-linear, trinodular shape with a total length of 47.5 nm [12]. The two equal end domains of rather irregular shape are approximated by spheres having the diameter of 6.5 nm; and the middle domain has a diameter of 5 nm. However, it should be mentioned that these dimensions were determined under dry or vacuum conditions, where the molecule is likely to change its native conformations occurring in the electrolyte solutions, because of a considerable dehydration.

This was confirmed in recent works [22], [23] where conformations of fibrinogen molecule in electrolyte solution at various pHs and ionic strength have been studied using the DLS, dynamic viscosity and micro-electrophoretic measurements. These results, supplement by theoretical modeling, allowed one to confirm that presence of two major fibrinogen conformation: (i) the semi-collapsed conformation prevailing at pH 7.4, and (ii) the expanded conformation prevailing at lower pHs (see Table 2). Additionally, in these works [22], [23] it was predicted that the length the side arms is equal to 18 nm and that they are positively charged in the αC domains for the broad range of pHs from 3.5 to 9.5.

Numerous experimental studies have also been devoted to fibrinogen adsorption on various substrates. They were mainly focused on determining adsorption isotherms for various conditions by measuring the maximum coverage as a function of the bulk concentration of the protein [21], [24], [25], [26], [27], [28], [29]. In all of the works experiments were performed at pH 7.4, ionic strength of 0.15 M, pertinent to physiological conditions [20], [21], [24], [25], [26], [27], [28], [29] and for flat interfaces. For example, precise kinetic measurements of fibrinogen adsorption on silicon and modified glass surfaces forming parallel-plate channels were conducted by Malmsten [27] using the in situ ellipsometric measurements and by Santore et al. [28], [29] using the TIRF and AFM techniques.

The influence of pH and ionic strength on fibrinogen adsorption was systematically studied by Ortega-Vinuesa et al. [16], [17] by ellipsometry.

Lindon et al. [30] showed that platelet adhesion to artificial surfaces increases with the surface coverage of fibrinogen if the adsorbed fibrinogen attained such conformation that functional domains are accessible.

Analogously, Massa et al. [31] experimentally demonstrated that fibrinogen immobilized in the correct conformation could mediate the adhesion of platelets.

Chinn et al. [32] showed that binding of a monoclonal antibody to fragment D upon fibrinogen adsorption to polystyrene is attributed to changes in the conformation of fibrinogen after adsorption.

However, in only few works the important issue of fibrinogen molecule orientations in monolayers formed on solid substrates was considered. One of the exceptions is the work of Santore and Wertz [29] who measured spreading kinetics of fibrinogen molecules adsorbed on hydrophobic and hydrophilic surfaces at pH 7.4 using the adsorption probe technique. A significant increase in the footprint area of molecules adsorbed on hydrophobized surfaces was observed. This was interpreted in terms of the transition from end-on to side-on orientation of adsorbed fibrinogen molecules.

Analogously, in the work of Dyr et al. [33] it was shown via SPR (surface plasmon resonance) measurements that the results of antibody binding to fibrinogen monolayers on gold can only be explained assuming the end-on orientation of molecules. However, quantitative evidences of this effect were not provided.

Malmsten [27] suggested that the increase in the ellipsometric fibrinogen monolayer thickness on oxidized silica from 10 to 25 nm (for fibrinogen coverage changing between 1.0 and 3.5 mg m−2) is due to the transition from the side-on to end-on orientations. However, given the much larger length of fibrinogen molecules (48 nm) one cannot quantitatively explain the observed monolayer thickness in terms of the perpendicular end-on orientation.

In Ref. [34] the thickness of fibrinogen monolayers on mica was evaluated under wet, in situ conditions using the colloid deposition method. The technique consist in non-specific, electrostatically driven, deposition of colloid particles (latex microspheres) on protein monolayers under precisely regulated ionic strength that controls the approach distance between colloids and surfaces. It was revealed that at pH 3.5 fibrinogen molecules are adsorbed in the end-on orientation that promoted an efficient deposition of negatively charged latex particles on negatively charged surfaces. The thickness of the monolayer was estimated to be 50–75 nm that suggests an extended conformation of molecules at this pH. However, in Ref. [34] only the case of pH 3.5 was studied for a rather limited range of fibrinogen coverage.

Therefore, in this work, the colloid deposition method is used for determining fibrinogen molecule orientation at different pHs of 3.5 and 7.4. Transitions between pH 7.4 and 3.5 are also studied that provides additional information about the reversibility of molecule conformation changes. A quantitative interpretation of these results is facilitated by the measurements of zeta potential of fibrinogen monolayers performed by the streaming potential method for a broad range of ionic strength. In this way essential clues on fibrinogen adsorption mechanisms at different pHs can be acquired.

It should also be mentioned that the colloid particle deposition processes on fibrinogen monolayers can mimic the biofilm formation, an important phenomenon pertinent to thrombosis, angiogenesis, inflammatory response, fouling of artificial organs, etc.

Besides the significance for basic science, the obtained results allow one to specify conditions for preparing fibrinogen monolayers of precisely known coverage and orientations of molecules having potential applications for biosensing and immunological assays.

Section snippets

Materials and methods

Fibrinogens from human blood plasmas, in the form of crystalline powders containing 50–70% protein, (Sigma F3879) was used in this work.

Fibrinogen solutions were prepared according to the procedure elsewhere described [22] by dissolving an appropriate amount of the crystalline fibrinogen powder under gentle stirring at pH 3.5 and 310 K in a high purity distilled water. Afterward, the suspension was filtered through the Millex®-GS 0.45 μm filter to eliminate aggregates and impurities and the bulk

Bulk characteristics of fibrinogen latex and mica

Initially, basic physicochemical characteristics of fibrinogen molecules and latex particles were determined. These comprised the diffusion coefficient, electrophoretic mobility and zeta potential.

It was determined by DLS that the diffusion coefficient D of fibrinogen molecules was 2.3 × 10−7 cm2 s−1 for the ionic strength range of 10−3–10−2 M and pH 3.5 (T = 298 K). Slightly higher value of 2.4 × 10−7 cm2 s−1 was obtained for pH 7.4. The hydrodynamic diameter of fibrinogen df calculated from the Stokes

Concluding remarks

The electrokinetic properties of fibrinogen monolayers on mica for various ionic strength and pHs were characterized in terms of the streaming potential measurements, enabling one to determine the mean-field zeta potentials. It was established that at pH 3.5 fibrinogen adsorption is irreversible for ionic strength varied between 3 × 10−4 and 0.15 M. On the other hand, for pH 7.4, a partial desorption of fibrinogen was observed for ionic strength below 10−2 M.

The orientation of adsorbed molecules

Acknowledgment

This work was supported by the NCN Grant UMO-2012/07/B/ST4/00559.

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