Adsorption of the blood protein fibrinogen (Fg) on the surface of biomaterials is a critical early event during the interaction of blood with implanted vascular grafts. Because of its rapid adsorption and the ability to support adhesion of platelets, Fg is generally viewed as a culprit responsible for the development of surface- induced thrombosis, especially in small-diameter vascular prostheses. The perception of Fg as a foe is perplexing in view of the fact that implanted vascular grafts are invariably coated with a fibrin layer which, like Fg, can support efficient platelet adhesion. Nevertheless, in humans, this fibrin layer remains largely without platelets, maintaining its characteristic a cellular appearance over the years. The discrepancy between the ability of immobilized Fg to support platelet adhesion, which was primarily inferred from in vitro experiments, and the situation in vivo, attests to a clear need for a greater understanding of the mechanisms that regulate the balance between adhesive and nonadhesive functions of Fg. We have recently identified a new nanoscale phenomenon whereby Fg dramatically reduces cell adhesion, and which may explain this discrepancy. Specifically, adsorption of Fg at high concentrations results in the formation of a multilayered extensible matrix (~2-10 nm thick) characterized by low adhesion forces. Conversely, adsorption of Fg at low density produces a monolayer, in which the molecules are directly attached to hard surfaces, resulting in high adhesion forces. Consistent with their distinct physical properties, a monolayer induces strong integrin- mediated signaling in platelets resulting in their firm adhesion and spreading. In contrast, a multilayered Fg matrix is nonadhesive due to its inability to induce a strong mechanotransduction response. The central hypothesis of this application is that the origin of the nonadhesive properties of Fg matrices is the formation of an extensible multilayer incapable of transducing strong mechanical forces via platelet integrins, resulting in weak signaling and cell spreading.
Specific Aim 1 is to establish the structural features that enable the formation of an extensible Fg multilayer. Nanotechnology was developed to study the mechanical and adhesive properties of the fibrinogen matrices by single-cell and molecular force spectroscopy and AFM imaging. It will be used to determine how enzymatic crosslinking alters the mechanical and adhesive properties of Fg multilayer, as well as the role of several structural regions of Fg in the increased extensibility of Fg multilayer. This will be accomplished by using recombinant Fgs carrying selected mutations.
Specific Aim 2 is to examine how the surfaces of several contemporary biomaterials trigger the formation of the mono- and multilayer Fg matrices and to characterize their mechanical and adhesive properties. Since the lack of endothelium on the blood surface of implanted vascular grafts has substantial medical importance, the possibility that multilayer Fg is incapable of supporting firm attachment of endothelial cells and endothelial progenitor cells under flow will be explored in Specific Aim 3.

Public Health Relevance

Adsorption of the blood protein fibrinogen on implanted vascular grafts and its ability to interact with circulating blood cells plays a central role in their clinical performance. We have discovered a new nanoscale phenomenon whereby deposition of fibrinogen on surfaces creates a nonadhesive multilayer. The proposed studies will define the molecular mechanisms involved in the formation of fibrinogen multilayer and its role in controlling adhesion of platelets and endothelial progenitor cells. This knowledge should be translatable into the design of better vascular grafts.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Biomaterials and Biointerfaces Study Section (BMBI)
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Kindzelski, Andrei L
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Arizona State University-Tempe Campus
Other Basic Sciences
Schools of Arts and Sciences
United States
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Podolnikova, Nataly P; Yakovlev, Sergiy; Yakubenko, Valentin P et al. (2014) The interaction of integrin *IIb*3 with fibrin occurs through multiple binding sites in the *IIb *-propeller domain. J Biol Chem 289:2371-83
Christenson, W; Yermolenko, I; Plochberger, B et al. (2014) Combined single cell AFM manipulation and TIRFM for probing the molecular stability of multilayer fibrinogen matrices. Ultramicroscopy 136:211-5
Lishko, V K; Yermolenko, I S; Owaynat, H et al. (2012) Fibrinogen counteracts the antiadhesive effect of fibrin-bound plasminogen by preventing its activation by adherent U937 monocytic cells. J Thromb Haemost 10:1081-90