The Role of Putative Fibrinogen Aα-, Bβ-, and γA-chain Integrin Binding Sites in Endothelial Cell-mediated Clot Retraction

Abstract

In this study, endothelial cell-mediated clot retraction was supported by fibrin generated from several purified fractions of plasma fibrinogen, purified proteolytic fragments of plasma fibrinogen, recombinant normal fibrinogen, and recombinant variant fibrinogen. These results were surprising because some of these fibrinogens lack domains that are known binding sites for the integrin receptors that support clot retraction. Specifically, fibrinogens lacking A␣-chain RGD residues at 572-574 or lacking the ␥-chain residues AGDV 408 -411 supported endothelial cell-mediated clot retraction as well as intact fibrinogen. Thus, clot retraction mediated by endothelial cells is not dependent on either of these sites. A variety of monoclonal antibodies against the integrin ␣ v ␤ 3 partially inhibited the endothelial cell-mediated retraction of clots formed from plasma fibrinogen. As expected, an antibody to the platelet integrin ␣ IIb ␤ 3 did not inhibit endothelial cell-mediated clot retraction. These results indicate that this retraction is mediated at least in part by ␣ v ␤ 3 . These results support the conclusion that (a) neither of the two fibrinogen cell binding sites described above is required to support clot retraction or that (b) either site alone or in conjunction with other fibrin(ogen) region(s) can support clot retraction. Thus, endothelial cell-mediated clot retraction appears to be dependent on fibrinogen cell binding sites other than those required to support adhesion of resting platelets to immobilized fibrinogen and platelet aggregation.This study was undertaken to evaluate the role in clot retraction mediated by endothelial cells of presumptive endothelial cell and platelet binding sites on fibrinogen. Platelets, fibroblasts, melanoma cells, and endothelial cells are known to support clot retraction (1-4). However, it is not known whether clot retraction mediated by endothelial cells is dependent on either the presumptive endothelial cell fibrinogen A␣-chain binding site, the 572-574 RGD residues (5), or the fibrinogen ␥A-chain carboxyl-terminal AGDV sequence as is resting platelet adhesion to immobilized fibrinogen (6, 7) and platelet aggregation (8, 9).Clot retraction is dependent on fibrin binding to activated ␣ IIb ␤ 3 in platelets (10,11) or to the homologous integrin ␣ v ␤ 3 (12, 13) in nucleated cells (3,4). Katagiri et al. (3) used monoclonal antibodies and immunoelectron microscopy to show that clot retraction mediated by melanoma cells is dependent on fibrin binding to unstimulated ␣ v ␤ 3 . In their study, clot retraction mediated by melanoma cells was blocked by RGD-containing peptides and anti-␤ 3 as well as anti-␣ v ␤ 3 mAbs 1 but not by an ␣ IIb ␤ 3 -specific inhibitor. The conclusion that ␣ v ␤ 3 can support clot retraction mediated by nucleated cells was confirmed by Chen et al. (4). Alemany et al. (14) provided evidence that a fibrinogen ␥A-chain binding region of the platelet integrin ␣ IIb ␤ 3 is on its ␤ 3 subunit and that ligand binding to this site is independent of platelet ac...

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

“…The mAb inhibited clot retraction in a dose-dependent manner; 50% inhibition was attained at ϳ15 g/ml mAb, and 50 g/ml produced complete inhibition. The potency of mAb 2G5 was similar to that of Fab 7E3, which binds platelet integrins ␣ IIb ␤ 3 and ␣ V ␤ 3 and inhibits clot retraction (10,25,26). In addition, the effect of mAb 2G5 was similar to that of mAb 4A5 (IC 50 ϳ10 g/ml) directed against the binding site for ␣ IIb ␤ 3 at ␥408 -411 (21), which inhibits platelet adhesion (27) and clot retraction (26).…”

Unique Pathway of Thrombin-induced Platelet Aggregation Mediated by Glycoprotein Ib

“…3A (b)). A recombinant mutant fibrinogen (␥407), lacking the AGDV sequence in the ␥-chain required for GP IIb/IIIa-fibrinogen interactions (40,41), still supported ␣-thrombin-induced platelet aggregation with normal kinetics, as did recombinant wild type fibrinogen (data not shown). Aggregation via the PAR-1 pathway was again initially blocked by the addition of RGDS when ␥407 replaced normal fibrinogen, until ␥407 presumably began to polymerize (Fig.…”

Role of β1 and β3 Integrins in Human Smooth Muscle Cell Adhesion to and Contraction of Fibrin Clots In Vitro

“…Thus, we need to consider the possible sites of fibrin that may be binding sites for integrins. It should be noted that Suehiro et al 27 determined that endothelial cells bind the C terminal RGD site on the ␣ chain of fibrinogen, while Smith et al 64 demonstrated that this RGD site is not required to support endothelial cell-mediated clot retraction.…”