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Tumor or tumor-associated cells cleave circulating plasminogen into three or four kringle-containing antiangiogenic fragments, collectively referred to as angiostatin. Angiostatin blocks tumor growth and metastasis by preventing the growth of endothelial cells that are critical for tumor vascularization. Here, we show that cancer and normal cells convert plasminogen into a novel 22 kDa fragment (p22). Production of this plasminogen fragment in a cell-free system has allowed characterization of the structure and activity of the protein. p22 consists of amino acid residues 78-180 of plasminogen and therefore embodies the first plasminogen kringle (residues 84-162) as well as additional N- and C-terminal residues. Circular dichroism and intrinsic fluorescence spectrum analysis have defined structural differences between p22 and recombinant plasminogen kringle 1 (rK1), therefore suggesting a unique conformation for kringle 1 within p22. Proliferation of capillary endothelial cells but not cells of other lineages was selectively inhibited by p22 in vitro. In addition, p22 prevented vascular growth of chick chorioallantoic membranes (CAMs) in vivo. Furthermore, administration of p22 at low dose suppressed the growth of murine Lewis lung carcinoma (LLC) metastatic foci in vivo. This is the first identification of a single kringle-containing antiangiogenic plasminogen fragment produced under physiological conditions.

Plasmin, a broad spectrum proteinase, is inactivated by an autoproteolytic reaction that results in the destruction of the heavy and light chains of the protein. Recently we demonstrated that a 61-kDa plasmin fragment was one of the major products of this autoproteolytic reaction (Fitzpatrick, S. L., Kassam, G., Choi, K. S., Kang, H. M., Fogg, D. K., and Waisman, D. M. (2000)Biochemistry 39, 1021–1028). In the present communication we have identified the 61-kDa plasmin fragment as a novel four kringle-containing protein consisting of the amino acid sequence Lys78–Lys468. To avoid confusion with the plasmin(ogen) fragment, angiostatin® (Lys78-Ala440), we have named this protein A61. Unlike angiostatin, A61 was producedin vitro from plasmin autodigestion in the absence of sulfhydryl donors. A61 bound to lysine-Sepharose and also underwent a large increase in fluorescence yield upon binding of the lysine analogue, trans-4-aminomethylcyclohexanecarboxylic acid. Circular dichroism suggested that A61 was composed of 21% β-strand, 14% β-turn, 18% 31-helix and 8% 310-helix. A61 was an anti-angiogenic protein as indicated by the inhibition of bovine capillary endothelial cell proliferation. Plasminogen was converted to A61 by HT1080 cells and bovine capillary endothelial cells. Furthermore, a plasminogen fragment similar to A61 was present in the serum of humans as well as normal and tumor-bearing mice. These results establish that plasmin turnover can generate anti-angiogenic plasmin fragments in a nonpathological setting.

Fucoidan, a sulfated fucopolysaccharide, mimics the fucosylated glycans of glycoproteins and has therefore been used as a probe for investigating the role of membrane polysaccharides in cell-cell adhesion. In the present report we have characterized the interaction of fucoidan with the Ca(2+)- and phospholipid-binding protein annexin II tetramer (AIIt). AIIt bound to fucoidan with an apparent K(d) of 1.24 +/- 0.69 nM (mean +/- SD, n = 3) with a stoichiometry of 0.010 +/- 0.001 mol of fucoidan/mol of AIIt (mean +/- SD, n = 3). The binding of fucoidan to AIIt was Ca(2+)-independent. Furthermore, in the presence but not the absence of Ca(2+), the binding of fucoidan to AIIt caused a decrease in the alpha-helical content from 32% to 7%. A peptide corresponding to a region of the p36 subunit of AIIt, F(306)-S(313), which contains a Cardin-Weintraub consensus sequence for heparin binding, was shown to undergo a conformational change upon fucoidan binding. This suggests that heparin and fucoidan bound to this region of AIIt. The binding of fucoidan but not heparin by AIIt also inhibited the ability of AIIt to bind to and aggregate phospholipid liposomes. These results suggest that the binding of AIIt to the carbohydrate conjugates of certain membrane glycoproteins may have profound effects on the structure and biological activity of AIIt.

The enzymatic cascade triggered by activation of plasminogen has been implicated in a variety of normal and pathologic events, such as fibrinolysis, wound healing, tissue remodeling, embryogenesis, and the invasion and spread of transformed tumor cells. Recent data established that the Ca(2+)- and phospholipid-binding protein, annexin II heterotetramer (AIIt) binds tissue-type plasminogen activator (tPA), plasminogen, and plasmin, and dramatically stimulates the tPA-dependent conversion of plasminogen to plasmin in vitro. Interestingly, the binding of plasmin to AIIt can inhibit the activity of the enzyme, suggesting that plasmin bound to the cell surface is regulated by AIIt. The existing experimental evidence suggests that AIIt is the key physiological receptor for plasminogen on the extracellular surface of endothelial cells.

In this paper, we have characterized the regulation of plasmin activity by annexin II tetramer (AIIt). Plasmin activity was measured by a fibrin lysis assay in which a fibrin polymer was produced from purified components and the extent of polymer lysis was determined by following changes in turbidity. Extrinsic lysis of the fibrin polymer, initiated by addition of tissue plasminogen activator (t-PA), was totally blocked if AIIt was present during fibrin polymer formation. Furthermore, fibrin polymer formed in the presence of AIIt was resistant to extrinsic lysis initiated by addition of plasmin. AIIt bound to fibrin polymer under conditions in which polymer lysis was inhibited. Plasmin-dependent extrinsic lysis of the fibrin polymer was also blocked if AIIt was present in the incubation medium, and under these conditions the amidolytic activity of plasmin, measured with an artificial substrate, was inhibited about 5-fold. In contrast, in the absence of fibrin, and at an AIIt/plasmin molar ratio of 526, the amidolytic activity of plasmin was inhibited by only 22.3% +/- 7.4% (mean +/- SD, n = 5) by AIIt. Plasmin-dependent fibrinolysis was only slightly inhibited if fibrin polymer was formed in the presence of annexins I, II, V, or VI. These results identify AIIt as an in vitro regulator of plasmin activity.

In this report, we have characterized the interaction of heparin with the Ca2+- and phospholipid-binding protein annexin II tetramer (AIIt). Analysis of the circular dichroism spectra demonstrated that the Ca2+-dependent binding of AIIt to heparin caused a large decrease in the alpha-helical content of AIIt from approximately 44 to 31%, a small decrease in the beta-sheet content from approximately 27 to 24%, and an increase in the unordered structure from 20 to 29%. The binding of heparin also decreased the Ca2+ concentration required for a half-maximal conformational change in AIIt from 360 to 84 microM. AIIt bound to heparin with an apparent Kd of 32 +/- 6 nM (mean +/- S.D., n = 3) and a stoichiometry of 11 +/- 0.9 mol of AIIt/mol of heparin (mean +/- S.D., n = 3). The binding of heparin to AIIt was specific as other sulfated polysaccharides did not elicit a conformational change in AIIt. A region of the p36 subunit of AIIt (Phe306-Ser313) was found to contain a Cardin-Weintraub consensus sequence for glycosaminoglycan recognition. A peptide to this region underwent a conformational change upon heparin binding. Other annexins contained the Cardin-Weintraub consensus sequence, but did not undergo a substantial conformational change upon heparin binding.

Annexin II tetramer (AIIt) is a Ca2+-dependent, phosphatidylserine-binding, and F-actin-bundling phosphoprotein which is localized to both the extracellular and cytoplasmic surfaces of the plasma membrane. The tetramer is composed of two p36 heavy chains and two p11 light chains. We have produced prokaryotic cDNA expression constructs for both p36 and p11. Both proteins were expressed in large amounts in Escherichia coli upon induction with IPTG. Electrospray ionization mass spectrometry and amino acid sequence analysis of purified recombinant p36 (rp36) and recombinant p11 (rp11) suggested that the recombinant proteins were identical to their native counterparts except for the lack of N-terminal acetylation of rp36. Furthermore, the non-acetylated rp36 bound rp11 and formed AIIt. The circular dichroism spectra and urea denaturation profiles of acetylated AIIt and non-acetylated rAIIt were identical. In addition, both the acetylated AIIt and non-acetylated rAIIt were similar in their Ca2+ dependence and concentration dependence of phospholipid liposome aggregation, chromaffin granule aggregation, and F-actin bundling. These results suggest that N-terminal acetylation of p36 is not in fact necessary for binding of the protein to p11 and that N-terminal acetylation does not affect the conformational stability of AIIt or the in vitro activities of AIIt. The availability of large amounts of rAIIt will facilitate further characterization of the structure-function relationships of the protein.

In this study, we investigated the effects of testosterone and a nonaromatizable androgen, 11 beta-hydroxyandrosterone, on maturational gonadotropin (GtH-II) subunit gene expression in the goldfish pituitary. While testosterone treatment at physiological doses resulted in stimulation of GtH-II-alpha and -beta subunit mRNA production, time-course and dose-response studies performed on sexually immature goldfish of mixed sex, using a wider dose range exceeding physiological levels, demonstrated a biphasic response to in vivo androgen treatment. Time-related treatment with testosterone and 11 beta-hydroxyandrosterone (20 micrograms/fish) resulted in an initial inhibition of GtH-II subunit mRNA production (12-24 h) followed by stimulation at 72-96 h. In dose-response studies, treatment for 24 h with testosterone resulted in a significant stimulation at the low physiological doses of 0.2 and 2 micrograms/fish. At the supraphysiological level of 20 micrograms/fish, testosterone treatment resulted in no stimulation or in decreased GtH-II subunit mRNA levels compared to the control values. Similarly, treatment with 11 beta-hydroxyandrosterone resulted in a significant stimulation of GtH-II subunit mRNA levels at low physiological concentrations (0.2 microgram/ fish) and an inhibition, or no stimulation, at higher concentrations (2-20 micrograms/fish). In sexually mature goldfish of mixed sex, the biphasic effect of testosterone was not observed in vivo, and treatment with this steroid resulted in stimulation of GtH-II subunit mRNA production in a dose-related manner. To investigate the direct action of testosterone, studies were carried out using isolated goldfish pituitary fragments from goldfish of mixed sex in vitro. Treatment with testosterone at various concentrations was found to stimulate GtH-II subunit mRNA production in pituitary glands obtained from both sexually immature and sexually mature goldfish. Overall, the present study demonstrates a stimulatory effect of testosterone on GtH-II subunit mRNA levels in goldfish. The observed stimulation of basal GtH-II subunit mRNA production by testosterone occurs, in part, through a direct action at the level of the pituitary in both sexually immature and mature goldfish.

In a previous report we showed that plasmin-dependent lysis of a fibrin polymer, produced from purified components, was totally blocked if annexin II heterotetramer (AIIt) was present during fibrin polymer formation. Here, we show that AIIt inhibits fibrin clot lysis by stimulation of plasmin autodegradation, which results in a loss of plasmin activity. Furthermore, the C-terminal lysine residues of its p11 subunit play an essential role in the inhibition of fibrin clot lysis by AIIt. We also found that AIIt binds to fibrin with a K(d) of 436 nm and a stoichiometry of about 0.28 mol of AIIt/mol of fibrin monomer. The binding of AIIt to fibrin was not dependent on the C-terminal lysines of the p11 subunit. Furthermore, in the presence of plasminogen, the binding of AIIt to fibrin was increased to about 1.3 mol of AIIt/mol of fibrin monomer, suggesting that AIIt and plasminogen do not compete for identical sites on fibrin. Immunohistochemical identification of p36 and p11 subunits of AIIt in a pathological clot provides important evidence for its role as a physiological fibrinolytic regulator. These results suggest that AIIt may play a key role in the regulation of plasmin activity on the fibrin clot surface.

Annexin II tetramer (AIIt) is an important endothelial cell surface protein receptor for plasminogen and t-PA. AIIt, a heterotetramer, is composed of two p36 subunits (called annexin II) and two p11 subunits. In this report, we have compared the ability of the isolated p36 and p11 subunits to stimulate t-PA-dependent [Glu]plasminogen activation. The fluid-phase recombinant p11 subunit stimulated the rate of t-PA-dependent activation of [Glu]plasminogen about 46-fold compared to an approximate stimulation of 2-fold by the recombinant p36 subunit and 77-fold by recombinant AIIt. The stimulation of t-PA-dependent activation of [Glu]plasminogen by the p11 subunit was Ca2+-independent and inhibited by epsilon-aminocaproic acid. [Glu]Plasminogen bound to a p11 subunit affinity column and could be eluted with epsilon-aminocaproic acid. Both AIIt and the p11 subunit protected t-PA and plasmin from inactivation by PAI-1 and alpha2-antiplasmin, respectively. A peptide to the C terminus of the p11 subunit (85-Y-F-V-V-H-M-K-Q-K-G-K-K-96) inhibited the p11-dependent stimulation of t-PA-dependent plasminogen activation. In addition, a deletion mutant of the p11 subunit, missing the last two C-terminal lysine residues, retained only about 15% of the activity of the wild-type p11 subunit. Similarly, a mutant AIIt composed of the wild-type p36 subunit and the p11 subunit deletion mutant possessed about 12% of the wild-type activity. These results, therefore, suggest that the C-terminal lysine residues of the p11 subunit bind plasminogen and participate in the stimulation of t-PA-dependent activation of plasminogen by AIIt.

Annexin II tetramer (AIIt) is a major Ca2+-binding protein of endothelial cells which has been shown to exist on both the intracellular and extracellular surfaces of the plasma membrane. In this report, we demonstrate that AIIt stimulates the activation of plasminogen by facilitating the tissue plasminogen activator (t-PA)-dependent conversion of plasminogen to plasmin. Fluid-phase AIIt stimulated the rate of activation of [Glu]plasminogen about 341-fold compared with an approximate 6-fold stimulation by annexin II. AIIt bound to [Glu]plasminogen(S741C-fluorescein) with a Kd of 1. 26 +/- 0.04 microM (mean +/- S.D., n = 3) and this interaction resulted in a large conformational change in [Glu]plasminogen. Kinetic analysis established that AIIt produces a large increase of about 190-fold in the kcat, app and a small increase in the Km,app which resulted in a 90-fold increase in the catalytic efficiency (kcat/Km) of t-PA for [Glu]plasminogen. AIIt also stimulated the t-PA-dependent activation of [Lys]plasminogen about 28-fold. Furthermore, other annexins such as annexin I, V, or VI did not produce comparable activation of t-PA-dependent conversion of [Glu]plasminogen to plasmin. The stimulation of the activation of [Glu]plasminogen by AIIt was Ca2+-independent and inhibited by epsilon-aminocaproic acid. AIIt bound to human 293 cells potentiated t-PA-dependent plasminogen activation. AIIt that was bound to phospholipid vesicles or heparin also stimulated the activation of [Glu]plasminogen 5- or 11-fold, respectively. Furthermore, immunofluorescence labeling of nonpermeabilized HUVEC revealed a punctated distribution of AIIt subunits on the cell surface. These results therefore identify AIIt as a potent in vitro activator of plasminogen.

La presente etude a eu pour but d'examiner les effets de la testosterone et d'un androgene non aromatisable, la 11β-hydroxyandrosterone, sur l'expression genique de l'hormone de croissance dans l'hypophyse du poisson rouge. Chez le poisson rouge sexuellement immature, un traitement avec des doses physiologiques de testosterone et de 11β-hydroxyandrosterone a significativement augmente la production d'ARNm de l'hormone de croissance au-dela des taux de base. Toutefois, un traitement avec des doses supra-physiologiques de ces androgenes a induit une plus faible stimulation ou une inhibition des taux d'ARNm de l'hormone de croissance. Chez le poisson rouge sexuellement mature, le traitement a la testosterone a stimule les taux d'ARNm de l'hormone de croissance de maniere dose-dependante et avec un plus faible degre de sensibilite comparativement a ce qui a ete observe chez le poisson sexuellement immature. Pour etudier l'action directe de la testosterone au niveau de l'hypophyse, des hypophyses isolees provenant de poissons sexuellement matures et immatures ont ete examinees in vitro. Une stimulation dose-dependante du taux d'ARNm de l'hormone de croissance hypophysaire a ete observee chez les deux groupes de poissons rouges. Dans l'ensemble, les presents resultats indiquent que des concentrations physiologiques d'androgenes stimulent la synthese de l'hormone de croissance hypophysaire chez les poissons rouges sexuellement matures et immatures. L'effet stimulateur de la testosterone se produit directement au niveau de l'hypophyse ; toutefois, la testosterone pourrait aussi exercer des effets indirects par biais d'autres facteurs neuroendocriniens.