Deposition of fibrinogen on the surface of in vitro thrombi prevents platelet adhesion.

The initial accumulation of platelets after vessel injury is followed by thrombin-mediated generation of fibrin which is deposited around the plug. While numerous in vitro studies have shown that fibrin is highly adhesive for platelets, the surface of experimental thrombi in vivo contains very few platelets suggesting the existence of natural anti-adhesive mechanisms protecting stabilized thrombi from platelet accumulation and continuous thrombus propagation. We previously showed that adsorption of fibrinogen on pure fibrin clots results in the formation of a nonadhesive matrix, highlighting a possible role of this process in surface-mediated control of thrombus growth. However, the deposition of fibrinogen on the surface of blood clots has not been examined. In this study, we investigated the presence of intact fibrinogen on the surface of fibrin-rich thrombi generated from flowing blood and determined whether deposited fibrinogen is nonadhesive for platelets. Stabilized fibrin-rich thrombi were generated using a flow chamber and the time that platelets spend on the surface of thrombi was determined by video recording. The presence of fibrinogen and fibrin on the surface of thrombi was analyzed by confocal microscopy using specific antibodies. Examination of the spatial distribution of two proteins revealed the presence of intact fibrinogen on the surface of stabilized thrombi. By manipulating the surface of thrombi to display either fibrin or intact fibrinogen, we found that platelets adhere to fibrin- but not to fibrinogen-coated thrombi. These results indicate that the fibrinogen matrix assembled on the outer layer of stabilized in vitro thrombi protects them from platelet adhesion.

Fibrinogen matrix deposited on the surface of biomaterials acts as a natural anti-adhesive coating.

Adsorption of fibrinogen on the luminal surface of biomaterials is a critical early event during the interaction of blood with implanted vascular graft prostheses which determines their thrombogenicity. We have recently identified a nanoscale process by which fibrinogen modifies the adhesive properties of various surfaces for platelets and leukocytes. In particular, adsorption of fibrinogen at low density promotes cell adhesion while its adsorption at high density results in the formation of an extensible multilayer matrix, which dramatically reduces cell adhesion. It remains unknown whether deposition of fibrinogen on the surface of vascular graft materials produces this anti-adhesive effect. Using atomic force spectroscopy, single cell force spectroscopy, and standard adhesion assays with platelets and leukocytes, we have characterized the adhesive and physical properties of the contemporary biomaterials, before and after coating with fibrinogen. We found that uncoated PET, PTFE and ePTFE exhibited high adhesion forces developed between the AFM tip or cells and the surfaces. Adsorption of fibrinogen at the increasing concentrations progressively reduced adhesion forces, and at ≥2 µg/ml all surfaces were virtually nonadhesive. Standard adhesion assays performed with platelets and leukocytes confirmed this dependence. These results provide a better understanding of the molecular events underlying thrombogenicity of vascular grafts.

The assembly of nonadhesive fibrinogen matrices depends on the αC regions of the fibrinogen molecule.

Adsorption of fibrinogen on fibrin clots and other surfaces strongly reduces integrin-mediated adhesion of platelets and leukocytes with implications for the surface-mediated control of thrombus growth and blood compatibility of biomaterials. The underlying mechanism of this process is surface-induced aggregation of fibrinogen, resulting in the assembly of a nanoscale multilayered matrix. The matrix is extensible, which makes it incapable of transducing strong mechanical forces via cellular integrins, resulting in insufficient intracellular signaling and weak cell adhesion. To determine the mechanism of the multilayer formation, the physical and adhesive properties of fibrinogen matrices prepared from human plasma fibrinogen (hFg), recombinant normal (rFg), and fibrinogen with the truncated αC regions (FgAα251) were compared. Using atomic force microscopy and force spectroscopy, we show that whereas hFg and rFg generated the matrices with a thickness of ∼8 nm consisting of 7-8 molecular layers, the deposition of FgAα251 was terminated at two layers, indicating that the αC regions are essential for the multilayer formation. The extensibility of the matrix prepared from FgAα251 was 2-fold lower than that formed from hFg and rFg. In agreement with previous findings that cell adhesion inversely correlates with the extensibility of the fibrinogen matrix, the less extensible FgAα251 matrix and matrices generated from human fibrinogen variants lacking the αC regions supported sustained adhesion of leukocytes and platelets. The persistent adhesiveness of matrices formed from fibrinogen derivatives without the αC regions may have implications for conditions in which elevated levels of these molecules are found, including vascular pathologies, diabetes, thrombolytic therapy, and dysfibrinogenemia.

High-resolution visualization of fibrinogen molecules and fibrin fibers with atomic force microscopy.

We report an atomic force microscopy (AFM) study of fibrinogen molecules and fibrin fibers with resolution previously achieved only in few electron microscopy images. Not only are all objects triads, but the peripheral D regions are resolved into the two subdomains, apparently corresponding to the ßC and γC domains. The conformational analysis of a large population of fibrinogen molecules on mica revealed the two most energetically favorable conformations characterized by bending angles of ∼100 and 160 degrees. Computer modeling of the experimental images of fibrinogen molecules showed that the AFM patterns are in good agreement with the molecular dimensions and shapes detected by other methods. Imaging in different environments supports the expected hydration of the fibrinogen molecules in buffer, whereas imaging in humid air suggests the 2D spreading of fibrinogen on mica induced by an adsorbed water layer. Visualization of intact hydrated fibrin fibers showed cross-striations with an axial period of 24.0 ± 1.6 nm, in agreement with a pattern detected earlier with electron microscopy and small-angle X-ray diffraction. However, this order is clearly detected on the surface of thin fibers and becomes less discernible with the fiber's growth. This structural change is consistent with the proposal that thinner fibers are denser than thicker ones, that is, that the molecule packing decreases with the increasing of the fibers' diameter.

Origin of the nonadhesive properties of fibrinogen matrices probed by force spectroscopy.

The deposition of a multilayered fibrinogen matrix on various surfaces results in a dramatic reduction of integrin-mediated cell adhesion and outside-in signaling in platelets and leukocytes. The conversion of a highly adhesive, low-density fibrinogen substrate to the nonadhesive high-density fibrinogen matrix occurs within a very narrow range of fibrinogen coating concentrations. The molecular events responsible for this transition are not well understood. Herein, single-cell and molecular force spectroscopy were used to determine the early steps in the formation of nonadhesive fibrinogen substrates. We show that the adsorption of fibrinogen in the form of a molecular bilayer coincides with a several-fold reduction in the adhesion forces generated between the AFM tip and the substrate as well as between a cell and the substrate. The subsequent deposition of new layers at higher coating concentrations of fibrinogen results in a small additional decrease in adhesion forces. The poorly adhesive fibrinogen bilayer is more extensible under an applied tensile force than is the surface-bound fibrinogen monolayer. Following chemical cross-linking, the stabilized bilayer displays the mechanical and adhesive properties characteristic of a more adhesive fibrinogen monolayer. We propose that a greater compliance of the bi- and multilayer fibrinogen matrices has its origin in the interaction between the molecules forming the adjacent layers. Understanding the mechanical properties of nonadhesive fibrinogen matrices should be of importance in the therapeutic control of pathological thrombosis and in biomaterials science.

Control of integrin alphaIIb beta3 outside-in signaling and platelet adhesion by sensing the physical properties of fibrin(ogen) substrates.

The physical properties of substrates are known to control cell adhesion via integrin-mediated signaling. Fibrin and fibrinogen, the principal components of hemostatic and pathological thrombi, may represent biologically relevant substrates whose variable physical properties control adhesion of leukocytes and platelets. In our previous work, we have shown that binding of fibrinogen to the surface of fibrin clot prevents cell adhesion by creating an antiadhesive fibrinogen layer. Furthermore, fibrinogen immobilized on various surfaces at high density supports weak cell adhesion whereas at low density it is highly adhesive. To explore the mechanism underlying differential cell adhesion, we examined the structural and physical properties of surfaces prepared by deposition of various concentrations of fibrinogen using atomic force microscopy and force spectroscopy. Fibrinogen deposition at high density resulted in an aggregated multilayered material characterized by low adhesion forces. In contrast, immobilization of fibrinogen at low density produced a single layer in which molecules were directly attached to the solid surface, resulting in higher adhesion forces. Consistent with their distinct physical properties, low- but not high-density fibrinogen induced strong alpha(IIb)beta(3)-mediated outside-in signaling in platelets, resulting in their spreading. Moreover, while intact fibrin gels induced strong signaling in platelets, deposition of fibrinogen on the surface of fibrin resulted in diminished cell signaling. The data suggest that deposition of a multilayered fibrinogen matrix prevents stable cell adhesion by modifying the physical properties of surfaces, which results in reduced force generation and insufficient signaling. The mechanism whereby circulating fibrinogen alters adhesive properties of fibrin clots may have important implications for control of thrombus formation and thrombogenicity of biomaterials.