Myeloperoxidase-induced fibrinogen unfolding and clotting

Abstract

AbstractFibrinogen is a major protein of blood coagulation system and is a promising component of biomaterials and protein matrixes. Conformational changes of fibrinogen underlie the important mechanism of thrombin mediated fibrinogen clotting but also may induce the loss of its biological activity and (amyloid) aggregation. Understanding and controlling of fibrinogen unfolding is important for the development of fibrinogen based materials with tunable properties. We have discovered that myeloperoxidase induces denaturation of fibrinogen molecules followed by fibrinogen clotting, which is not thrombin-dependent. This is the first example of ATP-independent, non-targeted protein-induced protein denaturation. The morphological structure of unfolded fibrinogen molecules and “non-conventional” fibrinogen clots has been characterized using high-resolution atomic force microscopy and scanning electron microscopy techniques. Circular dichroism (CD) spectroscopy has shown no significant changes of the secondary structure of the fibrinogen clots. The absorbance spectrophotometry has demonstrated that the kinetics of myeloperoxidase induced fibrinogen clotting strongly decays with growth of ionic strength indicating a major role of the Debye screening effect in regulating of this process. The obtained results provide with the novel concepts of protein unfolding and open new insights into fibrinogen clotting. Moreover, they give new possibilities in biotechnological and biomedical applications, e.g., for regulation of fibrinogen clotting and platelet adhesion and for the development of fibrinogen-based matrices.The organization of a protein molecule is characterized by different hierarchical levels such as primary, secondary, tertiary and quaternary structure. Protein unfolding or denaturation, i.e. its transformation to a lower order structure (and loss of a higher order structure), is a biologically and biotechnologically relevant process. Protein unfolding is a prerequisite for an alternative folding pathway including amyloid aggregation 1,2. Unfolded proteins may be used in development of protein films and coatings with special properties such as enhanced mechanical stability 3–5, resistance to protein adsorption or platelet adhesion 6,7 and other advantages 8. Unfolding of a protein molecule may lead to the loss of its biological function 9 that has important consequences in biosensor 10,11 and pharmaceutical applications 12.