The relationship between platelet adhesion on surfaces and the structure versus the amount of adsorbed fibrinogen

Abstract

While platelet adhesion to biomaterial surfaces is widely recognized to be related to adsorbed fibrinogen (Fg), it has remained controversial whether platelet adhesion is in response to the adsorbed amount or the adsorbed conformation of this protein. To address this issue, we designed a series of platelet adhesion studies to clearly separate these two factors, thus enabling us to definitively determine whether it is the amount or the conformation of adsorbed Fg that mediates platelet response. Fg was adsorbed to a broad range of surface chemistries from a wide range of solution concentrations, with the amount and conformation of adsorbed Fg determined by absorbance and circular dichroism (CD) spectropolarimetry, respectively. Platelet adhesion response was determined by lactate dehydrogenase (LDH) assay and scanning electron microscopy (SEM). Our results show that platelet adhesion is strongly correlated with the degree of adsorption-induced unfolding of Fg (r 2 = 0.96) with essentially no correlation with the amount of Fg adsorbed (r 2 = 0.04). Platelet receptor inhibitor studies using an RGDS peptide reduced platelet adhesion by only about 50%, and SEM results show that adherent platelets after RGDS blocking were much more rounded with minimal extended filopodia compared with the unblocked platelets. These results provide definitive proof that the conformation of adsorbed Fg is the critical determinant of platelet adhesion, not the amount of Fg adsorbed, with adsorption-induced unfolding potentially exposing two distinctly different types of platelet binding sites in Fg; one that induces platelet adhesion alone and one that induces both platelet adhesion and activation.

Are there sufficient standards for the in vitro hemocompatibility testing of biomaterials?

“…In the complex and dynamic process of blood-material interaction, the composition of immediately adsorbed protein layers, but also conformational changes of surface adsorbed proteins are considered to substantially influence the cellular interactions (by the exposure of neo/cryptic epitopes) [14][15][16][17][18]. Key elements of the latter are blood platelets, which play a central role in physiological and pathological processes of hemostasis, inflammation, tumormetastasis, wound healing, and host defence [19][20][21][22][23][24].…”

Response of osteoblasts and preosteoblasts to calcium deficient and Si substituted hydroxyapatites treated at different temperatures

“…It is widely accepted that the amount of adsorbed fibrinogen and, more specifically, its conformation are important factors for platelet adhesion which determines the hemocompatibility of a given biomaterial [47]. On the other hand, recent studies demonstrate a reduced in vitro bioactivity of HA surface coated with BSA in comparison to the uncoated surface [48].…”

Single-molecule resolution of protein structure and interfacial dynamics on biomaterial surfaces

“…Importantly, surface interactions that perturb protein structure can inactivate proteins, as has widely been observed in the case of surface-immobilized enzymes (1)(2)(3)(4)(5)(6). Such interactions, by inducing unfolding and subsequent accumulation of freely absorbing proteins on biomaterial surfaces, may trigger unfavorable cellular responses (7)(8)(9)(10). However, experimental methods to elucidate both protein structure and interfacial dynamics (e.g., adsorption, diffusion, desorption), particularly in heterogeneous near-surface environments, are virtually nonexistent.…”