Plasminogen activator(PA) and plasminogen activator inhibitor(PAI) are involved in many physiological or pathological events. The Sertoli cells, the important elements within the seminiferous epithelium, are thought to play a key role in spermatogenesis. The Sertoli cells secrete PA and PAI. The levels of them are modulated by hormonal and cell-mediated influences. They play a fundamental role in the maintenance of spermatogenesis, sperm motility and fertilization.
Humans ; Male ; Plasminogen Activators ; metabolism ; physiology ; Plasminogen Inactivators ; metabolism ; physiology ; Sertoli Cells ; metabolism ; physiology ; Testis ; cytology

OBJECTIVE: To assess the potential of transient expression of recombinant human plasminogen activator (rhPA) in plants as a cost-effective approach for recombinant rhPA production. METHODS: Tobacco mosaic virus-based expression vector pTMV rhPA-NSK and plant binary expression vector pJ Zera-rhPA were constructed by sequence synthesis and subcloning. The two vectors were inoculated on either or leaves agroinfiltration. The expression of recombinant rhPA in leaves was examined using Western blotting and ELISA, and the fibrinolysis activity of plant-produced rhPA was assessed by fibrin agarose plate assay (FAPA). RESULTS: Five to nine days after infiltration with an inoculum containing pTMV rhPA-NSK, necrosis appeared in the infiltrated area on the leaves of both plants, but intact recombinant rhPA was still present in the necrotic leaf tissues. The accumulation level of recombinant rhPA in infiltrated leaves was significantly higher than that in leaves ( < 0.05). The yield of recombinant rhPA was up to 0.6% of the total soluble protein (or about 60.0 μg per gram) in the fresh leaf biomass at 7 days post-inoculation. The plant-derived rhPA was bioactive to convert inactive plasminogen to active plasmin. No necrosis occurred in pJ Zera-rhPA-infiltrated leaves. The Zera-rhPA protein was partially cleaved between the site of Zera tag and rhPA sequence in both leaves. We speculated that the formation of Zera tags-induced particles in the plant cells was a dynamic process of progressive aggregation in which some of the soluble polypeptides were encapsulated in these particles. CONCLUSIONS Enzymatically active recombinant rhPA can be rapidly expressed in tobacco plants using the plant viral ampliconbased system, which offers a promising alternative for cost-effective production of recombinant rhPA.
Humans ; Plant Leaves ; Plants, Genetically Modified ; Plasminogen ; Plasminogen Activators ; metabolism ; Recombinant Proteins ; Tobacco

OBJECTIVETo observe the urokinase receptor (uPAR) expression in atherosclerotic plaques of human femoral arteries.

METHODSHuman femoral artery samples were collected from patients underwent femoral endarterectomy. Normal internal mammary artery samples were taken from bypass surgery served as control. uPAR protein distribution at shoulders, lipid pool and rupture sites of a plaque and the association with macrophages and smooth muscle cells (SMCs) were detected by immunohistochemistry methods.

RESULTSThere was no uPAR expression in intima or tunica media of normal internal mammary arteries. In atherosclerotic lesions of femoral artery, the mean optical density (A) of uPAR was 92 +/- 37 in intima and 46 +/- 28 in tunica media (P < 0.05). The intimal uPAR was coexisted with macrophages and SMCs. uPAR expression was observed at plaque shoulders and lipid pool, while the maximal expression was found at rupture sites.

CONCLUSIONThe increased expression of uPAR in atherosclerotic lesion and uPAR distribution at shoulders, lipid pool, as well as rupture sites of plaques suggest a role of uPAR in plaque rupture process.

Atherosclerosis ; metabolism ; pathology ; Endarterectomy ; Femoral Artery ; pathology ; Humans ; Receptors, Urokinase Plasminogen Activator ; metabolism ; Urokinase-Type Plasminogen Activator ; metabolism

OBJECTIVEThe purpose of this work was to investigate the distribution pattern of fibrinolytic factors and their inhibitors in rabbit tissues.

METHODSThe components of the fibrinolytic system in extracts from a variety of rabbit tissues, including tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), plasminogen (Plg), plasmin (Pl) and alpha(2) plasmin inhibitor (alpha(2)PI), were determined by colorimetric assay.

RESULTSThe tissue extracts in renal, small intestine, lung, brain and spleen demonstrated strong fibrinolytic function, in which high activity of tPA, Plg and Pl was manifested; whereas in skeletal muscle, tongue and stomach, higher activity of PAI-1 and alpha(2)PI showed obviously. Also excellent linear correlations were found between levels of tPA and PAI-1, Pl and alpha(2)PI, Plg and Pl. In related tissues, renal cortex and renal marrow showed distinctly higher activity of tPA and lower activity of PAI-1, with the levels of Plg and Pl in renal cortex being higher than those in renal marrow, where the alpha(2)PI level was higher than that in renal cortex. Similarly, the levels of tPA, Plg and Pl in small intestine were higher than those in large intestine, but with respect to PAI-1 and alpha(2)PI, the matter was reverse. In addition, the fibrinolytic activity in muscle tissue was lower, however, the levels of tPA, Plg, and Pl in cardiac muscle were obviously higher than those in skeletal muscles, and the levels of PAI-1 and alpha(2)PI were significantly lower than those in skeletal muscle.

CONCLUSIONOur data demonstrate that a remarkable difference of the fibrinolytic patterns exists in rabbit tissues, which has probable profound significance in understanding the relationship between the function of haemostasis or thrombosis and the physiologic function in tissues.

Animals ; Female ; Fibrinolysin ; metabolism ; Fibrinolysis ; Gastric Mucosa ; metabolism ; Gastrointestinal Tract ; metabolism ; Intestinal Mucosa ; metabolism ; Male ; Organ Specificity ; Plasminogen ; metabolism ; Plasminogen Activator Inhibitor 1 ; metabolism ; Rabbits ; Tissue Extracts ; metabolism ; Tissue Plasminogen Activator ; metabolism ; alpha-2-Antiplasmin ; metabolism

OBJECTIVETo explore the relationship between phlegm-stasis syndrome (PSS) and the fibrinolytic status in patients with non-alcoholic fatty liver (NAFL).

METHODSSeventy patients with NAFL were divided into the PSS group and non-PSS group according to TCM Syndrome typing, and a control group consisted of 28 healthy subjects was set up. Levels of plasminogen (PLG), tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1) and D-dimer were determined and compared.

RESULTSThe activity of t-PA in NAFL patients was significantly lower than that in the control group (P<0.05), and PLG and PAI-1 were significantly higher than those in the control group (P<0.05). In respect to the TCM Syndrome typing, in patients of PSS, t-PA was significantly lower and PLG, PAI-1 were significantly higher than those in patients of non-PSS (P<0.05 or P<0.01), while D-dimer was insignificantly different between patients of the two Syndrome types (P>0.05).

CONCLUSIONNAFL patients of PSS type shows significant lower of fibrinolytic activity, indicating that there is certain degree of microcirculatory disturbance and hyper viscosity state, so the application of dissolving phlegm and dispelling stasis principle in treating NAFL is significant.

Adult ; Aged ; Diagnosis, Differential ; Fatty Liver ; blood ; diagnosis ; Female ; Fibrinolysis ; Humans ; Male ; Medicine, Chinese Traditional ; Middle Aged ; Plasminogen ; metabolism ; Plasminogen Activator Inhibitor 1 ; blood ; Tissue Plasminogen Activator ; blood

AIMTo investigate the influences of testosterone with varied concentrations on the functions of HUVEC.

METHODSHuman umbilical vein endothelial cells within 2-3 passages were cultured with testosterone (3 x 10(-10) to 3 x 10(-8), 3 x 10(-6), 3 x 10(-5) mol/ L), and the control confluent cells were cultured in the same medium without steroid. MTT experiment was repeated for 7 days to investigate each groups' cell proliferation. The values of NO were tested as recommended. The tPA and PAI-1 antigen levels were assayed with ELISA Kits.

RESULTSTestosterone at physiologic or lower concentrations (3 x 10(-10) to 3 x 10(-8) mol/L ) had no adverse effect on A490 and NO level, meanwhile, stimulated the secretion of tPA (P < 0.01). However, tPA levels markedly reduced at larger dose (3 x 10(-6) to 3 x 10(-5) mol/L). On the other hand, PAI-1 antigen levels decreased significantly at the testosterone concentrations ranging from 3 x 10(-10) to 3 x 10(-5) mol/L (P < 0.05).

CONCLUSIONTestosterone at physiologically relevant concentrations affectively decreased PAI-1, while increased tPA levels, which suggested that testosterone might have beneficial effects on the Human umbilical vein endothelial cells and cardiovascular system to prevent atherosclerosis.

Cells, Cultured ; Human Umbilical Vein Endothelial Cells ; drug effects ; metabolism ; Humans ; Nitric Oxide ; metabolism ; Plasminogen Activator Inhibitor 1 ; metabolism ; Testosterone ; pharmacology ; Tissue Plasminogen Activator ; metabolism

OBJECTIVETo elucidate how chronic prostatitis affects the expression and activity of the plasminogen activator (PA) system and relates to male infertility.

METHODSTwenty-three normal fertile males and 80 chronic prostatitis patients (40 fertile and 40 infertile) were included in this research. SDS polyacrylamide gel electrophoresis and fibrin overlay method were used to estimate the total PA, and tissue PA (tPA), urokinase type PA (uPA) in semen.

RESULTSTotal PA, tPA and uPA highly expressed in normal males, but decreased in the semen of the chronic prostatitis patients of both the fertile and infertile groups. However, there was no significant difference in total PA between the fertile and infertile patients.

CONCLUSIONChronic prostatitis reduces the secretory function and PA synthesis and secretion of the prostate, but the decrease of PA alone does not cause infertility. PA may be one of the tools for estimating the function of the prostate.

Adult ; Case-Control Studies ; Chronic Disease ; Humans ; Infertility, Male ; metabolism ; Male ; Prostatitis ; metabolism ; Semen ; metabolism ; Tissue Plasminogen Activator ; biosynthesis ; Urokinase-Type Plasminogen Activator ; biosynthesis

In order to further investigate the effect of annexin II (Ann-II) on tissue plasminogen activator (t-PA)-dependent plasminogen (PLG) activation and its interactive mechanism, recombinant native Ann-II bound t-PA, PLG and plasmin with high affinity was examined. The flow cytometric assay showed that the ann-II expression rate was higher in the human umbilical vein endothelial cell (HUVEC) (87.65%) than in the HL-60 cells as controls (35.79%). Two irrelevant proteins, bovine serum albumin (BSA) and equine IgG (EIG) had no effect on the production of plasmin. Ann-II-mediated enhancement of t-PA-dependent PLG activation was inhibited by epsilon-aminocaproic acid or by pretreatment of Ann-II with carboxypeptidase B with the inhibitive rate being 77.8% and 77.0%, respectively. It was revealed that the effect of Ann-II on PLG activation was specific for t-PA. Urokinase didn't bind to Ann-II, demonstrating the role of receptor-related lysine residues on activation of PLG, showing that the Ann-II-PLG interaction was dependent upon carboxyl-terminal lysine residues. These findings suggest that annexin II-mediated co-assembly of t-PA and PLG may promote plasmin generation and play a key role in modulating fibrinolysis on the endothelial surface.
Annexin A2 ; pharmacology ; Cells, Cultured ; Endothelium, Vascular ; cytology ; metabolism ; Fibrinolysis ; Humans ; Plasminogen ; metabolism ; Recombinant Proteins ; pharmacology ; Tissue Plasminogen Activator ; metabolism ; Umbilical Veins ; cytology

In order to further investigate the effect of annexin II (Ann-II) on tissue plasminogen activator (t-PA)-dependent plasminogen (PLG) activation and its interactive mechanism, recombinant native Ann-II bound t-PA, PLG and plasmin with high affinity was examined. The flow cytometric assay showed that the ann-II expression rate was higher in the human umbilical vein endothelial cell (HUVEC) (87.65%) than in the HL-60 cells as controls (35.79%). Two irrelevant proteins, bovine serum albumin (BSA) and equine IgG (EIG) had no effect on the production of plasmin. Ann-II-mediated enhancement of t-PA-dependent PLG activation was inhibited by epsilon-aminocaproic acid or by pretreatment of Ann-II with carboxypeptidase B with the inhibitive rate being 77.8% and 77.0%, respectively. It was revealed that the effect of Ann-II on PLG activation was specific for t-PA. Urokinase didn't bind to Ann-II, demonstrating the role of receptor-related lysine residues on activation of PLG, showing that the Ann-II-PLG interaction was dependent upon carboxyl-terminal lysine residues. These findings suggest that annexin II-mediated co-assembly of t-PA and PLG may promote plasmin generation and play a key role in modulating fibrinolysis on the endothelial surface.
Annexin A2/*pharmacology ; Cells, Cultured ; Endothelium, Vascular/cytology ; Endothelium, Vascular/*metabolism ; Fibrinolysis ; Plasminogen/*metabolism ; Recombinant Proteins/pharmacology ; Tissue Plasminogen Activator/*metabolism ; Umbilical Veins/cytology

As a therapeutic agent in thrombosis the fibrinolytic enzymes are of interest and the search for a new enzyme continues. A novel fibrinolytic enzyme was produced from Rhizopus chinensis 120, which was screened from the starter for brewing rice wine in the South of China, by solid fermentation, and purified through ammonium sulfate precipitation, hydrophobic interaction, ionic exchange and gel filtration chromatographies. The purified enzyme hydrolyzed fibrin, it cleaved the alpha-, beta- and gamma-chains of fibrinogen simultaneously, and it also activated plasminogen to plasmin. The enzyme hydrolyzed N-Succinyl-Ala-Ala- Pro-Phe-pNA, and Km was 0.23 mmol/L and Kcat 16.36 s(-1). The optimal temperature of the enzyme for hydrolying fibrin was 45 degrees C, and the optimal pH range of 6.8 - 8.8. The isoelectric point of the enzyme estimated by isoelectric focusing electrophoresis was 8.5 +/- 0.1. The enzyme was a glycoprotein. EDTA, PCMB, PMSF inhibited the activety of the enzyme, and SBTI, Lys, TPCK, Aprotinine had none obvious inhibition, which suggested that the activity centre of the enzyme had hydrosulfuryl, metal and serine. The first 12 amino acids of the N-termimal sequence of the enzyme were NH2-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly, and had none homology with that of other fibrinolytic enzyme from other microbes. The novel fibrinolytic enzyme from Rhizopus chinensis 12# has potential to become a therapeutic agent in thrombosis.
Enzyme Stability ; Fermentation ; Fibrinolysin ; metabolism ; Fibrinolysis ; Fibrinolytic Agents ; chemistry ; Humans ; Plasminogen ; metabolism ; Rhizopus ; enzymology