Solution Structure and Interaction with Basic and Acidic Fibroblast Growth Factor of a 3-kDa Human Platelet Factor-4 Fragment with Antiangiogenic Activity

Abstract

Platelet factor-4 is a protein belonging to the family of ELR-negative CXC chemokines which binds to fibroblast growth factor and inhibits its mitogenic activity. Platelet factor-4 also inhibits tumor growth by mechanisms involving antiangiogenesis. Antiangiogenic activity in vitro has also been shown for the 24-residue Cterminal fragment of the protein, which decreases the affinity between basic fibroblast growth factor and its cell-surface receptor. In this study, the preferential conformation of this fragment in solution has been determined and has been found to be composed of two helical subdomains. In addition, we show that the fragment forms a specific 1:1 complex with acidic and basic fibroblast growth factors and that both subdomains are probably required for inhibition of fibroblast growth factordriven mitogenesis. Finally, we show that the binding of the fragment alters the structure of the fibroblast growth factors, although some of such alterations do not seem related with the inhibition of mitogenic activity. Since this fragment has recently been shown to inhibit fibroblast growth factor-induced angiogenesis in vivo when injected intraperitoneally, these results are relevant for developing new antiangiogenic treatments.Angiogenesis plays an important role during embryonic development and in many pathologies including cancer and cardiovascular and inflammatory diseases. Fibroblast growth factors (FGFs) 1 and vascular endothelial growth factor (VEGF) family members constitute the major class of angiogenic regulators. These factors induce endothelial cell proliferation, migration, and angiogenesis in vitro and in vivo (1, 2). FGFs and VEGFs induce specific cellular responses by binding selectively to cell-surface receptors. The FGF family is presently known to include 22 closely related proteins. Acidic fibroblast growth factor (aFGF) and the subsequently discovered basic fibroblast growth factor (bFGF) are considered to be representative of the whole family (3-5). FGFs bind to two receptor classes with high and low affinity at the cell surface. The high affinity receptors (FGFR) belong to the class of the transmembrane receptor tyrosine kinases (6). FGFRs are widely distributed in all cells of mesodermal and neuroectodermal origin studied thus far, which accounts for the wide spectrum of in vitro mitogenic targets of this protein family (7,8). The low affinity receptor of FGFs is the glycosidic moiety of the cell coat and basal membranes (9). Angiogenesis promoted by VEGF has been reported to be dependent on the endogenous expression of bFGF by endothelial cells and is therefore blocked by antibodies that neutralize to bFGF (10, 11). Consequently, there is considerable pharmacological interest in developing inhibitors with the mitogenic activity of FGFs. Platelet factor-4 (PF4) is a CXC chemokine with high affinity for sulfated glycosaminoglycans like heparin and heparan sulfate. PF4 inhibits endothelial cell proliferation, migration, and angiogenesis in vitro and in vivo (12)(13)(14). Furthermore, th...

Leads for Development of New Naphthalenesulfonate Derivatives with Enhanced Antiangiogenic Activity

“…Because proteins often undergo conformational changes when they bind to other molecules or macromolecular structures, the decrease in flexibility of the ␤8/␤9 loop of aFGF upon 5-amino-2-NMS binding should probably be considered an additional unfavorable contribution of 5-amino-2-NMS to the conformational fitting of aFGF to its receptor binding site. Slight changes on this loop have been shown to deeply affect the specific mitogenic activity of aFGF (52). Hindered heparin binding and poor fitting onto its receptor may both account for the low mitogenesis inducing capacity of aFGF complexed with 5-amino-2-NMS.…”

Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis

“…PF4 binds FGF1 [171] and FGF2 [170]. In endothelial cells, PF4 inhibits FGF2 interaction with HSPGs and FGFR1, FGF2 internalization and mitogenic activity [170].…”

“…Since then, 22 structurally-related members of the FGF family have been identified [8]. FGFs are pleiotropic factors acting on different cell types, including www.elsevier.com/locate/cytogfr Cytokine & Growth Factor Reviews 16 (2005) [159][160][161][162][163][164][165][166][167][168][169][170][171][172][173][174][175][176][177][178] endothelial cells, following interaction with heparan-sulfate proteoglycans (HSPGs) and tyrosine kinase FGF receptors (FGFRs). To date, more than 1200 PubMed-referenced papers related to FGFs and FGFRs in endothelial cells and during neovascularization have been published.…”

The COOH-Terminal Peptide of Platelet Factor-4 Variant (CXCL4L1/PF-4var47-70) Strongly Inhibits Angiogenesis and Suppresses B16 Melanoma Growth In vivo

“…In addition, CXCL4/PF-4 inhibits epidermal growth factorstimulated proliferation of endothelial cells and interferes with the cell cycle by impaired downregulation of p21 CIP1/WAF1 (43). Further, it has been shown that different CXCL4/PF-4 peptides compete with FGF-2 or VEGF by inhibiting their binding to FGF or VEGF receptors, respectively, or reduce heparin-induced FGF-2 dimerization, resulting in the angiostatic activity (21,31,44,45). However, other mechanisms seem to be involved as well because an analogue of CXCL4/PF-4 that lacks affinity for heparin still retains its angiostatic activity (46), and CXCL4/PF-4 also inhibits the function of VEGF-121, an isoform with deficient heparin-binding ability (47).…”