PLATELET FACTOR-IV INHIBITS THE MITOGENIC ACTIVITY OF VEGF(121) AND VEGF(165) USING SEVERAL CONCURRENT MECHANISMS

The 121-amino acid form of vascular endothelial growth factor (VEGF(121)) and the 165-amino acid form (VEGF(165)) are mitogenic for vascular endothelial cells and induce angiogenesis in vivo. VEGF(165) possesses a heparin binding ability and in the absence of heparin-like molecules does not bind efficiently to the VEGF receptors of vascular endothelial cells, The binding of I-125-VEGF(165) to the VEGF receptors of endothelial cells, and the heparin dependent binding of I-125-VEGF(165) to a soluble extracellular domain of the VEGF receptor KDR/flk-1, were inhibited by the angiogenesis inhibitor platelet factor-4 (PF4). In contrast, PF4 was not able to inhibit the binding of VEGF(121), a VEGF isoform which lacks a heparin binding capacity, to the VEGF receptors of the cells or to KDR/flk-1. These results indicate that PF4 may inhibit VEGF(165) binding to VEGF receptors by disrupting the interaction of VEGF(165) with cell surface heparan sulfates, Since PF4 mutants lacking a heparin binding ability retain their anti-angiogenic activity, alternative inhibitory mechanisms were also examined. I-125-PF4 bound with high affinity (K-d 5 x 10(-9) M) to VEGF(165)-coated wells, The binding of I-125-PF4 to the VEGF(165)-coated wells was inhibited by several types of heparin binding proteins, including unlabeled PF4 and unlabeled VEGF(165). The binding was not inhibited by proteins which lack a heparin binding capacity, nor was it inhibited by VEGF(121). Heparinase did not inhibit the binding of I-125-PF4 to VEGF(165), indicating that heparin-like molecules are not required. These experiments suggest that PF4 can bind to heparin binding proteins such as VEGF(165) leading to an inhibition of their receptor binding ability, In agreement with these results, we have observed that PF4 inhibits efficiently the VEGF(165) induced proliferation of vascular endothelial cells. Unexpectedly, PF4 also inhibited efficiently the VEGF(121)-induced proliferation of the cells, indicating that PF4 can disrupt VEGF receptor mediated signal transduction using an unknown mechanism which does not interfere with VEGF(121) binding.