Cleaved high molecular weight kininogen binds directly to the integrin CD11b/CD18 (Mac-1) and blocks adhesion to fibrinogen and ICAM-1.
High molecular weight kininogen (HK) and its cleaved form (HKa) have been shown to bind to neutrophils. Based on studies using monoclonal antibodies (mAbs), we postulated that CD11b/CD18 (Mac-1) might be the receptor on the neutrophils for binding to HK/HKa. However, the direct interaction of HK/HKa and Mac-1 had not been demonstrated. We therefore transfected HEK 293 cells with human Mac-1. Cell binding assays using fluorescein isothiocyanate-labeled HKa showed increased binding to the Mac-1 transfected cells compared with the control transfected cells. The binding was specific because unlabeled HKa, Mac-1-specific antibody, and fibrinogen can inhibit the binding of biotin-HKa to Mac-1 transfected cells. HKa bound to Mac-1 transfected cells (20 000 molecules/cell) with a K(d) = 62 nmol/L. To demonstrate directly the formation of a complex between HKa and Mac-1, we examined the interaction of HKa and purified Mac-1 in a cell-free system using an IAsys resonant mirror optical biosensor. The association and dissociation rate constants (k(on) and k(off), respectively) were determined, and they yielded a dissociation constant (K(d)) of 3.2x10(-9) mol/L. The functional significance of direct interaction of HKa to Mac-1 was investigated by examining the effect of HKa on cellular adhesion to fibrinogen and intercellular adhesion molecule-1 (ICAM-1), molecules abundant in the injured vessel wall. HKa blocked the adhesion of Mac-1 transfected cells to fibrinogen and ICAM-1 in a dose-dependent manner. Thus, HKa may interrupt Mac-1-mediated cell-extracellular matrix and cell-cell adhesive interactions and may therefore influence the recruitment of circulating neutrophils/monocytes to sites of vessel injury. (Blood. 2000;95:3788-3795)
Sheng
N
N
Sol Sherry Thrombosis Research Center and the Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA 19140, USA. nsheng@thunder.ocis.temple.edu
Fairbanks
M B
MB
Heinrikson
R L
RL
Canziani
G
G
Chaiken
I M
IM
Mosser
D M
DM
Zhang
H
H
Colman
R W
RW
D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D005340:Fibrinogen / Q000502:physiology*; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000502:physiology*; D007668:Kidney; D007700:Kinetics; D007704:Kininogens / Q000737:chemistry / Q000494:pharmacology*; D016177:Macrophage-1 Antigen / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D014162:Transfection
D007704:Kininogens; D016177:Macrophage-1 Antigen; D011994:Recombinant Proteins; D018799:Intercellular Adhesion Molecule-1; D005340:Fibrinogen; D016650:Fluorescein-5-isothiocyanate