Prothrombin Kringle-2 Domain Has a Growth Inhibitory Activity against Basic Fibroblast Growth Factor-stimulated Capillary Endothelial Cells

Recently, O'Reilly et al.(O'Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R. A., Moses, M., Lane, W. S., Cao, Y., Sage, E. H., and Folkman, J. (1994) Cell 79, 315–328; O'Reilly, M. S., Boehm, T., Shing, Y., Fukai, N., Vasios, G., Lane, W. S., Flynn, E., Birkhead, J. R., Olsen, B. R., and Folkman, J. (1997) Cell 88, 277–285) developed a simple in vitro angiogenesis assay system using bovine capillary endothelial cell proliferation and purified potent angiogenic inhibitors, including angiostatin and endostatin. Using a simplein vitro assay for angiogenesis, we purified a protein molecule that showed anti-endothelial cell proliferative activity from the serum of New Zealand White rabbits, which was stimulated by lipopolysaccharide. The purified protein showed only bovine capillary endothelial cell growth inhibition and not any cytotoxicity. This molecule was identified as a prothrombin kringle-2 domain (fragment-2) using Edman degradation and the amino acid sequence deduced from the cloned cDNA. Both the prothrombin kringle-2 domain released from prothrombin by factor Xa cleavage and the angiogenic inhibitor purified from rabbit sera exhibited anti-endothelial cell proliferative activity. The recombinant rabbit prothrombin kringle-2 domain showed potent inhibitory activity with half-maximal concentrations (ED50) of 2 μg/ml media. As in angiostatin, the recombinant rabbit prothrombin kringle-2 domain also inhibited angiogenesis in the chorioallantoic membrane of chick embryos. Recently, O'Reilly et al.(O'Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R. A., Moses, M., Lane, W. S., Cao, Y., Sage, E. H., and Folkman, J. (1994) Cell 79, 315–328; O'Reilly, M. S., Boehm, T., Shing, Y., Fukai, N., Vasios, G., Lane, W. S., Flynn, E., Birkhead, J. R., Olsen, B. R., and Folkman, J. (1997) Cell 88, 277–285) developed a simple in vitro angiogenesis assay system using bovine capillary endothelial cell proliferation and purified potent angiogenic inhibitors, including angiostatin and endostatin. Using a simplein vitro assay for angiogenesis, we purified a protein molecule that showed anti-endothelial cell proliferative activity from the serum of New Zealand White rabbits, which was stimulated by lipopolysaccharide. The purified protein showed only bovine capillary endothelial cell growth inhibition and not any cytotoxicity. This molecule was identified as a prothrombin kringle-2 domain (fragment-2) using Edman degradation and the amino acid sequence deduced from the cloned cDNA. Both the prothrombin kringle-2 domain released from prothrombin by factor Xa cleavage and the angiogenic inhibitor purified from rabbit sera exhibited anti-endothelial cell proliferative activity. The recombinant rabbit prothrombin kringle-2 domain showed potent inhibitory activity with half-maximal concentrations (ED50) of 2 μg/ml media. As in angiostatin, the recombinant rabbit prothrombin kringle-2 domain also inhibited angiogenesis in the chorioallantoic membrane of chick embryos. fibroblast growth factor basic FGF lipopolysaccharide prothrombin kringle prothrombin kringle-2 domain plasminogen tumor necrosis factor recombinant human TNF polyacrylamide gel electrophoresis Dulbecco's modified Eagle's medium bovine capillary endothelial cells phosphate-buffered saline d-phenylalanyl-l-prolyl-l-arginine chroromethyl ketone chorioallantoic membrane fast protein liquid chromatography. Angiogenesis is a process of blood vessel formation in which new vessels sprout from existing blood vessels (1Folkman J. D'Amore P.A. Cell. 1996; 87: 1153-1155Abstract Full Text Full Text PDF PubMed Scopus (1098) Google Scholar). Blood capillaries are primarily composed of endothelial cells that are normally quiescent in adult mammals under physiological conditions (2Hanahan D. Folkman J. Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6066) Google Scholar). Angiogenesis is required for a variety of physiological processes such as embryonic development, wound healing, tissue regeneration, and organ regeneration. Outgrowth of new blood vessels under pathological conditions can lead to the development and progression of diseases such as tumor growth, diabetic retinopathy, tissue and organ malformation, and cardiovascular disorders (3Folkman J. Shing Y. J. Biol. Chem. 1992; 267: 10931-10934Abstract Full Text PDF PubMed Google Scholar). The switch of the angiogenesis phenotype depends upon the net balance between the up-regulation of angiogenic stimulators and the down-regulation of angiogenic suppressors (2Hanahan D. Folkman J. Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6066) Google Scholar). A variety of growth factors can stimulate angiogenesis in vitro and in vivo (4Folkman J. N. Engl. J. Med. 1995; 333: 1757-1763Crossref PubMed Scopus (2243) Google Scholar). Of the known angiogenic factors, fibroblast growth factors (FGFs)1 and vascular endothelial growth factors are most commonly expressed (5Nguyen M. Watanabe H. Budson A.E. Richie J.P. Hayes D.F. Folkman J. J. Natl. Cancer Inst. 1994; 86: 356-361Crossref PubMed Scopus (449) Google Scholar, 6Ferrara N. Henzel W.J. Biochem. Biophys. Res. Commun. 1989; 161: 851-858Crossref PubMed Scopus (2005) Google Scholar, 7Senger D.R. Connoly D.T. Van de Water L. Feder J. Dvorak H.F. Cancer Res. 1990; 50: 1774-1778PubMed Google Scholar, 8Gospodarowicz D. Abraham J.A. Schilling J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7311-7315Crossref PubMed Scopus (555) Google Scholar, 9Cao Y. Chen H. Zhou L. Chiang M.-K. Anand-Apte B. Weatherbee J.A. Wang Y. Fang F. Flanagan J.G. Tsang M. J. Biol. Chem. 1996; 271: 3154-3162Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). These factors may function synergistically in promoting tumor growth when angiogenic inhibitors are simultaneously down-regulated (10Good D.J. Polverini P.J. Rastinejad F. Lebeau M.M. Lemons R.S. Frazier W.A. Bouck N.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6624-6628Crossref PubMed Scopus (893) Google Scholar, 11Van Meir E.G. Polverini P.J. Chazin V.R. Huang H.J. de Tribolet N. Cavenee W.K. Nat. Genet. 1994; 8: 171-176Crossref PubMed Scopus (301) Google Scholar). Multiple factors, including an angiogenic stimulator (FGF) and an inhibitor (transforming growth factor-β), are called into play during wound healing to bring about regrowth of damaged tissues and a functional vascular bed (12Lynch S.E. Colvin R.B. Antoniades H.N. J. Clin. Invest. 1989; 84: 640-646Crossref PubMed Scopus (453) Google Scholar). The strategy to discover a new angiogenic factor depends upon the generation of neovascularization in the chick chorioallantoic membrane assay and in the corneal assay in vivo and in the endothelial cell tube formation and migration assay in vitro. Recently, O'Reilly et al. (13O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W.S. Cao Y. Sage E.H. Folkman J. Cell. 1994; 79: 315-328Abstract Full Text PDF PubMed Scopus (3162) Google Scholar, 14O'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 (4236) Google Scholar) developed a simplein vitro angiogenesis assay system using bovine capillary endothelial (BCE) cell proliferation and purified potent angiogenic inhibitors, including angiostatin and endostatin. These proteins suppressed neovascularization in the mouse corneal assay and growth of tumor metastases. In this report we describe the identification of an angiogenic inhibitor from rabbit serum treated with lipopolysaccharide (LPS) by using a BCE cell proliferation assay. Also, we report that this molecule is identical to the prothrombin kringle-2 domain (PtK2). New Zealand White rabbits were challenged intravenously with 150 μg of LPS (serotype 055: B5; Sigma) and were bled 1 h later. The blood was allowed to stand at room temperature for 1 h and then overnight at 4 °C. The blood was centrifuged at 1,000 × g for 30 min to remove hard clots. L929 cells were maintained in Earle's modified Eagle's medium. The medium contained 10% fetal bovine serum, 2 mml-glutamine, 10 units/ml penicillin G, and streptomycin sulfate. All media components were from Life Technologies, Inc. Capillary endothelial cells of the adrenal cortex were cultured from bovine adrenal grands according to the methods of Folkman et al. (15Folkman J. Haudenschild C.C. Zetter B.R. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 5217-5221Crossref PubMed Scopus (616) Google Scholar) and Gospodarowicz et al. (16Gospodarowicz D. Massoglia J.C. Fujii D.K. J. Cell. Physiol. 1986; 127: 121-136Crossref PubMed Scopus (215) Google Scholar). Briefly, five adrenal glands were obtained in as sanitary conditions as possible from slaughtered calves. The adrenal cortex was extricated from the glands and cut into 1-mm pieces. The sliced tissues were then incubated in 0.5% collagenase at room temperature for 1 h. The detached capillary segment and endothelial cell aggregates were suspended in a culture medium (DMEM containing 10% bovine calf serum, 2 mml-glutamine, 10 units/ml penicillin G, and streptomycin sulfate) and plated onto gelatinized dishes. After 2–4 days, colonies of endothelial cells were mechanically removed using a pipette equipped with a microtip. Endothelial cell aggregates were seeded onto a gelatinized dish and grown in a culture medium containing 3 ng/ml recombinant human bFGF (R & D Systems, Inc.). The cells over 30 passages were used for the endothelial cell proliferation assay. Endothelial cell proliferation assay was performed according to the method of O'Reilly et al. (13O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W.S. Cao Y. Sage E.H. Folkman J. Cell. 1994; 79: 315-328Abstract Full Text PDF PubMed Scopus (3162) Google Scholar). BCE cells were maintained in DMEM containing 10% heat-inactivated bovine calf serum and 3 ng/ml recombinant human bFGF. Cells growing in gelatin-coated six-well plates were dispersed in a 0.05% trypsin solution and resuspended with DMEM containing 10% bovine calf serum. Approximately 12,500 cells in 0.5 ml were to of gelatinized plates and incubated at 10% for h. The media were with ml of DMEM containing bovine calf serum, and were to After 30 min of media were to a of 0.5 ml of DMEM containing bovine calf serum and bFGF at 1 After h of the cells were and using a that any inhibition was not to of the BCE cells from the of the assay were for of cell under an to cell activity was by using mouse L929 fibroblast cells as by and J. Google Scholar). the rabbit angiogenic we used TNF assay and BCE cell proliferation the endothelial cell growth inhibitory activity with fibroblast cell activity Briefly, was to rabbit serum to were 10 containing The proteins were to a with a The was at 4 with a of in 10 The were and at 4 onto a with The were at room temperature onto a with The was with a of in The from the were a polyacrylamide gel Inc.). After electrophoresis for h at and 4 the were sliced to and in phosphate-buffered saline and the were for BCE cell proliferation and L929 cell cytotoxicity. amino acid sequence was performed with 10 μg of the protein onto a membrane protein were in of and was performed with and at an to of for h at °C. The proteins were then to a for the of The were a protein amino acid the purified angiogenic inhibitor was incubated with at a for and 30 was then onto a amino acid the amino acid of the purified angiogenic the proteins were in ml of acid solution in an The was then under and at for h. The amino acid of the was using a amino acid prothrombin was purified according to the modified method of et al. Res. 1990; Full Text PDF PubMed Scopus Google Scholar). The blood was treated with and centrifuged at 1,000 for 30 min to remove blood cells and and chroromethyl were to a of and 1 of were to ml of and the was at room temperature for 1 h. After the to the was and the were with a 1 The proteins were with a containing The was and was to a of ml of 1 was and the was at 4 for 1 h. After at × g for the was resuspended in and then overnight at 4 the The proteins were to a 1 × of and then from the with a of using an were identified with and were for 4 h at 4 a containing and to a After with the prothrombin was with a containing The purified was at 4 overnight the 150 1 and incubated with factor Xa at overnight a The were to a polyacrylamide gel Inc.). After electrophoresis for h in at 4 the were sliced to and in The containing the were and The was by and for BCE cell plasminogen was purified according to the modified method of and PubMed Scopus Google Scholar). of blood were treated with and centrifuged at 1,000 × g for 30 min to remove blood cells and The was with and to a with The was by containing 3 and then with were identified with The was with an of and the was removed and using was from the purified by a according to the method of O'Reilly et al. M.S. Holmgren L. Chen C. Folkman J. Nat. Med. 1996; PubMed Scopus Google Scholar). Briefly, was to of in The solution was incubated at for h and then onto a that with The was and with The was and onto a gel with The angiostatin containing was by and angiostatin was by and by were with μg of recombinant rabbit in by μg in at the After 4 formation was by and was by μg of recombinant rabbit in After 1 the were and the of the was by assay. the were and to The was in The was by a G, was from the rabbit using the method by and N. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). from a New Zealand White rabbit was used in of The used for were a of the amino acid of the angiogenic inhibitor by Edman degradation of and The were to the and by the method F. S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). was expressed in using a The was onto a that with 10 containing were with an containing were and a with The purified was by and and were performed according to al. Y. D. J. D. S. O'Reilly M.S. M. Folkman J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). μg of purified protein in ml of DMEM in the of serum were incubated at room temperature with of 0.5 for After 30 of 0.5 was to the The protein solution was at 4 for h of After the were under conditions and for inhibitory BCE cell were incubated at in and a was the of M. Shing Y. Folkman J. Res. 1994; PubMed Scopus Google Scholar). After 2 of a containing was to the of embryos. After a was into the of the and were we a of a factor from the serum of New Zealand White rabbits, which was stimulated by including and cell that the factor a of PubMed Scopus Google Scholar). a purified factor was purified by the were into by BCE cell proliferation assay 1 The that showed BCE cell growth inhibition and L929 cell were to recombinant human under conditions 1 This was with the report that potent BCE cell growth inhibitory activity L. B. Gospodarowicz D. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar). the the that only BCE cell growth inhibition not with 1 This that the that are from the were to under a was by Using a we of angiogenic inhibitor from rabbit serum containing of The half-maximal inhibition in BCE cell proliferation was when a 2 μg/ml of this angiogenic was The amino acid sequence of the angiogenic inhibitor was by Edman degradation 3 Approximately 10 μg of the purified protein was to a membrane and a protein In an amino acid sequence was identified that only of protein molecule was in the the μg of the protein was with The were by liquid and by Edman degradation a protein The acid were identified from the of by amino not and from of the not sequence of the that the rabbit angiogenic inhibitor of amino we not a sequence to the of we the of the angiogenic inhibitor by this showed a with a of and a with a of The of the rabbit angiogenic inhibitor to an of of amino in the protein N. 1992; PubMed Scopus Google Scholar). a of the of the molecule may also for the E. N. PubMed Scopus Google Scholar). The amino acid sequence of the angiogenic inhibitor showed with the human and mouse the rabbit prothrombin was cloned and the sequence kringle-2 domain was not M. J. F. Res. Full Text PDF PubMed Scopus Google we obtained rabbit from rabbit The sequence of the was by the method 3 The amino acid sequence deduced from was with the sequence of the angiogenic inhibitor as and was to the known human and bovine sequence The amino acid of as by the factor Xa cleavage was to when the angiogenic inhibitor was by the released amino acid was 3 This that the of was in the angiogenic the between the angiogenic inhibitor and at the protein we purified rabbit prothrombin and obtained the kringle-2 domain from prothrombin by factor Xa cleavage The of from prothrombin was under conditions and a protein and a protein not the amino acid sequence of this protein was by Edman a sequence was identified were to the of a 3 A sequence the at the amino acid was also These between the can the by The from prothrombin also showed anti-endothelial cell proliferative activity as the angiogenic inhibitor purified from rabbit serum. These that the angiogenic inhibitor is the molecule as the the by The not only with prothrombin also with the kringle-2 domain containing prothrombin such as the not with the kringle 1 domain Both the kringle 2 domain purified from factor rabbit prothrombin and the recombinant showed to the the not with rabbit angiostatin, the domain by were plasminogen and angiostatin, were to of the were also in the that incubated with the recombinant rabbit in an E. Using and the recombinant was purified to from E. The purified a growth inhibitory BCE and the was by an recombinant Also, the activity of for BCE cells was and of the protein This that by to inhibitory activity. The anti-endothelial cell proliferative activity of was also with that of from factor As in the concentrations of half-maximal inhibition (ED50) for and are about 2 μg/ml and 4 the activity of is that of this the of recombinant to in of endothelial cell growth inhibitory activity of with human purified from factor rabbit prothrombin and human angiostatin were BCE cells in the of 1 ng/ml bFGF in a proliferation the of as a of et al. (13O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W.S. Cao Y. Sage E.H. Folkman J. Cell. 1994; 79: 315-328Abstract Full Text PDF PubMed Scopus (3162) Google Scholar) that angiostatin, a of including kringle was a potent inhibitor of BCE cell growth in vitro and of tumor cell metastases. to the inhibitory and the angiostatin were for of BCE cell The of half-maximal inhibition (ED50) for angiostatin is about 2 This is to is for A of BCE cells by angiostatin was not These that the of the BCE cell by to that by the of to in by using the chick assay Y. D. J. D. S. O'Reilly M.S. M. Folkman J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). of the showed potent inhibition of angiogenesis purified the angiogenic an inhibitor of endothelial cell from rabbit serum. The amino acid sequence of the purified angiogenic inhibitor by Edman degradation showed with the from the the kringle-2 domain from rabbit and the The amino acid sequence deduced from the sequence of the cloned was with that of the angiogenic inhibitor by Edman the released by factor Xa cleavage from purified rabbit prothrombin a under conditions and In the amino acid sequence of the proteins was to we that the of was from the angiogenic that of the of the angiogenic inhibitor by a during All the that the angiogenic inhibitor is the molecule as the kringle-2 domain from et al. S. T. M. G. D. PubMed Scopus Google Scholar) the of TNF from rabbit serum. that the protein activity L929 cells and a amino acid sequence with that of the angiogenic inhibitor 3 not this molecule as a prothrombin kringle-2 In this we that this molecule not activity L929 cells and was not to 1 Also, the amino acid sequence of the angiogenic inhibitor not any sequence to that of this molecule was at the which contained L929 cell activity in The angiogenic inhibitor from gel as a in a to by et al. S. T. M. G. D. PubMed Scopus Google Scholar) not The migration of this molecule under conditions may to of the protein such as the of a of in the The factor and angiogenic inhibitor to BCE cell In the of the of BCE cells to that of the In inhibition by TNF in an L. B. Gospodarowicz D. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar) and not These that to BCE cell growth inhibition by from that of TNF was from the cleavage of prothrombin by factor Xa and during blood et H. H. J. Biochem. J. 1997; PubMed Scopus Google Scholar) that may as a of the by prothrombin of recombinant rabbit expressed in E. we the inhibition of prothrombin by recombinant The recombinant showed a inhibition of prothrombin T. and S. S. This is with the report of et al. H. H. J. Biochem. J. 1997; PubMed Scopus Google Scholar). at that the of recombinant was with that of the domain of tissue plasminogen A. R.A. M. J. Biol. Chem. Full Text PDF PubMed Google These that the recombinant in E. LPS is by B. A. N. Engl. J. Med. PubMed Scopus Google we the of in serum with that of serum by As in the of from serum was from serum. This that by LPS is with is a protein domain that The kringle was in such as prothrombin D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google plasminogen Blood & 1992; PubMed Scopus Google D. W.J. E. Scopus Google growth factor P.J. 1994; PubMed Scopus Google and W.J. Chen PubMed Scopus Google Scholar). These to a functional is not In the of the plasminogen and were known to in the of plasminogen to L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). These kringle are to with by to an that prothrombin kringle not to in of a amino acid sequence A. PubMed Scopus Google Scholar). Recently, was that angiostatin, an angiogenesis suppressed endothelial cell growth in vitro and tumor in vivo (13O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W.S. Cao Y. Sage E.H. Folkman J. Cell. 1994; 79: 315-328Abstract Full Text PDF PubMed Scopus (3162) Google Scholar). of the kringle of plasminogen kringle is from cleavage of plasminogen by R. Cell. 1997; 88: Full Text Full Text PDF PubMed Scopus (449) Google Scholar) from and a S. M.S. D. J. F. R.A. Bouck N. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). recombinant and the angiostatin were for of endothelial cell proliferation in vitro and chick neovascularization in recombinant were with of In to a kringle is to angiostatin in that proteins are from degradation of proteins in of blood that kringle angiostatin and the kringle domain of plasminogen BCE cell growth as as This inhibition is not to Y. D. J. D. S. O'Reilly M.S. M. Folkman J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Chen A. D. Cao Y. M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The kringle domain of angiostatin endothelial cell growth the kringle of also a migration kringle of angiostatin under conditions Y. D. J. D. S. O'Reilly M.S. M. Folkman J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). is to the kringle domain of angiostatin in that the of the proteins are by under not that the kringle of plasminogen and angiostatin in blood In the of the of BCE cells to that of the cells this the of angiostatin and kringle endothelial cell growth to that the of endothelial cell by to that of angiostatin and is possible that a kringle domain endothelial cell growth inhibitory activity In that the kringle domain a A. Sci. 1994; PubMed Scopus Google Scholar). is known that the and angiogenic stimulators can in the of cell including endothelial factors are released by degradation of the J. M. J. M. D. J. Google Scholar). the angiogenic inhibitors also to in a that may of that are not inhibitors (2Hanahan D. Folkman J. Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6066) Google Scholar). A of angiogenic inhibitors as of In to angiostatin (13O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W.S. Cao Y. Sage E.H. Folkman J. Cell. 1994; 79: 315-328Abstract Full Text PDF PubMed Scopus (3162) Google Scholar) and the prothrombin kringle-2 are 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 (4236) Google which is a of of D.J. W.A. Polverini P.J. Bouck N. J. Cell Biol. PubMed Scopus Google of B. Y. Cell. 1989; Full Text PDF PubMed Scopus Google and a of D. B. R. J. Google Scholar). The to inhibitor from as of proteins may to the of the and to angiogenic processes such as and wound This may also to the of the by diseases with angiogenic components including and diabetic In this we that the prothrombin kringle-2 domain a potent endothelial cell growth inhibitory activity that of of this inhibitor is to such as of and the of this by of prothrombin kringle-2 domain in in vivo Cao for BCE cells proliferation assay. the protein for protein and amino acid also for of and for angiostatin and kringle also for the