Peeling Effect of Cells on Fibrinogen Multilayer as an Anti-Adhesive Control Mechanism for Hemostatic Thrombus Formation

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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

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.
Date Created
2022
Agent

Isolation of Recombinant IgV-CD47 Fragment to Probe the Interaction Between CD47 and Mac-1 Integrin

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Description

CD47 is a cell surface receptor expressed on many cells in the body. It has many immune functions such as marking host cells as “self” and the activation of apoptosis through phagocytosis. Mac-1 is a major integrin on myeloid cells

CD47 is a cell surface receptor expressed on many cells in the body. It has many immune functions such as marking host cells as “self” and the activation of apoptosis through phagocytosis. Mac-1 is a major integrin on myeloid cells and has been implicated in several different macrophage immune functions. Previous studies from Dr. Ugarova’s lab demonstrated CD47 may form a complex with Mac-1 through the cis-interaction and could regulate Mac-1-dependent macrophage functions. To localize the binding site for Mac-1 in CD47, the extracellular domain of CD47 IgV was isolated as GST-fusion protein from E. coli cells. The recombinant fusion protein is being used in current studies with cell adhesion assays and immunoprecipitation to determine the complementary binding site in Mac-1.

Date Created
2021-05
Agent

Creating Paramagnetically-Labeled PF4 Mutants to Evaluate Interactions with Mac-1 in NMR

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Description
PF4 (CXCL4) is a cationic platelet chemokine that has been identified as a ligand for the integrin Mac-1 (αMβ2). The interaction between PF4 and Mac-1 has been shown to cause leukocyte migration, improve phagocytosis, and trigger the up-regulation of Mac-1

PF4 (CXCL4) is a cationic platelet chemokine that has been identified as a ligand for the integrin Mac-1 (αMβ2). The interaction between PF4 and Mac-1 has been shown to cause leukocyte migration, improve phagocytosis, and trigger the up-regulation of Mac-1 expression in leukocytes, thereby increasing leukocytic adhesion. Though Mac-1 is known to serve as the site of interaction between PF4 and the leukocyte, the PF4 binding site of Mac-1 remains unknown. 1H-15N HSQC NMR spectroscopy of the interaction between PF4 and Mac-1’s binding site, the αMI domain, can provide this data. This project seeks to create PF4 mutants with site-directed spin labels to enhance the sensitivity of NMR for future experiments that seek to locate the PF4-Mac-1 binding site. It was hypothesized that the mutants created would adopt the native conformation and accept an MTSL label. Two mutants were successfully created and harvested, PF4 S17C and PF4 S26C. Both were soluble and the Sanger sequencing results show that primary structure was conserved except for the substitutions of structurally similar residues indicating the protein folds and likely adopts native conformation. PF4 S26C was labeled with MTSL, and 1H-15N HSQC NMR spectroscopy was performed on unlabeled PF4 S26C (at pH 3.40), MTSL-labeled PF4 S26C (at pH 3.15), and MTSL-labeled PF4 S26C exposed to ascorbic acid (at pH 3.15) to evaluate if the mutant accepts the label and, resultantly, experiences reduced signal intensity. Significant change in signal intensity occurred without change in location of the peaks between the unlabeled and labeled spectra, showing that PF4 S26C accepts the spin label without changing the protein structure and that the label works as expected; however, no change occurred after reducing the spin label with ascorbic acid, preventing confirmation that signal changes were exclusively caused by the MTSL-label. Therefore, though these mutants show potential for future titration with the αMI domain and the hypothesis is supported, a future attempt to reduce MTSL-labeled PF4 S26C at a higher pH (approximately pH 5) is required. Additionally, PF4 S17C should also be evaluated with the methodology used to assess PF4 S26C before its employment in future projects.
Date Created
2018-05
Agent

Expression of the Fusogenic Protein Syncytin in Macrophages

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Description
Cell fusion is a process that occurs in normal cells as well as in pathological cells. This process does not occur spontaneously, fusogens are required to mediate the process. Syncytin is one of the proteins that was determined to have

Cell fusion is a process that occurs in normal cells as well as in pathological cells. This process does not occur spontaneously, fusogens are required to mediate the process. Syncytin is one of the proteins that was determined to have fusogenic properties. Syncytin is a newly discovered transmembrane protein that is generally expressed in mammalian placenta and it is known for its role in cell fusion during placentation. The recent studies in Ugarova’s laboratory suggest syncytin is expressed in macrophages, thus it may be involved in macrophage cells fusion. This paper provides a literature review of syncytin protein; it also contains an experimental study conducted to determine syncytin expression on both RNA and protein level. The study was conducted on RNA and protein isolated from macrophages isolated from mouse peritoneum. Agarose gel electrophoresis and Western blot analysis were used to determine syncytin expression on RNA and protein level respectively. Using these methods, syncytin expression was determined at different time points during macrophage fusion. The results show that syncytin is not expressed in freshly isolated macrophages, but its expression is initiated during macrophage adhesion in the presence of IL-4.
Date Created
2019-05
Agent

Determining Magnesium Metal Affinity of Alpha L- Id in pH 5

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Description
Integrin is a protein in cells that manage cell adhesion. They are crucial to the biochemical functions of cells. L 2 is one type of integrin. Its I domain is responsible for ligand binding. Scientists understand how Alpha L I

Integrin is a protein in cells that manage cell adhesion. They are crucial to the biochemical functions of cells. L 2 is one type of integrin. Its I domain is responsible for ligand binding. Scientists understand how Alpha L I domain binds Mg2+ at a pH of 7 but not in acidic environments. Knowing the specificity of integrin at a lower pH is important because when tissues become inflamed, they release acidic compounds. We have cloned, expressed, and purified L I-domain and using NMR analysis, we determined that wild type Alpha L I domain does not bind to Mg2+ at a pH of 5.
Date Created
2017-05
Agent

The Association of Cytoplasmic Molecules with Integrin During Bidirectional Signaling

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Description
Platelets are specialized blood cells that play crucial role in normal physiologic and pathologic processes such as hemostasis, inflammation, wound healing, and host defense. Activation of platelets is essential for platelet function and it includes a complex interplay of adhesion

Platelets are specialized blood cells that play crucial role in normal physiologic and pathologic processes such as hemostasis, inflammation, wound healing, and host defense. Activation of platelets is essential for platelet function and it includes a complex interplay of adhesion and intracellular signaling molecules. Platelets are known to be activated during vessel injury by a complex interaction of soluble agonists and once activated, they adhere to sub-endothelial matrix to aggregate and secrete granules leading to the formation of platelet aggregate that is necessary for thrombus formation. Platelet integrin plays a central role in platelet adhesive reactions by serving as a receptor for fibrinogen involved in bidirectional transmembrane signaling. In order to elucidate the interaction of integrin with cytoplasmic signaling molecules during inside-out and outside-in signaling, we have studied the kinetics of the recruitment of talin, kindling, filmin-A, skelemin, Scr and syk to the B3 cytoplasmic tails. Platelets were isolated from human blood and activated with ADP/Epinephrine for different times. The complexes of *** with signaling proteins were obtained by immunoprecipitation of platelet lysates with anit-*** monoclonal antibody and then analyzed by Western blotting using antibodies directed against selected signaling proteins. Our results show different kinetics in recruitment of signaling molecules to the B3 integrin cytoplasmic tail during inside-out and outside in signaling.
Date Created
2012-05
Agent

Identification of Human Cathelicidin Peptide LL-37 as a Ligand for Macrophage Integrin αMβ2 (Mac-1, CD11b/CD18) That Promotes Phagocytosis by Opsonizing Bacteria

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Description

LL-37, a cationic antimicrobial peptide, has numerous immune-modulating effects. However, the identity of a receptor that mediates the responses in immune cells remains uncertain. We have recently demonstrated that LL-37 interacts with the αMI-domain of integrin αMβ2 (Mac-1), a major

LL-37, a cationic antimicrobial peptide, has numerous immune-modulating effects. However, the identity of a receptor that mediates the responses in immune cells remains uncertain. We have recently demonstrated that LL-37 interacts with the αMI-domain of integrin αMβ2 (Mac-1), a major receptor on the surface of myeloid cells, and induces a migratory response in Mac-1-expressing monocyte/macrophages as well as activation of Mac-1 on neutrophils. Here, we show that LL-37 and its C-terminal derivative supported strong adhesion of various Mac-1-expressing cells, including human embryonic kidney cells stably transfected with Mac-1, human U937 monocytic cells, and murine IC-21 macrophages. The cell adhesion to LL-37 was partially inhibited by specific Mac-1 antagonists, including monoclonal antibody against the αM integrin subunit and neutrophil inhibitory factor, and completely blocked when anti-Mac-1 antibodies were combined with heparin, suggesting that cell surface heparan sulfate proteoglycans act cooperatively with integrin Mac-1. Coating both gram-negative and gram-positive bacteria with LL-37 significantly potentiated their phagocytosis by macrophages, and this process was blocked by a combination of anti-Mac-1 monoclonal antibody and heparin. Furthermore, phagocytosis by wild-type murine peritoneal macrophages of LL-37-coated latex beads, a model of foreign surfaces, was several fold higher than that of untreated beads. In contrast, LL-37 failed to augment phagocytosis of beads by Mac-1-deficient macrophages. These results identify LL-37 as a novel ligand for integrin Mac-1 and demonstrate that the interaction between Mac-1 on macrophages and bacteria-bound LL-37 promotes phagocytosis.

Date Created
2016-07-07
Agent

Ligand Recognition Specificity of Leukocyte Integrin alpha(M)beta(2) (Mac-1, CD11b/CD18) and Its Functional Consequences

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Description

The broad recognition specificity exhibited by integrin αMβ2 (Mac-1, CD11b/CD18) has allowed this adhesion receptor to play innumerable roles in leukocyte biology, yet we know little about how and why αMβ2 binds its multiple ligands. Within αMβ2, the αMI-domain is

The broad recognition specificity exhibited by integrin αMβ2 (Mac-1, CD11b/CD18) has allowed this adhesion receptor to play innumerable roles in leukocyte biology, yet we know little about how and why αMβ2 binds its multiple ligands. Within αMβ2, the αMI-domain is responsible for integrin’s multiligand binding properties. To identify its recognition motif, we screened peptide libraries spanning sequences of many known protein ligands for αMI-domain binding and also selected the αM I-domain recognition sequences by phage display. Analyses of >1400 binding and nonbinding peptides derived from peptide libraries showed that a key feature of the αMI-domain recognition motif is a small core consisting of basic amino acids flanked by hydrophobic residues. Furthermore, the peptides selected by phage display conformed to a similar pattern. Identification of the recognition motif allowed the construction of an algorithm that reliably predicts the αMI-domain binding sites in the αMβ2 ligands. The recognition specificity of the αMI-domain resembles that of some chaperones, which allows it to bind segments exposed in unfolded proteins. The disclosure of the αMβ2 binding preferences allowed the prediction that cationic host defense peptides, which are strikingly enriched in the αMI-domain recognition motifs, represent a new class of αMβ2 ligands. This prediction has been tested by examining the interaction of αMβ2 with the human cathelicidin peptide LL-37. LL-37 induced a potent αMβ2-dependent cell migratory response and caused activation of αMβ2 on neutrophils. The newly revealed recognition specificity of αMβ2 toward unfolded protein segments and cationic proteins and peptides suggests that αMβ2 may serve as a previously proposed “alarmin” receptor with important roles in innate host defense.

Date Created
2015-02-17
Agent

The Opioid Peptide Dynorphin A Induces Leukocyte Responses Via Integrin Mac-1 (alpha(M)beta(2), CD11b/CD18)

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Description

Background: Opioid peptides, including dynorphin A, besides their analgesic action in the nervous system, exert a broad spectrum of effects on cells of the immune system, including leukocyte migration, degranulation and cytokine production. The mechanisms whereby opioid peptides induce leukocyte

Background: Opioid peptides, including dynorphin A, besides their analgesic action in the nervous system, exert a broad spectrum of effects on cells of the immune system, including leukocyte migration, degranulation and cytokine production. The mechanisms whereby opioid peptides induce leukocyte responses are poorly understood. The integrin Mac-1 (alpha(M)beta(2), CD11b/CD18) is a multiligand receptor which mediates numerous reactions of neutrophils and monocyte/macrophages during the immune-inflammatory response. Our recent elucidation of the ligand recognition specificity of Mac-1 suggested that dynorphin A and dynorphin B contain Mac-1 recognition motifs and can potentially interact with this receptor.

Results: In this study, we have synthesized the peptide library spanning the sequence of dynorphin AB, containing dynorphin A and B, and showed that the peptides bound recombinant alpha I-M-domain, the ligand binding region of Mac-1. In addition, immobilized dynorphins A and B supported adhesion of the Mac-1-expressing cells. In binding to dynorphins A and B, Mac-1 cooperated with cell surface proteoglycans since both anti-Mac-1 function-blocking reagents and heparin were required to block adhesion. Further focusing on dynorphin A, we showed that its interaction with the alpha I-M-domain was activation independent as both the alpha 7 helix-truncated (active conformation) and helix-extended (nonactive conformation) alpha I-M-domains efficiently bound dynorphin A. Dynorphin A induced a potent migratory response of Mac-1-expressing, but not Mac-1-deficient leukocytes, and enhanced Mac-1-mediated phagocytosis of latex beads by murine IC-21 macrophages.

Conclusions: Together, the results identify dynorphins A and B as novel ligands for Mac-1 and suggest a role for the Dynorphin A-Mac-1 interactions in the induction of nonopiod receptor-dependent effects in leukocytes.

Date Created
2015-06-03
Agent

Skelemin Association With Alpha(IIb)Beta(3) Integrin: A Structural Model

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Description

Over the last two decades, our knowledge concerning intracellular events that regulate integrin’s affinity to their soluble ligands has significantly improved. However, the mechanism of adhesion-induced integrin clustering and development of focal complexes, which could further mature to form focal

Over the last two decades, our knowledge concerning intracellular events that regulate integrin’s affinity to their soluble ligands has significantly improved. However, the mechanism of adhesion-induced integrin clustering and development of focal complexes, which could further mature to form focal adhesions, still remains under-investigated. Here we present a structural model of tandem IgC2 domains of skelemin in complex with the cytoplasmic tails of integrin α[subscript IIb]β[subscript 3]. The model of tertiary assembly is generated based upon NMR data and illuminates a potential link between the essential cell adhesion receptors and myosin filaments. This connection may serve as a basis for generating the mechanical forces necessary for cell migration and remodeling.

Date Created
2014-11-04
Agent