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 s hown to bind to neutrophils, Based on studies using monoclonal antibodies ( mAbs), we postulated that CD11b/CD18 (Mac-1) might be the receptor on the n eutrophils for binding to HK/HKa, However, the direct interaction of HK/HKa and Mac-1 had not been demonstrated. We therefore transfected HEK 293 cell s with human Mac-1. Cell binding assays using fluorescein isothiocyanate-la beled HKa showed increased binding to the Mac-1 transfected cells compared with the control transfected cells. The binding was specific because unlabe led 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 cell s (20 000 molecules/cell) with a K-d = 62 nmol/L, To demonstrate directly t he formation of a complex between HKa and Mac-1, we examined the interactio n of HKa and purified Mac-1 in a cell-free system using an IAsys resonant m irror optical biosensor, The association and dissociation rate constants (k (on) and k(off), respectively) were determined, and they yielded a dissocia tion constant (K-d) of 3.2 x 10(-9) mol/L, The functional significance of d irect interaction of HKa to Mac-1 was investigated by examining the effect of HKa on cellular adhesion to fibrinogen and intercellular adhesion molecu le-1 (ICAM-1), molecules abundant in the injured vessel wall, HKa blocked t he adhesion of Mac-1 transfected cells to fibrinogen and ICAM-1 in a dose-d ependent 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. (C) 2000 by The American Society of Hematology.