Differential Binding of Plasminogen, Plasmin, and Angiostatin4.5 to Cell Surface B-Actin: Implications for Cancer-Mediated Angiogenesis

Research Article

Hao Wang,1 Jennifer A. Doll,1 Keyi Jiang,1 Deborah L. Cundiff,1 Jarema S. Czarnecki,1 Mindy Wilson,2 Karen M. Ridge,2 and Gerald A. Soff1,3

1Division of Hematology/Oncology and 2Division of Pulmonary and Critical Care Medicine, Department of Medicine and Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois and 3Division of Hematology/ Oncology, State University of New York Downstate Medical Center, Brooklyn, New York

Abstract Several angiostatin isoforms have been reported, differing in Angiostatin4.5 (AS4.5) is the product of plasmin autoproteol- kringle content. Angiostatins consisting of plasminogen kringles 1 f to 3, kringles 1 to 4, or angiostatin4.5 (AS4.5), which includes ysis and consists of kringles 1 to 4 and 85% of kringle 5. In 78 529 culture, cancer cell surface globular B-actin mediates plasmin kringles 1 to 4 plus 85% of kringle 5 (Lys -Arg ), have been autoproteolysis to AS4.5. We now show that plasminogen generated in vitro by a variety of proteinases. However, it is still not binds to prostate cancer cells and that the binding colocalizes well understood which angiostatin isoforms are present naturally with surface B-actin, but AS4.5 does not bind to the cell in the human body (5, 8–13). We have shown previously that surface. Plasminogen and plasmin bind to immobilized AS4.5 is a naturally occurring human form (14). AS4.5 is generated B-actin similarly, with a K of f140 nmol/L. The binding is from plasminogen in a two-step process (12, 14). Plasminogen d activators cleave plasminogen to form plasmin. Plasmin can then inhibited by E-aminocaproic acid (EACA), indicating the requirement for a lysine-kringle domain interaction. Using a be converted to AS4.5 via autoproteolysis by cleavage within series of peptides derived from B-actin in competitive binding kringle 5 (12, 14). In vitro, plasmin autoproteolysis to AS4.5 may studies, we show that the domain necessary for plasminogen be mediated by an alkaline pH, phosphoglycerate kinase, or a free binding is within amino acids 55 to 69 (GDEAQSKRGILTLKY). sulfhydryl donor (12, 15, 16). Substitution of Lys61 or Lys68 with arginine results in the loss We showed recently that PC-3 (human prostate cancer), HT1080 of the ability of the peptide to block plasminogen binding, (human fibrosarcoma), and MDA-MB231 (human breast cancer) 61 68 cells express globular h-actin on the surface of the cells (17, 18). We indicating that Lys and Lys are essential for plasminogen h binding. Other actin peptides, including peptides with lysine, further showed that -actin can mediate plasmin autoproteolysis did not inhibit the plasminogen-actin interaction. AS4.5 did to generate AS4.5 on the cancer cell surfaces in the absence of a not bind actin at concentrations up to 40 Mmol/L. Plasmin- free sulfhydryl donor (18). The mechanism of actin trafficking and binding to the cell surface is not well understood. In this study, we ogen, plasmin, and AS4.5 all contain kringles 1 to 4; however, h kringle 5 is truncated in AS4.5. Isolated kringle 5 binds to show colocalization of plasminogen to surface -actin on PC-3 cells. Further, we characterized the binding of plasminogen, actin, suggesting intact kringle 5 is necessary for plasminogen and plasmin to bind to cell surface B-actin, and the truncated plasmin, and AS4.5 to actin. kringle 5 in AS4.5 results in its release from B-actin. These Materials and Methods data may explain the mechanism by which AS4.5 is formed locally on cancer cell surfaces and yet acts on distant sites. Reagents. Plasminogen and AS4.5 were prepared as described previously (Cancer Res 2006; 66(14): 7211-5) (13). Pure plasmin was from Enzyme Research Laboratories (South Bend, IN). Recombinant kringle 5 was a gift from Donald Davidson of Abbott Laboratories (Abbott Park, IL). Synthetic peptides were made by NeoMPS Introduction (San Diego, CA). Purified nonmuscle actin was from Cytoskeleton (Denver, CO) and contains 80% h-actin. Anti-kringles 1 to 3 monoclonal antibodies Plasmin is a two-chain serine proteinase formed by cleavage of (VAP) was a kind gift from V. Ploplis and F. Castellino of Notre Dame the zymogen plasminogen by urokinase-type plasminogen activator University (Notre Dame, IN). Goat anti-mouse IgG conjugated with alkaline (uPA) or tissue plasminogen activator (tPA) through hydrolysis of phosphatase was from Kirkegaard & Perry Laboratories, Inc. (Gaithersburg, 561 562 the Arg -Val peptide bond (1). In addition to being the primary MD). U-Nitrophenyl phosphate and N-acetyl-L-cysteine (NAC) were from fibrinolytic enzyme, plasmin cleaves matrix proteins, such as Sigma-Aldrich (St. Louis, MO). EZ-Link Sulfo-NHS-LC-Biotin was from laminin, and activates matrix metalloproteinases (2, 3), which may Pierce Biotechnology, Inc. (Rockford, IL). promote angiogenesis and invasion by tumors. Angiostatin, first Immunofluorescent staining of PC-3 cells. PC-3 cells were plated on described by O’Reilly et al. in 1994, is an internal fragment of untreated glass coverslips for 2 days. Cells were washed twice with PBS and A plasminogen (4, 5). Angiostatin is a potent inhibitor of vascular then treated with 40 mol/L pefabloc (Roche, Indianapolis, IN) for 1 hour at 37jC followed by 2 mmol/L q-aminocaproic acid (qACA; Sigma-Aldrich) for endothelial cell proliferation, migration, and tube formation and 1 hour at 37jC and a final wash of PBS. Cells were then treated with 1 Amol/ induces endothelial cell apoptosis (6, 7). L human Glu-plasminogen or AS4.5 for 1 hour at 37jC. Cells were washed twice with PBS followed by fixation in freshly prepared 2% formaldehyde. Cells were rinsed thrice with PBS. Coverslips were blocked with normal goat serum (NGS) for 30 minutes at 37jC followed by 30 minutes of incubation Requests for reprints: Gerald A. Soff, Division of Hematology/Oncology, State at 37jC with 1:100 dilution (in NGS/PBS) of primary antibody, a polyclonal University of New York Downstate Medical Center, 450 Clarkson Avenue, Box 20, Suite rabbit anti-actin (Sigma-Aldrich). Coverslips were next rinsed with 0.05% 850, Brooklyn, NY 11203. Phone: 718-270-2785; E-mail: [email protected]. I2006 American Association for Cancer Research. Tween 20/PBS, twice with PBS, followed by 30 minutes of incubation doi:10.1158/0008-5472.CAN-05-4331 at 37jC with a 1:200 dilution of secondary antibody, Alexa Fluor goat www.aacrjournals.org 7211 Cancer Res 2006; 66: (14). July 15, 2006

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Cancer Research anti-rabbit 568 (Molecular Probes, Carlsbad, CA). The procedure was AS4.5, and kringle 5 were buffer exchanged as above to remove unreacted repeated with a primary mouse monoclonal antibody to plasminogen biotin reagent. kringles (GMA-086, Green Mountain Antibodies, Burlington, VT) or VAP Microtiter plate wells were coated with 100 ALof80Ag/mL nonmuscle (a kind gift from V.A. Ploplis and F. Castellino) and a secondary antibody, actin in PBS overnight at room temperature and then washed thrice with Alexa Fluor goat anti-mouse 488 (Molecular Probes). Coverslips mounted on PBS/BSA. Wells were blocked with PBS/BSA for 1 hour at room glass slides with gelvatol mounting medium containing 1.5 mg/mL DABCO temperature. Biotinylated plasminogen, plasmin, AS4.5, or kringle 5 was (Sigma-Aldrich). Images of fixed, stained preparations were taken with a added to duplicate wells. After 1 hour of incubation at room temperature, Zeiss LSM 510 microscope (Carl Zeiss, Thornwood, NY). wells were washed as above. Phosphatase-labeled streptavidin (100 ALof Solid-phase ELISA for plasminogen binding to immobilized actin. 0.001 mg/mL in PBS/BSA) was added to the wells. After 1 hour of All steps were conducted at room temperature. Microtiter plates (Nalgene incubation at room temperature, the wells were washed as above, and Nunc International, Rochester, NY) were coated with 100 AL/well of phosphatase substrate solution (200 AL/well) was added. A405 was read after 80 Ag/mL nonmuscle actin or bovine serum albumin (BSA, Sigma-Aldrich) 20 minutes of incubation. in PBS overnight and washed thrice with PBS with 1% BSA (PBS/BSA). A Wells were blocked with PBS/BSA for 1 hour. Plasminogen (100 L) at 10 Results to 500 nmol/L in PBS/BSA was added to duplicate wells coated with actin or BSA as control. After 1 hour of incubation, the wells were washed thrice Binding of plasminogen and AS4.5 to PC-3 cells in culture. with PBS/BSA, and VAP antibody (0.01 mg/mL in PBS/BSA) was added to Using immunofluorescent staining, we compared plasminogen and the wells. After 1 hour of incubation, the wells were washed as above. Goat AS4.5 binding to fixed PC-3 cells (Fig. 1). The cells had been anti-mouse IgG conjugated with alkaline phosphatase (0.01 mg/mL diluted pretreated with pefabloc to neutralize any serine proteinase activity in PBS/BSA) was added to the wells, incubated as above, and wells were on the cells and with qACA to remove any plasminogen-derived washed as above. Phosphatase substrate solution (1 mg/mL U-nitrophenyl protein that may be present in the serum. Surface h-actin was phosphate in 0.05 mol/L NaHCO with 1 mmol/L MgCl ) was added to the 3 2 observed on nonpermeabilized PC-3 cells as reported previously wells. A was read after 20 minutes of incubation. To test the influence 405 (18). Following treatment with plasminogen, there was an avid of other agents on binding of plasminogen, putative binding inhibitors (qACA, synthetic actin-derived peptides, purified recombinant kringle 5, or binding of GMA-086 to the nonpermeabilized cells, which h AS4.5) were coincubated with 50 nmol/L plasminogen. colocalized with -actin. In contrast, control cells and cells treated Measurement of AS4.5 generation. AS4.5 generation was measured by with AS4.5 did not bind two different antibodies to plasminogen ELISA as described previously (18). For cell studies, PC-3 cells were cultured kringles, GMA-086 (or VAP; data not shown), indicating absence of and fixed with glutaraldehyde as described previously (12, 18). Fixed PC-3 binding of AS4.5. These data confirmed that plasminogen does cells were incubated with 440 nmol/L (40 Ag/mL) plasminogen alone or bind to the h-actin on the PC-3 cell surface and that AS4.5 does not with synthetic peptide C, C1, C2, or qACA. In a cell-free system, plasminogen bind. (440 nmol/L) was incubated alone or with urokinase (uPA), NAC, Plasminogen-B-actin binding in a solid-phase ELISA. To j nonmuscle actin, and/or synthetic peptide C at 37 C as described characterize the plasminogen-actin interaction, we used a solid- previously (12, 18). phase ELISA to measure the binding efficiency. When microtiter Binding of biotinylated plasminogen, plasmin, AS4.5, or kringle 5 to actin. Plasmin was inactivated with 10 mmol/L pefabloc for 5 minutes. plates were coated with actin, plasminogen binding was dose f Plasminogen, plasmin, AS4.5, and recombinant kringle 5 were buffer dependent up to 500 nmol/L where it reached a maximum exchanged thrice with PBS. EZ-Link Sulfo-NHS-LC-Biotin was added (Fig. 2A). Using Scatchard analysis, the Kd of plasminogen binding to plasminogen, plasmin, AS4.5, or kringle 5 at a 10:1 molar ratio. After to actin was 140 nmol/L. When plates were coated with BSA, 30 minutes of incubation at room temperature, plasminogen, plasmin, plasminogen did not bind, indicating specific binding to actin.

Figure 1. Immunofluorescent staining of h-actin, plasminogen, and AS4.5 on nonpermeabilized PC-3 cells. PC-3 cells were treated with Glu-plasminogen, AS4.5, or buffer as control. Surface h-actin was visualized by rabbit polyclonal antibody to h-actin and with Alexa Fluor goat anti-rabbit 568. A monoclonal antibody to the plasminogen kringle domain (GMA-086) and a secondary Alexa Fluor goat anti-mouse 488 were used to visualize plasminogen or AS4.5, if bound. Merged, colocalization of surface plasminogen and actin. AS4.5 did not bind to the cells. Phase, distribution of the staining.

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Figure 3. Peptides A, B, and C in actin crystal structure. Peptide A consists of amino acids 241 to 255 of the actin sequence (ELPDGQVITIGNERF), peptide B consists of amino acids 106 to 120 (TEAPLNPKANREKMT), and peptide C consists of amino acids 55 to 69 (GDEAQSKRGILTLKY). This was made with the public sequence of h-actin using Cn3D4.1 software.

Because plasminogen contains several kringle domains, which are known to interact with lysines, we assessed the ability of qACA (1 mmol/L), a lysine analogue known to inhibit kringle binding, to inhibit plasminogen binding to actin. qACA inhibited plasminogen binding to actin by 84% to 91% (Fig. 2A). These data indicate that plasminogen binding to actin is kringle dependent. Influences of actin-derived peptides on plasminogen-B-actin binding. Previous studies by Lu et al. suggested that Lys61 and Lys113, which are conserved in all actin isoforms, are on the surface of globular actin (19). Therefore, we tested three synthetic, actin- derived peptides to determine if these or other surface lysine residues contributed to plasminogen binding to actin (Fig. 3). Actin peptide A (amino acids 241-255) was used as a presumed negative control because, although a surface epitope, it did not contain a lysine residue. Peptide B consisted of amino acids 106 to 120, including Lys113 and Lys118. Peptide C consisted of amino acids 55 to 69, including Lys61 and Lys68. These peptides, as well as qACA, were tested for their ability to competitively inhibit plasminogen binding to h-actin. qACA, at 0.2 and 1 mmol/L, inhibited plasminogen binding to h-actin by f74% (P < 0.02) and 91% (P <0.01),respectively(Table1).PeptideCalsoinhibited plasminogen binding to h-actin; 37% inhibition (P < 0.05) at 0.2 mmol/L and 79% inhibition at 1 mmol/L (P < 0.01), respectively (Table 1). Peptide A and B did not show significant inhibition (Table 1). These data indicate that Lys61 and Lys68 are important in plasminogen binding. 61 68 Figure 2. A, plasminogen binding to h-actin in solid-phase ELISA. Plasminogen To further confirm the role of Lys and/or Lys ,two (0-500 nmol/L) was added to duplicate wells coated with h-actin or BSA additional synthetic peptides were made, substituting each lysine (80 Ag/mL). Plasminogen binding was specific for h-actin. qACA (1 mmol/L) was used as an inhibitor of plasminogen binding to wells coated with actin. residue in peptide C to an arginine residue. Peptide C1 68 Points, mean of duplicate wells; data, SE. B, differential binding of plasminogen, (GDEAQSKRGILTLRY) has Lys replaced with an arginine, and plasmin, and AS4.5 to h-actin. Biotin-labeled plasminogen, plasmin, or AS4.5 peptide C2 (GDEAQSRRGILTLKY) has Lys61 replaced with (0-500 nmol/L) was added to duplicate wells of microtiter plates coated with 80 Ag/mL actin. Binding was detected by phosphatase-labeled streptavidin, arginine. At 1 mmol/L, peptides C1 and C2 did not show and A 405 was read after incubation with phosphatase substrate solution for significant inhibition of plasminogen binding to actin (Table 1), 20 minutes. Plasminogen and plasmin bound efficiently, but AS4.5 did not bind 61 68 the actin. C, binding of kringle 5 or AS4.5 to h-actin. Recombinant kringle 5 or indicating that both Lys and Lys may be necessary for the AS4.5 (0-40 Amol/L) was added to duplicate wells coated with actin or BSA interaction between plasminogen and actin. (80 Ag/mL), respectively. Kringle 5 showed specific binding to immobilized Influences of actin-derived peptides on cell surface AS4.5 globular actin, whereas AS4.5 did not show any binding to actin. This directly q supports the conclusion that kringle 5 is necessary for binding of plasminogen generation. Because peptide C and ACA inhibited plasminogen and plasmin to actin and for the inability of AS4.5 to bind. binding to h-actin in a cell-free system, we investigated their ability www.aacrjournals.org 7213 Cancer Res 2006; 66: (14). July 15, 2006

Table 1. Inhibition of plasminogen: h-actin binding support a critical role for kringle 5, but not kringles 1 to 4, in plasminogen-plasmin binding to h-actin. Inhibitor Inhibitor concentration* Discussion (0.2 mmol/L) (1 mmol/L) We and others have shown that cell surface actin can bind plasminogen (18, 20–22), but the nature and functions of this q ACA 73.7 +/À 1.2; P < 0.02 90.9 +/À 0.4; P < 0.01 binding were uncharacterized previously. Further, we have Peptide A 15.8 +/À 5.7 18.5 +/À 0.6 published recently that h-actin on the surface of cells plays a key Peptide B 12.2 +/À 2.3 4.9 +/À 1.2 Peptide C 37.1 +/À 1.5; P < 0.05 79.4 +/À 0.8; P < 0.01 role in the proteolysis of plasminogen to form AS4.5 by cancer cells Peptide C1 1.5 +/À 6.4 19.5 +/À 3.8 (18). Plasminogen contains 5 kringle domains, which are known to Peptide C2 0.4 +/À 0.2 0.4 +/À 3.6 interact with lysines, and kringle-containing proteins can be isolated using lysine-binding methods (13). Here, we have shown that qACA inhibited plasminogen binding to actin, indicating that NOTE: Data presented as percentage inhibition. this interaction is lysine-kringle dependent. We also showed that *Ps, where statistical significance was achieved. Lys61 and Lys68 in actin are critical for binding plasminogen/ plasmin because when either was substituted for an arginine in a synthetic peptide, the ability to inhibit plasminogen binding to actin was lost. Lu and Szilagyi have shown that Lys61 is a surface to inhibit plasminogen conversion to AS4.5 on the surface of PC-3 lysine residue in actin molecules (19), and because Lys61 and Lys68 cells. qACA inhibited AS4.5 generation by 88% (P < 0.001), and are conserved in all actin isoforms, this explains the ability of peptide C inhibited AS4.5 generation by 49% (P < 0.01), whereas plasminogen to bind to different actin isoforms (18, 20–22). peptides C1 and C2 had a negligible effect on AS4.5 generation Plasminogen and plasmin have intact kringles 1 to 5 and bind (Fig. 4). This further supports that both Lys61 and Lys68 have an actin efficiently (K , f140 nmol/L), whereas AS4.5 with intact important role in binding plasminogen to actin and also suggests d kringles 1 to 4 but a truncated kringle 5 does not bind actin at that they function in the actin-mediated plasmin autoproteolysis concentrations as high as 40 Amol/L. We thus conclude that kringle that results in AS4.5 generation. 5 is the key domain of plasminogen and plasmin, which binds to Effects of actin-derived peptides on cell-free AS4.5 genera- actin. Kringle 5 of plasminogen/plasmin contains 80 amino acids tion. In a cell-free system, h-actin or a small-molecule free that are structurally similar to kringles 1 to 4 (23). However, our sulfhydryl donor can mediate plasmin autoproteolysis to generate studies showed that kringle 5 alone could bind to actin and, in AS4.5 (12, 18). To determine if peptide C could substitute for competitive binding studies, could inhibit plasminogen binding to h-actin in this reaction, AS4.5 generation was measured in a cell- actin. We propose a model of plasminogen/plasmin interaction free system with or without this peptide. Peptide C did not with actin where plasminogen/plasmin is bound, but on autopro- significantly increase AS4.5 generation over the level of uPA alone teolysis of plasmin, which cleaves plasmin within kringle 5, the (data not shown). These data indicate that, although peptide C was generated AS4.5 is released from actin, thus, from the cell surface able to compete with h-actin for plasminogen binding, it was not (Fig. 5). sufficient to replace the full h-actin protein in AS4.5 generation. Interestingly, Dudani et al. proposed that endothelial cell surface Differential binding of plasminogen, plasmin, and AS4.5 to actin can bind both plasminogen and angiostatin (24). As they B-actin. Plasminogen and plasmin contain five kringle domains, used a different angiostatin isoform (kringles 1-4 produced by which bind to lysines. AS4.5 shares the first four of the kringles, but limited enzymatic proteolysis of plasminogen, by Hematologic kringle five is cleaved and truncated by the plasmin autoproteolysis. Therefore, we compared the different h-actin binding affinities of plasminogen, plasmin, and AS4.5 in a solid-phase ELISA. To compare the binding affinity, h-actin was coated onto plates, and biotinylated plasminogen, plasmin, or AS4.5 was added. Prelimi- nary experiments indicated that biotinylation did not affect plasminogen, plasmin, or AS4.5 binding to h-actin (data not shown). When bound protein was measured, plasminogen and plasmin both bound to h-actin comparably and in a dose-dependent manner (Fig. 2B). AS4.5, however, did not show any significant binding to h-actin (Fig. 2B). Therefore, intact kringle 5 in plasminogen and plasmin may be necessary for actin binding because AS4.5 contains kringles 1 to 4 but has a truncated kringle 5. Purified kringle 5-B-actin binding in a solid-phase ELISA. To test the ability of kringle 5 to bind actin, we obtained purified recombinant kringle 5 protein. Kringle 5 bound to h-actin in a dose-dependent manner (Fig. 2C). In contrast, AS4.5 did not bind h A to -actin in concentrations as high as 40 mol/L (Fig. 2B and C). Figure 4. Inhibition of AS4.5 generation by peptides and qACA. Plasminogen Furthermore, when tested for its ability to competitively inhibit (440 nmol/L of 40 Ag/mL) was incubated with glutaraldehyde-fixed PC-3 cells. plasminogen binding to h-actin, intact kringle 5 inhibited plasmi- Peptide C, C1, or C2, or qACA at 1 mmol/L was used as an inhibitor. AS4.5 f generation was measured by ELISA. qACA and peptide C significantly inhibited nogen binding by 40%, whereas AS4.5 did not inhibit plasminogen- plasminogen binding (P < 0.001 and P < 0.01, respectively). Peptide C1 and C2 actin binding at the same concentrations. These data further exhibited no significant inhibition of plasminogen binding.

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tPA on endothelial cells results in a 2.5- to 3.0- fold increase in endothelial cell penetration of matrix (25). In another study, Tarui et al. observed that plasmin binds to integrin avh3 via the kringle domains and that this interaction induces the migration of endothelial cells (26). This migration required both plasmin binding to integrin avh3 and the plasmin catalytic activity (26). Angiostatin can inhibit both plasmin-induced endothelial cell invasion and migration and has been reported to bind to avh3 (25, 26). The proangiogenic activity of plasmin may also be mediated through induction of other angiogenic factors. Vascular endothelial growth factors [VEGF; (VEGF-C and VEGF-D)] are potent angiogenesis inducers (27, 28). Plasmin can activate VEGF-C and VEGF-D by propeptide cleavage, generating mature forms that have markedly higher binding affinity to their receptors (29). Figure 5. Model of plasminogen, plasmin, and AS4.5 interaction with h-actin on the cell surface. Plasminogen (PLGN) binds to the cell surface actin via Therefore, in the setting of cancer, plasminogen activation to kringle 5. Urokinase (uPA), bound to its receptor (uPAR), cleaves plasminogen to plasmin may have complex effects. The locally generated plasmin (PLSMN). Plasmin remains tightly bound to actin. Plasmin, when bound plasmin may induce angiogenesis by either inducing migration to actin, undergoes autoproteolysis within kringle 5 to form AS4.5. AS4.5, with truncation of kringle 5, can no longer bind to actin, is released from the cell and invasion and/or activating other angiogenic inducers surface, and can enter the systemic circulation. Thus, plasmin, with (25, 26, 29). However, if the cancer cell surface also expresses h- proangiogenic activity, remains surface bound to act locally, whereas AS4.5, a actin, then the generation and release of AS4.5 may inhibit potent angiogenesis inhibitor, acts systemically. metastatic spread as has been postulated (25, 26). The plasminogen-h-actin system may thus help explain the paradigm that Technologies, Essex Junction, VT) and not the native AS4.5 human angiogenesis inducers act locally, whereas angiogenesis inhibitors isoform, direct comparison of their results with ours is not possible. act systemically. It is possible that endothelial cell actin may have a role in differential angiostatin binding to endothelial and PC-3 cells. Acknowledgments However, we have not been able to show AS4.5 binding to actin in Received 12/5/2005; revised 3/30/2006; accepted 5/8/2006. either a cell-dependent or a cell-free system. Grant support: National Cancer Institute grants P50 CA90386 and P50 CA89018-02. Our model may explain some important observations related The costs of publication of this article were defrayed in part by the payment of page to angiogenesis and cancer. Although AS4.5 is known to inhibit charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. angiogenesis, plasmin is believed to promote angiogenesis We thank Donald Davidson for the gift of recombinant kringle 5 protein and valued (5, 25, 26). For example, plasminogen activation to plasmin by discussions on this article.

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