The Role of the αC Domains in the Formation of Nonadhesive Fibrinogen Matrices

Abstract 1194

Fibrinogen strongly reduces adhesion of platelets and leukocytes to the surface of fibrin clots, highlighting a possible role for this abundant plasma protein in surface-mediated control of thrombus growth, stability and timely dissolution. We have previously shown that the underlying mechanism by which fibrinogen adsorprtion on various surfaces decreases cell adhesion is its aggregation, leading to the formation of an extensible multilayered matrix. This matrix is incapable of transducing strong mechanical forces via integrins resulting in insufficient intracellular signaling and weak cell adhesion. To further characterize the properties of the nonadhesive fibrinogen multilayer and determine the structural basis for its formation, we compared the physical and adhesive properties of fibrinogen matrices prepared from human plasma fibrinogen (hFg), recombinant normal fibrinogen (rFg) and fibrinogen with the truncated αC domains (FgAα251). Using atomic force microscopy (AFM) and force spectroscopy we determined the thickness, adhesion forces, extensibility and the energy of the AFM tip-fibrinogen matrix interactions of various matrices. All three fibrinogens adsorbed on mica in a concentration-dependent manner. However, while hFg and rFg formed the matrix with a maximal thickness of ∼8 nm corresponding to 8–9 molecular layers, the deposition of FgAα251 was terminated after 2–3 layers, indicating the αC domains are involved in the formation of the multilayer. Consistently, the extensibility of the full multilayered matrix prepared from hFg and rFg was 2-fold higher than that of the matrix formed from FgAα251 (57±6 nm and 28±4 nm for hFg and FgAα251, respectively). The formation of the multilayered matrix upon adsorption of the increasing concentrations of hFg and FgAα251 was associated with a decrease in the adhesion forces generated between the AFM tip and the matrix. However, the energy required to disrupt the AFM tip-matrix interactions was 1.6 nN·nm for hFg and 0.88 nN·nm for FgAα251, indicating that the αC domains contribute about half of the total energy to the fibrinogen-fibrinogen interactions during the formation of the multilayer. Since cell adhesion inversely correlates with the growth of the fibrinogen matrices, we examined adhesion of U937 monocytic cells to the substrates prepared from different concentrations of hFg and FgAα251. As expected, adhesion sharply declined with the increase in the coating concentration of hFg, with only 2–5% of the cells adhering to the full multilayer. At the same time, 40–45% of the cells remained adherent on the matrices prepared from FgAα251. These results indicate that the inability of FgAα251 to assemble a highly extensible multilayer correlates with enhanced cell adhesion. The increased adhesiveness of matrices formed from fibrinogen with truncated αC domains may have implications for situations where the formation of fibrinogen degradation products with truncated αC domains such as X-fragment occurs, for example during thrombolytic therapy and pathological fibrinogenolysis, as well as in the cases of disfibrinogenemia.