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-A(121) 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 VEGFA(165) and VEGF-A(121), but binding kinetics of the other two pro- and all anti-angiogenic splice variants are not known. We measured the binding kinetics for VEGF-A(165), -A(165b), and -A(121) 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-A(121):VEGFR2 as K(D) = 0.66 nM. We found that VEGF-A(121) 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.