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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:1968.)
© 2007 American Heart Association, Inc.

Vascular Biology

Kininostatin Associates With Membrane Rafts and Inhibits vβ3 Integrin Activation in Human Umbilical Vein Endothelial Cells

Yi Wu; Victor Rizzo; Yuchuan Liu; Irma M. Sainz; Noah G. Schmuckler; Robert W. Colman

From The Sol Sherry Thrombosis Research Center (Y.W., Y.L., I.M.S., N.G.S., R.W.C.) and the Cardiovascular Research Center and Department of Anatomy and Cell Biology (V.R.), Temple University School of Medicine, Philadelphia, Pa.

Correspondence to Robert W. Colman, MD, or Yi Wu, MD, PhD, The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, 3400 N Broad Street, OMS 418, Philadelphia, PA 19140. E-mail colmanr{at}temple.edu; or yiwu@temple.edu

Objective— The cleaved form of high molecular weight kininogen (HKa) is a potent inhibitor of angiogenesis and tumor growth in vivo; the functional domain has been identified as domain 5 (D5, named as kininostatin). We now identify the subcellular targeting site for D5 on endothelial cells (ECs), and investigate D5 inhibition of integrin functions.

Methods and Results— Endothelial membrane rafts were isolated using sucrose density gradient centrifugation. D5, bound to ECs, was predominantly associated with membrane rafts, in which uPAR, a HKa receptor, was also localized. In contrast, other HKa receptors, cytokeratin-1 and gC1q receptor, were not detected in membrane rafts. Colocalization of D5 with caveolin-1 was demonstrated on ECs by confocal microscopy. Disruption of membrane rafts by cholesterol removal decreased D5 binding to ECs. On stimulation with vascular endothelial growth factor, vβ3 integrin formed a complex with uPAR and caveolin-1, which was accompanied by an increase in ligand binding affinity of vβ3 integrin. These events were inhibited by D5. Consistently, D5 suppressed specific vβ3 integrin-mediated EC adhesion and spreading as well as small guanosine triphosphatase Rac1 activation.

Conclusions— D5 binds to ECs via membrane rafts and downregulates vβ3 integrin bidirectional signaling and the downstream Rac1 activation pathway.

The cleaved form of high molecular weight kininogen and its functional domain (D5) are potent inhibitors of angiogenesis. This study indicates that D5 binding to endothelial cells depends on membrane rafts, by which it inhibits vβ3 integrin bi-directional signaling and downstream Rac1 activation.

Key Words: kininogen • membrane rafts • integrin • uPAR • angiogenesis

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