Abstract
Monocytic cells bind fibrinogen (fg) through integrin alphaMbeta2. fg-bound monocytic cells demonstrate an enhanced adhesion to endothelial cells, which is dependent on intercellular adhesion molecule-1 (ICAM-1). Our studies differentiate fg interactions with stimulated and resting endothelial cells, which are ICAM-1 dependent and independent, respectively. This report documents a direct interaction between fg and intact ICAM-1 and with a two-Ig domain form of ICAM-1. A small region within the first Ig domain of ICAM-1, ICAM-1-(8-21) (KVILPRGGSVLVTC), was identified to interact with fg in a specific and selective manner. ICAM-1-(8-21) bound to plasmin-derived fg fragments X, D100, and D80 but not to fragment E. Consistent with this finding, fg gamma-chain peptide, fg-gamma-117-133, blocked fg interaction with ICAM-1-(8-2 1. ICAM-1-(8-21) peptide and antibodies directed against ICAM-1-(8-21) also blocked the adhesion and binding of ICAM-1-bearing Raji cells with fg. ICAM-1-(8-21) and fg-gamma-117-133 are likely to be one of the contact pairs mediating fg-ICAM-1 interactions.
Highlights
While fibrinogen1 is a plasma protein and intracellular adhesion molecule 1 (ICAM-1) is primarily a cell surface protein, both play central roles in cell-cell interactions [1, 2]. fg, a dimeric 340-kDa molecule, circulates in blood at 2–3 mg/ml
The extreme COOH-terminal aspect of the ␥ chain, ␥-406 – 411, is involved in the recognition of fg by ␣IIb3 [6]. fg interacts with other integrins, including ␣v3 and ␣M2 [7, 8]. ␣v3 is expressed on many cell types, including endothelial cells (EC). fg recognition by this receptor is inhib
ICAM-1-Fibrinogen Interactions in Endothelial Cell Adhesion—Previous studies have yielded conflicting results regarding the roles of the ␣v3 integrin and ICAM-1 in mediating fg binding with EC [1, 38]
Summary
Reagents and Antibodies—TNF-␣ and interleukin-1 were purchased from Genzyme (Boston, MA). Anti-VCAM-1 (E1/6) and anti-ICAM-1 monoclonal antibodies (mAbs) (LB-2) were purchased from Becton Dickinson (San Jose, CA). The molecular masses of fragments X, D100, D80, and E were 245, 100, 80, and 45 kDa, respectively, as estimated from their mobilities relative to protein standards on nonreducing 5–15% gradient SDSpolyacrylamide gel electrophoresis [33]. Cells were resuspended in a staining medium of HBSS containing 2.0 mM CaCl2, 2.0 mM MgCl2, 10 mM HEPES (pH 7.4), and 0.1% BSA and incubated at 4 °C for 30 min with 5.0 g/ml of control mouse IgG, anti-␣v3 mAb LM 609, anti-ICAM-1 mAbs QE2, or LB-2. Cells were centrifuged through a cushion of fetal calf serum and resuspended in staining medium containing 50 g/ml fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibodies (Zymed Laboratories, South San Francisco, CA). The final structure was reached by using the conjugate gradient method until the maximum deviations were Ͻ0.1 kcal/mol Å
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