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Characterization of kringle domains of angiostatin as antagonists of endothelial cell migration, an important process in angiogenesis

^a Department of Oncology Drug Discovery, Bristol-Myers Squibb Pharmaceuticals, Inc., Princeton, New Jersey 08543, USA
^b Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213–2683, USA
^c Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA
^d Department of Chemistry and Biochemistry, University of Bern, CH-3012 Bern, Switzerland

	ABSTRACT

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Angiogenesis is a complex process that involves endothelial cell proliferation, migration, basement membrane degradation, and neovessel organization. Angiostatin, consisting of four homologous triple-disulfide bridged kringle domains, has previously been shown to exhibit profound inhibition of endothelial cell proliferation in vitro and angiogenesis in vivo. It was also demonstrated that angiostatin could suppress the growth of a variety of tumors via the blocking of angiogenesis. The primary aim of our study was to characterize the kringle domains of angiostatin for their inhibitory activities of endothelial cell migration in order to elucidate their contributions to the anti-angiogenic function of angiostatin. In this report, we demonstrate for the first time that the kringles of angiostatin play different roles in inhibiting endothelial cell migration, a crucial process in angiogenesis. Kringle 4, which has only marginal anti-proliferative activity, is among the most potent fragments in inhibiting endothelial cell migration (IC₅₀ of approximately 500 nM). In contrast, kringle 1–3, which is equivalent to angiostatin in inhibiting endothelial cell proliferation, manifests only a modest anti-migratory effect. The combination of kringle 1–3 and kringle 4 results in an anti-migratory activity comparable to that of angiostatin. When kringle 1 is removed from kringle 1–3, the resulting kringle 2–3 becomes more potent than kringle 1–3. This implies that kringle 1, although virtually ineffective in inhibiting endothelial cell migration, may influence the conformation of kringle 1–3 to alter its anti-migratory activity. We also show that disruption of the kringle structure by reducing/alkylating agents markedly attenuates the anti-migratory activity of angiostatin, demonstrating the significance of kringle conformation in maintaining the anti-angiogenic activity of angiostatin. Our data suggest that different kringle domains may contribute to the overall anti-angiogenic function of angiostatin by their distinct anti-migratory activities.—Ji, W. R., Castellino, F. J., Chang, Y., DeFord, M. E., Gray, H., Villarreal, X., Kondri, M. E., Marti, D. N., Llinás, M., Schaller, J., Kramer, R. A., and Trail, P. A. Characterization of kringle domains of angiostatin as antagonists of endothelial cell migration, an important process in angiogenesis. FASEB J. 12, 1731–1738 (1998)

Key Words: plasminogen • kringle, • BCE cells • fibroblast growth factor

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
ANGIOGENESIS is the process by which new capillaries are formed from preexisting blood vessels. In normal adults, angiogenesis is highly regulated and is involved in a wide array of physiological conditions, such as placenta development and embryogenesis. An imbalance of the angiogenic process has been shown to contribute to pathological disorders such as diabetic retinopathy, rheumatoid arthritis, and psoriasis (1, 2). In particular, both primary and metastatic tumors need to recruit angiogenic vessels for their growth (3, 4).

There is a mounting body of evidence supporting the concept that angiogenesis is a prerequisite for tumor growth. Blocking positive regulators of angiogenesis or utilizing negative regulators to suppress angiogenesis results in a delay or regression of experimental tumors. For instance, vascular endothelial growth factor (VEGF)³ and fibroblast growth factor (FGF) are potent mitogens and strong chemoattractants for endothelial cells (5–7). Their roles in inducing tumor angiogenesis have been demonstrated in a variety of human tumors (8, 9). Antibodies neutralizing VEGF or FGF caused a marked decrease of tumor growth via angiogenic inhibition (10, 11). Such anti-angiogenic and anti-tumor effects were also observed by antagonizing the corresponding receptors for these endothelial cell mitogens (12, 13). In addition, negative regulators of angiogenesis, such as angiostatin, endostatin, and antagonists for integrin vß3, displayed profound anti-tumor activities in vivo (14–16). TNP-470 and Interferon alfa-2a also manifested clinical evidence that tumor growth could be therapeutically intervened when using an anti-angiogenic approach (17, 18).

Angiostatin is an endogenous anti-angiogenic agent initially isolated from urine and sera of mice bearing Lewis lung carcinoma (14). It was found to inhibit endothelial cell proliferation in vitro and to block basic FGF-elicited angiogenesis in vivo in corneal micropocket assay. Angiostatin, produced by proteolytic cleavage, has been shown to be a 38 kDa internal fragment of plasminogen, which consists of four triple-disulfide bridged kringle structures. Kringles 1 and 4 have lysine binding sites, which are responsible for anchoring the plasminogen molecule on fibrin-rich blood clots (19, 20). Angiostatin was first produced from plasminogen by proteolytic cleavage with porcine pancreatic elastase (14). Subsequent studies have shown that angiostatin can be generated from plasminogen by a variety of physiological and pathological proteases, including macrophage-derived metalloelastases (21), members of the matrix metalloproteinase (MMP) family such as matrilysin (MMP-7), gelatinase B/type IV collagenase (MMP-9) (22), and urokinase (23). Angiostatin was shown to induce dormancy of several metastatic and primary tumors, including carcinomas of breast, prostate, colon, and lung (14, 24), and to reduce the growth of a murine hemangioendothelioma in vivo (25). Recombinant angiostatin also produced anti-tumor effects in vivo via the blocking of tumor angiogenesis (26, 27). These anti-tumor effects were accompanied by a marked reduction of microvessel density within the tumor mass, indicating that suppression of angiogenesis was associated with the inhibition of tumor growth.

The individual kringle domains of angiostatin were shown to exhibit distinct inhibitory activities of endothelial cell proliferation (28). Kringle 1–3 manifested a potent inhibition of endothelial cell proliferation, whereas kringle 4 had only a marginal effect. The kringle conformation was also shown to be essential for the potent anti-proliferative activity of angiostatin. Angiogenesis is a complex process that entails an orchestration of endothelial cell proliferation, migration, basement membrane degradation, and neovessel assembly. Thus, it is important to elucidate which of these processes is modulated by angiostatin and its related kringle fragments. In this report, we demonstrate for the first time that the kringle fragments of angiostatin display distinct inhibitory functions on endothelial cell migration.

	MATERIALS AND METHODS

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Gene construction, expression, and purification of recombinant human angiostatin
The human angiostatin fragment was amplified from a human plasminogen cDNA template (American Type Culture Collection, Rockville, Md.) by standard polymerase chain reaction (PCR) with the following two primers: 5'-GCGGATCCATGAAAGTGTATCTCTCAGAGTGCAAG (forward primer for residue 98–458) and 5'-GCGGATCCTCACTATTCTGTTCCTGAGCA TTTTTTCAG (reverse primer for residue 98–458). The amplified cDNA fragment was ligated into the BamHI site of the pMelBacA vector (InVitrogen, San Diego, Calif.). The pMelBac A vector containing angiostatin cDNA was then cotransfected into Sf9 cells with viral BaculoGold DNA (PharMingen, San Diego, Calif.). Briefly, 1 x 10⁶ Sf9 cells were seeded in a T25 tissue culture flask and incubated at 27°C with 1 ml of the transfection solution containing 2 µg of transfer vector DNA, 0.5 µg of BaculoGold DNA, and 6 µl of Cellfectin (Gibco BRL, Gaithersburg, Md.). The transfection solution was removed 4 h posttransfection and replaced with 3 ml of Sf900II medium (Gibco BRL). Four days after incubation, the viral supernatant was harvested and individual clones were identified by limiting dilution. The clone with the highest protein expression, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/Coomassie blue staining, was amplified in Sf9 cells for protein production. High Five insect cells (InVitrogen) (1.5x10⁶ cells/ml) were infected with approximately 1 x 10⁷ viral particles/ml of the recombinant virus. After 48 h, the culture supernatant was collected by centrifugation at 5000 x g for 30 min. The supernatant was then applied to a lysine Sepharose column and angiostatin protein eluted with -aminocapronic acid (E-ACA) as previously described (26).

Gene construction, expression, and purification of kringle fragments of angiostatin
The cDNA fragments encoding K1, K2, K3, K4, K1–3, and K2–3 were amplified from human plasminogen cDNA template (American Type Culture Collection) by standard PCR.

PCR fragments of K2, K3, and K2–3 were cloned into pQE8 vector and recombinant proteins expressed in bacterial strain M15 (K3 and K2–3) and BL21 (K2) as previously described (29, 30). The cysteine residue 169 of K2 and 297 of K3 were mutated to serines in order to eliminate interkringle disulfide bridge formation. Recombinant kringle 2–3 also contains a hexahistidine tag for purification. Recombinant K2, K3, and K2–3 were purified to homogeneity as described (29, 30).

The recombinant K1 and K4 were expressed in the Pichia pastoris system and purified to homogeneity as described elsewhere (31, 32). The K1–3 fragment was produced as previously reported (33). For production of the K1 and K4 fragments, the plasmid constructs, pPIC9K[K1_HPg-Xa-K5_HPg] and pPIC9K[K4_HPg-Xa-K5_HPg], were linearized with restriction endonuclease Sac I and used for transformation of the GS-115 and KM 71 strain of Pichia pastoris by electroporation, respectively. Selection procedures for isolation of appropriate clones and protocols for high bio-mass fermentation were performed as described in other reports (31, 32). The fermentation medium was passed over a lysine-Sepharose column as previously described (32). The K1_HPg-K5_HPg protein was extensively dialyzed against water, lyophilized, and subsequently digested with factor Xa (FXa) (Enzyme Research Labs., South Bend, Ind.) for 6 h at room temperature in 0.05 M Tris/5 mM CaCl₂ (pH 7.4) using a protein:FXa ratio of 1:50 (w/w). The K4_HPg-K5_HPg protein was digested with elastase for 16 h at room temperature in 0.1 M phosphate/15 mM E-ACA (pH 7.8) using a protein:elastase ratio of 1:250 (w/w). The digests were dialyzed against water to remove the E-ACA and then reapplied to a lysine-Sepharose column. The recombinant proteins were batch eluted from the column using 20 mM E-ACA, extensively dialyzed against water, and then lyophilized.

Endothelial cell migration assay
Bovine capillary endothelial (BCE) cells were harvested from bovine adrenal glands as previously described (34). BCE cells were maintained in Dulbecco's modified Eagle's medium (DMEM) in the presence of 10% bovine calf serum, 1% antibiotics, and 3 ng/ml of bFGF (PeproTech, Rockhill, N.J.). To evaluate endothelial cell migration, a Boyden Chamber-based assay was performed. First, the polycarbonate membranes with 8 µm pore sizes (Neuro Probe Inc., Cabin John, Md.) were coated with 100 µg/ml of collagen type I following the manufacturer's instructions (Becton Dickinson, Bedford, Mass.). BCE cells between passage 10 to 14 were harvested with 0.05% trypsin solution, washed, resuspended to a density of 75,000 cells/ml in DMEM containing 10% bovine calf serum and 10 ng/ml of bFGF, and incubated at 37°C for 30 min. During cell incubation, various concentrations of kringle fragments were loaded into the lower chambers. The collagen-coated membrane filter was placed on top of the lower chamber and the top chamber was then attached. After the 30 min incubation, BCE cells were loaded into the top chamber and incubated at 37°C for 4 h. The chemotaxis chamber was then dismantled and the filter membrane removed. The nonmigrated cells were scraped from the upper surface of the membrane with cotton swabs three times. After rinsing with phosphate-buffered saline, the membrane was fixed with 10% buffered formalin for 45 min and then stained with Gill No. 2 hematoxylin overnight (J. B. Baker, Phillipsburg, N.J.). The membrane was then rinsed with phosphate-buffered saline and mounted with Cytoseal (Stephens Scientific, Riverdale, N.J.). Each sample was tested in quadruplicate and a representative field in each well was counted at 100x magnification to determine the number of migrated cells.

Reduction and alkylation of angiostatin
Fifty micrograms of the recombinant angiostatin in 300 µl of serum-free DMEM was reduced by incubating with 15 µl of 0.5 M dithiothreitol for 15 min at room temperature. The reduced angiostatin protein was then alkylated with 30 µl of 0.5 M iodoacetamide. The reaction mixture was dialyzed against 500 volumes of DMEM at 4°C overnight and further dialyzed against another 500 volumes of fresh DMEM for an additional 4 h. The homogeneity of the reduced and alkylated angiostatin was analyzed by SDS-PAGE before being examined for its inhibition on BCE cell migration. The endotoxin concentrations of both native and reduced/alkylated angiostatin were evaluated according to the manufacturer's instructions (Associates of Cape Cod, Woods Hole, Mass.).

	RESULTS

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Characterization of recombinant kringle domains of human plasminogen
Human plasminogen (HPg) is the circulating proenzyme of plasmin, a serine protease that plays a pivotal role in fibrinolysis (35). HPg is present in circulation as a single-chain glycoprotein of 791 amino acids. It contains a signal leader sequence, an amino-terminal `preactivation' domain, five triple-disulfide bridged kringle domains, and a protease domain ( Fig. 1A). Angiostatin is an internal fragment of HPg and spans from K1 to K4. The four kringles of angiostatin share a high level of sequence homology. Each kringle has about 80 amino acids and consists of three pairs of disulfide bonds with two lysine binding sites on K1 and K4. To determine the anti-endothelial cell activities of individual kringles, recombinant angiostatin (K1–4), K1–3, K2–3, K1, K2, K3, and K4 were produced as described (see Materials and Methods). These kringle variants were characterized on SDS-PAGE and stained with Coomassie blue ( Fig. 1B, C). The recombinant angiostatin migrated with a greater molecular size of approximately 65 kDa. Amino-terminal sequence has verified the authenticity of the recombinant angiostatin. The higher molecular size is likely attributed to the heavy glycosylation of Baculovirus recombinant protein, a feature characteristic of the Baculovirus/insect cell expression system. K1–3 and K2–3 migrated with predicted molecular sizes of 38 and 25 kDa, respectively. Each individual kringle displayed predicted molecular sizes of 10 kDa. The only exception is K1, which manifested a greater molecular size of 17 kDa due to oligomerization (28).

View larger version (22K): [in this window] [in a new window]   Figure 1. Schematic structure and SDS-polyacrylamide gel electrophoresis analysis of kringle fragments of HPg. A) Schematic structure of human plasminogen and angiostatin. The human plasminogen molecule is composed of a signal sequence, a preactivation domain, 5 triple-disulfide bridged kringle structures, and a protease domain. Angiostatin consists of the first four kringle motifs. B) The purified recombinant angiostatin (AST), K1–3, and K2–3, were analyzed on 12% of SDS-polyacrylamide gel and stained with Coomassie blue. C) Purified recombinant K1, K2, K3, and K4 were analyzed on 12% of SDS-polyacrylamide gel and stained with Coomassie blue. The corresponding molecular size standard is indicated on the left of panels B, C.

Inhibition of endothelial cell migration by angiostatin and complementary anti-endothelial cell activities of K1–3 and K4 of HPg
Basic FGF (10 ng/ml) was used to stimulate the migration of BCE cells. Angiostatin inhibited the bFGF-induced BCE cell migration in a dose-dependent manner with an IC₅₀ of approximately 50 nM ( Fig. 2A). K1–3 displayed a marginal anti-migratory effect, whereas K4 exhibited a potent inhibitory activity with an IC₅₀ of 500 nM ( Fig. 2A and Table 1). On the other hand, K1–3 is a potent antagonist of endothelial cell proliferation whereas K4 has only negligible anti-proliferative activity (28). As K1–3 and K4 are structurally combined, the resulting angiostatin, or K1–4, becomes an effective inhibitor of both endothelial cell proliferation and migration ( Fig. 2B), suggesting that K1–3 and K4 are both required for the potent inhibitory functions of angiostatin with their complementary anti-endothelial cell activities.

View larger version (22K): [in this window] [in a new window]   Figure 2. Inhibition of endothelial cell migration by angiostatin and complementary anti-endothelial cell activities of K1–3 and K4 of HPg. A) The anti-migratory activities of angiostatin (AST), K1–3, and K4 were examined on endothelial cells in a Boyden chamber-based assay. bFGF (10 ng/ml) was used as a chemoattractant to elicit the migration of endothelial cells. The dashed line indicates the baseline migration of endothelial cells in the presence of bFGF. B) K1–3 and K4 are compared for their inhibitory efficacies of endothelial cell migration and proliferation. The black box and the hatched box represent K1–3 and K4, respectively. ++, very potent; +, potent; +/-, marginally active; -, inactive. Each data point represents the mean of three determinations (with standard error) as a percentage of inhibition.

Inhibition of endothelial cell migration with K1–3, K2–3, K1, K2, and K3
To determine the structural components that are responsible for the anti-migratory functions of angiostatin, we further examined the inhibitory activities of smaller kringle-containing fragments. K1–3 was fragmented into K1 and K2–3. As opposed to K1–3, K2–3 displayed a marked increase in the anti-migratory activity with an IC₅₀ of approximately 100 nM whereas K1 was only a modest inhibitor (IC₅₀>1000 nM) ( Fig. 3A and Table 1). These data suggest that the functional elements for the inhibition of migration may reside on K2–3. After K2–3 is further segmented into K2 and K3, both fragments manifest comparable dose-dependent anti-migratory activities relative to K2–3 ( Fig. 3B), implying that the anti-migratory motifs may be shared by both K2 and K3.

View larger version (22K): [in this window] [in a new window]   Figure 3. Inhibition of endothelial cell migration with K1–3, K1, K2–3, K2, and K3. The anti-migratory activities of K1–3, K1, and K2–3 (A) or K2–3, K2, and K3 (B) were examined on endothelial cells in a Boyden chamber-based assay. bFGF (10 ng/ml) was used as a chemoattractant to elicit the migration of endothelial cells. Dashed lines indicate the baseline migration of endothelial cells in the presence of bFGF.

Interactions among kringle fragments alter their anti-migratory activities
To elucidate the interactions among different kringles, we assessed various kringle combinations on bFGF-elicited endothelial cell migration. As compared with the potent inhibitory effect of angiostatin, K4 had a similar activity whereas K1–3 was remarkably less active ( Fig. 4A and Table 1). However, a combination of K1–3 and K4 resulted in an anti-migratory activity comparable to angiostatin, indicating that angiostatin requires the additive contributions of K1–3 and K4 for its potent anti-endothelial cell function. Contrarily, a combination of K1 and K2–3 failed to show any additive effect as compared with K2–3 alone ( Fig. 4B), which could result from two alternative mechanisms. First, the inhibitory activity of K2–3 on endothelial cells might have already been saturated so that K1 could not produce any additional anti-migratory effect. Second, K1 is too modest an antagonist to potentiate the inhibitory function of K2–3. Moreover, K2–3 had a similar anti-migratory effect as opposed to its smaller fragments, K2 or K3, or the combination of K2 and K3 ( Fig. 4C). It is conceivable that both K2 and K3 have the functional motifs for anti-migratory effects and that the interkringle interactions between K2 and K3 may alter their functions so that no additive effects could result from a combination of K2 and K3.

View larger version (13K): [in this window] [in a new window]   Figure 4. Anti-migratory effects of different combinations of kringle fragments of HPg. A) Anti-migratory effects of K1–4, K1–3, K4, and a combination of K1–3 and K4. B) Anti-migratory effects of K1–3, K1, K2–3, and a combination of K1 and K2–3. C) Anti-migratory effects of K2–3, K2, K3, and a combination of K2 and K3. A concentration of 100 nM for each kringle fragment was tested on bFGF (10 ng/ml) -elicited endothelial cell migration.

The important role of kringle structures in the potent anti-migratory function of angiostatin
Because angiostatin is composed of four triple-disulfide bridged kringle domains, it was of interest to determine the roles of these structures in suppressing endothelial cell migration. Angiostatin was thus reduced to disrupt the kringle conformation and the reduced product was alkylated to prevent refolding ( Fig. 5A). Relative to native product, the reduced angiostatin had a markedly decreased anti-migratory efficacy ( Fig. 5B), leading to the conclusion that the native kringle structures play a significant role in the functions of angiostatin.

View larger version (19K): [in this window] [in a new window]   Figure 5. Kringle structures are essential for the anti-migratory activities of angiostatin. A) Both native and reduced/alkylated angiostatin were analyzed on 12% SDS-polyacrylamide gel and stained with Coomassie blue. The corresponding molecular size standard is indicated on the left. B) Inhibition of endothelial cell migration by native and reduced/alkylated angiostatin. Dashed lines indicate the baseline migration of endothelial cells in the presence of bFGF.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Angiogenesis, the sprouting of new capillaries from preexisting vessels, plays a pivotal role in host development by supplying nutrients and oxygen and eliminating metabolic waste products. Most significant of all, angiogenesis has been substantiated as a prerequisite for tumor growth (3, 4). Inhibitors of angiogenesis have demonstrated remarkable anti-tumor effects by blocking angiogenic processes (10–18). Angiostatin was described as such a potent inhibitor of angiogenesis that it could markedly suppress the growth of a variety of tumors (14, 24, 25). Angiostatin has a complex structure including four triple-disulfide bridged kringle domains and lysine binding sites (14). It is important to identify the structural element(s) of angiostatin that contributes to its powerful anti-angiogenic effects. In this report, we demonstrate for the first time that different kringle motifs have discrete inhibitory functions on endothelial cell migration, an essential step of angiogenesis. Kringle fragments may interact with one another to alter their functions and to contribute to the potent anti-endothelial cell activities of angiostatin by their distinct and complementary activities. The triple-looped kringle structures are also reported to be crucial for angiostatin functions.

Angiogenesis requires the coordination of a series of steps, such as endothelial cell proliferation and migration. Although independent of each other, all these steps could serve as potential targets for the therapeutic intervention of angiogenesis. Despite their structural similarity and sequence homology (36, 37), kringle domains appear to have distinct inhibitory profiles on angiogenesis-associated endothelial cell activities. This observation is exemplified by the analysis of K1 and K4 of HPg. The K1 domain inhibits endothelial cell proliferation (28) but has only a modest effect on endothelial cell migration ( Table 1). In contrast, K4 is predominantly a potent anti-migratory agent with little activity on endothelial cell proliferation. Although they share high conformational uniformity and nearly 50% of sequence identity (36, 37), K1 and K4 manifest marked differences in their anti-endothelial cell activities. Additional studies are needed to identify the critical residues or structure motifs that lead to the functional discrepancies between K1 and K4.

Different kringles may interact with one another to affect their anti-endothelial cell functions. As opposed to angiostatin, K1–3 has a comparable anti-proliferative activity (28) and yet displays only a marginal effect on endothelial cell migration. K4, however, is a potent inhibitor of endothelial cell migration but has no effect on proliferation. In comparison, angiostatin, containing both K1–3 and K4, is a potent inhibitor of both endothelial cell proliferation and migration, implying that both K1–3 and K4 may be necessary for the potent anti-endothelial cell activities of angiostatin by their distinct and complementary functions. Moreover, although K1–3 is a weak anti-migratory agent, its internal fragment, K2–3, exhibited a marked increase of the anti-migratory activity. This suggests that the interactions between K1 and K2–3 may shield or suppress the functional elements on K2–3. After the K1 module is removed, the anti-endothelial elements on K2–3 may be adequately exposed and become readily accessible by endothelial cells, resulting in marked inhibition of endothelial cell migration. Additional studies will be conducted to determine the in vivo anti-angiogenic activities of these kringle-containing fragments.

Kringle structures were shown to have pronounced effects in the interactions between HPg and its effector molecules (20, 38). These domains are important in anchoring Pg on fibrinogen, fibrin, and their degradation products to activate the clot lysis cascade (39–41). The kringle-containing regions could also provide binding sites for Pg and plasmin receptors in a variety of cells (42). It was previously described that the disruption of kringle structure considerably alleviated the inhibition of endothelial cell proliferation by angiostatin (28). We now demonstrate that the integrity of kringle motifs is essential for potent anti-migratory activities of angiostatin. It was reported that another kringle-containing fragment of plasminogen, kringle 5, exhibited potent anti-proliferative effects on endothelial cells (43, 44). Taken together, the kringle domains could regulate the angiogenic process by their distinct interactions with endothelial cells. Like plasminogen, other proteins, such as tissue-type plasminogen activator and prothrombin (45, 46), also contain kringle structures and are involved in fibrinolysis. Thus, it is important to investigate these molecules for their anti-endothelial and anti-angiogenic functions.

	ACKNOWLEDGMENTS

 
We are grateful to Drs. Albert Bianchi, Joseph Fargnoli, Ji-zhong Gao, Maria Jure, Kate Lane, Joseph Naglich, and Mrs. Shirley Lasch for their invaluable suggestions on our research. We also acknowledge our gratitude to Dr. Judah Folkman for providing us BCE cells as a kind gift.

	FOOTNOTES

 
² Reprint requests.

¹ Correspondence: Department of Oncology Drug Discovery, Bristol-Myers Squibb Pharmaceuticals, Inc., Provinceline and Route 206, Princeton, NJ 08543, USA. E-mail: rji{at}ussmtp.bms.com

³ Abbreviations: E-ACA, -aminocapronic acid; FGF, fibroblast growth factor; FXa, factor Xa; VEGF: vascular endothelial growth factor; bFGF, basic fibroblast growth factor; BCE, bovine capillary endothelial cell; DMEM, Dulbecco's modified Eagle's medium; MMP, matrix metalloproteinase; PCR, polymerase chain reaction; HPg, human plasminogen; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Received for publication June 17, 1998. Revision received August 14, 1998.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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