Characterization of Endostatin Binding to Heparin and Heparan Sulfate by Surface Plasmon Resonance and Molecular Modeling

Endostatin (20 kDa) is a C-terminal proteolytic fragment of collagen XVIII that is localized in vascular basement membrane zones in various organs. It binds zinc, heparin/heparan sulfate, laminin, and sulfatides and inhibits angiogenesis and tumor growth. Here we determined the kinetics and affinity of the interaction of endostatin with heparin/heparan sulfate and investigated the effects of divalent cations on these interactions and on the biological activities of endostatin. The binding of human recombinant endostatin to heparin and heparan sulfate was studied by surface plasmon resonance using BIAcore technology and further characterized by docking and molecular dynamics simulations. Kinetic data, evaluated using a 1:1 interaction model, showed that heparan sulfate bound to and dissociated from endostatin faster than heparin and that endostatin bound to heparin and heparan sulfate with a moderate affinity (KD ∼ 2 μm). Molecular modeling of the complex between endostatin and heparin oligosaccharides predicted that, compared with mutagenesis studies, two further arginine residues, Arg47 and Arg66, participated in the binding. The binding of endostatin to heparin and heparan sulfate required the presence of divalent cations. The addition of ZnCl2 to endostatin enhanced its binding to heparan sulfate by ∼40% as well as its antiproliferative effect on endothelial cells stimulated by fibroblast growth factor-2, suggesting that this activity is mediated by the binding of endostatin to heparan sulfate. In contrast, no increase in the antiangiogenic and anti-proliferative activities of endostatin promoted by vascular endothelial growth factor was observed upon the addition of zinc. Endostatin (20 kDa) is a C-terminal proteolytic fragment of collagen XVIII that is localized in vascular basement membrane zones in various organs. It binds zinc, heparin/heparan sulfate, laminin, and sulfatides and inhibits angiogenesis and tumor growth. Here we determined the kinetics and affinity of the interaction of endostatin with heparin/heparan sulfate and investigated the effects of divalent cations on these interactions and on the biological activities of endostatin. The binding of human recombinant endostatin to heparin and heparan sulfate was studied by surface plasmon resonance using BIAcore technology and further characterized by docking and molecular dynamics simulations. Kinetic data, evaluated using a 1:1 interaction model, showed that heparan sulfate bound to and dissociated from endostatin faster than heparin and that endostatin bound to heparin and heparan sulfate with a moderate affinity (KD ∼ 2 μm). Molecular modeling of the complex between endostatin and heparin oligosaccharides predicted that, compared with mutagenesis studies, two further arginine residues, Arg47 and Arg66, participated in the binding. The binding of endostatin to heparin and heparan sulfate required the presence of divalent cations. The addition of ZnCl2 to endostatin enhanced its binding to heparan sulfate by ∼40% as well as its antiproliferative effect on endothelial cells stimulated by fibroblast growth factor-2, suggesting that this activity is mediated by the binding of endostatin to heparan sulfate. In contrast, no increase in the antiangiogenic and anti-proliferative activities of endostatin promoted by vascular endothelial growth factor was observed upon the addition of zinc. Endostatin (20 kDa), first identified by O'Reilly et al. (1.O'Reilly M.S. Boehm T. Shing Y. Fukai N. Vasios G. Lane W.S. Flynn E. Birkhead J.R. Olsen B.R. Folkman J. Cell. 1997; 88: 277-285Abstract Full Text Full Text PDF PubMed Scopus (4263) Google Scholar), is a proteolytic fragment from the C-terminal domain of collagen XVIII. It inhibits tumor angiogenesis by interfering at several levels with growth factor signaling (2.Ramchandran R. Karumanchi S.A. Hanai J. Alper S.L. Sukhatme V.P. Crit. Rev. Eukaryotic Gene Expression. 2002; 12: 175-191Crossref PubMed Scopus (20) Google Scholar). Two heparin-binding sites have been identified in endostatin involving two clusters of arginine residues, and a zinc binding site is located in the N-terminal part of the molecule (see Refs. 3.Marneros A.G. Olsen B.R. Matrix Biol. 2001; 20: 337-345Crossref PubMed Scopus (182) Google Scholar, 4.Sasaki T. Hohenester E. Timpl R. IUBMB Life. 2002; 53: 77-84Crossref PubMed Scopus (39) Google Scholar, 5.Ren B. Hotin N. Rabasseda X. Wang Y.Z. Wu M. Methods Find. Exp. Clin. Pharmacol. 2003; 25: 215-224Crossref PubMed Scopus (19) Google Scholar for reviews). The possibility that its anti-angiogenic effect might be related to displacement of angiogenic factors from the surface of endothelial cells through binding of heparan sulfate has prompted several investigations of its interaction with heparin and heparan sulfate (6.Sasaki T. Fukai N. Mann K. Gohring W. Olsen B.R. Timpl R. EMBO J. 1998; 17: 4249-4256Crossref PubMed Scopus (327) Google Scholar, 7.Sasaki T. Larsson H. Kreuger J. Salmivirta M. Claesson-Welsh L. Lindahl U. Hohenester E. Timpl R. EMBO J. 1999; 18: 6240-6248Crossref PubMed Scopus (201) Google Scholar, 8.Karumanchi S.A. Jha V. Ramchandran R. Karihaloo A. Tsiokas L. Chan B. Dhanabal M. Hanai J.I. Venkataraman G. Shriver Z. Keiser N. Kalluri R. Zeng H. Mukhopadhyay D. Chen R.L. Lander A.D. Hagihara K. Yamaguchi Y. R. L. Sukhatme V.P. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, J. T. M. T. Timpl R. Claesson-Welsh L. D. Lindahl U. EMBO J. 2002; PubMed Scopus Google Scholar, M. Folkman J. K. J. 2003; PubMed Google Scholar). The of zinc in the biological activity of endostatin binding has been to be for the anti-angiogenic activity of endostatin T. O'Reilly M.S. K. J. R. Folkman J. 1998; PubMed Scopus Google Scholar). of in zinc to its the to of T. O'Reilly M.S. K. J. R. Folkman J. 1998; PubMed Scopus Google Scholar). the zinc binding to be for the effects of endostatin on vascular endothelial growth fibroblast growth resonance surface plasmon and tumor growth in N. B. M. T. Fukai N. R. Timpl R. Olsen B.R. EMBO J. 1999; 18: PubMed Scopus Google Scholar). is of to the that in the binding of endostatin to heparin binding is required for the of angiogenesis in the membrane T. Larsson H. Kreuger J. Salmivirta M. Claesson-Welsh L. Lindahl U. Hohenester E. Timpl R. EMBO J. 1999; 18: 6240-6248Crossref PubMed Scopus (201) Google Scholar, J. Larsson H. T. K. L. A. Timpl R. R. L. PubMed Google Scholar), for the of activity and of endothelial cells J. Larsson H. T. K. L. A. Timpl R. R. L. PubMed Google Scholar). The of this was to the interactions of endostatin with heparin and its heparan sulfate, to the affinity of these interactions and the interaction by surface plasmon resonance the of heparin and heparan sulfate in the binding to endostatin using binding and molecular and the effect of divalent cations on the interaction of endostatin with heparin/heparan sulfate and on its anti-angiogenic and anti-proliferative activities in and of human endostatin was by human cells was for of endostatin with the of at the N-terminal N. B. M. T. Fukai N. R. Timpl R. Olsen B.R. EMBO J. 1999; 18: PubMed Scopus Google Scholar). The was through and to a heparin in Endostatin was by a of from to and further by on a in endostatin was by and at human endostatin in was as well as endostatin from BIAcore on a BIAcore and on a BIAcore in was to the of its BIAcore at a of and as B. D. J. M. E. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). to heparin and heparan sulfate. and heparan sulfate at A. A. A. M. M. M. H. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google and in and at a of between and resonance was was by from the with heparin heparan sulfate to binding B. D. J. M. E. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). at in and BIAcore Endostatin was this and at several and The surface was with a of The and and using a the at of the to a for of as the of the of the surface was the The is a of the of It the of the and and oligosaccharides of a by of heparin with by on a R. A. A. H. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). sulfate oligosaccharides by of heparan sulfate by by for of the of the and of the heparin of the of the and a of heparin of the of the and a of the heparin of the of the and a of the M. G. M. J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). heparin of the of the and of the of human endostatin was from the of the K. R. Boehm T. J. Y. R. Hohenester E. Timpl R. Folkman J. U. A. 1998; PubMed Scopus Google from the J. Z. G. H. PubMed Scopus Google Scholar). with the to the and using the M. T. W. M. H. J. in Google Scholar). of was using the R. J. 1998; Scopus Google Scholar). to no for the zinc was in the predicted binding and was at the of the that a of to the and the clusters to of was for the from the between the and the and the in its and 2 and for for the from a of the and to the to the R. A. A. H. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, A. E. M. K. M. N. in of and Scholar). of interaction and the using the in with a of at a of from the and to of docking was using a of and of 2 of two heparin that in the of the from the B. J. PubMed Scopus Google Scholar). as of the of the docking the docking was using a and the that be was a was from the surface of the in a with the binding site of was using a of and of Molecular the a molecular dynamics on the complex was at K. The M. N. J. 1998; was with to the for these of R. A. A. H. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, A. E. M. K. M. N. in of and for the heparin The was the The molecular dynamics was by a at to the complex a The complex from the Two molecular dynamics using heparin with the in from to in a as D. R. A. H. G. E. 88: PubMed Google Scholar, D. T. A. E. U. A. 1997; PubMed Scopus Google Scholar). collagen by the with and two further in and with a at a of and a Y. D. 2001; PubMed Scopus Google Scholar). The of the was as collagen and Y. D. 2001; PubMed Scopus Google Scholar). in the presence of first with endostatin for at in collagen of angiogenesis was by of endothelial cells by Y. D. 2001; PubMed Scopus Google Scholar). effect of endostatin on was on human endothelial cells stimulated by as well as on the human and human in for in a The was for the cells with endostatin in the presence of zinc for was by a a of Endostatin to and by binding that human recombinant endostatin from bound to binding was by heparin and to a by sulfate from and by sulfate from in was observed for heparin with molecular and sulfate from was a compared with of the interaction at a of In contrast, heparan sulfate from the binding of endostatin to heparin and heparan sulfate of and to and heparan sulfate oligosaccharides to the of these two required for endostatin binding using The binding of endostatin to heparin heparan sulfate with of heparin and heparan sulfate oligosaccharides The of heparin at of binding was a a of heparan sulfate was required to a of was observed with the heparan sulfate oligosaccharides and the heparin oligosaccharides of the endostatin binding to heparin by and and to heparan sulfate by and The by heparin and heparan sulfate oligosaccharides of endostatin binding to heparan sulfate was than the of endostatin binding to and of in the to with to the sulfate of heparin in the interaction with endostatin. for with endostatin heparin heparan sulfate a heparin its to with the of the complex in of endostatin binding to heparin heparan sulfate. In this a that the sulfate in the binding. the to the binding of endostatin. The of to endostatin binding was that of was Endostatin to and with a was by of endostatin heparan sulfate heparin The of the of endostatin with heparin heparan sulfate was by the to to using two interaction for the of endostatin binding to a 1:1 a involving of with endostatin. The addition of a that the of the was as a for the of recombinant endostatin from bound to heparan sulfate with a moderate The affinity was to be using the 1:1 to the The and and affinity was for the interaction between endostatin and heparin (KD 1:1 model, the for and for sulfate a complex than heparin with as by the faster to heparin and heparan sulfate well to the in the the effects of of In this model, endostatin to two sites on heparin heparan sulfate. The two of affinity and for heparin and for heparan sulfate The affinity was in the as the to the 1:1 to the affinity of the interaction between endostatin in cells and heparin heparan sulfate in the of zinc. In binding a to the model, the affinity was and for heparan and for Molecular of of with heparin and oligosaccharides and endostatin the docking the on the endostatin a of clusters was for for and for The the of on the surface of the binding zones the of the surface for the The docking further by the of between and of endostatin The that to be in binding through Arg66, and in two clusters from located between the two clusters at the surface of endostatin of predicted to between (1.O'Reilly M.S. Boehm T. Shing Y. Fukai N. Vasios G. Lane W.S. Flynn E. Birkhead J.R. Olsen B.R. Folkman J. Cell. 1997; 88: 277-285Abstract Full Text Full Text PDF PubMed Scopus (4263) Google Scholar, R. Karumanchi S.A. Hanai J. Alper S.L. Sukhatme V.P. Crit. Rev. Eukaryotic Gene Expression. 2002; 12: 175-191Crossref PubMed Scopus (20) Google Scholar, and 3.Marneros A.G. Olsen B.R. Matrix Biol. 2001; 20: 337-345Crossref PubMed Scopus (182) Google and of human endostatin docking simulations. from the first of human endostatin to as from the first of the C-terminal domain of collagen and modeling on the of the arginine of endostatin in its binding to required to of endostatin from a heparin with as the first of of predicted to between the for the and and human endostatin from the first of of required to of endostatin from a heparin with as the first of of predicted to between the for the and and human endostatin from the first of in a heparin was for a docking on endostatin. fragment was to the two clusters of identified as heparin binding sites T. Larsson H. Kreuger J. Salmivirta M. Claesson-Welsh L. Lindahl U. Hohenester E. Timpl R. EMBO J. 1999; 18: 6240-6248Crossref PubMed Scopus (201) Google Scholar). this is the of the in of of heparin on the endostatin surface and clusters for the with in the and The to the of the with to the the two arginine clusters on the endostatin The to the of the in the the oligosaccharides with the the two In contrast, for the in two to this The oligosaccharides the a of with the surface than the the Molecular of the between Endostatin and for and in the with the in that of was observed the was in the was further for molecular dynamics simulations. In the two a of the was observed the first of the The was in the binding in the and in the from the to The was for the with the of at the of the that by interaction with endostatin. The in the for the two dynamics with the two of the of the In the two the heparin the two clusters of The surface the of arginine and the molecular dynamics The binding in a between the and the clusters of endostatin. the of the binding with of the heparin be The of Endostatin to and on human recombinant endostatin from two endostatin by cells was for heparin and heparan sulfate binding in the for the of endostatin in In these endostatin from cells bound to heparan sulfate heparin The of binding might be of the of the biological activity of endostatin from cells the as in the this endostatin angiogenesis and in and was possibility to the binding of the endostatin was the of a endostatin binds of zinc K. R. Boehm T. J. Y. R. Hohenester E. Timpl R. Folkman J. U. A. 1998; PubMed Scopus Google Scholar), and on the of zinc in the interaction of endostatin with heparin have been T. O'Reilly M.S. K. J. R. Folkman J. 1998; PubMed Scopus Google Scholar, N. B. M. T. Fukai N. R. Timpl R. Olsen B.R. EMBO J. 1999; 18: PubMed Scopus Google Scholar), we to further the of zinc and divalent cations in endostatin binding to heparin heparan sulfate. The addition of several divalent cations and the binding of endostatin from to heparin and heparan sulfate and The binding to heparin and heparan sulfate observed for of endostatin by cells was by and The addition of is a and the binding of the two to heparin and heparan sulfate than to using a this bound in the and we to the addition of divalent to endostatin from cells increase its binding to heparin heparan sulfate, we in to endostatin its The addition of and the binding of endostatin from cells to heparin and heparan sulfate to the and the and a was for heparan sulfate than for The addition of zinc the binding of endostatin from to heparan sulfate, and no increase in binding to heparin was observed increase in have further of the addition of several divalent cations to endostatin on its binding to heparin and heparan sulfate The as of increase in binding and on two and with of heparin and heparan heparan ZnCl2 cells ZnCl2 cells in a the addition of zinc to of the binding of endostatin and of the binding of endostatin to heparin and heparan sulfate, The addition of of zinc to of endostatin binding activities to heparin and heparan sulfate. The heparin J. PubMed Scopus Google endostatin K. R. Boehm T. J. Y. R. Hohenester E. Timpl R. Folkman J. U. A. 1998; PubMed Scopus Google to the enhanced The of on heparin and heparan sulfate to the of endostatin its binding to the suggesting that the divalent to heparin heparan sulfate was with endostatin. of in the of of to observed by and in endostatin from and from cells The was observed using by endostatin from endostatin from The addition of zinc to endostatin to its the interaction of endostatin with of on the of in of we evaluated the of endostatin by by cells to endothelial by endostatin the endothelial by for the of endothelial and for and and for the Endostatin in to be than that from cells and of The in the presence and of ZnCl2 and The addition of zinc a further of angiogenesis by endostatin in this and in the and presence of zinc of on the of of human endothelial cells stimulated by by was by human endostatin from cells the cells stimulated by the addition of zinc to endostatin in a further in this the effects of endostatin and zinc The of cells stimulated by was further upon the addition of zinc to endostatin compared with endostatin and in to the with the of was than the effect of endostatin and zinc of recombinant human endostatin from with zinc on The as of of human endothelial human human stimulated by stimulated by in a Endostatin the of a human by The addition of zinc a further in zinc by In contrast, recombinant human endostatin no effect on the of the The of endostatin and zinc the as zinc the first of endostatin binding to heparin and heparan sulfate. Endostatin binds to these with a moderate affinity in the of further the the The by to a using the was is with that for endostatin binding to heparin as evaluated by (KD ∼ T. Larsson H. Kreuger J. Salmivirta M. Claesson-Welsh L. Lindahl U. Hohenester E. Timpl R. EMBO J. 1999; 18: 6240-6248Crossref PubMed Scopus (201) Google Scholar). sulfate binds to and from endostatin faster than The is for heparan sulfate, suggesting that the complex of endostatin with heparan sulfate is in than the The BIAcore be to a of involving between and endostatin. The to this be by the of heparin and heparan sulfate. heparin and heparan sulfate the and of several that endostatin with oligosaccharides from to in bound to no binding to endostatin was observed for the heparan sulfate oligosaccharides and of heparin than a binding to endostatin. is with using that heparin of required for binding to endostatin T. Larsson H. Kreuger J. Salmivirta M. Claesson-Welsh L. Lindahl U. Hohenester E. Timpl R. EMBO J. 1999; 18: 6240-6248Crossref PubMed Scopus (201) Google Scholar). is required for endostatin binding to its heparan sulfate. with the of binding of heparan sulfate and the that heparan sulfate oligosaccharides a of and no J. T. M. T. Timpl R. Claesson-Welsh L. D. Lindahl U. EMBO J. 2002; PubMed Scopus Google Scholar). that and of heparin in the binding to endostatin. is for the interaction of heparin with endostatin T. Larsson H. Kreuger J. Salmivirta M. Claesson-Welsh L. Lindahl U. Hohenester E. Timpl R. EMBO J. 1999; 18: 6240-6248Crossref PubMed Scopus (201) Google and is in the binding of endostatin to the heparan sulfate of S.A. Jha V. Ramchandran R. Karihaloo A. Tsiokas L. Chan B. Dhanabal M. Hanai J.I. Venkataraman G. Shriver Z. Keiser N. Kalluri R. Zeng H. Mukhopadhyay D. Chen R.L. Lander A.D. Hagihara K. Yamaguchi Y. R. L. Sukhatme V.P. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), have been by M. Folkman J. K. J. 2003; PubMed Google as for the interaction of endostatin with heparan sulfate. In the of of heparin in the binding to endostatin to be in to by affinity T. Larsson H. Kreuger J. Salmivirta M. Claesson-Welsh L. Lindahl U. Hohenester E. Timpl R. EMBO J. 1999; 18: 6240-6248Crossref PubMed Scopus (201) Google by a binding M. Folkman J. K. J. 2003; PubMed Google Scholar). The of human and and of the arginine in in the human the a by E. T. Olsen B.R. Timpl R. EMBO J. 1998; 17: PubMed Scopus Google Scholar). of from the of the human K. R. Boehm T. J. Y. R. Hohenester E. Timpl R. Folkman J. U. A. 1998; PubMed Scopus Google and E. T. Olsen B.R. Timpl R. EMBO J. 1998; 17: PubMed Scopus Google a of from mutagenesis on endostatin T. Larsson H. Kreuger J. Salmivirta M. Claesson-Welsh L. Lindahl U. Hohenester E. Timpl R. EMBO J. 1999; 18: 6240-6248Crossref PubMed Scopus (201) Google be compared with of the human for heparin binding modeling with T. Larsson H. Kreuger J. Salmivirta M. Claesson-Welsh L. Lindahl U. Hohenester E. Timpl R. EMBO J. 1999; 18: 6240-6248Crossref PubMed Scopus (201) Google in in binding and with the and the endostatin a affinity for heparin T. Larsson H. Kreuger J. Salmivirta M. Claesson-Welsh L. Lindahl U. Hohenester E. Timpl R. EMBO J. 1999; 18: 6240-6248Crossref PubMed Scopus (201) Google Scholar). in the binding for the that as in the binding and in identified in human endostatin by the as in heparin binding and two further arginine residues, Arg47 and Arg66, for no mutagenesis by the molecular modeling to be in the binding of endostatin to is located in the of of the heparin binding sites Arg47 is between the two binding sites identified by mutagenesis and the The of Arg47 and as of endostatin in heparin and heparan sulfate binding the by arginine in these that the binding of endostatin to heparin and heparan sulfate upon divalent cations. for divalent cations was in to the of binding to heparin and heparan sulfate of endostatin by of affinity on a heparin of the of cells was to as by the presence in of molecular with endostatin from the heparin affinity of by was and is that the divalent required for binding to the was this In of the endostatin that we the addition of zinc in a increase in binding to heparin and heparan sulfate. we observed a increase in binding in endostatin to in the presence of zinc and to in the presence of that of the divalent to be zinc, as from to and the of endostatin binding to heparin in and human endostatin of to 2 to to in bound to a heparin T. Larsson H. Kreuger J. Salmivirta M. Claesson-Welsh L. Lindahl U. Hohenester E. Timpl R. EMBO J. 1999; 18: 6240-6248Crossref PubMed Scopus (201) Google Scholar). The in zinc at in for the and of the biological activities of have been to to between from The for a divalent in endostatin binding to heparin at with the that heparin binding to endostatin is by the of zinc binding T. Larsson H. Kreuger J. Salmivirta M. Claesson-Welsh L. Lindahl U. Hohenester E. Timpl R. EMBO J. 1999; 18: 6240-6248Crossref PubMed Scopus (201) Google Scholar). be this was by mutagenesis to the zinc binding site and by to the interaction as we and et al. T. Larsson H. Kreuger J. Salmivirta M. Claesson-Welsh L. Lindahl U. Hohenester E. Timpl R. EMBO J. 1999; 18: 6240-6248Crossref PubMed Scopus (201) Google we the human in this might be of zinc in endostatin E. T. Mann K. Timpl R. J. Biol. PubMed Scopus Google Scholar). and to the zinc is a of human endostatin in and is in a 1:1 T. O'Reilly M.S. K. J. R. Folkman J. 1998; PubMed Scopus Google Scholar, K. R. Boehm T. J. Y. R. Hohenester E. Timpl R. Folkman J. U. A. 1998; PubMed Scopus Google Scholar), we that the required in for binding to heparin heparan sulfate is zinc. The of divalent cations from heparin and heparan sulfate by of to endostatin in in suggesting that the is with endostatin than with the The of the of the between endostatin and heparin heparan sulfate in the presence of divalent suggesting that might the The binding of endostatin to observed by in the presence of zinc is with the of as observed in of human endostatin K. R. Boehm T. J. Y. R. Hohenester E. Timpl R. Folkman J. U. A. 1998; PubMed Scopus Google Scholar). The addition of zinc to endostatin was required for the anti-angiogenic activity of endostatin from cells in a of angiogenesis by for its antiproliferative activity on tumor cells and on endothelial cells stimulated by that these two biological activities of endostatin mediated by its interaction with heparan sulfate is the growth The of effect of zinc on the biological activities of endostatin by is with the that endostatin with no zinc binding to the of endothelial cells by N. B. M. T. Fukai N. R. Timpl R. Olsen B.R. EMBO J. 1999; 18: PubMed Scopus Google and to tumor growth N. B. M. T. Fukai N. R. Timpl R. Olsen B.R. EMBO J. 1999; 18: PubMed Scopus Google Scholar, W. H. K. O'Reilly M.S. 1999; PubMed Scopus Google Scholar). In to the with the addition of zinc to endostatin its anti-proliferative activity on endothelial cells stimulated by that is to the anti-proliferative activity of endostatin is at in by its binding to heparan sulfate. the of two and for the anti-proliferative activity of endostatin on the growth factor to The of heparin binding for the anti-angiogenic activity of endostatin for is that endostatin its anti-angiogenic at in by two on the growth factor to and by Yamaguchi et al. N. B. M. T. Fukai N. R. Timpl R. Olsen B.R. EMBO J. 1999; 18: PubMed Scopus Google that endostatin inhibits angiogenesis of its to the heparin binding is required for the of angiogenesis in the membrane T. Larsson H. Kreuger J. Salmivirta M. Claesson-Welsh L. Lindahl U. Hohenester E. Timpl R. EMBO J. 1999; 18: 6240-6248Crossref PubMed Scopus (201) Google Scholar, J. Larsson H. T. K. L. A. Timpl R. R. L. PubMed Google Scholar). human endostatin from cells inhibits to a the of endothelial cells by and of the The that human endostatin is to the of a tumor is in with the that endostatin inhibits the growth of and H. H. Z. M. H. Gene 2003; PubMed Scopus Google Scholar). Endostatin might anti-proliferative effect on tumor endostatin in has been to the of cells (1.O'Reilly M.S. Boehm T. Shing Y. Fukai N. Vasios G. Lane W.S. Flynn E. Birkhead J.R. Olsen B.R. Folkman J. Cell. 1997; 88: 277-285Abstract Full Text Full Text PDF PubMed Scopus (4263) Google Scholar). that is that human endostatin inhibits the of a human of a the effect of endostatin has been for the J. PubMed Scopus Google Scholar). In endostatin is in a of interactions with the of the and with membrane as M. T. E. H. M. T. K. K. U. A. 2001; PubMed Scopus Google Scholar, S.A. K. J. 2002; Google Scholar). at the to the interaction of endostatin with the heparan sulfate of the surface to its biological the that located at the surface of endothelial cells and endostatin and its sulfate and to with and these interactions in the anti-angiogenic activity of endostatin. for heparin and heparan sulfate and for of and J. for of the