Kininostatin Associates With Membrane Rafts and Inhibits αvβ3 Integrin Activation in Human Umbilical Vein Endothelial Cells

Abstract

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, ␣v␤3 integrin formed a complex with uPAR and caveolin-1, which was accompanied by an increase in ligand binding affinity of ␣v␤3 integrin. These events were inhibited by D5. Consistently, D5 suppressed specific ␣v␤3 integrin-mediated EC adhesion and spreading as well as small guanosine triphosphatase Rac1 activation. Key Words: kininogen Ⅲ membrane rafts Ⅲ integrin Ⅲ uPAR Ⅲ angiogenesis C aveolae are specialized components of membrane rafts, 1 which are plasmalemmal invaginations formed by the sequestration of cholesterol and glycosphingolipids with selfassociating molecules named caveolins. The major structural protein of endothelial caveolae is caveolin-1. Because the definitive identification of caveolae in experimental protocols can be only made by electron microscopy, we use the term membrane rafts in most of this paper and indicate when they contain caveolin. Caveolae are abundant on the surface of endothelial cells (ECs). 2 Caveolae and caveolin-1 are widely involved in the regulation of EC function, including EC proliferation, migration, and angiogenesis. Angiogenesis is the process of formation of new blood vessels from the existing microvessels, initiated by stimulation of vascular ECs with growth factors and cytokines. The functional significance of caveolae in angiogenesis has been suggested by defective nitric oxide-dependent angiogenesis in caveolin-1-null mice, 3 and by reduced tube formation in the absence of caveolin-1 in vivo and in vitro. 4 Integrins, particularly the ␣v integrin family, are major players in each step of angiogenesis. 5 Interaction of integrins with extracellular matrix (ECM) is required for EC function and maturation of newly formed blood vessels. ␣v integrins, highly expressed on activated ECs during angiogenesis, colocalize with caveolin-1 in membrane rafts. 6 Downstream effectors of integrins such as Src family kinases, and small guanosine triphosphatases are localized in membrane rafts and are involved in regulation of integrin activation as well as integrin-mediated cytoskeletal rearrangement. 7 Human plasma high molecular weight kininogen (HK) is a major component (660 nmol/L) of th...

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