Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Monocytes and monocytoid cell lines previously have been shown to express receptors for plasminogen and urokinase (u-PA). In the present study, the monocytoid cell lines, U937 and THP-1, are shown to bind tissue plasminogen activator (t-PA) in a specific, saturable, and reversible manner. These cells bound t-PA with low affinity (kd = 0.67 to 0.97 mumol/L) and high capacity (0.71 to 3.3 x 10(6) receptors/cell). Human peripheral blood monocytes bound t-PA with a kd (0.9 mumol/L) similar to that of the monocytoid cells but with a lower capacity (0.17 x 10(6) sites/cell). These binding parameters also were similar to the low-affinity interaction of t-PA with endothelial cells as measured with the cells in suspension (kd = 0.73 mumol/L and 1.1 x 10(6) sites/cell). Lysine analogues and active or diisopropylfluorophosphate-inactivated u-PA inhibited t-PA binding to monocytes, monocytoid cells, and endothelial cells with similar IC50 (concentration producing 50% inhibition) values, suggesting that the same recognition specificity mediates t-PA binding to all of these cell types. The existence of a high-affinity binding site for t-PA on monocytoid cells was also explored in detail. Unlike endothelial cells where plasminogen activator inhibitor-1 has been implicated in mediating a high-affinity interaction of t-PA with the cells, no evidence for a role of this inhibitor in ligand binding to the monocytoid cells was found. Furthermore, using both high and low 125I-t-PA concentrations, competition analyses with lysine analogues or u-PA, or treatment of the cells with carboxypeptidase B, failed to indicate the presence of distinguishable classes of t-PA binding sites. In sum, low-affinity receptors for t-PA are expressed at high density on monocytes and monocytoid cells, identifying a new element in the fibrinolytic arsenal of these cells.
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PMID:Binding of tissue plasminogen activator to human monocytes and monocytoid cells. 193 47

Mononuclear phagocytes regulate the generation of plasmin by secreting urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-2 (PAI-2). We investigated the production of plasminogen activator (PA) and PA inhibitor by the human monocytic leukemia cell line, THP-1. Similar to U937 monoblast-like cells and peripheral blood monocytes (PBM), THP-1 cells produce a PA that is specifically neutralized by anti-uPA antibody and comigrates with human high molecular mass uPA (54 kDa) on casein-plasminogen zymogaphy. PA activity could be dissociated from intact THP-1 cells by brief treatment with a weak acid-glycine buffer, indicating that the uPA is secreted and bound to receptors on the plasma membrane. Regulation of uPA proceeds normally in THP-1 cells, with cell-associated PA activity increasing from 77 +/- 20 to 163 +/- 26 and 325 +/- 30 mPU/10(6) cells in response to PMA and LPS, respectively; parallel increases in steady state levels of uPA mRNA were observed. In contrast to normal expression of uPA activity, functional PAI-2 could not be demonstrated in either the conditioned media or cell lysates of THP-1 under basal or stimulated conditions. Both U937 and PBM secrete low levels of PA inhibitor activity that increase substantially in response to stimulation with PMA and LPS. Immunoreactive PAI-2, measured by ELISA, was undetectable in THP-1 lysates or conditioned medium, but was consistently present in U937 and PBM, paralleling the presence of PA inhibitor activity. THP-1 cells express low levels of an abnormally sized mRNA for PAI-2 and demonstrate a regulatory defect whereby steady state levels of PAI-2 mRNA are markedly reduced upon stimulation with PMA or LPS. By contrast, U937 and PBM respond to identical stimulation with increases in PAI-2 mRNA. We conclude that THP-1 cells express a structurally abnormal species of PAI-2 mRNA, with complete loss of inhibitory activity as well as altered function of PMA- and LPS-responsive regulatory elements.
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PMID:The THP-1 cell line is a urokinase-secreting mononuclear phagocyte with a novel defect in the production of plasminogen activator inhibitor-2. 230 45

Nanoparticles were prepared from methoxy poly(ethylene glycol)poly(d,l-lactic acid) block copolymers (Me.PEG-PLA) or blends of Me.PEG-PLA and PLA by the precipitation-solvent diffusion method. These nanoparticles, labeled by introducing [14C]PLA in the formulation, were shown to be more slowly captured by cultured THP-1 monocytes than F68-coated PLA nanoparticles, in a PEG chain-length-dependent manner. In vivo, the half-life in plasma of the Me.PEG-PLA nanoparticles that were intravenously administered to rats is increased by a factor 180 compared with the F68-coated PLA nanoparticles. This mononuclear phagocytes system avoidance was explained according to a conformation model in which the PEG density at the surface of the particles is a key parameter.
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PMID:Stealth Me.PEG-PLA nanoparticles avoid uptake by the mononuclear phagocytes system. 762 43

Plasminogen activator inhibitor-1 (PAI-1) is the most important inhibitor of tissue-type plasminogen activator (t-PA) in plasma and plays a major role in the regulation of fibrinolysis. Plasma t-PA/PAI-1 complexes are cleared via a receptor-dependent mechanism in hepatocytes, while the fate of complexes formed in the extracellular matrix and in thrombi is less well understood. In this study, the degradation of t-PA/PAI-1 complexes by monocytes was examined. THP-1 monocytoid cells and freshly isolated human monocytes internalize and degrade [125I]t-PA/PAI-1 complexes at rates of 11.4 +/- 5.9 (mean +/- S.D.) and 44.6 +/- 6.3 ng/10(6) cells/h, respectively. Degradation is blocked by receptor-associated protein (RAP), indicating a member of the low density lipoprotein (LDL) receptor family is involved in the uptake/degradation of t-PA/PAI-1 complexes by monocytes. Degradation of t-PA/PAI-1 complexes is also inhibited by chloroquine and by pepstatin A, suggesting that a lysosomal aspartyl protease is likely involved. SDS-PAGE and Western blotting demonstrated that the purified lysosomal aspartyl protease, cathepsin D, is capable of digesting t-PA (t1/2 15 min), active PAI-1 (t1/2 2 h), and t-PA/PAI-1 complex (t1/2 30 min). Cathepsin D sequentially cleaves PAI-1 after hydrophobic amino acids, yielding lower molecular weight fragments. PAI-1 conformation influences the degradative efficiency of cathepsin D, with vitronectin-bound PAI-1 and latent PAI-1 exhibiting resistance to proteolysis and > 10-fold prolongation in t1/2. These data provide evidence that t-PA/PAI-1 complexes are internalized by human monocytes via a member of the low density lipoprotein (LDL) receptor family, and identifies cathepsin D-like aspartyl protease activity as largely responsible for the degradation of these complexes. Furthermore, vitronectin-bound PAI-1 and latent PAI-1 are relatively resistant to degradation by cathepsin D, which may be of importance in complex physiological environments.
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PMID:Cathepsin D-like aspartyl protease activity mediates the degradation of tissue-type plasminogen activator/plasminogen activator inhibitor-1 complexes in human monocytes. 766 1

Fibrin(ogen) (FGN) is important for hemostasis and wound healing and is cleared from sites of injury primarily by the plasminogen activator system. However, there is emerging evidence in plasminogen activator-deficient transgenic mice that nonplasmin pathways may be important in fibrin(ogen)olysis, as well. Given the proximity of FGN and monocytes within the occlusive thrombus at sites of vascular injury, we considered the possibility that monocytes may play an ancillary role in the degradation and clearance of fibrin. We found that monocytes possess an alternative fibrinolytic pathway that uses the integrin Mac-1, which directly binds and internalizes FGN, resulting in its lysosomal degradation. At 4 degrees C, FGN binds to U937 monocytoid cells in a specific and saturable manner with a kd of 1.8 mumol/L. Binding requires adenosine diphosphate stimulation and is calcium-dependent. At 37 degrees C, FGN and fibrin monomer (FM) are internalized and degraded at rates of 0.37 +/- 0.13 and 0.55 +/- 0.03 microgram/10(6) cells/h by U937 cells, 1.38 +/- 0.02 and 1.20 +/- 0.30 microgram/10(6) cells/h by THP-1 cells, and 2.10 +/- 0.20 and 2.52 +/- 0.18 micrograms/10(6) cells/h by human peripheral blood mononuclear cells, respectively. The serine protease inhibitors, PPACK and aprotinin, and the specific elastase inhibitor, AAPVCK, do not significantly inhibit degradation. However, degradation is inhibited by chloroquine, suggesting that a lysosomal pathway is involved. Factor X, a competitive ligand with FGN for the Mac-1 receptor, also blocks degradation, as does a monoclonal antibody to the alpha-subunit of Mac-1. Autoradiography of radioiodinated, internalized FGN shows that FGN proteolysis by the pathway produces a unique degradation pattern distinct from that observed with plasmin. In a fibrin clot lysis assay, Mac-1-mediated fibrinolysis contributed significantly to total fibrinolysis. In summary, FGN is internalized and degraded by activated human monocytoid cells via Mac-1 in the absence of plasmin, thereby providing an alternative fibrinolytic pathway. Thus, in addition to the function of cell adhesion, integrins may also act as receptors that mediate the internalization and degradation of bound ligands.
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PMID:Fibrin(ogen) is internalized and degraded by activated human monocytoid cells via Mac-1 (CD11b/CD18): a nonplasmin fibrinolytic pathway. 840 Feb 91

The low density lipoprotein receptor-related protein (LRP) is a multiligand clearance receptor that removes free tissue-type plasminogen activator (t-PA) or complexes of t-PA with plasminogen activator inhibitor type 1 (PAI-1) from the blood circulation or the pericellular space. Co-receptors are essential for LRP-mediated clearance of several ligands (e.g. glycosaminoglycans for thrombin/protease nexin and lipoprotein lipase, and the urokinase receptor for urokinase/PAI-1 complexes). The present study was undertaken to investigate whether LRP-mediated t-PA clearance requires a co-receptor as well. In five cell lines from different organs and species degradation of t-PA and t-PA/PAI-1 was mediated by LRP (or LRP-like receptors). No degradation of t-PA and t-PA/PAI-1 occurred in THP-1 or U-937 human monocyte-like cells, despite the presence of functional LRP. As glycosaminoglycans can bind t-PA and PAI-1 we investigated whether they are involved in t-PA/PAI-1 degradation. Pre-treatment of COS cells or HT1080 cells with chlorate, an inhibitor of glycosaminoglycan sulfation, did not decrease t-PA/PAI-1 degradation. Furthermore, CHO cells genetically deficient in glycosaminoglycans efficiently degraded t-PA/PAI-1. Thus it is unlikely that glycosaminoglycans are co-receptors for degradation of t-PA or t-PA/PAI-1. This study indicates that THP-1 and U-937 cells lack a critical component (co-receptor?) for the LRP-mediated degradation of t-PA.
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PMID:Cellular degradation of free and inhibitor-bound tissue-type plasminogen activator--requirement for a co-receptor? 1073 88

The chemokine macrophage inflammatory protein (MIP)-2alpha was identified as a plasminogen binding protein by phage display analysis. MIP-2alpha and a truncated form lacking 5 lysine residues in the COOH-terminal region (mut-MIP-2alpha) were expressed in E. coli and purified to apparent homogeneity. Purified MIP-2alpha but not mut-MIP-2alpha bound specifically to plasminogen, with K(A) of 3.7 X 10(5) M(-1) for the interaction of plasminogen with surface-bound MIP-2alpha. Binding and competition experiments indicated that the interaction involves the region comprising the first 3 kringles of plasminogen and the COOH-terminal lysine-rich domain of MIP-2alpha. Activation of plasminogen bound to surface-associated MIP-2alpha by two-chain urokinase-type plasminogen activator (tcu-PA) was about 2.5-fold more efficient than in solution (catalytic efficiency k(cat)K(M) of 0.1 microM(-1)s(-1), as compared to 0.04 microM(-1)s(-1). In contrast, binding of plasminogen to MIP-2alpha in solution was very weak, as evidenced by the absence of competition of MIP-2alpha with lysine-Sepharose or with human THP-1 cells for binding of plasminogen. In agreement with this finding, addition of excess MIP-2alpha did not affect the main functional properties of plasmin(ogen) in solution, as indicated by unaltered activation rates of plasminogen by tcu-PA or tissue-type plasminogen activator (t-PA), t-PA-mediated fibrinolysis, and inhibition rate of plasmin by alpha2-antiplasmin. Thus, association of MIP-2alpha with surfaces exposes its COOH-terminal plasminogen-binding site, and may result in enhanced local plasmin generation.
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PMID:Plasminogen binding properties of macrophage inflammatory protein (MIP)-2alpha. 1092 73

Hemostatic and immunologic factors have been implicated in future cardiac events in patients with coronary artery disease. The role of these factors and their interaction is less established in cardiac transplant recipients. We sought to characterize the role of these factors in these patients. Cardiac transplant patients who presented for surveillance coronary angiography and/or endomyocardial biopsy were eligible for enrollment. Ninety-nine consecutive patients were enrolled. Plasma levels of tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1, von Willebrand factor, fibrin D-dimer, and anti-t-PA antibody were determined by enzyme-linked immunosorbent assays. Anti-THP-1 cell antibodies directed against a monocytic leukemia cell line were detected by incubating patient plasma with THP-1 cells. Bound antibody was detected using goat peroxidase-labeled immunoglobulin G directed against human immunoglobulins. Lipids were measured by enzymatic methods. Multivariate analysis identified the presence of anti-THP-1 cell antibodies (risk ratio 4.41, p = 0.002), t-PA antigen (risk ratio 1.10, p = 0.033), donor age 20 to 26 years (risk ratio 8.83, p = 0.042), and donor age >36 years (risk ratio 15.53, p = 0.009) as predictors of allograft failure. Altered hemostatic function, as demonstrated by elevated plasma t-PA antigen levels, is predictive of subsequent allograft failure in cardiac transplant recipients. In addition, the presence of anti-THP-1 cell antibodies in these patients is predictive of allograft failure.
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PMID:Elevated plasma tissue plasminogen activator and anti-THP-1 antibodies are independently associated with decreased graft survival in cardiac transplant recipients. 1142 54

It is shown that the release of matrix metalloproteinase-9 (gelatinase B) by THP-1 and U937 cells into conditioned media is increased under the action of recombinant single-chain urokinase. This effect is not accompanied by proteolytic activation of gelatinase B and is related to release of a pro-form of the enzyme. The action of urokinase on monocytes is time-dependent and becomes significant 12-24 h after the beginning of cell incubation. The dependence of the effect on the concentration of urokinase is characterized by half-maximum at about 20 nM and saturation at about 200 nM. The urokinase-induced gelatinase B release is not dependent on the action of plasmin because plasmin inhibitors aprotinin and alpha2-antiplasmin do not abolish this action. Additionally, tissue type plasminogen activator does not induce gelatinase B release by monocytes as observed under the action of urokinase. Nevertheless, the catalytic activity of urokinase participates in the development of the observed effect because it is significantly depressed by the natural urokinase inhibitor PAI-1. The effect of urokinase is completely abolished by actinomycin D and cycloheximide, indicating the participation of transcription and translation processes in its development.
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PMID:Plasmin-independent gelatinase B (matrix metalloproteinase-9) release by monocytes under the influence of urokinase. 1170 74

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha), one of the three subtypes of cPLA(2) (alpha, beta and gamma), is thought to be a rate-limiting enzyme in eicosanoid biosynthesis. We developed a novel and potent cPLA(2)alpha inhibitor with an optically active pyrrolidine, termed pyrrophenone, and characterized this compound in detail using enzyme and cellular assay systems. Pyrrophenone, which shows strong inhibition of cPLA(2)alpha activity, is one of the most potent cPLA(2)alpha inhibitors reported to date. Similar inhibitory potencies for cPLA(2)alpha were obtained from three different assays. The inhibitory activity of pyrrophenone is two or three orders of magnitude more potent than arachidonyl trifluoromethyl ketone (AACOCF(3)) under the same assay conditions. Pyrrophenone shows reversible inhibition of cPLA(2)alpha and displays no characteristics of the slow-binding inhibition observed for AACOCF(3). Pyrrophenone also inhibited the esterase and lysophospholipase activities of cPLA(2)alpha. However, the inhibition by pyrrophenone of 14 kDa secretory PLA(2)s, types IB and IIA, was over two orders of magnitude less potent than that for cPLA(2)alpha. Pyrrophenone strongly inhibited arachidonic acid release in calcium ionophore (A23187)-stimulated human monocytic cells (THP-1 cells) in a dose-dependent manner with an IC(50) value of 0.024 microM, followed by suppression of eicosanoid synthesis, and also showed dose-dependent inhibition for interleukin-1-induced prostaglandin E(2) synthesis in human renal mesangial cells with an IC(50) value of 0.0081 microM. The mechanism of inhibition of eicosanoid synthesis in these cell-based assays was due to inhibition of only one step of arachidonic acid release without any effect on cyclo-oxygenase or lipoxygenase pathways. These results suggest that pyrrophenone could be a potential therapeutic agent for inflammatory diseases.
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PMID:Characterization of a novel inhibitor of cytosolic phospholipase A2alpha, pyrrophenone. 1196 73


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