Role of Fibrinogen Conformation in Platelet Activation

Abstract

Platelet adhesion and activation induced by fibrinogen (Fbg) coating on polysaccharide layers of hyaluronic acid (Hyal) and its sulfated derivative (HyalS) were analyzed. Hyal or HyalS was coated and grafted on the glass substrate using a photolithographic method. The Fbg coating was achieved by two different routes: the immobilization of Fbg by means of covalent bond to the polysaccharide layers and the mere adsorption of Fbg to Hyal and HyalS surfaces. Platelet adhesion and activation to the surfaces were evaluated using, respectively, scanning electron microscopy (SEM) and quantifying the release of Platelet Factor 4 by ELISA. The method used for the coating of the surfaces with the Fbg influenced the platelet response. In fact, platelet adhesion and activation took place on surfaces covered by bound Fbg but not on those containing adsorbed Fbg. To explain this difference, the molecular mechanism involved in the Fbg--platelet interaction was investigated blocking platelet membrane receptors by monoclonal antibodies. Because the interaction between Fbg and the GPIIb/IIIa platelet membrane receptor was the only molecular pathway involved, Fbg conformation after the interaction (adsorption or binding) with the Hyal and the HyalS chains and the role of serum proteins adsorbed on the Fbg containing surfaces were accurately analyzed. Both adsorbed and bound Fbg prevented the adsorption of further serum proteins; consequently, a direct interaction between Fbg and platelets was supposed and the different platelet behavior was ascribed to the different conformational changes that occurred after the adsorption and the chemical binding of the Fbg to the Hyal and HyalS surfaces.

The effect of topography of polymer surfaces on platelet adhesion

“…Although a strong correlation between the amount of adsorbed fibrinogen and adhered platelets is found in this work (Fig. 3a), more research is required to determine this relationship further because not only the amount of adsorbed Hfg is important, but also its conformation on the surface [51]. In other words, fibrinogen adsorption may be necessary but is not the only factor required for platelet adhesion.…”

The effect of surface microtopography of poly(dimethylsiloxane) on protein adsorption, platelet and cell adhesion

“…Massa et al [29] also reported that the distribution of Fg correlates to the platelet adhesion pattern on fluorinated surface-modified polyetherurethane. Additionally, the conformation of adsorbed Fg adopted on surface topography should be responsible for platelet adhesion [28,30]. In our case, Fg adsorbed on the flanks of protrusions may have favorable conformation for the interaction between ligands (Arg-Gly-Asp sequence) in Fg and receptors (Glycoprotein IIb/IIIa) on platelet membrane [31,32].…”

Proteins, platelets, and blood coagulation at biomaterial interfaces

“…It allows for examination of multiple proteins at the molecular scale 144,145 , and also probes the conformation, orientation, and activity of adsorbed proteins with appropriate choice of antibodies 146 . As the γ-chain dodecapeptide sequence in fibrinogen is the primary ligand for platelet adhesion to adsorbed fibrinogen, the availability of the dodecapeptide can be viewed as an indicator of the functional activity of fibrinogen as related to the conformation and/or orientation of the adsorbed protein 18,41,147 .…”

“…For example, platelet adhesion and activation on biomaterial surfaces is influenced by surface properties such as energy 14 , charge 15 , and composition 16 . However, platelet adhesion and activation are largely mediated by proteins such as fibrinogen 17,18 . As plasma proteins can rapidly adsorb onto the material surface to form a “conditioning film” following blood contact, this adsorbed protein layer may minimize the direct effect of biomaterial surface properties on cell responses, and the surface-biology interactions are mediated by proteins (Figure 1).…”