The alternatively spliced αEC domain of human fibrinogen-420 is a novel ligand for leukocyte integrins αMβ2 and αXβ2

Abstract

The interaction of human plasma fibrinogen with leukocyte integrins ␣ M ␤ 2 (CD11b/ CD18, Mac-1) and ␣ X ␤ 2 (CD11c/CD18, p150,95) is an important component of the inflammatory response. Previously, it was demonstrated that binding of fibrinogen to these integrins is mediated by ␥C, the globular C-terminal domain of the ␥ chain. In this study, evidence was found of another fibrinogen domain that can serve as a ligand for the 2 leukocyte integrins: ␣ E C, a homologous domain that extends the ␣ chains in a recently discovered subclass of fibrinogen known as fibrinogen-420. Recombinant ␣ E C supported strong adhesion and migration of cells expressing ␣ M ␤ 2 and ␣ X ␤ 2 , including nonactivated and activated U937 and THP-1 monocytoid cells, and neutrophils. Cells transfected with complementary DNA for these integrins also bound ␣ E C. The specificity of interaction was substantiated by inhibition of cell adhesion with antibodies against ␣ M , ␣ X , and ␤ 2 subunits. Also, neutrophil inhibitory factor, a specific inhibitor of ␣ M ␤ 2 and ␣ X ␤ 2 function, efficiently blocked cell adhesion to ␣ E C. In ␣ M ␤ 2 and ␣ X ␤ 2 , the I domain is the binding site for ␣ E C, since ␣ E C bound to recombinant ␣ M I and ␣ X I domains in a dose-dependent and saturable manner. Synthetic peptides that duplicated sequences ␥190 to 202 and ␥377 to 395, previously considered putative binding sites in ␥C, effectively inhibited ␣ M ␤ 2 -and ␣ X ␤ 2 -mediated adhesion to ␣ E C, suggesting that recognition of ␣ E C by the I domain involves structural features in common with those of ␥C. These findings identify ␣ E C as a second domain in fibrino IntroductionIn addition to its function in the coagulation and hemostatic systems, plasma protein fibrinogen (Fg) also participates in inflammatory responses. This function of Fg depends on its ability to interact with leukocyte receptors. On inflammatory challenge, Fg mediates adhesive and migratory reactions of leukocytes, such as leukocyte attachment to the vessel wall and subsequent transmigration through the endothelium into a subendothelial matrix. [1][2][3] In addition, fibrinogen and fibrin deposited at sites of vascular injury and within tissues 4,5 promote accumulation of inflammatory cells. 6,7 The accessory role of Fg in inflammation was documented by in vivo studies in which congenital afibrinogenemia in patients or experimental depletion of Fg from the circulation in animals altered the manifestation of inflammatory responses. [8][9][10][11][12] The interaction of leukocytes with Fg is mediated by 2 transmembrane receptors that belong to the integrin gene superfamily: ␣ M ␤ 2 (CD11b/CD18, Mac-1) and ␣ X ␤ 2 (CD11c/CD18, p150,95). Abundantly expressed on monocytes and neutrophils and induced on lymphocytes during their activation, integrin ␣ M ␤ 2 is primarily responsible for leukocyte adhesion to Fg. 13 Studies in mice deficient in ␣ M ␤ 2 demonstrated that fibrinogen-dependent inflammatory reactions were significantly curtailed in these animals. 14 Specifically, the mice failed to...

Multiple Binding Sites in Fibrinogen for Integrin αMβ2 (Mac-1)

“…In RLTYAYFAG (Fig. 6A, spot 8), mutation of Arg to Glu reduced binding (spot 9) and additional mutations of Leu and Tyr created the completely inactive EATAAYFAG peptide (spots [11][12][13][14][15][16]. Taken together, these analyses identified several new ␣ M I-domain recognition sequences in ␥C and ␤C and provided preliminary insights into the ␣ M I-domain binding preferences.…”

“…Second, deletion of P2 in ␥C decreased the ␣ M I-domain binding by only ϳ50% suggesting that one or more alternative sites participate in recognition (10). Third, the ␤C and ␣ E C domains of Fg, which share with ␥C only 39 and 40% sequence identity, respectively, and do not contain P2, bind the ␣ M I-domain (10,11). This further suggests that ␤C and ␣ E C also contain ␣ M I-domain recognition sites.…”

“…Fourth, although the P2 sequence accounts for the partial ␣ M I-domain binding by ␥C, the P2 peptide inhibits ␣ M ␤ 2 -mediated cell adhesion to immobilized ␥C almost completely. It also efficiently inhibits cell adhesion to Fg and its fragments D and ␣ E C (6,11). Taken together, these data imply that Fg binding by ␣ M ␤ 2 cannot be explained by a simple model in which P2 is the single site in Fg and prompt us to reconsider the molecular basis for ␣ M ␤ 2 -Fg recognition.…”

Integrin αDβ2, an adhesion receptor up-regulated on macrophage foam cells, exhibits multiligand-binding properties

“…Further evidence for the similarity of ligand specificity of integrins ␣ D ␤ 2 and ␣ M ␤ 2 was obtained in inhibition experiments using soluble peptide P2-C. We previously reported that P2-C inhibits ␣ M ␤ 2 -mediated cell adhesion not only to the ␥C domain of fibrinogen, from which this sequence is derived, but also to other fibrinogen domains 32 and to many unrelated proteins (Valeryi Lishko, unpublished data, June 2002). 27,33 P2-C was also an effective inhibitor of ␣ D ␤ 2 -mediated cell adhesion to vitronectin, fibrinogen, plasminogen, and VCAM-1 (Table 1).…”

Fibrinogen splice variation and cross-linking: Effects on fibrin structure/function and role of fibrinogen γ′ as thrombomobulin II

“…Its structure has been elucidated through crystallography [6], but its functional characteristics have been poorly described. The EC region is susceptible to early proteolysis by plasmin [7] and can act as a ligand for neutrophils and monocytes via  M  2 and  X  2 integrins [8]. Fibrinogen EC has been shown to produce clots with thinner, more branched fibres with increased maximum amplitude in thromboelastometry, which is an indication of increased clot stiffness [9].…”