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Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes
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Cleaved high molecular weight kininogen binds directly to the integrin CD11b/CD18 (Mac-1) and blocks adhesion to

N Sheng1, M B Fairbanks, R L Heinrikson

  • 1Sol 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

Blood
|June 14, 2000
PubMed

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)

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