Abstract
Human and murine fibroblasts were found to spread far more avidly on fibrin monomer monolayers than on immobilized fibrinogen, indicating that removal of fibrinopeptides by thrombin is a prerequisite for the fibrin-mediated augmentation of cell spreading. In fact, cell spreading was not efficiently augmented on monolayers of a thrombin-treated dysfibrinogen lacking the release of fibrinopeptide A due to an Aalpha Arg-16 --> Cys substitution. Since a synthetic Arg-Gly-Asp (RGD)-containing peptide inhibited the fibrin-mediated cell spreading, subsequent dissociation of the carboxyl-terminal globular domain of the Aalpha-chains appears to render the RGD segments accessible to the cell-surface integrins. In support of this, fibrin-augmented cell spreading was inhibited by an antibody recognizing a 12-kDa peptide segment with gamma Met-89 at its amino terminus, which is located in close association with the RGD segment at Aalpha 95-97 in the helical coiled-coil interdomainal connector. The fibrin-mediated augmentation of cell spreading was inhibited not only by an antibody against human vitronectin receptor (LM 609) but also by an antibody against the beta1 subunit of integrin (mAb13), suggesting that the beta1-class integrin together with a vitronectin receptor, alphavbeta3, is mobilized onto the surface of fibroblasts upon contact with the fibrin monomer monolayer.
Highlights
Fibrinogen is a 340-kDa glycoprotein consisting of three pairs of polypeptide subunits, A␣, B, and ␥, linked together by multiple disulfide bonds [1]
We describe the binding of cultured human and murine fibroblasts to immobilized fibrin monomer, but not to immobilized fibrinogen, by focusing on the conformational changes induced in the fibrinogen molecule upon its conversion to fibrin
Coating Efficiency of Fibrin Monomer and Fibrinogen—Microtiter wells (96-well) were coated with 50 l of various concentrations of fibrin monomer and fibrinogen for 4 h at 22 °C, and the wells were washed with TBS followed by blocking with TBS containing 1% Bovine serum albumin (BSA)
Summary
Materials—All chemical reagents were of the highest analytical grade commercially available and were purchased from the sources shown below. Fibrinogen fractions prepared were found to be Ͼ96% pure as determined by SDSPAGE They were found to contain neither von Willebrand factor nor fibronectin as determined by an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies directed against respective proteins. 2-cm well (24-well Costar plates) polystyrene plates were coated with various concentrations of fibrinogen, fibrin monomer, fragment X, XDP, or heat-denatured BSA in TBS containing a synthetic antipolymerant GPRP peptide at 1 mM. Adhesion Inhibition Assay—After incubation of fibrin monomer, fibrinogen, fragments D, E, X, and XDP with DMEM and 0.2% BSA containing 0.5 mM GPRP peptide, cells were mixed with these solutions, and were added to the wells coated with fibrinogen or fibrin monomer. All experiments were performed at least three times with two independent isolates
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