VEGF-A splice variants bind VEGFRs with differential affinities

Vascular endothelial growth factor A (VEGF-A) and its binding to VEGFRs is an important angiogenesis regulator, especially the earliest-known isoform, VEGF-A_165a. Yet several additional splice variants play prominent roles in regulating angiogenesis in health and in vascular disease, including VEGF-A₁₂₁ and an anti-angiogenic variant, VEGF-A_165b. Few studies have attempted to distinguish these forms from their angiogenic counterparts, experimentally. Previous studies of VEGF-A:VEGFR binding have measured binding kinetics for VEGFA₁₆₅ and VEGF-A₁₂₁, but binding kinetics of the other two pro- and all anti-angiogenic splice variants are not known. We measured the binding kinetics for VEGF-A₁₆₅, -A_165b, and -A₁₂₁ with VEGFR1 and VEGF-R2 using surface plasmon resonance. We validated our methods by reproducing the known affinities between VEGF-A_165a:VEGFR1 and VEGF-A_165a:VEGFR2, 1.0 pM and 10 pM respectively, and validated the known affinity VEGF-A₁₂₁:VEGFR2 as K_D = 0.66 nM. We found that VEGF-A₁₂₁ also binds VEGFR1 with an affinity K_D = 3.7 nM. We further demonstrated that the anti-angiogenic variant, VEGF-A_165b selectively prefers VEGFR2 binding at an affinity = 0.67 pM while binding VEGFR1 with a weaker affinity-K_D = 1.4 nM. These results suggest that the - A_165b anti-angiogenic variant would preferentially bind VEGFR2. These discoveries offer a new paradigm for understanding VEGF-A, while further stressing the need to take care in differentiating the splice variants in all future VEGF-A studies.