To obtain a recombinant human plasminogen (hPLG) with potential anti-platelet aggregation activity, we cloned the cDNA coding Pro544 to Asn791 of hPLG, a kringle-deficit derivative (hPLG-deltaK). The Pro559 in activation loop was then mutated into Asp559 to provide Arg-Gly-Asp (RGD) motif. The constructed pPICZalphaA-RGD-HPLG-deltaK plasmid was expressed in yeast Pichia pastoris GS115, which produced RGD-hPLG-deltaK about 0.160 g/L broth. After affinity chromatography, the purity of the recombinant protein reached above 90%. Western blotting test confirmed that it retained the immunological reaction capability as human PLG. Its urokinase activation rate in 24 hours and its fibrinolytic activity made no deference against native hPLG-deltaK (P=0.630, n=5). Importantly, after activation by urokinase, RGD-hPLG-deltaK showed a significantly higher platelet aggregation inhibition rate (Ri) (21.8% +/- 1.57%) than hPLG-deltaK (3.8% +/- 0.33%) (P=0.000, n=5). These results proved that we constructed an hPLG mutant with anti-platelet aggregation activity, which made a foundation for developing innovative thrombolytic drugs with multifunction.
Humans ; Kringles ; genetics ; Oligopeptides ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Plasminogen ; biosynthesis ; genetics ; Platelet Aggregation Inhibitors ; isolation & purification ; pharmacology ; Point Mutation ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; pharmacology

Kringle 1-3 domain is a recently found angiogenesis inhibitor with anti-angiogenesis and anti-tumor activity. The kringle 1-3 gene was amplified by PCR technique using angiostatin gene as template. After DNA sequencing, the PCR product was cloned into pPIC9K resulting in recombinant plasmid pPIC9K13 which was then transformed into Pichia pastoris GS115. The high copy integration transformants screened by PCR and G418 methods were cultivated in flasks. The K1-3 was expressed and secreted to the medium and has immunogenic activity as shown by SDS-PAGE and Western blotting. High cell density culture was carried out in 30-liter and 80-liter bioreactor, the biomass reaches 300 OD after methanol induction, and the expressed product is 200 mg/L. The fermentation supernatant was purified by Streamline SP and Phenyl Sepharose Chromatography, the product appears as a single band on SDS-PAGE, with a purity of 95%-96%. The purified product has anti-angiogenesis and anti-tumor activity.
Bioreactors ; Cloning, Molecular ; Fermentation ; Humans ; Kringles ; genetics ; Pichia ; genetics ; Plasmids ; Plasminogen ; genetics ; isolation & purification ; pharmacology ; Recombinant Proteins ; biosynthesis ; isolation & purification ; pharmacology

OBJECTIVETo assess the atherogenicity of lipoprotein(a), the effect of the heterogeneity of lysine binding of apolipoprotein(a) [apo(a)], a plasminogen-like glycoprotein component on the proliferation of human arterial smooth muscle cells (SMCs).

METHODSBoth wild type (wt) Trp72 and mutant (mut) Trp72-->Arg forms of apo(a) kringle IV-10 were expressed by employing a GST-gene fusion system into E. coli. The proliferation of SMCs was determined by flow cytometry and MTT colorimetry. Enzyme-linked immunosorbent assay (ELISA) assay was used to detect the active form of transforming growth factor beta(1) (TGF-beta(1)).

RESULTSApo(a) wt-kringle IV-10 that has lysine binding properties possessed a growth-stimulating activity to SMCs on a dose-dependence manner by stimulating cells in the G(1)/G(0) phase of cell cycle to S and G(2)/M phase, and reduced significantly the amounts of endogenous active TGF-beta(1) in culture when compared with the control medium and the GST group (2.4 +/- 0.5 vs 8.6 +/- 1.6 and 9.1 +/- 1.7 ng/ml, P < 0.01). The growth-stimulating effect of apo(a) mut-kringle IV-10 deficient in lysine binding was negligible.

CONCLUSIONSApo(a) induces SMCs growth by inhibiting the activation of latent TGF-beta(1), an activity that may involve the ability of apo(a) kringle IV-10 to bind lysine. The mitogenic effect of apo(a) wt-kringle IV-10 on SMCs might play an active role in the atherogenic function of lipoprotein(a).

Apolipoproteins ; genetics ; metabolism ; Apoprotein(a) ; Cell Division ; physiology ; Humans ; In Vitro Techniques ; Kringles ; genetics ; Lipoprotein(a) ; genetics ; metabolism ; Muscle, Smooth, Vascular ; cytology ; Transforming Growth Factor beta ; metabolism

Angiostatin is a potent angiogenesis inhibitor that is composed of the first four kringles of plasminogen fragment. Angiostatin with one less kringle molecule (kringle 1 to 3) was recently demonstrated to be an effective angiogenic inhibitor. To determine whether recombinant plasminogen kringle 1-3 (rPK1-3) can inhibit the corneal neovascularization induced by potent angiogenic factors; angiogenin, bFGF, or VEGF, hydron polymer discs each containing 2.0 microg of angiogenin, 500 ng of bFGF, or 500 ng of VEGF respectively were implanted into the corneal stroma of 138 rabbit eyes, and then discs each containing 10 microg, 12.5 microg, 20 microg or 30 microg of rPK1-3 were implanted randomly. Discs containing phosphate buffered saline were also implanted as a control. The angiogenesis score on number and length of newly formed vessels on the each of the rabbit's cornea were recorded daily by two observers (blinded). The treated corneas were also examined histologically. Recombinant PK1-3 treated corneas showed less neovascularization induced by all angiogenic factors (p < 0.05). and the extent of inhibition of neovascularization was proportional to the concentration of rPK1-3 (p < 0.05). Histologic examination showed leukocyte infiltration into the corneal stroma on the PBS treated eyes whereas rPK1-3 treated eyes showed only traces of leukocytes. These results of the effective rPK1-3 inhibition of corneal neovascularization induced by angiogenin, bFGF, or VEGF suggest that this angiostatin related fragment, rPK1-3, may be useful in the treatment of various neovascular diseases. Copyright 2000 Academic Press.
Angiogenesis Inhibitors/pharmacology* ; Angiogenesis Inhibitors/genetics ; Animal ; Chick Embryo ; Chorion/drug effects ; Chorion/blood supply ; Cornea/pathology ; Cornea/drug effects ; Cornea/blood supply* ; Endothelial Growth Factors/pharmacology ; Fibroblast Growth Factor, Basic/pharmacology ; Kringles/genetics ; Lymphokines/pharmacology ; Microscopy/methods ; Neovascularization, Pathologic/drug therapy* ; Plasminogen/pharmacology* ; Plasminogen/genetics* ; Rabbits ; Recombinant Proteins/pharmacology ; Recombinant Proteins/genetics ; Ribonuclease, Pancreatic/pharmacology

The serine protease urokinase-type plasminogen activator (uPA) is implicated in pericellular proteolysis in a variety of physiological and pathological processes including angiogenesis and tumor metastasis. The kringle domain of uPA (UK1) has proven to be an anti-angiogenic molecule with unknown mechanism and amino terminal fragment of uPA (u-ATF) with additional growth factor-like domain can be used for blocking interaction of uPA and uPA receptor. Here, we compared anti-angiogenic activities of these two molecules in vitro and in vivo. The recombinant u-ATF from E. coli and refolded in vitro was found to bind to uPAR with high affinity, whereas E. coli-derived UK1 showed no binding by Biacore analysis. In contrast to UK1 having potent inhibitory effect, u-ATF exhibited low inhibitory effect on bovine capillary endothelial cell growth (ED(50)>320 nM). Furthermore, u-ATF inhibition of VEGF-induced migration of human umbilical vein endothelial cell was far less sensitive (IC(50)= 600 nM) than those observed with UK1, and angiogenesis inhibition was marginal in chorioallantoic membrane. These results suggest that kringle domain alone is sufficient for potent anti- angiogenic activity and additional growth factor-like domain diverts this molecule in undergoing different mechanism such as inhibition of uPA/uPAR interaction rather than undergoing distinct anti- angiogenic mechanism driven by kringle domain.
Animals ; Biosensing Techniques ; Cattle ; Cell Division/drug effects ; Cell Movement/*drug effects ; Cells, Cultured ; Chickens ; Cricetinae ; Endothelial Cells/*cytology/*drug effects ; Humans ; Kinetics ; *Kringles ; Ligands ; Peptide Fragments/*chemistry/genetics/metabolism/*pharmacology ; Protein Binding ; Receptors, Cell Surface/metabolism ; Receptors, Urokinase Plasminogen Activator ; Urokinase-Type Plasminogen Activator/*chemistry/genetics/pharmacology ; Vascular Endothelial Growth Factor A/pharmacology