Molecular characterization of a carboxy-terminal eukaryotic-cell-binding domain of intimin from enteropathogenic Escherichia coli.

Plasma fibronectin has been shown to increase the binding of fibrin monomer to macrophages in vitro. In the present study we began characterization of the mechanism underlying this fibronectin activity. Fragments of fibronectin containing the amino terminus enhanced macrophage fibrin binding to the same extent as intact fibronectin on an equimolar basis. However, fibronectin fragments containing the gelatin-binding domain or the cell-binding domain, but lacking the amino terminus, had no effect on fibrin binding. Fibronectin enhanced fibrin binding was not affected by the addition of synthetic peptides containing the RGD adhesion sequence. The ability of fibronectin to augment fibrin binding remained after paraformaldehyde fixation of macrophage monolayers. Fixation did not alter the basal levels of fibrin binding by macrophages. Preincubation of macrophages with exogenous fibronectin did not increase the binding of fibrin. Fibronectin enhanced fibrin binding remained unaltered after the removal of endogenous cell surface fibronectin by capping with F(ab')2 fragments of antibodies to fibronectin. These results suggest that the amino terminus of fibronectin supports the attachment of fibrin to macrophages by an initial fluid-phase interaction that precedes cellular binding and does not require a cellular response.

Download Full-text

Fibronectin dependent macrophage fibrin binding

Blood ◽

10.1182/blood.v78.11.2900.2900 ◽

1991 ◽

Vol 78 (11) ◽

pp. 2900-2907 ◽

Cited By ~ 9

Author(s):

SD Blystone ◽

LK Weston ◽

JE Kaplan

Keyword(s):

Synthetic Peptides ◽

Cellular Response ◽

Fluid Phase ◽

Binding Domain ◽

Amino Terminus ◽

Cell Binding ◽

Fibronectin Fragments ◽

Cell Binding Domain

Abstract Plasma fibronectin has been shown to increase the binding of fibrin monomer to macrophages in vitro. In the present study we began characterization of the mechanism underlying this fibronectin activity. Fragments of fibronectin containing the amino terminus enhanced macrophage fibrin binding to the same extent as intact fibronectin on an equimolar basis. However, fibronectin fragments containing the gelatin-binding domain or the cell-binding domain, but lacking the amino terminus, had no effect on fibrin binding. Fibronectin enhanced fibrin binding was not affected by the addition of synthetic peptides containing the RGD adhesion sequence. The ability of fibronectin to augment fibrin binding remained after paraformaldehyde fixation of macrophage monolayers. Fixation did not alter the basal levels of fibrin binding by macrophages. Preincubation of macrophages with exogenous fibronectin did not increase the binding of fibrin. Fibronectin enhanced fibrin binding remained unaltered after the removal of endogenous cell surface fibronectin by capping with F(ab')2 fragments of antibodies to fibronectin. These results suggest that the amino terminus of fibronectin supports the attachment of fibrin to macrophages by an initial fluid-phase interaction that precedes cellular binding and does not require a cellular response.

Download Full-text

The Carboxy-Terminal Cell-Binding Domain of Thrombospondin Is Essential for Sickle Red Blood Cell Adhesion

Blood ◽

10.1182/blood.v94.1.302.413k38_302_309 ◽

1999 ◽

Vol 94 (1) ◽

pp. 302-309 ◽

Cited By ~ 15

Author(s):

Cheryl A. Hillery ◽

J. Paul Scott ◽

Ming C. Du

Keyword(s):

Cell Adhesion ◽

Matrix Protein ◽

Binding Domain ◽

Terminal Cell ◽

Heparin Binding ◽

Cell Binding ◽

Adhesive Site ◽

Carboxy Terminal ◽

Cell Binding Domain

Sickle red blood cells (SS-RBCs) have enhanced adhesion to the plasma and subendothelial matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. TSP has at least four domains that mediate cell adhesion. The goal of this study was to map the site(s) on TSP that binds SS-RBCs. Purified TSP proteolytic fragments containing either the N-terminal heparin-binding domain, or the type 1, 2, or 3 repeats, failed to sustain SS-RBC adhesion (<10% adhesion). However, a 140-kD thermolysin TSP fragment, containing the carboxy-terminal cell-binding domain in addition to the type 1, 2, and 3 repeats fully supported the adhesion of SS-RBCs (126% ± 25% adhesion). Two cell-binding domain adhesive peptides, 4N1K (KRFYVVMWKK) and 7N3 (FIRVVMYEGKK), failed to either inhibit or support SS-RBC adhesion to TSP. In addition, monoclonal antibody C6.7, which blocks platelet and melanoma cell adhesion to the cell-binding domain, did not inhibit SS-RBC adhesion to TSP. These data suggest that a novel adhesive site within the cell binding domain of TSP promotes the adhesion of sickle RBCs to TSP. Furthermore, soluble TSP did not bind SS-RBCs as detected by flow cytometry, nor inhibit SS-RBC adhesion to immobilized TSP under conditions of flow, indicating that the adhesive site on TSP that recognizes SS-RBCs is exposed only after TSP binds to a matrix. We conclude that the intact carboxy-terminal cell-binding domain of TSP is essential for the adhesion of sickle RBCs under flow conditions. This study also provides evidence for a unique adhesive site within the cell-binding domain that is exposed after TSP binds to a matrix.

Download Full-text

Activation-dependent recognition by hematopoietic cells of the LDV sequence in the V region of fibronectin.

The Journal of Cell Biology ◽

10.1083/jcb.116.2.489 ◽

1992 ◽

Vol 116 (2) ◽

pp. 489-497 ◽

Cited By ~ 86

Author(s):

E A Wayner ◽

N L Kovach

Keyword(s):

Cell Adhesion ◽

Active Form ◽

Variable Region ◽

Cell Interaction ◽

Binding Domain ◽

Terminal Cell ◽

High Avidity ◽

Cell Binding ◽

Carboxy Terminal ◽

Cell Binding Domain

It has been shown that the alpha 4 beta 1 integrin is the lymphocyte receptor for the carboxy terminal cell-binding domain of fibronectin which comprises adhesion sites in Hep 2 and a high affinity site, CS-1, in the type III connecting segment or V (for variable) region. In the present studies, using a series of peptides derived from CS-1, we identify the tripeptide leu-asp-val (LDV), as the minimal peptide capable of supporting stable lymphocyte or melanoma cell adhesion. However, only cells which expressed an active form of the alpha 4 beta 1 complex were capable of attaching to and spreading on LDV peptide. On a molar basis, LDV minimal peptides were either not active or 10-20 times less active than intact CS-1 in promoting the adhesion of lymphocytes expressing the resting form of the receptor. In cells which express the high avidity form of the receptor, LDV and CS-1 were equally effective in promoting cell adhesion and spreading. The avidity of the alpha 4 beta 1 complex could be altered with mAbs to beta 1 which specifically activate beta 1 dependent function. The high avidity form of the alpha 4 beta 1 complex could be induced on U937 cells, T, and B lymphoblastoid cell lines, or PHA-stimulated T cell blasts. Resting PBL could not be induced to bind LDV peptide conjugates by activating antibodies to beta 1 implying that two signals are required for LDV recognition by T cells. In conclusion, these data show clearly that the minimal peptide for the alpha 4 beta 1 complex in CS-1 is the LDV sequence. Although numerous cell populations can interact with intact CS-1 only cells which express an active alpha 4 beta 1 complex can bind the LDV sequence. This implies that cell interaction with the carboxy terminal cell-binding domain of fibronectin can be regulated at several levels: (a) alpha 4 beta 1 expression; (b) activation of the alpha 4 beta 1 complex; and (c) alternate splicing of CS-1 into V+ isoforms of fibronectin.

Download Full-text