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

Abstract

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. 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, alpha(v)beta3 integrin formed a complex with uPAR and caveolin-1, which was accompanied by an increase in ligand binding affinity of alpha(v)beta3 integrin. These events were inhibited by D5. Consistently, D5 suppressed specific alpha(v)beta3 integrin-mediated EC adhesion and spreading as well as small guanosine triphosphatase Rac1 activation. D5 binds to ECs via membrane rafts and downregulates alpha(v)beta3 integrin bidirectional signaling and the downstream Rac1 activation pathway.