VEGF(165) is a key regulator of angiogenesis and a potent vascular permeability factor. Using snake venom proteins as tools, we demonstrate the enhanced vascular leakage of VFGF by KDR-binding proteins. The snake venom-derived KDR-specific VEGF, vammin, potently enhanced vascular leakage compared with other known permeability-enhancing factors including VEGF(165), while KDR-bp from snake venom, a KDR antagonist of enclothelial cell growth was a very weak permeability enhancer. Unexpectedly when co-administrated, KDR-bp synergistically enhanced either vammin or VEGF(165)-stimulated vascular leakage, despite its antagonistic effect on cell growth. This augmenting effect was specifically observed in the combined administration of KDR-bp with either VEGF(165) or vammin, but not other combination of known permeability-enhancing factors. We further demonstrated a similar increased vascular leakage by the combined administration of VEGF(165) and TIM-3, the only known endogenous antagonist of KDR. Our findings implicate TIMP-3 as a critical player in the vascular leakage-enhancing effect of VEGF(165) in vivo. (c) 2007 Elsevier Inc. All rights reserved.