fibrin and glycyl-arginyl-glycyl-aspartyl-seryl-proline

Upon conversion of fibrinogen into fibrin, fibrinogen αC-domains containing the RGD recognition motif form ordered αC polymers. Our previous study revealed that polymerisation of these domains promotes integrin-dependent adhesion and spreading of endothelial cells, as well as integrin-mediated activation of the FAK and ERK1/2 signalling pathways. The major goal of this study was to test the impact of αC-domain polymerisation on endothelial cell migration and proliferation during wound healing, and to clarify the mechanism underlying superior activity of αC polymers toward endothelial cells. In an in vitro wound healing assay, confluent endothelial cell monolayers on tissue culture plates coated with the αC monomer or αC polymers were wounded by scratching and wound closure was monitored by time-lapse videomicroscopy. Although the plates were coated with equal amounts of αC species, as confirmed by ELISA, wound closure by the cells occurred much faster on αC polymers, indicating that αC-domain polymerisation promotes cell migration and proliferation. In agreement, endothelial cell proliferation was also more efficient on αC polymers, as revealed by cell proliferation assay. Wound closure on both types of substrates was equally inhibited by the integrin-blocking GRGDSP peptide and a specific antagonist of the ERK1/2 signalling pathway. In contrast, blocking the FAK signaling pathway by a specific antagonist decreased wound closure only on αC polymers. These results indicate that polymerisation of the αC-domains enhances integrin-dependent endothelial cell migration and proliferation mainly through the FAK signalling pathway. Furthermore, clustering of integrin-binding RGD motifs in αC polymers is the major mechanism triggering these events.

Topics: Cell Movement; Cell Proliferation; Cells, Cultured; Extracellular Signal-Regulated MAP Kinases; Fibrin; Fibrinogen; Focal Adhesion Kinase 1; Human Umbilical Vein Endothelial Cells; Humans; Microscopy, Video; Oligopeptides; Peptide Fragments; Protein Interaction Domains and Motifs; Protein Kinase Inhibitors; Protein Multimerization; Protein Structure, Tertiary; Signal Transduction; Time Factors; Time-Lapse Imaging; Wound Healing

Fibroblast chemotaxis is implicated in many physiological processes, including wound healing and morphogenesis. We present a novel assay for chemotaxis of fibroblasts (and other slow-moving tissue cells) in a direct-viewing chamber containing a physiologically relevant three-dimensional fibrin or collagen gel in which long-lasting, spatially continuous gradients have been sustained for at least 24 h, long enough for significant fibroblast migration. This combination of features is not available in any alternative assay of comparable setup simplicity. Using a putative fibroblast chemotactic factor, the fibronectin peptide GRGDSP, we measured human foreskin fibroblast alignment in the direction along the gradient, which followed a biphasic dependence on GRGDSP concentration with an optimal concentration of about 10 nM. Time-lapse video microscopy revealed that cell migration was up the soluble GRGDSP gradient, confirming positive chemotaxis to GRGDSP and rejecting the possibility of dominant haptotaxis down the soluble GRGDSP gradient, that is, up a putative gradient of integrin-mediated adhesion induced by the soluble GRGDSP gradient.

Topics: Cell Line; Cells, Cultured; Chemotaxis; Collagen; Fibrin; Fibroblasts; Fibronectins; Gels; Humans; Oligopeptides

Platelet integrin alpha IIb beta 3 (GPIIb-IIIa) plays important roles in platelet-mediated clot retraction. However, little is known about the mechanisms of clot retraction mediated by nucleated cells. In this report, we demonstrate that another member of the beta 3 integrin family, alpha v beta 3, is involved in clot retraction mediated by nucleated cells. Retraction of fibrin clots was observed using a human melanoma cell line, C32TG, which contains no alpha IIb beta 3 complex. This retraction was inhibited by RGD-containing peptide, monoclonal anti-beta 3, and anti-alpha v beta 3 antibodies. Immunoelectron microscopic studies revealed a direct interaction between beta 3 integrin and fibrin fibers at an early stage of clot retraction. We found that another human embryonal cell line, 293, which is known to express alpha v beta 1, but no alpha v beta 3, lacks fibrin gel retractile activity. Upon transfection of beta 3 DNA into 293 cells, the beta 3 subunit formed a complex with an endogenous alpha v subunit. The beta 3-bearing transfectants were found to retract fibrin gels, which was specifically inhibited by anti-beta 3 antibody. In addition, a point mutation at Asp119 in the beta 3 ligand binding domain abolished the clot retractile activity of 293 transfectants, indicating the requirement of alpha v beta 3 ligand-binding activity. Our findings suggest that alpha v beta 3 is involved in mediating the interaction between the three-dimensional fibrin network and nucleated cells and in promoting "post-receptor occupancy" events.

Topics: Antibodies, Monoclonal; Blood Platelets; Cell Line; Clot Retraction; Embryo, Mammalian; Fibrin; Humans; Integrins; Melanoma; Microscopy, Electron; Oligopeptides; Platelet Glycoprotein GPIIb-IIIa Complex; Platelet Membrane Glycoproteins; Receptors, Cytoadhesin; Receptors, Vitronectin; Recombinant Proteins; Transfection; Tumor Cells, Cultured

Centrifuged human platelets bound soluble 125I-labelled fibrin, mediated by a plasma factor. Binding was inhibited by D-phenylalanyl-L-prolyl-L-arginyl- chloromethane (PPACK), which specifically blocks thrombin. As the binding-promoting principle was adsorbed to barium citrate, it was tentatively characterized as prothrombin, suggesting that it might be converted to thrombin at the cell surface. The peptide GRGDSP failed to inhibit binding, thus eliminating the glycoprotein IIb/IIIa complex as a receptor. Most likely, a thrombin - fibrin complex is recognized by a cell receptor, possibly protease-nexin I. In a platelet concentrate, the cells also internalized 125I-labelled fibrin, providing evidence that platelets are involved in the clearance of circulating fibrin - monomer complexes. Engulfment was again inhibited by PPACK or hirudin but not by an antibody against the glycoprotein IIb/IIIa complex.

Topics: Amino Acid Chloromethyl Ketones; Amino Acid Sequence; Blood Platelets; Endocytosis; Fibrin; Humans; Molecular Sequence Data; Oligopeptides; Prothrombin; Thrombin