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1.
J R González Rey M Cuevas Valdespino M Cardona A Díaz C Gongora B Pérez J Sánchez 《Folia haematologica (Leipzig, Germany : 1928)》1988,115(1-2):171-176
The pharmacokinetic characteristics of APP were studied in 10 male New Zealand rabbits. An initial dose of 15,000 IU of APP per kg of weight was administered. The respective fibrinolytic activity was estimated by fibrin plates and transformed according to theoretical considerations. Knorre's criterion was used for the determination of the number of compartments. In every case the values were below 0.2 which indicates a one-compartment open model. The data were fitted to the proposed model in two steps; first the stripping method was used, afterwards the values obtained by the stripping were used as initial values for nonlinear fitting by means of nonlin programs. 相似文献
2.
We investigated the effect of agents which raise intracellular levels of cyclic AMP (cAMP) on the secretion of tissue-type plasminogen activator (t-PA) and type 1 plasminogen activator inhibitor (PAI-1) by cultured human umbilical-vein endothelial cells. Significant inhibition of baseline (unstimulated) t-PA and PAI-1 secretion was observed in response to several agents which, when added exogenously, cause increased intracellular cAMP: cholera toxin, 1-methyl-3-isobutylxanthine (MIX), dibutyryl-cAMP, and prostaglandin E1. These agents also significantly reduced or abolished the previously reported stimulatory effects of thrombin and histamine on t-PA secretion, and, with the exception of MIX, significantly reduced the previously reported stimulatory effect of thrombin on PAI-1 secretion. MIX at a concentration (10 microM) below that required to inhibit t-PA and PAI-1 secretion when tested alone, significantly increased the inhibitory effects of cholera toxin, dibutyryl-cAMP, and prostaglandin E1 on both t-PA and PAI-1 secretion. The data suggest that elevated intracellular levels of cAMP inhibit both spontaneous endothelial secretion of t-PA and PAI-1, and secretion induced by agents (thrombin and histamine) which stimulate endothelial phosphoinositide metabolism, consistent with bidirectional regulation of endothelial fibrinolytic protein secretion by the adenylate cyclase and phosphoinositide signal transduction pathways. The inhibitory effects of cAMP do not appear to be specific for t-PA and PAI-1, since cholera toxin and MIX also inhibited endothelial secretion of the adhesive protein, fibronectin. Significant inhibition of baseline endothelial t-PA and PAI-1 secretion was also caused by the stable prostacyclin analogue iloprost (ZK 36 374) and by arachidonic acid, which is converted by endothelial cells to prostacyclin, suggesting that prostacyclin produced endogenously by endothelial cells may inhibit secretion of fibrinolytic proteins by increasing intracellular cAMP. 相似文献
3.
A plasminogen activator is induced during goldfish optic nerve regeneration. 总被引:2,自引:0,他引:2
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The use of purified piscine plasminogen in a chromogenic solution assay enabled us to detect plasminogen activator (PA) activity in crude homogenates of goldfish optic nerve following nerve injury. In contrast, no activity was detected in the homogenates of uninjured nerve. Under conditions allowing regeneration of the optic axons (optic nerve crush), PA activity peaked 8 days after crush, and decreased to undetectable levels by 60 days. Under conditions allowing only degeneration of the axons (enucleation), the activity peaked at 8 days but decreased more rapidly. Casein zymography of samples after fractionation in SDS-PAGE showed that PA activity migrated as a doublet at Mr = 60-65 kd. Using this assay, activity was also observed in uninjured control nerves. This plasminogen-dependent activity migrated as three bands of higher molecular weight (Mr = 75, 95 and 120 kd) and was undetectable in solution assays of unfractionated extracts, suggesting complex formation with an inhibitor(s). Fibrin overlay assay of retinal explants and isolated primary cells in culture suggest that the goldfish PA is associated with the glial cells of the goldfish visual pathway. 相似文献
4.
Stempien-Otero A Plawman A Meznarich J Dyamenahalli T Otsuka G Dichek DA 《The Journal of biological chemistry》2006,281(22):15345-15351
Human hearts with end-stage failure and fibrosis have macrophage accumulation and elevated plasminogen activator activity. However, the mechanisms that link macrophage accumulation and plasminogen activator activity with cardiac fibrosis are unclear. We previously reported that mice with macrophage-targeted overexpression of urokinase plasminogen activator (SR-uPA+/o mice) develop cardiac macrophage accumulation by 5 weeks of age and cardiac fibrosis by 15 weeks. We used SR-uPA+/o mice to investigate mechanisms through which macrophage-expressed uPA causes cardiac macrophage accumulation and fibrosis. We hypothesized that: 1) macrophage accumulation and cardiac fibrosis in SR-uPA+/o mice are dependent on localization of uPA by the uPA receptor (uPAR); 2) activation of plasminogen by uPA and subsequent activation of transforming growth factor-beta1 (TGF-beta1) and matrix metalloproteinase (MMP)-2 and -9 by plasmin are critical pathways through which uPA-expressing macrophages accumulate in the heart and cause fibrosis; and 3) uPA-induced cardiac fibrosis can be attenuated by treatment with verapamil. To test these hypotheses, we bred the SR-uPA+/o transgene into mice deficient in either uPAR or plasminogen and measured cardiac macrophage accumulation and fibrosis. We also measured cardiac TGF-beta1 protein (total and active), Smad2 phosphorylation, and MMP activity after the onset of macrophage accumulation but before the onset of cardiac fibrosis. Finally, we treated mice with verapamil. Our studies revealed that plasminogen is necessary for uPA-induced cardiac fibrosis and macrophage accumulation but uPAR is not. We did not detect plasmin-mediated activation of TGF-beta1, MMP-2, or MMP-9 in hearts of SR-uPA+/o mice. However, verapamil treatment significantly attenuated both cardiac fibrosis and macrophage accumulation. 相似文献
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6.
Phorbol myristate acetate (PMA) added to human synovial fibroblast cultures caused a dose-dependent increase in the production of plasminogen activator inhibitor-type 1 (PAI-1). In addition, PMA inhibited endogenous and interleukin-1 (IL-1) induced plasminogen activator (PA) activity, while increasing mRNA PAI-1 levels. Other protein kinase C (PKC) activators, mezerein and teleocidin B4, caused similar effects. The simultaneous addition of the PKC antagonists, H-7 or staurosporine, prevented the inhibition of PA activity by PMA. This study shows that activation of PKC inhibits PA and stimulates PAI production in human synovial fibroblasts. These results suggest that activation of PKC may play an important role in regulating increased PA production associated with joint destruction in rheumatoid arthritis (RA). 相似文献
7.
Human tissue-type plasminogen activator. Production in continuous serum-free cell culture and rapid purification. 总被引:1,自引:0,他引:1
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A simplified procedure for the production and purification of human tissue-type plasminogen activator (t-PA) is described. Bowes-melanoma cells were maintained in continuous serum-free culture. The cell nutrient consisted of Dulbecco's modified Eagle's medium (DMEM) supplemented with insulin (5 mg/litre), transferrin (5 mg/litre), progesterone (1 nM), cortisol (10 nM), aprotinin (2 X 10(4) units/litre) and a mixture of trace elements. t-PA accumulated in the culture medium at a rate of 40 units/day per ml and was harvested every third day. Cell losses during each harvest, leading to a steady decline of enzyme yields, were compensated for by treating the cells with 5% (v/v) fetal-bovine serum in DMEM every 6-8 weeks. t-PA was rapidly purified by a combination of cation-exchange chromatography and gel filtration. The procedure yielded mainly single-chain t-PA of a specific activity of 80 000 to 100 000 units/mg. 相似文献
8.
Bidder's organ (BO, a vestigeal organ), present in toad Bufo melanostictus (Schenider), is a characteristic feature of all male bufo. Its possible anaphylactic properties are investigated on experimental animals. BO extract produced both in vivo and in vitro anaphylactic reaction in guineapig. Dyspnoea and bronchoconstriction was a major cause of anaphylactic death. Blood histamine level was significantly increased in the anaphylactic animals. BO extract significantly released histamine from chopped lung preparation, an action antagonised by disodium chromoglycate. BO extract degranulated peritoneal mast cell in vitro. Passive cutaneous anaphylactic reactions were enhanced by BO extract and were significantly inhibited by disodium chromoglycate. Anaphylotoxin (identity not known) present in bidder's organ is probably involved in toad defence. 相似文献
9.
A component extracted from endothelium and partially purified has been found to have a capacity to enhance the rate of plasminogen activation by tissue-type plasminogen activator. The mechanism of action of this cofactor differs from that of others, such as fibrin. 相似文献
10.
Recombinant tissue-type plasminogen activator (rt-PA) from cultures of a genetically manipulated Bowes melanoma cell line (TRBM6) was purified in batches of average volume 451 using an autoclavable, reusable, continuous chromatography system comprising zinc chelate-Sepharose CL4B and lysine-Sepharose CL4B. After eight successive purifications the rt-PA was ultrafiltered to yield a preparation containing 4.9 mg protein/ml and 2.7 X 10(6) IU/ml. Analysis by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie brilliant blue R250 showed major protein bands at Mr = 63,000 and 65,000; most of the material was in the 1-chain form. The potential usefulness of a simple, rapid continuous chromatography system that can be operated under aseptic conditions is discussed. 相似文献
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12.
Lin Z Jiang L Yuan C Jensen JK Zhang X Luo Z Furie BC Furie B Andreasen PA Huang M 《The Journal of biological chemistry》2011,286(9):7027-7032
Plasminogen activator inhibitor-1 (PAI-1), together with its physiological target urokinase-type plasminogen activator (uPA), plays a pivotal role in fibrinolysis, cell migration, and tissue remodeling and is currently recognized as being among the most extensively validated biological prognostic factors in several cancer types. PAI-1 specifically and rapidly inhibits uPA and tissue-type PA (tPA). Despite extensive structural/functional studies on these two reactions, the underlying structural mechanism has remained unknown due to the technical difficulties of obtaining the relevant structures. Here, we report a strategy to generate a PAI-1·uPA(S195A) Michaelis complex and present its crystal structure at 2.3-Å resolution. In this structure, the PAI-1 reactive center loop serves as a bait to attract uPA onto the top of the PAI-1 molecule. The P4–P3′ residues of the reactive center loop interact extensively with the uPA catalytic site, accounting for about two-thirds of the total contact area. Besides the active site, almost all uPA exosite loops, including the 37-, 60-, 97-, 147-, and 217-loops, are involved in the interaction with PAI-1. The uPA 37-loop makes an extensive interaction with PAI-1 β-sheet B, and the 147-loop directly contacts PAI-1 β-sheet C. Both loops are important for initial Michaelis complex formation. This study lays down a foundation for understanding the specificity of PAI-1 for uPA and tPA and provides a structural basis for further functional studies. 相似文献
13.
R Maran A Kadouri S Floru A Gelvan A M Cohen 《Biochemical medicine and metabolic biology》1990,44(2):106-113
PtdIns liposomes, at a concentration of 40 microM, induced in FLF the synthesis of t-PA-Ag, and enhanced 45Ca2+ uptake. The induction of t-PA-Ag biosynthesis by PtdIns liposomes in FLF was inhibited by 5-15 microM verapamil, an inhibitor of Ca2+ uptake via the so-called "slow channels" by 0.5-10 microM TFP, an inhibitor of Ca2+ transport ATPase, and by 10-90 microM TMB-8, an inhibitor of intracellular Ca2+ mobilization. t-PA-Ag secretion was inhibited by decreasing the Ca2+ concentration less than 1.2 mM. On the other hand, addition of 0.08 microM of calcium ionophore A23187 increased t-PA-Ag biosynthesis after 72 hr of incubation by 247% (P less than 0.01). These data support previous results and indicate that the synthesis of t-PA in FLF is Ca2+ dependent. Thus, it is suggested that PtdIns liposomes increase t-PA biosynthesis by affecting calcium metabolism. 相似文献
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15.
Dhungana Sandra Madhyastha Radha Madhyastha Harishkumar Nakajima Yuichi Omura Sayuri Maruyama Masugi 《Phytomedicine》2010,17(1):42-46
Urokinase plasminogen activator (uPA) system, comprising of uPA, its receptor uPAR and inhibitor, type 1 plasminogen activator inhibitor (PAI-1), plays a vital role in various biological processes involving extracellular proteolysis, fibrinolysis, cell migration and proliferation. The timely occurence of these processes are essential for normal wound healing. This study examines the regulation of uPA and PAI-1 by a natural polyphenol-rich compound, grape seed extract (GSE). GSE is reported to have beneficial effects in promoting wound healing. Fibroblast cells exposed to different doses of GSE for 18 hours were processed for further studies such as ELISA, RT-PCR, western blotting, fibrinolytic assay, cell surface plasmin activity assay and in vitro wound healing assay. GSE treatment caused a significant downregulation of uPA and PAI-1 expression, both at the RNA and protein levels. ELISA also revealed a dose-dependent decrease in uPA and PAI-1 activities. Functional significance of the downregulation was evident in decreased fibrinolytic activity, concomittant with decreased cell-surface plasmin activity. In vitro wound healing studies showed that GSE also retarded the migration of cells towards the wounded region. 相似文献
16.
J Keijer H J Ehrlich M Linders K T Preissner H Pannekoek 《The Journal of biological chemistry》1991,266(16):10700-10707
The "serpin" plasminogen activator inhibitor 1 (PAI-1) is the fast acting inhibitor of plasminogen activators (tissue-type (t-PA) and urokinase type-PA) and is an essential regulatory protein of the fibrinolytic system. Its P1-P1' reactive center (R346 M347) acts as a "bait" for tight binding to t-PA/urokinase-type PA. In vivo, PAI-1 is encountered in complex with vitronectin, an interaction known to stabilize its activity but not to affect the second-order association rate constant (k1) between PAI-1 and t-PA. Nevertheless, by using PAI-1 reactive site variants (R346M, M347S, and R346M M347S), we show that the binding of vitronectin to the PAI-1 mutant proteins improves plasminogen activator inhibition. In the absence of vitronectin the PAI-1 R346M mutants are virtually inactive toward t-PA (k1 less than 1 x 10(3) M-1 s-1). In contrast, in the presence of vitronectin the rate of association increases about 1,000-fold (k1 of 6-8 x 10(5) M-1 s-1). This inhibition coincides with the formation of serpin-typical, sodium dodecyl sulfide-stable t-PA.PAI-1 R346M (R346M M347S) complexes. As evidenced by amino acid sequence analysis, the newly created M346-M/S347 peptide bond is susceptible to attack by t-PA, similar to the wild-type R346-M347 peptide bond, indicating that in the presence of vitronectin M346 functions as an efficient P1 residue. In addition, we show that the inhibition of t-PA and urokinase-type PA by PAI-1 mutant proteins is accelerated by the presence of the nonprotease A chains of the plasminogen activators. 相似文献
17.
Kinetic analysis of the interaction between plasminogen activator inhibitor-1 and tissue-type plasminogen activator. 总被引:4,自引:0,他引:4
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The kinetics of inhibition of tissue-type plasminogen activator (t-PA) by the fast-acting plasminogen activator inhibitor-1 (PAI-1) was investigated in homogeneous (plasma) and heterogeneous (solid-phase fibrin) systems by using radioisotopic and spectrophotometric analysis. It is demonstrated that fibrin-bound t-PA is protected from inhibition by PAI-1, whereas t-PA in soluble phase is rapidly inhibited (K1 = 10(7) M-1.s-1) even in the presence of 2 microM-plasminogen. The inhibitor interferes with the binding of t-PA to fibrin in a competitive manner. As a consequence the Kd of t-PA for fibrin (1.2 +/- 0.4 nM) increases and the maximal velocity of plasminogen activation by fibrin-bound t-PA is not modified. From the plot of the apparent Kd versus the concentration of PAI-1 a Ki value of 1.3 +/- 0.3 nM was calculated. The quasi-similar values for the dissociation constants between fibrin and t-PA (Kd) and between PAI-1 and t-PA (Ki), as well as the competitive type of inhibition observed, indicate that the fibrinolytic activity of human plasma may be the result of an equilibrium distribution of t-PA between both the amount of fibrin generated and the concentration of circulating inhibitor. 相似文献
18.
Urokinase-type plasminogen activator is induced in migrating capillary endothelial cells 总被引:7,自引:10,他引:7
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《The Journal of cell biology》1987,105(6):2535-2541
Cellular migration is an essential component of invasive biological processes, many of which have been correlated with an increase in plasminogen activator production. Endothelial cell migration occurs in vivo during repair of vascular lesions and angiogenesis, and can be induced in vitro by wounding a confluent monolayer of cells. By combining the wounded monolayer model with a substrate overlay technique, we show that cells migrating from the edges of an experimental wound display an increase in urokinase-type plasminogen activator (uPA) activity, and that this activity reverts to background levels upon cessation of movement, when the wound has closed. Our results demonstrate a direct temporal relationship between endothelial cell migration and uPA activity, and suggest that induction of uPA activity is a component of the migratory process. 相似文献
19.
Barbara D. Wilson Caecilia J. Huang Vernon L. Moore Nickolas J. Calvanico 《Cellular immunology》1982,67(1):90-100
A rabbit model of hypersensitivity pneumonitis (HP) was employed to evaluate the release of plasminogen activator (PA) as a method for monitoring the degree of pulmonary inflammation. PA release from alveolar macrophages (AM) was shown to coincide with inflammation and was maximal at approximately 2 weeks of aerosol challenge. PA release could also be induced in normal AM by peripheral lymphocytes obtained from sensitized animals after incubation with antigen. Unseparated peripheral blood mononuclear cells from experimental animals also exhibited antigen-induced PA release. These results suggest that the measurement of PA release using several different cell populations can be used to evaluate pulmonary inflammation in HP. 相似文献
20.
Ahmed W Malik M Saeed I Khan AA Sadeque A Kaleem U Ahmed N Ajmal M Azam M Qamar R 《Molecular biology reports》2011,38(4):2541-2548
A case–control association study on 229 Myocardial Infarction (MI) patients and 217 healthy controls was carried out to determine
the role of tissue-plasminogen activator (t-PA) (Alu-repeat insertion (I)/deletion (D)) and plasminogen activator inhibitor
(PAI-1) (4G/5G insertion/deletion) polymorphisms with MI in the Pakistani population. In MI patients the genotype distribution
of the PAI-1 gene was not found to be different when compared with the unaffected controls (P > 0.05, χ2 = 1.03). The risk allele 4G was also not associated with MI (P > 0.05, χ2 = 0.46, odds ratio (OR) = 1.1 (95% confidence interval (CI) = 0.84–1.43), P > 0.05). Similarly, the genotype frequencies of t-PA I/I, I/D and D/D were not different from the unaffected controls (P > 0.05, χ2 = 1.60), and the risk allele “I” was not found to be associated with MI (P > 0.05, χ2 = 1.35, OR = 0.86 (95% CI = 0.66–1.11), P > 0.05). However, when the data were distributed along the lines of gender a significant association of the 4G/4G PAI-1 genotype
was observed with only the female MI patients (P < 0.05, z-test = 2.21). When the combined genotypes of both the polymorphisms were analyzed, a significant association of
MI was observed with the homozygous DD/4G4G genotype (P < 0.01, z-test = 2.61), which was specifically because of the female samples (P = 0.01, z-test = 2.53). In addition smoking (P < 0.001, χ2 = 13.52, OR = 3.45 (95% CI = 1.77–6.94)), diabetes (P < 0.001, χ2 = 22.45, OR = 8.89 (95% CI = 2.96–29.95)), hypertension (OR = 7.76 (95% CI = 2.88–22.68), P < 0.001) family history (P < 0.001, χ2 = 13.72, OR = 3.7 (95% CI = 1.71–8.18)) and lower HDL levels (P < 0.05) were found to be significantly associated with the disease. In conclusion the PAI-1 gene polymorphism was found to
have a gender specific role in the female MI patients. 相似文献