Matching Items (3)
Description
Adsorption of fibrinogen on various surfaces, including biomaterials, dramatically reduces the adhesion of platelets and leukocytes. The mechanism by which fibrinogen renders surfaces non-adhesive is its surface-induced self-assembly leading to the formation of a nanoscale multilayer matrix. Under the applied tensile force exerted by cellular integrins, the fibrinogen matrix extends as a result of the separation of layers which prevents the transduction of strong mechanical forces, resulting in weak intracellular signaling and feeble cell adhesion. Furthermore, upon detachment of adherent cells, a weak association between fibrinogen molecules in the superficial layers of the matrix allows integrins to pull fibrinogen molecules out of the matrix. Whether the latter mechanism contributes to the anti-adhesive mechanism under the flow is unclear. In the present study, using several experimental flow systems, it has been demonstrated that various blood cells as well as model HEK293 cells expressing the fibrinogen receptors, were able to remove fibrinogen molecules from the matrix in a time- and cell concentration-dependent manner. In contrast, insignificant fibrinogen dissociation occurred in a cell-free buffer, and crosslinking fibrinogen matrix significantly reduced cell-mediated dissociation of adsorbed fibrinogen. Surprisingly, cellular integrins contributed minimally to fibrinogen dissociation since function-blocking anti-integrin antibodies did not significantly inhibit this process. In addition, erythrocytes that are not known to express functional fibrinogen receptors and naked liposomes caused fibrinogen dissociation, suggesting that the removal of fibrinogen from the matrix may be caused by nonspecific low-affinity interactions of cells with the fibrinogen matrix. These results indicate that the peeling effect exerted by flowing cells upon their contact with the fibrinogen matrix is involved in the anti-adhesive mechanism.
ContributorsMursalimov, Aibek (Author) / Ugarova, Tatiana (Thesis advisor) / Chandler, Douglas (Committee member) / Podolnikova, Nataly (Committee member) / Ros, Robert (Committee member) / Arizona State University (Publisher)
Created2022
Description
Thrombus (blood clot) formation is at the roots of hemostasis and pathological thrombosis. Although many studies have successfully elucidated the cellular and molecular mechanisms underlying thrombus formation, there is still a void in understanding the processes limiting thrombus growth beyond that needed for stabilization. As a hemostatic thrombus grows, its surface consisting primarily of platelets changes to that composed of fibrin, which mechanically stabilizes the thrombus. Formation of fibrin ceases after some time; however, it is unclear why this fibrin is non-thrombogenic. This is puzzling since fibrin is known to support strong integrin-mediated adhesion of both platelets and leukocytes in vitro. Therefore, it would be expected that the fibrin surface of hemostatic thrombi in the circulation also support accumulation of these cells and thus continuous thrombus growth or degradation. Nevertheless, many in vivo studies did not detect any accumulation of blood cells including platelets at the fibrin surfaces of thrombi. This finding suggests the existence of natural processes that modulate the adhesive properties of fibrin to ensure proper regulation of thrombus growth, stability and degradation. In this dissertation, I document and discuss the findings supporting the existence of anti-adhesive mechanisms and their physiological relevance in surface-mediated control of thrombus growth and stability. The studies discussed in my dissertation have the potential to establish a novel aspect of hemostasis. Furthermore, it may provide new insights into the intricate and dynamic interplay between the mechanisms underlying hemostatic balance, which is essential to understanding the dysfunction of this process during pathological conditions.
ContributorsOwaynat, Hadil (Author) / Chandler, Douglas E. (Thesis advisor) / Wilson-Rawls, Norma J (Committee member) / Lake, Douglas F (Committee member) / Baluch, Debra P (Committee member) / Arizona State University (Publisher)
Created2016
Description
Cell adhesion is an important aspect of many biological processes. The atomic force microscope (AFM) has made it possible to quantify the forces involved in cellular adhesion using a technique called single cell force spectroscopy (SCFS). AFM based SCFS offers versatile control over experimental conditions for probing directly the interaction between specific cell types and specific proteins, surfaces, or other cells. Transmembrane integrins are the primary proteins involved in cellular adhesion to the extra cellular matix (ECM). One of the chief integrins involved in the adhesion of leukocyte cells is αMβ2 (Mac-1). The experiments in this dissertation quantify the adhesion of Mac-1 expressing human embryonic kidney (HEK Mac-1), platelets, and neutrophils cells on substrates with different concentrations of fibrinogen and on fibrin gels and multi-layered fibrinogen coated fibrin gels. It was shown that multi-layered fibrinogen reduces the adhesion force of these cells considerably. A novel method was developed as part of this research combining total internal reflection microscopy (TIRFM) with SCFS allowing for optical microscopy of HEK Mac-1 cells interacting with bovine serum albumin (BSA) coated glass after interacting with multi-layered fibrinogen. HEK Mac-1 cells are able to remove fibrinogen molecules from the multi-layered fibrinogen matrix. An analysis methodology for quantifying the kinetic parameters of integrin-ligand interactions from SCFS experiments is proposed, and the kinetic parameters of the Mac-1 fibrinogen bond are quantified. Additional SCFS experiments quantify the adhesion of macrophages and HEK Mac-1 cells on functionalized glass surfaces and normal glass surfaces. Both cell types show highest adhesion on a novel functionalized glass surface that was prepared to induce macrophage fusion. These experiments demonstrate the versatility of AFM based SCFS, and how it can be applied to address many questions in cellular biology offering quantitative insights.
ContributorsChristenson, Wayne B (Author) / Ros, Robert (Thesis advisor) / Beckstein, Oliver (Committee member) / Lindsay, Stuart (Committee member) / Ugarova, Tatiana (Committee member) / Arizona State University (Publisher)
Created2016