Platelet interactions with fibronectin: divalent cation-independent platelet adhesion to the gelatin-binding domain of fibronectin

Divalent cation-dependent platelet adhesion to fibronectin (FN) is mediated by the integrin receptors alpha 5 beta 1 (GP Ic-IIa) and alpha IIb beta 3 (GP IIb-IIIa), which recognize the RGD (Arg-Gly-Asp) sequence in the cell-binding domain. However, FN can also support divalent cation-independent platelet adhesion. To determine which domain of FN mediates divalent cation-independent adhesion, proteolysis with thermolysin and affinity chromatography were used to isolate the cell-binding, gelatin-binding, and heparin-binding domains of FN. Unactivated and thrombin-activated platelets adhered to intact FN and the 45-Kd gelatin-binding domain in the presence of either Ca2+ or EDTA. Platelet spreading was mediated only by the 105-Kd cell-binding domain and required divalent cations. The heparin-binding domains did not support platelet adhesion. Reduction of intrachain disulfide bonds or removal of carbohydrate side chains on the gelatin-binding domain did not alter the ability to support platelet adhesion. Divalent cation- independent adhesion to the 45-Kd gelatin-binding domain was not inhibited by RGDS (Arg-Gly-Asp-Ser) synthetic peptides or monoclonal antibodies (MoAbs) directed against known platelet receptors. We conclude that platelets can adhere but not spread on the gelatin- binding domain of FN by a novel divalent cation-independent mechanism.

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Platelet interactions with fibronectin: divalent cation-independent platelet adhesion to the gelatin-binding domain of fibronectin

Blood ◽

10.1182/blood.v81.7.1778.1778 ◽

1993 ◽

Vol 81 (7) ◽

pp. 1778-1786 ◽

Cited By ~ 8

Author(s):

KJ Winters ◽

JJ Walsh ◽

BG Rubin ◽

SA Santoro

Keyword(s):

Divalent Cation ◽

Disulfide Bonds ◽

Platelet Adhesion ◽

Divalent Cations ◽

Binding Domain ◽

Heparin Binding ◽

Cell Binding ◽

Binding Domains ◽

Platelet Spreading ◽

Cell Binding Domain

Abstract Divalent cation-dependent platelet adhesion to fibronectin (FN) is mediated by the integrin receptors alpha 5 beta 1 (GP Ic-IIa) and alpha IIb beta 3 (GP IIb-IIIa), which recognize the RGD (Arg-Gly-Asp) sequence in the cell-binding domain. However, FN can also support divalent cation-independent platelet adhesion. To determine which domain of FN mediates divalent cation-independent adhesion, proteolysis with thermolysin and affinity chromatography were used to isolate the cell-binding, gelatin-binding, and heparin-binding domains of FN. Unactivated and thrombin-activated platelets adhered to intact FN and the 45-Kd gelatin-binding domain in the presence of either Ca2+ or EDTA. Platelet spreading was mediated only by the 105-Kd cell-binding domain and required divalent cations. The heparin-binding domains did not support platelet adhesion. Reduction of intrachain disulfide bonds or removal of carbohydrate side chains on the gelatin-binding domain did not alter the ability to support platelet adhesion. Divalent cation- independent adhesion to the 45-Kd gelatin-binding domain was not inhibited by RGDS (Arg-Gly-Asp-Ser) synthetic peptides or monoclonal antibodies (MoAbs) directed against known platelet receptors. We conclude that platelets can adhere but not spread on the gelatin- binding domain of FN by a novel divalent cation-independent mechanism.

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C-terminal heparin-binding domain of fibronectin regulates integrin-mediated cell spreading but not the activation of mitogen-activated protein kinase

Biochemical Journal ◽

10.1042/bj3600239 ◽

2001 ◽

Vol 360 (1) ◽

pp. 239-245 ◽

Cited By ~ 7

Author(s):

Jungyean KIM ◽

Innoc HAN ◽

Yeonhee KIM ◽

Seungin KIM ◽

Eok-Soo OH

Keyword(s):

Cell Spreading ◽

Binding Domain ◽

Mitogen Activated Protein Kinase ◽

Heparin Binding ◽

Cell Binding ◽

Rat Embryo ◽

Mapk Activation ◽

Mitogen Activated Protein ◽

Heparin Binding Domain ◽

Cell Binding Domain

Fibronectin (FN) stimulates multiple signalling events including mitogen-activated protein kinase (MAPK) activation. During cell spreading, both the cell-binding domain and the C-terminal heparin-binding domain (HepII) of FN co-operatively regulate cytoskeleton organization. However, in comparison with the large number of studies on the functions of cell-binding domain, there is little information about the role of HepII. We therefore investigated the effect of HepII on integrin-mediated cell spreading and adhesion on FN and MAPK activation. In contrast with cells on FN substrates, rat embryo fibroblasts on FN120, which lacks HepII, were less spread, had weaker adhesion to FN and failed to form focal adhesions and actin stress fibres. Phosphotyrosine was present in the focal contacts of rat embryo fibroblasts on FN within 30min but was absent from cells on FN120. Overall, tyrosine phosphorylation was much less in cell lysates from cells on FN120, with decreased phosphorylation of focal adhesion kinase (‘pp125FAK’) on tyrosine-397, implying additional regulation of tyrosine phosphorylation by HepII. Nevertheless, adhesion-mediated MAPK activity was similar in cells on FN and on FN120. Furthermore, cells spread on FN and on FN120 substrates showed similar MAPK activation in response to treatment with epidermal growth factor and with platelet-derived growth factor. Consistently, overexpression of syndecan-4, which binds to HepII, enhanced cell spreading and adhesion on FN but did not affect integrin-mediated MAPK activation. We therefore conclude that both HepII and syndecan-4 regulate integrin-mediated cell spreading but not MAPK activation.

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Fibronectin Facilitates Mycobacterium tuberculosis Attachment to Murine Alveolar Macrophages

Infection and Immunity ◽

10.1128/iai.70.3.1287-1292.2002 ◽

2002 ◽

Vol 70 (3) ◽

pp. 1287-1292 ◽

Cited By ~ 25

Author(s):

Rajamouli Pasula ◽

Paul Wisniowski ◽

William J. Martin

Keyword(s):

Mycobacterium Tuberculosis ◽

Alveolar Macrophages ◽

Pulmonary Infection ◽

Polyclonal Antibodies ◽

Binding Domain ◽

Heparin Binding ◽

Cell Binding ◽

Adhesive Protein ◽

Heparin Binding Domain ◽

Cell Binding Domain

ABSTRACT Mycobacterium tuberculosis remains a major cause of pulmonary infection worldwide. Attachment of M. tuberculosis organisms to alveolar macrophages (AMs) represents the earliest phase of primary infection in pulmonary tuberculosis. In this study fibronectin (Fn), an adhesive protein, is shown to bind M. tuberculosis organisms and facilitates attachment of M. tuberculosis to murine AMs. A monoclonal antibody (MAb) specific to the heparin binding domain (HBD) of Fn decreases 125I-Fn binding to M. tuberculosis; whereas MAbs specific to either the cell binding domain (CBD) or the gelatin binding domain (GBD) have no effect on Fn binding to M. tuberculosis. In the presence of exogenous Fn (10 μg/ml) M. tuberculosis attachment to AMs increased significantly from control levels (means ± standard errors of the means) of 11.5% ± 1.1% to 44.2% ± 4.2% (P < 0.05). Fn-enhanced attachment was significantly decreased from 44.2% ± 4.2% to 10.8% ± 1.2% (P < 0.05) in the presence of anti-Fn polyclonal antibodies. The attachment is also inhibited in the presence of MAbs specific for the HBD and CBD, whereas MAbs specific to GBD did not affect the attachment. Further, an Fn cell binding peptide, Arg-Gly-Asp-Ser (RGDS), decreased the attachment from 44.2% ± 4.2% to 15.3% ± 1.2% (P < 0.05), whereas addition of a control peptide, Arg-Gly-Glu-Ser (RGES) did not affect the attachment (40.5% ± 1.8%). These results suggest that Fn-mediated attachment of M. tuberculosis can occur through the binding of Fn to the AM via the CBD and to M. tuberculosis organisms via the HBD.

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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.

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Rat embryo fibroblasts require both the cell-binding and the heparin-binding domains of fibronectin for survival

Biochemical Journal ◽

10.1042/bj3560531 ◽

2001 ◽

Vol 356 (2) ◽

pp. 531-537 ◽

Cited By ~ 15

Author(s):

Jinsook JEONG ◽

Innoc HAN ◽

Yangmi LIM ◽

Jungyean KIM ◽

Ilseon PARK ◽

...

Keyword(s):

Cell Survival ◽

Binding Domain ◽

Heparin Binding ◽

Cell Binding ◽

Rat Embryo ◽

Nuclear Condensation ◽

Embryo Fibroblasts ◽

Induced Apoptosis ◽

Domain Peptide ◽

Cell Binding Domain

Fibronectin (FN) is known to transduce signal(s) to rescue cells from detachment-induced apoptosis (anoikis) through an integrin-mediated survival pathway. However, the functions of individual FN domains have not been studied in detail. In the present study we investigated whether the interaction of the cell-binding domain of FN with integrin is sufficient to rescue rat embryo fibroblasts (REFs) from detachment-induced apoptosis. REFs attached and spread normally after plating on substrates coated with either intact FN or a FN fragment, FN120, that contains the cell-binding domain but lacks the C-terminal heparin-binding domain, HepII. REFs on FN maintained a well-spread fibroblastic shape and even proliferated in serum-free medium at 20h after plating. In contrast, previously well-spread REFs on FN120 started losing fibroblastic shape with time and detached from FN120-coated plates after approx. 8h. Nuclear condensation indicated apototic cell death. This was due to the decreased activity/stability of focal adhesion kinase (pp125FAK) in the absence of HepII domain. A peptide in the HepII domain [peptide V, WQPPRARI (single-letter amino acid codes)], which has previously been implicated in cytoskeletal organization, rescued apoptotic changes. Consistently, pp125FAK phosphorylation was increased, and both cleavage of pp125FAK and activation of caspase 3 on FN120 were partly blocked by peptide V. Thus the interaction of the cell-binding domain with integrin has a major role in cell survival but is itself not sufficient for cell survival. One or more additional survival signals come from the HepII domain to regulate pp125FAK activity/stability.

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Modulation of matrix adhesive responses of human neuroblastoma cells by neighboring sequences in the fibronectins.

The Journal of Cell Biology ◽

10.1083/jcb.106.3.931 ◽

1988 ◽

Vol 106 (3) ◽

pp. 931-943 ◽

Cited By ~ 28

Author(s):

G Mugnai ◽

K Lewandowska ◽

B Carnemolla ◽

L Zardi ◽

L A Culp

Keyword(s):

Heparan Sulfate ◽

Neurite Outgrowth ◽

Neuroblastoma Cells ◽

Binding Domain ◽

Heparin Binding ◽

Cell Binding ◽

Human Neuroblastoma Cells ◽

Human Neuroblastoma ◽

Dependent Cell ◽

Cell Binding Domain

Attachment and neurite extension have been measured when Platt or La-N1 human neuroblastoma cells respond to tissue culture substrata coated with a panel of complementary fragments from the individual chains of human plasma (pFN) or cellular fibronectins (cFN) purified from thermolysin digests. A 110-kD fragment (f110), which contains the Arg-Gly-Asp-Ser sequence (RGDS)-dependent cell-binding domain but no heparin-binding domains and whose sequences are shared in common by both the alpha- and beta-subunits of pFN, facilitated attachment of cells that approached the level observed with either intact pFN or the heparan sulfate-binding platelet factor-4 (PF4). This attachment on f110 was resistant to RGDS-containing peptide in the medium. Neurite outgrowth was also maximal on f110, and half of these neurites were also resistant to soluble RGDS peptide. Treatment of cells with glycosaminoglycan lyases failed to alter these responses on f110. Therefore, there is a second "cell-binding" domain in the sequences represented by f110 that is not RGDS- or heparan sulfate-dependent and that facilitates stable attachment and some neurite outgrowth; this domain appears to be conformation-dependent. Comparisons were also made between two larger fragments generated from the two subunits of pFN-f145 from the alpha-subunit and f155 from the beta-subunit--both of which contain the RGDS-dependent cell-binding domain and the COOH-terminal heparin-binding domain but which differ in the former's containing some IIICS sequence at its COOH terminus and the latter's having an additional type III homology unit. Heparin-binding fragments (with no RGDS activity) of f29 and f38, derived from f145 or f155 of pFN, respectively, and having the same differences in sequence, were also compared with f44 + 47 having the "extra domain" characteristic of cFN. Attachment on f145 was slightly sensitive to soluble RGDS peptide; attachment on f155 was much more sensitive. There were also differences in the percentage of cells with neurites on f145 vs. f155 but neurites on either fragment were completely sensitive to RGDS peptide. Mixing of f29, f38, or PF4 with f110 could not reconstitute the activities demonstrated in f145 or f155, demonstrating that covalently linked sequences are critical in modulating these responses. However, mixing of f44 + 47 from cFN with f110 from pFN increased the sensitivity to RGDS peptide.(ABSTRACT TRUNCATED AT 400 WORDS)

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Requirement for two different cell-binding domains in fibronectin for neurite extension of neuronal derivative cells

Journal of Cell Science ◽

10.1242/jcs.95.1.75 ◽

1990 ◽

Vol 95 (1) ◽

pp. 75-83

Author(s):

K. Lewandowska ◽

E. Balza ◽

L. Zardi ◽

L.A. Culp

Keyword(s):

Neuroblastoma Cells ◽

Binding Domain ◽

Dorsal Root Ganglion Neurons ◽

Cell Binding ◽

Human Neuroblastoma ◽

Neurite Extension ◽

Binding Domains ◽

C Terminus ◽

Ganglion Neurons ◽

Cell Binding Domain

Some neuron-derived cells, such as neuroblastoma cells, adhere and extend neurites on fibronectin (FN) substrata by processes that can be independent of binding to the Arg-Gly-Asp-Ser sequence (RGDS in FN) and independent of proteoglycan/ganglioside-binding activities of FN. Proteolytic fragments of various FNs have been used in this study to map a new adhesion-promoting domain in FNs that may be neural cell-specific. A thermolysin-generated fragment of human plasma FN (F110 containing the RGDS domain) or the analagous fragment from transformed human cell FN (F120, also containing the alternately spliced extra domain b[EDb]) facilitate RGDS-independent adherence and neurite extension of human neuroblastoma cells and an F11 hybrid neuronal line (by fusion of mouse neuroblastoma cells with rat dorsal root ganglion neurons) as effectively as adherence and neurite extension on intact FN. Since neither F110 nor F120 contains sequences from the alternately spliced IIICS region of FN, neurite-promoting activity in these fragments cannot be ascribed to a recently discovered cell-binding domain in this region. Furthermore, F120 could be cleaved into two subfragments retaining virtually all the sequence of the parent fragment: F35 from the C terminus of F120 containing the RGDS domain, and F90 from the N terminus containing most of the EDb region bordering the thermolysin cleavage site. These neuronal cells could adhere but not extend neurites on substrata coated with either F35 or F90 alone while 3T3 cells could adhere only on F35. Mixtures of F35 and F90 on substrata could reconstitute some, but not nearly all, of the neurite-promoting activity of F120. Therefore, these data identify a new cell-binding domain in common sequences of FNs on the N-terminal side of EDb and demonstrate cooperativity between this RGDS-independent domain and the RGDS-dependent domain for maximal differentiation of these neuron-derived cells. Several possibilities for a receptor directed to this new domain are discussed.

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Fibronectin/integrin interaction induces tyrosine phosphorylation of a 120-kDa protein.

Cell Regulation ◽

10.1091/mbc.2.11.951 ◽

1991 ◽

Vol 2 (11) ◽

pp. 951-964 ◽

Cited By ~ 353

Author(s):

J L Guan ◽

J E Trevithick ◽

R O Hynes

Keyword(s):

Tyrosine Phosphorylation ◽

Concanavalin A ◽

Cell Spreading ◽

Binding Domain ◽

Heparin Binding ◽

Cell Binding ◽

Heparin Binding Domain ◽

Coated Surfaces ◽

Cell Binding Domain ◽

In Cells

We describe a 120-kDa protein (pp120) that is phosphorylated on tyrosine in cells attached to fibronectin-coated surfaces. The protein appears to be located in focal contacts where it codistributes with beta 1 integrins. pp120 is distinct from the beta 1 subunit of integrins and from vinculin and alpha-actinin. pp120 is rapidly dephosphorylated in cells suspended by trypsinization but becomes rapidly phosphorylated in cells attaching and spreading on fibronectin. Attachment of cells to RGD-containing peptides, polylysine, or concanavalin A is not sufficient to induce phosphorylation of pp120. The 120-kDa cell-binding domain of fibronectin can induce some phosphorylation of pp120, but further phosphorylation occurs in the presence also of the heparin-binding domain of fibronectin. Phosphorylation of pp120 precedes, but is correlated with, subsequent cell spreading. Phosphorylation of pp120 can also be triggered by attachment of cells to anti-integrin antibodies, and this requires the cytoplasmic domain of the integrin beta 1 subunit. Thus interaction of beta 1 integrins with extracellular ligands (fibronectin or antibodies) triggers phosphorylation of an intracellular 120-kDa protein, pp120, that may be involved in the responses of cells to attachment.

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