Abstract
The leukocyte-restricted integrin CD11b/CD18 (alpha M beta 2, Mac-1) is a receptor for fibrinogen on stimulated monocytes and neutrophils. At variance with platelet alpha IIb beta 3 or endothelial cell alpha v beta 3 integrins, CD11b/CD18 interacts with a approximately 30-kDa plasmic fragment D (D30) of fibrinogen that lacks the Arg-Gly-Asp sequences in the A alpha chain and the carboxyl terminus of the gamma chain. Using epitope-mapped antibodies and synthetic peptidyl mimicry, we have now identified a unique linear sequence in fibrinogen that mediates ligand binding to CD11b/CD18. Anti-fibrinogen antibodies directed to the gamma chain region 95-264 inhibited 125I-fibrinogen or 125I-D30 binding to chemoattractant-stimulated neutrophils or monocytic THP-1 cells in a dose-dependent fashion. Partially overlapping synthetic peptides reproducing this gamma chain region were tested for their ability to inhibit fibrinogen binding to leukocytes. A synthetic peptide designated P1, duplicating gamma chain Gly190-Val202, inhibited 125I-fibrinogen binding to stimulated neutrophils or THP-1 cells and blocked adhesion of these cells to immobilized fibrinogen in a dose-dependent fashion. Increasing concentrations of P1 inhibited 125I-fibrinogen binding to isolated CD11b/CD18 in a cell-free system. Consistent with genuine peptidyl mimicry, 125I-P1 bound saturably to THP-1 cells in a reaction inhibited by molar excess of unlabeled peptide, fibrinogen, or D30. Finally, immobilized P1 effectively supported adhesion of THP-1 cells in a CD11b/CD18-dependent manner. These data suggest that the fibrinogen gamma chain region Gly190-Val202 functions as a minimal recognition sequence for the leukocyte integrin CD11b/CD18. Given the participation of fibrinogen:leukocyte interaction in inflammation and atherogenesis, antagonists based on this unique structural motif would effectively interfere with aberrant leukocyte adhesion mechanisms without affecting Arg-Gly-Asp-directed vascular integrins.
Highlights
The Structural Motif Glycine 190-Valine 202 of the Fibrinogen y Chain Interacts with CDllb/CD18 Integrin (a!Mp2,Mac-1) and Promotes Leukocyte Adhesion*
AII& or endothelial cell avo3integrins, CDllb/CD18 to form theheterodimersLFA-1(CDlla/CD18)M, ac-1 interacts with a -30-kDa plasmic fragment D (D30)of (CDllb/CD18), and p150,95 (CDllc/CD18) [3]
Using epitope-mapped antibodies and synthetic peptidyl mimicry, we have identified a unique linear sequence in fibrinogenthat mediates ligand binding to CDllb/CD18
Summary
95-264 anti-^'^-'^^) [18], inhibited the binding of1251-D30to terminus) blocked binding of lZ5I-fibrinogento fMLP-stimuchemoattractant (fMLP)-stimulated PMN in a dose-depend- lated THP-1cells in a dose-dependent manner (Fig. 2). 108.3 (Fig. 1).Both anti-y95-26a4ntibody and mAb 108.3 imental conditionsdescribed above, a control peptide syntheinhibited '251-fibrinogen binding to fMLP-stimulated PMN sized with a scrambled sequence(L-10-Y),or the L10 peptide, in a dose-dependent manner (not shown). Partiallyoverlapping synthetic below), none of the other peptides listed in Table I signifipeptides duplicating various aspects of this implicated region cantly reduced CDllb/CD18 interaction with fibrinogen or of the y chain (Table I) were initially tested for their ability D30when tested at concentrations ranging between 50 and t o inhibit binding of '251-fibrinogento stimulated THP-c1ells. In these experiments, increasing concentrations (3-300 p M ) In parallel experiments, increasing concentrations of P1 of P1 peptide (Gly'90-Va120w2ith Lys-Tyr residues at the NH, caused a dose-dependent inhibition (IC50= 70-80 p ~ of)the adhesion of fMLP-stimulatedTHP-1 cells to immobilized fibrinogen (Fig. 3).
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