Home About us Contact
|
Home About us Contact | ||
|
|
||
Plasminogen
Terms modified by Plasminogen Selected AbstractsProstromelysin-1 (proMMP-3) stimulates plasminogen activation by tissue-type plasminogen activatorFEBS JOURNAL, Issue 21 2000Begoña Arza Matrix metalloproteinase-3 (MMP-3 or stromelysin-1) specifically binds to tissue-type plasminogen activator (t-PA), without however, hydrolyzing the protein. Binding affinity to proMMP-3 is similar to single chain t-PA, two chain t-PA and active site mutagenized t-PA (Ka of 6.3 × 106 to 8.0 × 106 m,1), but is reduced for t-PA lacking the finger and growth factor domains (Ka of 2.0 × 106 m,1). Activation of native Glu-plasminogen by t-PA in the presence of proMMP-3 obeys Michaelis,Menten kinetics; at saturating concentrations of proMMP-3, the catalytic efficiency of two chain t-PA is enhanced 20-fold (kcat/Km of 7.9 × 10,3 vs. 4.1 × 10,4 µm,1·s,1). This is mainly the result of an enhanced affinity of t-PA for its substrate (Km of 1.6 µm vs. 89 µm in the absence of proMMP-3), whereas the kcat is less affected (kcat of 1.3 × 10,2 vs. 3.6 × 10,2 s,1). Activation of Lys-plasminogen by two chain t-PA is stimulated about 13-fold at a saturating concentration of proMMP-3, whereas that of miniplasminogen is virtually unaffected (1.4-fold). Plasminogen activation by single chain t-PA is stimulated about ninefold by proMMP-3, whereas that by the mutant lacking finger and growth factor domains is stimulated only threefold. Biospecific interaction analysis revealed binding of Lys-plasminogen to proMMP-3 with 18-fold higher affinity (Ka of 22 × 106 m,1) and of miniplasminogen with fivefold lower affinity (Ka of 0.26 × 106m,1) as compared to Glu-plasminogen (Ka of 1.2 × 106m,1). Plasminogen and t-PA appear to bind to different sites on proMMP-3. These data are compatible with a model in which both plasminogen and t-PA bind to proMMP-3, resulting in a cyclic ternary complex in which t-PA has an enhanced affinity for plasminogen, which may be in a Lys-plasminogen-like conformation. Maximal binding and stimulation require the N-terminal finger and growth factor domains of t-PA and the N-terminal kringle domains of plasminogen. [source] Zymogen activation in the streptokinase,plasminogen complexFEBS JOURNAL, Issue 13 2000Ile1 is required for the formation of a functional active site Plasminogen (Plgn) is usually activated by proteolysis of the Arg561,Val562 bond. The amino group of Val562 forms a salt-bridge with Asp740, which triggers a conformational change producing the active protease plasmin (Pm). In contrast, streptokinase (SK) binds to Plgn to produce an initial inactive complex (SK·Plgn) which subsequently rearranges to an active complex (SK·Plgn*) although the Arg561,Val562 bond remains intact. Therefore another residue must substitute for the amino group of Val562 and provide a counterion for Asp740 in this active complex. Two candidates for this counterion have been suggested: Ile1 of streptokinase and Lys698 of Plgn. We have investigated the reaction of SK mutants and variants of the protease domain of microplasminogen (µPlgn) in order to determine if either of these residues is the counterion. The mutation of Ile1 of SK decreases the activity of SK·Plgn* by 100-fold (Ile1Val) to ,,104 -fold (Ile1,Ala, Gly, Trp or Lys). None of these mutations perturb the binding affinity of SK, which suggests that Ile1 is not required for formation of SK·Plgn but is necessary for SK·Plgn*. The substitution of Lys698 of µPlgn decreases the activity of SK·Plgn* by only 10,60-fold. In contrast with the Ile1 substitutions, the Lys698 mutations also decreased the dissociation constant of the SK complex by 15,50-fold. These observations suggest that Lys698 is involved in formation of the initial SK·Plgn complex. These results support the hypothesis that Ile1 provides the counterion for Asp740. [source] Plasminogen on the surfaces of fibrin clots prevents adhesion of leukocytes and plateletsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 4 2010V. K. LISHKO Summary.,Background and Objectives: Although leukocytes and platelets adhere to fibrin with alacrity in vitro, these cells do not readily accumulate on the surfaces of fibrin clots in vivo. The difference in the capacity of blood cell integrins to adhere to fibrin in vivo and in vitro is striking and implies the existence of a physiologic antiadhesive mechanism. The surfaces of fibrin clots in the circulation are continually exposed to plasma proteins, several of which can bind fibrin and influence cell adhesion. Recently, we have demonstrated that adsorption of soluble fibrinogen on the surface of a fibrin clot results in its deposition as a soft multilayer matrix, which prevents attachment of blood cells. In the present study, we demonstrate that another plasma protein, plasminogen, which is known to accumulate in the superficial layer of fibrin, exerts an antiadhesive effect. Results: After being coated with plasminogen, the surfaces of fibrin clots became essentially non-adhesive for U937 monocytic cells, blood monocytes, and platelets. The data revealed that activation of fibrin-bound plasminogen by the plasminogen-activating system assembled on adherent cells resulted in the generation of plasmin, which decomposed the superficial fibrin layer, resulting in cell detachment under flow. The surfaces generated after the initial cell adhesion remained non-adhesive for subsequent attachment of leukocytes and platelets. Conclusion: We propose that the limited degradation of fibrin by plasmin generated by adherent cells loosens the fibers on the clot surface, producing a mechanically unstable substrate that is unable to support firm integrin-mediated cell adhesion. [source] Injections of Blood, Thrombin, and Plasminogen More Severely Damage Neonatal Mouse Brain Than Mature Mouse BrainBRAIN PATHOLOGY, Issue 4 2005Mengzhou Xue MD The mechanism of brain cell injury associated with intracerebral hemorrhage may be in part related to proteolytic enzymes in blood, some of which are also functional in the developing brain. We hypothesized that there would be an age-dependent brain response following intracerebral injection of blood, thrombin, and plasminogen. Mice at 3 ages (neonatal, 10-day-old, and young adult) received autologous blood (15, 25, and 50 ,l respectively), thrombin (3, 5, and 10 units respectively), plasminogen (0.03, 0.05, and 0.1 units respectively) (the doses expected in same volume blood), or saline injection into lateral striatum. Forty-eight hours later they were perfusion fixed. Hematoxylin and eosin, lectin histochemistry, Fluoro-Jade, and TUNEL staining were used to quantify changes related to the hemorrhagic lesion. Damage volume, dying neurons, neutrophils, and microglial reaction were significantly greater following injections of blood, plasminogen, and thrombin compared to saline in all three ages of mice. Plasminogen and thrombin associated brain damage was greatest in neonatal mice and, in that group unlike the other 2, greater than the damage caused by whole blood. These results suggest that the neonatal brain is relatively more sensitive to proteolytic plasma enzymes than the mature brain. [source] Efficient copackaging and cotransport yields postsynaptic colocalization of neuromodulators associated with synaptic plasticityDEVELOPMENTAL NEUROBIOLOGY, Issue 10 2008J.E. Lochner Abstract Recent data suggest that tissue plasminogen activator (tPA) influences long-term plasticity at hippocampal synapses by converting plasminogen into plasmin, which then generates mature brain-derived neurotrophic factor (mBDNF) from its precursor, proBDNF. Motivated by this hypothesis, we used fluorescent chimeras, expressed in hippocampal neurons, to elucidate (1) mechanisms underlying plasminogen secretion from hippocampal neurons, (2) if tPA, plasminogen, and proBDNF are copackaged and cotransported in hippocampal neurons, especially within dendritic spines, and (3) mechanisms mediating the transport of these neuromodulators to sites of release. We find that plasminogen chimeras traffic through the regulated secretory pathway of hippocampal neurons in dense-core granules (DCGs) and that tPA, plasminogen, and proBDNF chimeras are extensively copackaged in DCGs throughout hippocampal neurons. We also find that 80% of spines that contain DCGs contain chimeras of these neuromodulators in the same DCG. Finally, we demonstrate, for the first time, that neuromodulators undergo cotransport along dendrites in rapidly mobile DCGs, indicating that neuromodulators can be efficiently recruited into active spines. These results support the hypothesis that tPA mediates synaptic activation of BDNF by demonstrating that tPA, plasminogen, and proBDNF colocalize in DCGs in spines, where these neuromodulators can undergo activity-dependent release and then interact and/or mediate changes that influence synaptic efficacy. The results also raise the possibility that frequency-dependent changes in extents of neuromodulator release from DCGs influence the direction of plasticity at hippocampal synapses by altering the relative proportions of two proteins, mBDNF and proBDNF, that exert opposing effects on synaptic efficacy. © 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2008. [source] Independent signaling pathways in ATP-evoked secretion of plasminogen and cytokines from microgliaDRUG DEVELOPMENT RESEARCH, Issue 2-3 2001*Article first published online: 28 AUG 200, Kazuhide Inoue Abstract We investigated the action of ATP on the secretion of plasminogen, TNF-,, and IL-6 from microglia. ATP (10,100 ,M) stimulated the release of plasminogen from rat cultured microglia in a concentration-dependent manner with a peak response at 5,10 min after the stimulation. The release was dependent on extracellular Ca2+ and was blocked by pretreatment with oxidized ATP, a blocker of P2X7. UTP, an agonist of P2Y2, also stimulated the release of plasminogen from a subpopulation (about 20% of total cells) of cultured microglia. The release was also dependent on extracellular Ca2+, suggesting a role of stocker-operated calcium entry (SOC). ATP potently stimulated TNF-, release from 2 h after the stimulation with TNF-, mRNA expression in primary cultures of rat brain microglia. The TNF-, release was maximally elicited by 1 mM ATP and 2,- and 3,-O-(4-benzoylbenzoyl)-adenosine 5,-triphosphate (BzATP), a P2X7 selective agonist, suggesting the involvement of P2X7. This TNF-, release was correlated with a sustained Ca2+ influx. The release was inhibited by PD98059, an inhibitor of MEK1 which activates extracellular signal-regulated protein kinase (ERK), and SB203580, an inhibitor of p38 MAP kinase. However, both ERK and p38 were rapidly activated by ATP even in the absence of extracellular Ca2+. These results indicate that extracellular ATP triggers TNF-, release in rat microglia via P2X7 in a manner dependent on the sustained Ca2+ influx and via the ERK/p38 cascade independently of Ca2+ influx. ATP caused the mRNA expression and release of IL-6 in a concentration-dependent manner in MG-5. The physiological meaning of these independent release mechanisms is also discussed. Drug Dev. Res. 53:166,171, 2001. © 2001 Wiley-Liss, Inc. [source] Functional approach to investigate Lp(a) in ischaemic heart and cerebral diseasesEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 2 2003A. De La Peña-Díaz Abstract Background Lp(a), a major cardiovascular risk factor, contains a specific apolipoprotein, apo(a), which by virtue of structural homology with plasminogen inhibits the formation of plasmin, the fibrinolytic enzyme. A number of clinical reports support the role of Lp(a) as a cardiovascular or cerebral risk factor, and experimental data suggest that it may contribute to atherothrombosis by inhibiting fibrinolysis. Design A well-characterized model of a fibrin surface and an apo(a)-specific monoclonal antibody were used to develop a functional approach to detect pathogenic Lp(a). The assay is based on the competitive binding of Lp(a) and plasminogen for fibrin, and quantifies fibrin-bound Lp(a). High Lp(a) binding to fibrin is correlated with decreased plasmin formation. In a transversal case,control study we studied 248 individuals: 105 had a history of ischaemic cardiopathy (IC), 52 had cerebro-vascular disease (CVD) of thrombotic origin, and 91 were controls. Results The remarkably high apo(a) fibrin-binding in CVD (0·268 ± 0·15 nmol L,1) compared with IC (0·155 ± 0·12 nmol L,1) suggests the existence of peculiar and poorly understood differences in pro- or anti-thrombotic mechanisms in either cerebral and/or coronary arteries. Conclusions Our results demonstrated that Lp(a) fibrin-binding and small Apo(a) isoforms are associated with athero-thrombotic disease. [source] Staphylokinase reduces plasmin formation by endogenous plasminogen activatorsEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 1 2008Tao Jin Abstract Hyperfibrinolysis is a consequence of imbalance between fibrinolytic activators and their inhibitors. Increased levels of circulating plasminogen (Plg) activators such as tissue- or urokinase-type plasminogen activators (tPA or uPA respectively) are the most common causes of hyperfibrinolysis, occasionally causing major hemorrhages. We found that staphylokinase (SAK), a well-known Plg activator of bacterial origin, inhibits Plg activation mediated by endogenous tPA and uPA. Furthermore, mixture of SAK with tPA led to a significantly reduced Plg-dependent fibrinolysis. This inhibitory effect was exerted through direct action of SAK on Plg rather than indirectly on tPA or uPA. Inhibition of Plg activation by SAK is readily abrogated by interaction of SAK with human neutrophil peptides (HNPs). Finally, we show that NH2 -terminal residues of SAK are important for the inhibitory effect of SAK on tPA- and uPA-mediated Plg activation. In conclusion, SAK reduces tPA/uPA-mediated Plg activation by means of SAK.Plg complex formation, consequently downregulating tPA/uPA-induced fibrinolysis. [source] Tissue-type plasminogen activator-plasmin-BDNF modulate glutamate-induced phase-shifts of the mouse suprachiasmatic circadian clock in vitroEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2009Xiang Mou Abstract The mammalian circadian clock in the suprachiasmatic nucleus (SCN) maintains environmental synchrony through light signals transmitted by glutamate released from retinal ganglion terminals. Brain-derived neurotrophic factor (BDNF) is required for light/glutamate to reset the clock. In the hippocampus, BDNF is activated by the extracellular protease, plasmin, which is produced from plasminogen by tissue-type plasminogen activator (tPA). We provide data showing expression of proteins from the plasminogen activation cascade in the SCN and their involvement in circadian clock phase-resetting. Early night glutamate application to SCN-containing brain slices resets the circadian clock. Plasminogen activator inhibitor-1 (PAI-1) blocked these shifts in slices from wild-type mice but not mice lacking its stabilizing protein, vitronectin (VN). Plasmin, but not plasminogen, prevented inhibition by PAI-1. Both plasmin and active BDNF reversed ,2 -antiplasmin inhibition of glutamate-induced shifts. ,2 -Antiplasmin decreased the conversion of inactive to active BDNF in the SCN. Finally, both tPA and BDNF allowed daytime glutamate-induced phase-resetting. Together, these data are the first to demonstrate expression of these proteases in the SCN, their involvement in modulating photic phase-shifts, and their activation of BDNF in the SCN, a potential ,gating' mechanism for photic phase-resetting. These data also demonstrate a functional interaction between PAI-1 and VN in adult brain. Given the usual association of these proteins with the extracellular matrix, these data suggest new lines of investigation into the locations and processes modulating mammalian circadian clock phase-resetting. [source] Inhibition of urokinase receptor gene expression and cell invasion by anti-uPAR DNAzymes in osteosarcoma cellsFEBS JOURNAL, Issue 14 2005Charles E. De Bock The urokinase-type plasminogen activator (uPA) receptor (uPAR) has been implicated in signal transduction and biological processes including cancer metastasis, angiogenesis, cell migration, and wound healing. It is a specific cell surface receptor for its ligand uPA, which catalyzes the formation of plasmin from plasminogen, thereby activating the proteolytic cascade that contributes to the breakdown of extracellular matrix, a key step in cancer metastasis. We have synthesized three different DNA enzymes (Dz372, Dz483 and Dz720) targeting uPAR mRNA at three separate purine (A or G),pyrimidine (U or C) junctions. Two of these DNAzymes, Dz483 and Dz720, cleaved uPAR transcript in vitro with high efficacy and specificity at a molar ratio (uPAR to Dz) as low as 1 : 0.2. When analyzed over 2 h with a 200-fold molar excess of DNAzymes to uPAR transcript, Dz720 and Dz483 were able to decrease uPAR transcript in vitro by ,,93% and ,,84%, respectively. They also showed an ability to cleave uPAR mRNA in the human osteosarcoma cell line Saos-2 after transfection. The DNAzyme Dz720 decreased uPAR mRNA within 4 h of transfection, and inhibited uPAR protein concentrations by 55% in Saos-2 cells. The decrease in uPAR mRNA and protein concentrations caused by Dz720 significantly suppressed Saos-2 cell invasion as assessed by an in vitro Matrigel assay. The use of DNAzyme methodology adds a new potential clinical agent for decreasing uPAR mRNA expression and inhibiting cancer invasion and metastasis. [source] Nonlysine-analog plasminogen modulators promote autoproteolytic generation of plasmin(ogen) fragments with angiostatin-like activityFEBS JOURNAL, Issue 4 2004Shigeki Ohyama We recently discovered several nonlysine-analog conformational modulators for plasminogen. These include SMTP-6, thioplabin B and complestatin that are low molecular mass compounds of microbial origin. Unlike lysine-analog modulators, which increase plasminogen activation but inhibit its binding to fibrin, the nonlysine-analog modulators enhance both activation and fibrin binding of plasminogen. Here we show that some nonlysine-analog modulators promote autoproteolytic generation of plasmin(ogen) derivatives with its catalytic domain undergoing extensive fragmentation (PMDs), which have angiostatin-like anti-endothelial activity. The enhancement of urokinase-catalyzed plasminogen activation by SMTP-6 was followed by rapid inactivation of plasmin due to its degradation mainly in the catalytic domain, yielding PMD with a molecular mass ranging from 68 to 77 kDa. PMD generation was observed when plasmin alone was treated with SMTP-6 and was inhibited by the plasmin inhibitor aprotinin, indicating an autoproteolytic mechanism in PMD generation. Thioplabin B and complestatin, two other nonlysine-analog modulators, were also active in producing similar PMDs, whereas the lysine analog 6-aminohexanoic acid was inactive while it enhanced plasminogen activation. Peptide sequencing and mass spectrometric analyses suggested that plasmin fragmentation was due to cleavage at Lys615-Val616, Lys651-Leu652, Lys661-Val662, Lys698-Glu699, Lys708-Val709 and several other sites mostly in the catalytic domain. PMD was inhibitory to proliferation, migration and tube formation of endothelial cells at concentrations of 0.3,10 µg·mL,1. These results suggest a possible application of nonlysine-analog modulators in the treatment of cancer through the enhancement of endogenous plasmin(ogen) fragment formation. [source] Crystal structure of a staphylokinase variantFEBS JOURNAL, Issue 2 2002A model for reduced antigenicity Staphylokinase (SAK) is a 15.5-kDa protein from Staphylococcus aureus that activates plasminogen by forming a 1 : 1 complex with plasmin. Recombinant SAK has been shown in clinical trials to induce fibrin-specific clot lysis in patients with acute myocardial infarction. However, SAK elicits high titers of neutralizing antibodies. Biochemical and protein engineering studies have demonstrated the feasibility of generating SAK variants with reduced antigenicity yet intact thrombolytic potency. Here, we present X-ray crystallographic evidence that the SAK(S41G) mutant may assume a dimeric structure. This dimer model, at 2.3-Å resolution, could explain a major antigenic epitope (residues A72,F76 and residues K135-K136) located in the vicinity of the dimer interface as identified by phage-display. These results suggest that SAK antigenicity may be reduced by eliminating dimer formation. We propose several potential mutation sites at the dimer interface that may further reduce the antigenicity of SAK. [source] Prostromelysin-1 (proMMP-3) stimulates plasminogen activation by tissue-type plasminogen activatorFEBS JOURNAL, Issue 21 2000Begoña Arza Matrix metalloproteinase-3 (MMP-3 or stromelysin-1) specifically binds to tissue-type plasminogen activator (t-PA), without however, hydrolyzing the protein. Binding affinity to proMMP-3 is similar to single chain t-PA, two chain t-PA and active site mutagenized t-PA (Ka of 6.3 × 106 to 8.0 × 106 m,1), but is reduced for t-PA lacking the finger and growth factor domains (Ka of 2.0 × 106 m,1). Activation of native Glu-plasminogen by t-PA in the presence of proMMP-3 obeys Michaelis,Menten kinetics; at saturating concentrations of proMMP-3, the catalytic efficiency of two chain t-PA is enhanced 20-fold (kcat/Km of 7.9 × 10,3 vs. 4.1 × 10,4 µm,1·s,1). This is mainly the result of an enhanced affinity of t-PA for its substrate (Km of 1.6 µm vs. 89 µm in the absence of proMMP-3), whereas the kcat is less affected (kcat of 1.3 × 10,2 vs. 3.6 × 10,2 s,1). Activation of Lys-plasminogen by two chain t-PA is stimulated about 13-fold at a saturating concentration of proMMP-3, whereas that of miniplasminogen is virtually unaffected (1.4-fold). Plasminogen activation by single chain t-PA is stimulated about ninefold by proMMP-3, whereas that by the mutant lacking finger and growth factor domains is stimulated only threefold. Biospecific interaction analysis revealed binding of Lys-plasminogen to proMMP-3 with 18-fold higher affinity (Ka of 22 × 106 m,1) and of miniplasminogen with fivefold lower affinity (Ka of 0.26 × 106m,1) as compared to Glu-plasminogen (Ka of 1.2 × 106m,1). Plasminogen and t-PA appear to bind to different sites on proMMP-3. These data are compatible with a model in which both plasminogen and t-PA bind to proMMP-3, resulting in a cyclic ternary complex in which t-PA has an enhanced affinity for plasminogen, which may be in a Lys-plasminogen-like conformation. Maximal binding and stimulation require the N-terminal finger and growth factor domains of t-PA and the N-terminal kringle domains of plasminogen. [source] Plasma and urine levels of urinary trypsin inhibitor in patients with acute and fulminant hepatitisJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 2 2002SHI DE LIN Abstract Background and Aim Urinary trypsin inhibitor (UTI) is synthesized by hepatocytes and excreted into urine. Plasma and urine UTI levels have been measured to evaluate whether these levels may be useful markers in various pathological conditions. However, there has been no study on plasma and urine UTI levels in patients with acute liver diseases. The aim of the present study was to evaluate plasma and urine UTI levels and their relationship with the severity of hepatic damage in patients with acute liver diseases. Methods Plasma and urine UTI levels were measured by newly developed enzyme-linked immunosorbent assay in 15 patients with acute hepatitis (AH), 12 patients with acute severe hepatitis (ASH) and 10 patients with fulminant hepatitis (FH), as assessed on admission. The serial changes in plasma and urine UTI were also observed in some patients with AH and ASH. Results Plasma UTI levels (U/mL, median [25,75th percentile]) were: 11.0, (9.5,16.1) in patients with AH; 7.8 (5.6,11.5) in those with ASH; 6.5 (4.0,9.5) in patients with FH; and 9.7 (7.3,11.0) in normal controls. Plasma UTI levels in patients with FH were significantly lower than in those with AH. Plasma UTI levels showed significant positive correlations with the levels of prothrombin time (PT), hepaplastin test, antithrombin III, ,2-plasmin inhibitor, plasminogen (Plg) and fibrinogen. After the recovery of liver dysfunction, increased plasma UTI levels in patients with AH were decreased, whereas previously decreased plasma UTI levels in patients with ASH were increased. Urine UTI levels were significantly increased in patients with AH compared with those of normal controls. In patients with ASH and FH, urine UTI levels were increased but not significantly. Urine UTI levels significantly positively correlated with PT and Plg. After the recovery of liver dysfunction, previously increased urine UTI levels in patients with AH were decreased. The correlation between plasma UTI and urine UTI levels was not significant. Conclusions The findings of the present study suggested that the levels of plasma and urine UTI changed in patients with AH and were closely related to the abnormalities of coagulo-fibrinolysis, including PT. Further studies are needed to clarify whether these levels may be useful markers to predict the prognosis of acute hepatitis. [source] Urokinase-type plasminogen activator inhibits amyloid-, neurotoxicity and fibrillogenesis via plasminogenJOURNAL OF NEUROSCIENCE RESEARCH, Issue 2 2002H. Michael Tucker Abstract Amyloid-, (A,) appears central to Alzheimer's disease (AD), aggregates spontaneously, and is neurotoxic to neurons in vitro. Recently, several groups reported a familial AD locus on chromosome 10. Here, we note that urokinase-type plasminogen activator (uPA) is located within this locus. Previously, we reported that uPA and its functional homolog, tissue-type plasminogen activator, are induced by A, treatment of neurons in vitro as well as in a mouse model of A, accumulation in vivo. Moreover, the target of plasminogen activators, plasmin, degraded nonaggregated and aggregated A, and modulated A, toxicity and deposition. Here, we have evaluated the effects of uPA and plasminogen on A, fibril formation and neurotoxicity. We report that the combination of uPA and plasminogen, but neither alone, inhibits A, toxicity, reduces A, deposition in vitro, and inhibits A, fibrillogenesis. We interpret these observations as suggesting that uPA represents a possible candidate gene for the chromosome 10 familial AD locus. © 2002 Wiley-Liss, Inc. [source] Ethanol-Induced Up-Regulation of the Urokinase Receptor In Cultured Human Endothelial CellsALCOHOLISM, Issue 2 2001Edlue M. Tabengwa Background: Moderate alcohol consumption has been correlated to reduced coronary artery disease (CAD) risk and mortality. This alcohol effect may be mediated in part by an increased endothelial cell (EC) fibrinolysis. ECs synthesize fibrinolytic proteins, tissue plasminogen activator (t-PA), urokinase type plasminogen activator (u-PA), and plasminogen activator inhibitor type-1(PAI-1). In addition, they synthesize and regulate receptors for fibrinolytic proteins, namely (t-PA and plasminogen receptor) Annexin II and u-PA receptor (u-PAR). These receptors play an important role in the regulated expression of receptor-bound plasminogen activator conversion of receptor-bound plasminogen to receptor-bound plasmin on the EC surface (surface-localized fibrinolytic activity). Therefore, systemic factors, such as ethanol, that affect the level, or activity or interaction of one or more of these components, resulting in the increased expression of surface-localized EC fibrinolytic activity, will be expected to reduce the risk for thrombosis, CAD, and myocardial infarction (MI). We have previously shown that low ethanol up-regulates t-PA and u-PA gene transcription, while it down-regulates PAI-1, hence resulting in increased (sustained, 24 hr) surface-localized EC fibrinolytic activity. The current studies were carried out to determine whether low ethanol increased u-PAR expression in cultured human umbilical cord vein ECs (HUVECs). Methods: Cultured HUVECs were preincubated (1 hr) in the absence/presence of ethanol (0.025,0.2%, v/v); u-PAR mRNA (RT-PCR), antigen (western blot), and activity (125I-u-PA ligand binding/Scatchard analysis) levels were then measured after 0,24 hr. To determine whether the ethanol-induced changes in the u-PAR expression were transcriptional, transient transfection studies were carried out using a u-PAR/luciferase promoter construct (pu-PAR120/luc [1.2-kb u-PAR promoter fragment ligated to a promoterless luciferase vector]). Results: uPAR mRNA levels increased 2- to 3-fold and antigen levels (western blot) increased 2- to 4-fold while u-PA binding activity increased 36% (1.25 vs. 1.7 , 105 sites/cell, Bmax) without significantly affecting the Kd (1,2 nM). Transient transfection of cultured HUVECs with a pu-PAR120/luc construct resulted in a 2- to 3-fold increase in promoter activity in ethanol-induced cultures, compared with controls. Conclusion: These combined results demonstrate that low ethanol (,0.1%, v/v) induces the up-regulation of u-PAR gene transcription, resulting in increased u-PAR ligand binding activity. These results also further identify/define the contribution and role of another fibrinolytic protein in the overall ethanol-induced increase in surface-localized EC fibrinolysis that may underlie and contribute, in part, to the cardioprotection attributed to moderate alcohol consumption. [source] Ethanol-Induced Up-Regulation of Candidate Plasminogen Receptor Annexin II in Cultured Human Endothelial CellsALCOHOLISM, Issue 6 2000Edlue M. Tabengwa Introduction Epidemiological studies indicate that moderate alcohol consumption reduces the risk for coronary heart disease and that this cardioprotective benefit may be mediated, in part, by increased fibrinolysis. Endothelial cells (ECs) synthesize plasminogen activators, tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), receptors for plasminogen activators, and a receptor for plasminogen, annexin II (Ann-II). These receptors localize and facilitate receptor-bound plasminogen activator-mediated conversion of receptor-bound plasminogen to receptor-bound plasmin on the EC surface, which results in the regulated expression of surface-localized EC fibrinolytic activity. Ethanol is a systemic factor that affects these components, which increases EC fibrinolysis and hence reduces the risk for thrombosis, coronary heart disease, and myocardial infarction (MI). Methods: This study was carried out to determine whether low ethanol (0.1% v/v) increased plasminogen receptor, Ann-II antigen (western blot), messenger ribonucleic acid (mRNA) (reverse transcription polymerase chain reaction; RT-PCR) expression, activity (ligand binding/Scatchard analysis), and hence fibrinolysis (plasmin generation) in cultured human ECs. Results: Plasminogen receptor activity increased ,2-fold (2.5 vs. 5.6 × 106 sites/cell), as evidenced by increased 125I-labeled Glu-plasminogen ligand binding/Scatchard analysis. In addition, western blot analyses indicated an increase in Ann-II antigen, and mRNA levels increased ,2-fold (RT-PCR). This increase in Ann-II expression was concomitant with ,2- to 3-fold sustained increase (,24 hr) in surface-localized EC fibrinolytic activity. Nuclear transcription run-on assays showed an ,5- to 6-fold increase in new 32P-labeled Ann-II mRNA levels, compared with controls (no ethanol). Conclusions: These results demonstrated that low ethanol increased Ann-II antigen/mRNA levels and up-regulated Ann-II gene expression at the transcriptional level. The results further identify and define the contribution and role of the plasminogen receptor, Ann-II, in the ethanol-induced mechanism of increased EC fibrinolysis that may underlie and contribute, in part, to the cardioprotective benefit associated with moderate alcohol consumption. [source] Plasminogen on the surfaces of fibrin clots prevents adhesion of leukocytes and plateletsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 4 2010V. K. LISHKO Summary.,Background and Objectives: Although leukocytes and platelets adhere to fibrin with alacrity in vitro, these cells do not readily accumulate on the surfaces of fibrin clots in vivo. The difference in the capacity of blood cell integrins to adhere to fibrin in vivo and in vitro is striking and implies the existence of a physiologic antiadhesive mechanism. The surfaces of fibrin clots in the circulation are continually exposed to plasma proteins, several of which can bind fibrin and influence cell adhesion. Recently, we have demonstrated that adsorption of soluble fibrinogen on the surface of a fibrin clot results in its deposition as a soft multilayer matrix, which prevents attachment of blood cells. In the present study, we demonstrate that another plasma protein, plasminogen, which is known to accumulate in the superficial layer of fibrin, exerts an antiadhesive effect. Results: After being coated with plasminogen, the surfaces of fibrin clots became essentially non-adhesive for U937 monocytic cells, blood monocytes, and platelets. The data revealed that activation of fibrin-bound plasminogen by the plasminogen-activating system assembled on adherent cells resulted in the generation of plasmin, which decomposed the superficial fibrin layer, resulting in cell detachment under flow. The surfaces generated after the initial cell adhesion remained non-adhesive for subsequent attachment of leukocytes and platelets. Conclusion: We propose that the limited degradation of fibrin by plasmin generated by adherent cells loosens the fibers on the clot surface, producing a mechanically unstable substrate that is unable to support firm integrin-mediated cell adhesion. [source] Human plasminogen kringle 1,5 reduces atherosclerosis and neointima formation in mice by suppressing the inflammatory signaling pathwayJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 1 2010P. C. CHANG Summary.,Background:,Activation of vascular endothelial cells plays an important role in atherogenesis and plaque instability. Recent research has demonstrated that late-stage inhibition of plaque angiogenesis by angiostatin (kringle 1,4) reduces macrophage accumulation and slows the progression of advanced atherosclerosis. Kringle 1,5 (K1,5) is a variant of angiostatin that contains the first five kringle domains of plasminogen. Objective: To investigate whether K1,5 has an inhibitory effect on early-stage atherosclerosis, using the apolipoprotein E (ApoE)-deficient mouse model and a carotid artery ligation model. Methods: ApoE-deficient mice received K1,5 treatment for 4 weeks, and the severity of aortic atherosclerosis was measured. In the ligation model, the left common carotid arteries of C57BL/6 mice were ligated near the carotid bifurcation, and the mice received K1,5 for 4 weeks. Human umbilical vein endothelial cells were pretreated with K1,5 before tumor necrosis factor-, (TNF-,) treatment to explore the anti-inflammatory effect of K1,5. Results: The areas of the lesion in the aortas of ApoE-deficient mice that received K1,5 treatment were notably decreased, and the formation of carotid neointima in the C57BL/6 mice was decreased by treatment with K1,5. Expression of TNF-,-induced intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 was inhibited by K1,5 treatment, possibly via downregulation of translocation of nuclear factor-,B and expression of reactive oxygen species. Conclusions: K1,5 reduced atherosclerosis and neointima formation in mice, possibly through inhibition of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression in endothelial cells. [source] Apolipoprotein(a) inhibits the conversion of Glu-plasminogen to Lys-plasminogen: a novel mechanism for lipoprotein(a)-mediated inhibition of plasminogen activationJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 12 2008N. T. FERIC Summary.,Background:,Elevated plasma concentrations of lipoprotein(a) [Lp(a)] are associated with an increased risk for thrombotic disorders. Lp(a) is a unique lipoprotein consisting of a low-density lipoprotein-like moiety covalently linked to apolipoprotein(a) [apo(a)], a homologue of the fibrinolytic proenzyme plasminogen. Several in vitro and in vivo studies have shown that Lp(a)/apo(a) can inhibit tissue-type plasminogen activator-mediated plasminogen activation on fibrin surfaces, although the mechanism of inhibition by apo(a) remains controversial. Essential to fibrin clot lysis are a number of plasmin-dependent positive feedback reactions that enhance the efficiency of plasminogen activation, including the plasmin-mediated conversion of Glu-plasminogen to Lys-plasminogen. Objective:,Using acid,urea gel electrophoresis to resolve the two forms of radiolabeled plasminogen, we determined whether apo(a) is able to inhibit Glu-plasminogen to Lys-plasminogen conversion. Methods:,The assays were performed in the absence or presence of different recombinant apo(a) species, including point mutants, deletion mutants and variants that represent greater than 90% of the known apo(a) isoform sizes. Results:,Apo(a) substantially suppressed Glu-plasminogen conversion. Critical roles were identified for the kringle IV types 5,9 and kringle V; contributory roles for sequences within the amino-terminal half of the molecule were also observed. Additionally, with the exception of the smallest naturally-occurring isoform of apo(a), isoform size was found not to contribute to the inhibitory capacity of apo(a). Conclusion:,These findings underscore a novel contribution to the understanding of Lp(a)/apo(a)-mediated inhibition of plasminogen activation: the ability of the apo(a) component of Lp(a) to inhibit the key positive feedback step of plasmin-mediated Glu-plasminogen to Lys-plasminogen conversion. [source] Angiostatin K1-3 induces E-selectin via AP1 and Ets1: a mediator for anti-angiogenic action of K1-3JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 11 2008Y.-H. CHEN Summary.,Background:,Angiostatin, a circulating angiogenic inhibitor, is an internal fragment of plasminogen and consists of several isoforms, K1-3 included. We previously showed that K1-3 was the most potent angiostatin to induce E-selectin mRNA expression. The purpose of this study was to identify the mechanism responsible for K1-3-induced E-selectin expression and investigate the role of E-selectin in the anti-angiogenic action of K1-3. Methods and results:,Quantitative real time RT-PCR and Western blotting analyses confirmed a time-dependent increase of E-selectin mRNA and protein induced by K1-3. Subcellular fractionation and immunofluorescence microscopy showed the co-localization of K1-3-induced E-selectin with caveolin 1 (Cav1) in lipid rafts in which E-selectin may behave as a signaling receptor. Promoter-driven reporter assays and site-directed mutagenesis showed that K1-3 induced E-selectin expression via promoter activation and AP1 and Ets-1 binding sites in the proximal E-selectin promoter were required for E-selectin induction. The in vivo binding of both protein complexes to the proximal promoter was confirmed by chromatin immunoprecipitation (ChIP). Although K1-3 induced the activation of ERK1/2 and JNK, only repression of JNK activation attenuated the induction of E-selectin by K1-3. A modulatory role of E-selectin in the anti-angiogenic action of K1-3 was manifested by both overexpression and knockdown of E-selectin followed by cell proliferation assay. Conclusions:,We show that K1-3 induced E-selectin expression via AP1 and Ets-1 binding to the proximal E-selectin promoter (,356/+1), which was positively mediated by JNK activation. Our findings also demonstrate E-selectin as a novel target for the anti-angiogenic therapy. [source] Stromelysin-1 (MMP-3) is critical for intracranial bleeding after t-PA treatment of stroke in miceJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 8 2007Y. SUZUKI Summary.,Background:,Tissue-type plasminogen activator (t-PA) is approved for treatment of ischemic stroke patients, but it may increase the risk of intracranial bleeding (ICB). Matrix metalloproteinases (MMPs), which can be activated through the plasminogen/plasmin system, may contribute to ICB after ischemic stroke. Objectives:,To explore the contribution of plasminogen, MMP-3 and MMP-9 to ICB associated with t-PA treatment after ischemic stroke. Methods:,Using a thrombotic middle cerebral artery occlusion (MCA-O) model, ICB was studied in mice with genetic deficiencies of plasminogen (Plg,/,), stromelysin-1 (MMP-3,/,), or gelatinase B (MMP-9,/,) and their corresponding wild-type (WT) littermates. The induction of MMP-3 and MMP-9 was also studied in C57BL/6 WT mice. Results:,ICB induced by t-PA (10 mg kg,1) was significantly less than WT in Plg,/, (P < 0.05) and MMP-3,/, (P < 0.05) but not in MMP-9,/, mice. Furthermore, administration of the broad-spectrum MMP inhibitor GM6001 after t-PA treatment reduced ICB significantly (P < 0.05) in MMP-3+/+ mice, but had no effect on MMP-3,/, mice. MMP-3 expression was significantly enhanced at the ischemic hemisphere; with placebo treatment, it was expressed only in neurons, whereas it was up-regulated in endothelial cells with t-PA treatment. Although MMP-9 expression was also significantly enhanced at the ischemic brain, the amount and the distribution were comparable in mice with and without t-PA treatment. Conclusions:,Our data with gene-deficient mice thus suggest that plasminogen and MMP-3 are relatively more important than MMP-9 for the increased ICB induced by t-PA treatment of ischemic stroke. [source] The relative kinetics of clotting and lysis provide a biochemical rationale for the correlation between elevated fibrinogen and cardiovascular diseaseJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 6 2007P. Y. KIM Summary.,Background:,Elevated plasma fibrinogen is a well known risk factor for cardiovascular disease. The mechanistic rationale for this is not known.Objectives:,These studies were carried out to determine the fibrinogen concentration dependencies of clotting and lysis times and thereby determine whether these times rationalize the correlation between an increased risk of cardiovascular disease and elevated plasma fibrinogen.Methods:,The time courses of clot formation and lysis were measured by turbidity in systems comprising a) fibrinogen, thrombin and plasmin, or b) fibrinogen, thrombin, plasminogen and t-PA, or c) plasma, thrombin and t-PA. From the lysis times, kcat and Km values for plasmin action on fibrin were determined.Results:,The time to clot increased linearly from 2.9 to 5.6 minutes as the fibrinogen concentration increased from 1 to 9 ,M and did not increase further as the fibrinogen concentration was raised to 20 ,M. In contrast, the clot lysis time increased linearly over the input fibrinogen concentration range of 2 to 20 ,M. A similar linear trend was found in the two systems with t-PA and plasminogen. Apparent Km and kcat values for plasmin were 1.1 ± 0.6 ,M and 28 ± 2 min,1, respectively. Km values for plasmin in experiments initiated with t-PA and plasminogen were 1.6 ± 0.2 ,M in the purified system and 2.1 ± 0.9 ,M in plasma.Conclusion:,As the concentration of fibrinogen increases, especially above physiologic level, the balance between fibrinolysis and clotting shifts toward the latter, providing a rationale for the increased risk of cardiovascular disease associated with elevated fibrinogen. [source] Complete inhibition of fibrinolysis by sustained carboxypeptidase B activity: the role and requirement of plasmin inhibitorsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 6 2007J. B. WALKER Summary.,Background:,The antifibrinolytic effect of activated thrombin-activatable fibrinolysis inhibitor (TAFIa) and carboxypeptidase B (CPB) displays threshold behavior. When CPB was used to simulate conditions mimicking continuous TAFIa activity, it affected the lysis of plasma clots differently to clots formed from a minimal fibrinolytic system comprising fibrinogen, plasminogen and ,2 -antiplasmin. Whereas CPB saturably prolonged clot lysis in the purified system, the effect of CPB did not appear saturable in plasma clots. Methods:,To rationalize this difference, we investigated the effects of ,2 -antiplasmin, ,2 -macroglobulin, antithrombin and aprotinin on CPB-mediated antifibrinolysis. Results:,CPB alone prolonged fibrinolysis in a saturable manner and the efficacy of CPB increased with decreasing tissue-type plasminogen activator (t-PA) concentration. The inhibitors by themselves did not halt fibrinolysis and the potency of each inhibitor in the absence of CPB mirrored their solution-phase plasmin inhibitory potentials: ,2 -antiplasmin , aprotinin >> ,2 -macroglobulin >> antithrombin. With both CPB and inhibitor present, a synergistic effect was observed. The antifibrinolytic sensitivity to CPB was related to the plasmin inhibitory potential of the inhibitor. Conclusions:,Fibrinolysis could be completely inhibited by ,2 -antiplasmin, ,2 -macroglobulin and antithrombin, but not aprotinin, in the presence of CPB, and occurred only when the irreversible inhibitor or pool of inhibitors were in excess of plasminogen. Western blot analysis indicated that the CPB-mediated shutdown of fibrinolysis was a result of plasminogen consumption prior to clot lysis. The CPB concentration required for fibrinolytic shutdown was dependent on t-PA concentration and the inhibitory potential of the irreversible inhibitor pool. [source] What the structure of angiostatin may tell us about its mechanism of actionJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 1 2004J. H. Geiger Summary., Originally discovered in 1994 by Folkman and coworkers, angiostatin was identified through its antitumor effects in mice and later shown to be a potent inhibitor of angiogenesis. An internal fragment of plasminogen, angiostatin consists of kringle domains that are known to be lysine-binding. The crystal structure of angiostatin was the first multikringle domain-containing structure to be published. This review will focus on what is known about the structure of angiostatin and its implications in function from the current literature. [source] ,2 -Antiplasmin plays a significant role in acute pulmonary embolismJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 8 2003H. Matsuno Summary., The importance of pulmonary embolism (PE) due to venous thrombosis is recognized in the treatment of vascular diseases. We have investigated the physiological effects of plasmin generation in experimental acute PE using mice deficient in plasminogen (Plg,/,) or ,2 -antiplasmin (,2 -AP,/,). PE was induced by continuous induction of venous thrombus in the left jugular vein by endothelial injury due to photochemical reaction. The mortality of wild-type mice was 68.8% at 2 h after the initiation of venous thrombosis and it was significantly reduced in ,2 -AP,/, mice (41.7%). In contrast, Plg,/, mice did not survive. Histological evidence of thromboembolism in the lung was obtained in all mice. However, whereas a strict thromboembolism was observed in Plg,/, mice, only a few thrombi were detected in the lungs of ,2 -AP,/, mice. Plasma fibrinogen levels measured in mice were not different. When ,2 -AP was infused in ,2 -AP,/, mice, the mortality was indistinguishable from wild-type mice. Tissue-type plasminogen activator (tPA) did not reduce the mortality due to acute PE in wild-type mice. However, in ,2 -AP,/, mice, tPA (0.52 mg kg,1) significantly decreased the mortality compared with that of ,2 -AP,/, mice without tPA. The bleeding time was not significantly prolonged in either type of mice treated with tPA. The lack of plasminogen increases the mortality due to acute PE while a lack of ,2 -AP decreases the mortality rate, which can be further reduced by tPA administration. Therefore, the combination of inhibition of ,2 -AP with thrombolytic therapy could be beneficial in the treatment of acute PE. [source] Thrombin-activatable fibrinolysis inhibitor (TAFI, plasma procarboxypeptidase B, procarboxypeptidase R, procarboxypeptidase U)JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 7 2003B. N. Bouma Summary., Recently, a new inhibitor of fibrinolysis was described, which downregulated fibrinolysis after it was activated by thrombin, and was therefore named TAFI (thrombin-activatable fibrinolysis inhibitor; EC 3.4.17.20). TAFI turned out to be identical to the previously described proteins, procarboxypeptidase U, procarboxypeptidase R, and plasma procarboxypeptidase B. Activated TAFI (TAFIa) downregulates fibrinolysis by the removal of carboxy-terminal lysines from fibrin. These carboxy-terminal lysines are exposed upon limited proteolysis of fibrin by plasmin and act as ligands for the lysine-binding sites of plasminogen and tissue-type plasminogen activator (t-PA). Elimination of these lysines by TAFIa abrogates the fibrin cofactor function of t-PA-mediated plasminogen activation, resulting in a decreased rate of plasmin generation and thus downregulation of fibrinolysis. In this review, the characteristics of TAFI are summarized, with an emphasis on the pathways leading to activation of TAFI and the role of TAFIa in the inhibition of fibrinolysis. However, it cannot be ruled out that TAFI has other, as yet undefined, functions in biology. [source] A study on associations between antiprothrombin antibodies, antiplasminogen antibodies and thrombosisJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 4 2003M. J. A. Simmelink Summary., Anti-prothrombin antibodies are a frequent cause of lupus anticoagulant (LAC), a thrombotic risk factor. Prothrombin shares structural homology with plasminogen, a kringle protein with an important role in fibrinolysis. Cross-reactivity between antiprothrombin antibodies and plasminogen has been described. To study associations between LAC, IgG and IgM class antiprothrombin and antiplasminogen antibodies, plasminogen activity levels and thrombosis in selected patients with systemic autoimmune diseases. Patients included forty-six consecutive LAC-positive patients (29 with systemic lupus erythematosus (SLE); 33 with a thrombotic history), 38 patients without LAC (36 with SLE; seven with a history of thrombosis) and 40 healthy controls. In the total group of 84 patient samples, the prevalence of antiprothrombin and antiplasminogen antibodies was 30 and 38%, respectively. There was no significant relationship between the presence of these antibodies. In contrast to presence of antiplasminogen antibodies, presence of antiprothrombin antibodies was statistically significant related to thrombosis. Thirteen samples had antiprothrombin and antiplasminogen antibodies of similar isotype (IgG, n= 4; IgM, n= 9). Of these, all but one had LAC and 11/13 came from patients with a history of thrombosis. Simultaneous presence of IgM-class antiprothrombin and antiplasminogen antibodies had a significant association with thrombosis. Levels of plasminogen activity were similar in samples from healthy controls and patients (with or without antiplasminogen antibodies or thrombosis). Anti-prothrombin antibodies and antiplasminogen antibodies occur frequently in patients with systemic autoimmune disease. Anti-prothrombin antibodies, but not antiplasminogen antibodies are a risk factor for thrombosis. Anti-plasminogen are in most cases unrelated to antiprothrombin antibodies. [source] Fibrinolytic Poly(dimethyl siloxane) SurfacesMACROMOLECULAR BIOSCIENCE, Issue 9 2008Hong Chen Abstract PDMS surfaces have been modified to confer both resistance to non-specific protein adsorption and clot lyzing properties. The properties and chemical compositions of the surfaces have been investigated using water contact angle measurements, ATR FT-IR spectroscopy, and XPS. The ability of the PEG component to suppress non-specific protein adsorption was assessed by measurement of radiolabeled fibrinogen uptake from buffer. The adsorption of plasminogen from human plasma to the various surfaces was studied. In vitro experiments demonstrated that lysine-immobilized surfaces with free , -amino groups were able to dissolve fibrin clots, following exposure to plasma and tissue plasminogen activator. [source] The invasive behaviour of prostatic cancer cells is suppressed by inhibitors of tyrosine kinase,APMIS, Issue 1 2006HAAKON SKOGSETH Proteolytic enzymes, and especially urokinase plasminogen activator (uPA), play an important role in tumour invasion and metastasis. Previously we demonstrated that the production of urokinase plasminogen activator (uPA) was decreased by several tyrosine kinase inhibitors (TKI) in two prostatic carcinoma cell lines. The effect of the two TKI genistein and tyrphostin AG-1478 was investigated in the prostate carcinoma cell lines PC-3 and DU-145. A reconstituted basal lamina (Matrigel) was used as a migration barrier. The production of matrix metalloproteinases (MMP) was also measured. Roles of plasminogen and uPA were examined. Cell invasion was increased by plasminogen, but this enhanced cell migration was counteracted by TKI treatment. The increased cell invasion induced by plasminogen was decreased by at least 60% in both cell lines when ,-2 anti-plasmin was added to the assay. Cells in the absence of plasminogen were not affected by TKI. External uPA failed to regenerate the decreased cell invasion caused by TKI. The production of MMP was inhibited by both TKI. Our results indicate a possible role of TKI as inhibitors of cancer cell invasion by inhibiting uPA and MMP production. [source] | |||||||||||||