Fibroblasts Spread on Immobilized Fibrin Monomer by Mobilizing a β1-class Integrin, Together with a Vitronectin Receptor αvβ3 on Their Surface

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 A␣ Arg-16 3 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 A␣-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 ␥ Met-89 at its amino terminus, which is located in close association with the RGD segment at A␣ 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 ␤ 1 subunit of integrin (mAb13), suggesting that the ␤ 1 -class integrin together with a vitronectin receptor, ␣ v ␤ 3 , is mobilized onto the surface of fibroblasts upon contact with the fibrin monomer monolayer.Fibrinogen is a 340-kDa glycoprotein consisting of three pairs of polypeptide subunits, A␣, B␤, and ␥, linked together by multiple disulfide bonds (1). By structural studies including electron microscopic analysis together with biochemical data, there is now general agreement on the shape of the fibrinogen molecule (2-7). The fibrinogen molecule is composed of three major globular domains, i.e. one central E domain and two identical outer D domains connected by a three chain ␣-helical coiled-coil (4, 6). The distal part of the D domain is the carboxyl terminus of the ␥-chain, while the proximal part is the carboxyl terminus of the B␤-chain. The carboxyl-terminal two thirds of the A␣-chains fold back from the D domain and form two independently folded domains (␣C domains) at their carboxylterminal parts. In the native fibrinogen molecule, the ␣C domains interact with each other and form an additional small globular (␣C-␣C) domain that is closely associated with the central E domain (4, 6, 7). Upon thrombin cleavage of fibrinopeptides A and B, the globular ␣C-␣C domain is released from the E domain, and subsequently dissociated into individual ␣C domains (4, 6, 7). The fibrinogen molecule thus undergoes distinct conformational changes upon conversion to the fibrin monomer molecule (4), and thereby exposes several fibrin-specific regions that may participate in the functions of fibrin. The Arg-Gly-Asp (RGD) segments residing at A␣ 95-97 and A␣ 572-574, tentatively designated as RGD-1 and RGD-2, respectively, may also be categorized into this type of fibrin-specific segments. In this paper, we describe the binding of cultured human and murine fibroblasts to immobilized fibrin monome...

Fibrin Sealants from Fresh or Fresh/Frozen Plasma as Scaffolds forIn VitroArticular Cartilage Regeneration

“…43 Staining for a V b 3 integrin (Fig. 7e, f), a fibrin-binding receptor, 44 was also detected. Chondrocytes within articular cartilage stain positively for a 1 b 1 but not for a V b 3 integrin (Fig.…”

Identification of a Novel Binding Site for Platelet Integrins αIIbβ3 (GPIIbIIIa) and α5β1 in the γC-domain of Fibrinogen

“…Previous studies have demonstrated that ␣ 5 ␤ 1 binds ligands mainly through the RGD-dependent specificity (45), and the RGD-inhibitable binding of Fg to ␣ 5 ␤ 1 on endothelial cells has been shown (46). ␣ 5 ␤ 1 on other cells has also been reported to bind Fg (47)(48)(49). However, the role of ␣ 5 ␤ 1 in platelet adhesive reactions with Fg is not clear, since this integrin is present on the platelet surface at a considerably lower density than ␣ IIb ␤ 3 .…”

Multimeric vitronectin in ascites is involved in fibroblast spreading of EPS in patients on CAPD therapy

“…16 Briefly, microtiter plates (96-well) were coated with 50 µl of various concentrations (A280 ϭ 0.2) of ascites from the patients with EPS (EPS ascites), and the patient with minimal change nephrotic syndrome, in 50 mM Tris-HCl (pH 7.4), containing 0.135 M NaCl (TBS) at 4°C overnight. After the wells had been washed with TBS, followed by blocking with TBS containing 3% BSA (3% BSA-TBS), 5 ϫ 10 3 human fibroblast cells (TIG-3) were pipetted into the coated wells.…”