Gengrinovitch S, Greenberg S M, Cohen T, Gitay-Goren H, Rockwell P, Maione T E, Levi B Z, Neufeld G
Department of Biology, Technion, Israel Institute of Technology, Haifa.
J Biol Chem. 1995 Jun 23;270(25):15059-65. doi: 10.1074/jbc.270.25.15059.
The 121-amino acid form of vascular endothelial growth factor (VEGF121) and the 165-amino acid form (VEGF165) are mitogenic for vascular endothelial cells and induce angiogenesis in vivo. VEGF165 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 125I-VEGF165 to the VEGF receptors of endothelial cells, and the heparin-dependent binding of 125I-VEGF165 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 VEGF121, 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 VEGF165 binding to VEGF receptors by disrupting the interaction of VEGF165 with cell surface heparan sulfates. Since PF4 mutants lacking a heparin binding ability retain their anti-angiogenic activity, alternative inhibitory mechanisms were also examined. 125I-PF4 bound with high affinity (Kd 5 x 10(-9) M) to VEGF165-coated wells. The binding of 125I-PF4 to the VEGF165-coated wells was inhibited by several types of heparin binding proteins, including unlabeled PF4 and unlabeled VEGF165. The binding was not inhibited by proteins which lack a heparin binding capacity, nor was it inhibited by VEGF121. Heparinase did not inhibit the binding of 125I-PF4 to VEGF165, indicating that heparin-like molecules are not required. These experiments suggest that PF4 can bind to heparin binding proteins such as VEGF165 leading to an inhibition of their receptor binding ability. In agreement with these results, we have observed that PF4 inhibits efficiently the VEGF165 induced proliferation of vascular endothelial cells. Unexpectedly, PF4 also inhibited efficiently the VEGF121-induced proliferation of the cells, indicating that PF4 can disrupt VEGF receptor mediated signal transduction using an unknown mechanism which does not interfere with VEGF121 binding.
血管内皮生长因子的121个氨基酸形式（VEGF121）和165个氨基酸形式（VEGF165）对血管内皮细胞具有促有丝分裂作用，并在体内诱导血管生成。VEGF165具有肝素结合能力，在缺乏类肝素分子的情况下，不能有效地与血管内皮细胞的VEGF受体结合。血管生成抑制剂血小板因子-4（PF4）可抑制125I-VEGF165与内皮细胞VEGF受体的结合，以及125I-VEGF165与VEGF受体KDR/flk-1可溶性细胞外结构域的肝素依赖性结合。相反，PF4不能抑制VEGF121（一种缺乏肝素结合能力的VEGF异构体）与细胞VEGF受体或KDR/flk-1的结合。这些结果表明，PF4可能通过破坏VEGF165与细胞表面硫酸乙酰肝素的相互作用来抑制VEGF165与VEGF受体的结合。由于缺乏肝素结合能力的PF4突变体保留了它们的抗血管生成活性，因此也研究了其他抑制机制。125I-PF4以高亲和力（Kd 5×10(-9) M）与包被VEGF165的孔结合。125I-PF4与包被VEGF165的孔的结合受到几种肝素结合蛋白的抑制，包括未标记的PF4和未标记的VEGF165。缺乏肝素结合能力的蛋白质不能抑制这种结合，VEGF121也不能抑制。肝素酶不能抑制125I-PF4与VEGF165的结合，表明不需要类肝素分子。这些实验表明，PF4可以与肝素结合蛋白如VEGF165结合，导致其受体结合能力受到抑制。与这些结果一致，我们观察到PF4能有效抑制VEGF165诱导的血管内皮细胞增殖。出乎意料的是，PF4也能有效抑制VEGF121诱导的细胞增殖，这表明PF4可以通过一种未知机制破坏VEGF受体介导的信号转导，而这种机制不会干扰VEGF121的结合。
