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Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 3788-3795
Cleaved high molecular weight kininogen binds directly to the
integrin CD11b/CD18 (Mac-1) and blocks adhesion to fibrinogen and
ICAM-1
Nijing Sheng,
Michael B. Fairbanks,
Robert L. Heinrikson,
Gabriela Canziani,
Irwin M. Chaiken,
David M. Mosser,
Hong Zhang, and
Robert W. Colman
From the Sol Sherry Thrombosis Research Center and the Department of
Microbiology and Immunology, Temple University School of Medicine, and
the Department of Medicine, University of Pennsylvania School of
Medicine, Philadelphia, PA; and Pharmacia & Upjohn Inc, Kalamazoo, MI.
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 Kd = 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 (kon and
koff, respectively) were determined, and they yielded a
dissociation constant (Kd) of 3.2×10 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.
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