Ivan S. Yermolenko

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 αIIbβ3 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.

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

Background: Surface-induced aggregation of fibrinogen results in the assembly of an extensible multilayered matrix, which prevents integrin-mediated cell adhesion. Results: Without the αC regions, the fibrinogen molecules assemble a defective, poorly extensible matrix supporting sustained cell adhesion. Conclusion: The assembly of nonadhesive fibrinogen multilayer requires the αC regions of the molecule. Significance: The molecular mechanism for the assembly of the fibrinogen multilayer is identified. 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.

Plasminogen on the surfaces of fibrin clots prevents adhesion of leukocytes and platelets

Summary.  Background and Objectives: Although leukocytes and platelets adhere to fibrin with alacrity in vitro, these cells do not readily accumulate on the surfaces of fibrin clots in vivo. The difference in the capacity of blood cell integrins to adhere to fibrin in vivo and in vitro is striking and implies the existence of a physiologic antiadhesive mechanism. The surfaces of fibrin clots in the circulation are continually exposed to plasma proteins, several of which can bind fibrin and influence cell adhesion. Recently, we have demonstrated that adsorption of soluble fibrinogen on the surface of a fibrin clot results in its deposition as a soft multilayer matrix, which prevents attachment of blood cells. In the present study, we demonstrate that another plasma protein, plasminogen, which is known to accumulate in the superficial layer of fibrin, exerts an antiadhesive effect. Results: After being coated with plasminogen, the surfaces of fibrin clots became essentially non‐adhesive for U937 monocytic cells, blood monocytes, and platelets. The data revealed that activation of fibrin‐bound plasminogen by the plasminogen‐activating system assembled on adherent cells resulted in the generation of plasmin, which decomposed the superficial fibrin layer, resulting in cell detachment under flow. The surfaces generated after the initial cell adhesion remained non‐adhesive for subsequent attachment of leukocytes and platelets. Conclusion: We propose that the limited degradation of fibrin by plasmin generated by adherent cells loosens the fibers on the clot surface, producing a mechanically unstable substrate that is unable to support firm integrin‐mediated cell adhesion.

Combined single cell AFM manipulation and TIRFM for probing the molecular stability of multilayer fibrinogen matrices.

Adsorption of fibrinogen on various surfaces produces a nanoscale multilayer matrix, which strongly reduces the adhesion of platelets and leukocytes with implications for hemostasis and blood compatibility of biomaterials. The nonadhesive properties of fibrinogen matrices are based on their extensibility, ensuing the inability to transduce strong mechanical forces via cellular integrins and resulting in weak intracellular signaling. In addition, reduced cell adhesion may arise from the weaker associations between fibrinogen molecules in the superficial layers of the matrix. Such reduced stability would allow integrins to pull fibrinogen molecules out of the matrix with comparable or smaller forces than required to break integrin-fibrinogen bonds. To examine this possibility, we developed a method based on the combination of total internal reflection fluorescence microscopy, single cell manipulation with an atomic force microscope and microcontact printing to study the transfer of fibrinogen molecules out of a matrix onto cells. We calculated the average fluorescence intensities per pixel for wild-type HEK 293 (HEK WT) and HEK 293 cells expressing leukocyte integrin Mac-1 (HEK Mac-1) before and after contact with multilayered matrices of fluorescently labeled fibrinogen. For contact times of 500 s, HEK Mac-1 cells show a median increase of 57% of the fluorescence intensity compared to 6% for HEK WT cells. The results suggest that the integrin Mac-1-fibrinogen interactions are stronger than the intermolecular fibrinogen interactions in the superficial layer of the matrix. The low mechanical stability of the multilayer fibrinogen surface may contribute to the reduced cell adhesive properties of fibrinogen-coated substrates. We anticipate that the described method can be applied to various cell types to examine their integrin-mediated adhesion to the extracellular matrices with a variable protein composition.

Atomic Force Microscopy control system for electrostatic measurements based on mechanical and electrical modulation

Atomic Force Microscopy (AFM) has excellent potential ability for quantitative electrostatic measurements of surface potential and dielectric permittivity of materials with nanoscale resolution. Implementation of this ability, however, requires overcoming several challenges. The first task is developing an accurate computational model for electrostatic tip-sample interaction; the second - efficient instrumentation and a control system that supports the measurements. An analytical model of nanoscale tip-sample capacitance on thin dielectric films was introduced (Gomila, Toset, and Fumagalli, 2008). This model allows describing the electrostatic tip-sample interaction force in a form suitable for the AFM dynamic control model (Belikov, Magonov, 2009). This dynamic model contains integrals over the tip-sample forces that can be presented in a closed form for the Gomila-Toset-Fumagalli analytical model. These results allow for developing very efficient electrostatic AFM computational model. As for the second task (instrumentation and control), the above mentioned AFM dynamic model with (amplitude-phase) state variables can be used for the control system design that combines mechanical Amplitude Modulation mode and consequent electrical modulation of the mechanical phase cosine. The cosine is monitored by lock-in amplifiers at the electrical modulation frequency, and twice that frequency; surface potential and dielectric permittivity of the sample can then be mapped. This paper presents the model derivation, description of instrumentation and control schematics, and their implementation on NT-MDT microscopes. Results are illustrated with practical measurements on different materials.

Fibrinogen counteracts the antiadhesive effect of fibrin‐bound plasminogen by preventing its activation by adherent U937 monocytic cells

Summary.  Background:  Fibrinogen and plasminogen strongly reduce adhesion of leukocytes and platelets to fibrin clots, highlighting a possible role for these plasma proteins in surface‐mediated control of thrombus growth and stability. In particular, adsorption of fibrinogen on fibrin clots renders their surfaces non‐adhesive, while the conversion of surface‐bound plasminogen to plasmin by transiently adherent blood cells results in degradation of a superficial fibrin layer, leading to cell detachment. Although the mechanisms whereby these proteins exert their antiadhesive effects are different, the outcome is the same: the formation of a mechanically unstable surface that does not allow firm cell attachment.

Modeling and measurements in Atomic Force Microscopy Resonance Modes

Quantitative Nanoscale Mechanical (QNM) studies in Atomic Force Microscopy (AFM) are usually associated with the Contact and Non-Resonance modes. The extraction of QNM data in AFM Resonance modes, such as Amplitude and Frequency Modulation, is a more challenging task. This paper describes a novel modeling techniques for the AFM Resonance modes and related QNM applications. The approach is based on Krylov-Bogoliubov-Mitropolsky (KBM) asymptotic dynamics of AFM in resonance modes.

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.

A novel mechanism cont rolling the growth of hemostatic thrombi

Current knowledge of the mechanisms of blood coagulation does not provide an answer to one pivotal question: why is, in contrast to a pathological thrombus, the growth of normal hemostatic clot after blood vessel injury suddenly terminated? In the present paper, we summarize the results of our investigations that give an answer to this question. We show that the surface of fibrin clot in the circulation is coated with a thin metastable layer of fibrinogen which is not able to support adhesion of blood cells. Consequently, platelets and leukocytes, the cells expressing adhesive integrins, are incapable of consolidating their grip on the surface and washed away by blood flow, thereby preventing the thrombus propagation. The cells that escaped this fibrinogen shield and reached a solid fibrin matrix use an additional mechanism – the ability to activate plasminogen bound either to the surface of cells or to fibrin. Plasmin formed at the interface between the cells and the clot locally degrades fibrin resulting in the fragmentation of the surface rendering it unstable, non-adhesive and therefore non-thrombogenic. Thus, the growth of hemostatic thrombus is halted by two mechanisms, fibrinogenand plasminogen-dependent, both of which are based on the same principle – the generation of the mechanically unstable, non-adhesive surface.