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Biomedical subjects

R W Stephens

Publications and source records attributed to R W Stephens.

At least 19 recordsLinked to original sources
PubMed1992

Heparin binding to the urokinase kringle domain.

The binding of urokinase to immobilized heparin and dextran sulfate was studied using activity assays of the bound urokinase. The markedly higher binding observed with high M(r) urokinase compared to low M(r) urokinase indicated a role for the amino-terminal fragment (ATF). This was confirmed by the use of inactive truncated urokinase and monoclonal antibodies specific for the ATF in competition assays of urokinase binding. Antibody competition assays suggested a site in the kringle domain, and a synthetic decapeptide Arg-52-Trp-62 from the kringle sequence (kringle numbering convention) was competitive in assays of urokinase binding to dextran sulfate and heparin. Heparin binding to the urokinase kringle was unambiguously demonstrated via 1H NMR spectroscopy at 500 MHz. Effective equilibrium association constants (K(a)*) were determined for the interaction of isolated kringle fragment and low M(r) heparin at pH 7.2. The binding was strong in salt-free 2H2O (K(a)* approximately 57 mM-1) and remained significant in 0.15 M NaCl (K(a)* approximately 12 mM-1), supporting a potential physiological role for the interaction. This is the first demonstration of a function for the kringle domain of urokinase, and it suggests that while the classical kringle structure has specificity for lysine binding, there may also exist a class of kringles with affinity for polyanion binding.

Amino Acid Sequence↗
PubMed1992

Urokinase binding to laminin-nidogen. Structural requirements and interactions with heparin.

Recently we have shown that heparin and related sulfated polyanions are low-affinity ligands of the kringle domain in the amino-terminal region (ATF) of human urokinase (u-PA), and proposed that this may facilitate loading of u-PA onto its receptor at the focal contacts between adherent cells and their matrix. We have now tested other components of the cell matrix (fibronectin, vitronectin, thrombospondin and laminin-nidogen) for u-PA binding, and found that laminin-nidogen is also a ligand of the u-PA ATF. Direct binding assays and competition binding assays with defined fragments of laminin-nidogen showed that there are u-PA binding sites in fragment E4 of laminin as well as in nidogen. The long-arm terminal domain of laminin (fragment E3), which contains a heparin-binding site, competed for binding of u-PA to immobilised heparin. However nidogen, which does not bind to heparin, also inhibited binding of u-PA to heparin, and this effect was also observed with recombinant nidogen and with a fragment of nidogen lacking the carboxy-terminal domain. Direct binding assays confirmed that u-PA binds to nidogen through a site in the u-PA ATF. We conclude that u-PA binds to laminin-nidogen by interactions involving the ATF region of u-PA, the E4 domain of laminin and the rod or amino-terminal regions of nidogen. Since nidogen is suggested to be an important bridging molecule in the maintenance of the supramolecular organization in basement membranes, the presence of a binding site for u-PA in nidogen indicates a role for plasminogen activation in basement membrane remodelling.

Binding, Competitive↗
PubMed1992

Retinal pigment epithelial cells secrete urokinase-type plasminogen activator and its inhibitor PAI-1.

Secretion of plasminogen activators and their inhibitors was examined in cultures of human retinal pigment epithelial (RPE) cells. The methods employed were zymography and reverse zymography, solid-phase immunocapture assay, metabolic labeling followed by immunoprecipitation, and immunofluorescence. The results showed that these cells produce urokinase-type plasminogen activator (u-PA) and a plasminogen activator inhibitor (PAI) which is immunologically and biochemically similar to PAI-1. Tissue-type plasminogen activator activity (t-PA) was not detected, but we detected small amounts of t-PA in an inactive complex with inhibitor in RPE cell-conditioned media. We conclude that RPE cells have the potential to utilize u-PA-catalyzed plasminogen activation which is subject to regulation by PAI-1. These results may have a bearing on the pathogenesis of proliferative retinal diseases.

Antibodies, Monoclonal↗
PubMed1991

Stimulation of cell surface plasminogen activation by heparin and related polyionic substances.

The functional operation of the cell surface pro-u-PA and plasminogen activating system has previously been shown to depend on the assembly of u-PA receptors, plasminogen binding sites, and their respective ligands at the focal adhesions of cell extensions. We now show that additional factors operate that affect the persistence of functional activity and that evidently involve charge interactions mediated by polyanions, such as those found in the cell surface proteoglycans. Heparin-like compounds and protamine were identified as fast-acting stimulators of cell surface plasminogen activation. Heparin stabilized surface u-PA activity during plasminogen activation, and we propose that a heparin binding site exists in the kringle structure of u-PA. Heparin at 40 micrograms/ml could reduce u-PA loss to only 20% compared with 60% on control cells activating plasminogen. Protamine (25 micrograms/ml) exerted a strong stimulatory effect on the level of generated bound plasmin and notably prolonged the persistence of this activity, so that 100 minutes after addition of plasminogen the level of plasmin on protamine-treated cells was five times higher than on control-treated cells. The effect of protamine on plasmin clearance suggests that an unknown plasmin inhibitor may be produced by rhabdomyosarcoma cells, whose action is accelerated by endogenous polyanions, in an analogous manner to thrombin inactivation by antithrombin III and protease nexin on endothelial cells and fibroblasts, respectively. The stimulatory effects of heparin and protamine do not affect the inactivation of cell surface u-PA by recombinant PAI-2.

Fibrinolysin↗
PubMed1991

Alpha 2-macroglobulin restricts plasminogen activation to the surface of RC2A leukemia cells.

Human RC2A myelomonocytic leukemia cells are able to activate the prourokinase (pro-u-PA) they secrete so that active u-PA is present both in serum-free conditioned medium from these cells, as well as on the cell surface. When the cells are grown in serum-containing medium, no u-PA activity can be found in the medium but active u-PA is found bound to the cell surface where it can generate bound plasmin. This distribution of u-PA activity was shown to be, first, the net result of slow inactivation of free active u-PA by serum inhibitor(s) and simultaneous rapid uptake of u-PA onto the cell surface. Binding to cells was at least six times faster than inactivation by 10% serum. The principal serum inhibitor of u-PA was identified as alpha 2-macroglobulin (alpha 2M), and prior inactivation of u-PA by purified human alpha 2M was also shown to prevent uptake of u-PA activity onto cells. Second, although endogenous u-PA could form covalent complexes with purified alpha 2M in the culture medium of RC2A cells, covalent alpha 2M complexes were not formed by u-PA on the cell surface; the u-PA taken up in this compartment was protected against alpha 2M inhibition. u-PA anchored to plastic surfaces via monoclonal antibodies to the amino-terminal region of u-PA was also protected against alpha 2M, suggesting that the protection of cell surface u-PA results from a steric effect. These results provide evidence as to how the active u-PA produced by leukemia cells can contribute to proteolytic activity on their cell surface in the presence of serum inhibitors.

Antibodies, Monoclonal↗
PubMed1990

Prourokinase activation on the surface of human rhabdomyosarcoma cells: localization and inactivation of newly formed urokinase-type plasminogen activator by recombinant class 2 plasminogen activator inhibitor.

Recombinant class 2 plasminogen activator inhibitor (PAI-2) was used in an approach to probe the formation and location of enzymatically active urokinase-type plasminogen activator (u-PA) sites on the surface of cultured human rhabdomyosarcoma cells (RD cells). Activation of pro-u-PA on the cell surface and consequent binding of PAI-2 was dependent on the addition of native plasminogen to serum cultures of the cells. Inhibition of the enzyme activity of surface-bound u-PA by the added PAI-2 resulted in a 79% reduction in the capacity of the RD cells to generate cell surface-associated plasmin activity from bound plasminogen. Under these conditions, the PAI-2 probe was localized at focal adhesions of RD cells, where it colocalized with both extracellular u-PA and intracellular vinculin antigens in double immunofluorescence labeling. Specificity of the probe's interaction with cell surface-bound u-PA was confirmed by blocking with a monoclonal antibody to human u-PA, which could also inhibit the formation of bound plasmin activity. These results showed the assembly of the plasmin-generating system at focal adhesions and the accessibility of bound u-PA on which it depends to added PAI-2. Therefore, PAI-2 has the potential both to localize at sites of tumor expression of functionally active u-PA and simultaneously to inhibit cell surface plasminogen activation.

Cell Line↗
PubMed1990

Plasminogen activation by t-PA on the surface of human melanoma cells in the presence of alpha 2-macroglobulin secretion.

Several human melanoma cell lines produced tissue-type plasminogen activator (t-PA), as detected by zymography and immunocapture assay of culture media and cell lysates. Urokinase (u-PA) was found at only less than or equal to 1% the level of t-PA. Acid eluates of the cell surface indicated that the melanoma cells had t-PA bound on their surface, but no u-PA, and also had a very low capacity to bind exogenous u-PA. After incubation of the melanoma cells with 10% plasminogen-depleted fetal calf serum and human plasminogen, bound plasmin activity could be eluted from the cell surface with tranexamic acid, an analogue of lysine. This indicated that plasminogen was activated on the cell surface. The cell-surface plasmin formation was inhibited by an anti-catalytic monoclonal antibody to human t-PA, and not by an anti-catalytic antibody to u-PA. The melanoma cells also synthesized and secreted alpha 2-macroglobulin (alpha 2M), as shown by alpha 2M-specific mRNA in Northern blotting and detection of alpha 2M protein in conditioned cell culture media. The media were found to inhibit u-PA but not t-PA. This inhibition was related to their alpha 2M content, and immunoabsorption of alpha 2M removed the inhibitory activity. These studies suggest that t-PA can bind to the surface of melanoma cells and generate surface-bound plasmin. Because t-PA and cell-bound plasmin are unaffected by alpha 2M, t-PA may, in the case of melanoma cells, serve an analogous function to u-PA in supporting tumor cell invasion.

Antibodies, Monoclonal↗
PubMed1989

Induction of morphological differentiation of human neuroblastoma cells is accompanied by induction of tissue-type plasminogen activator.

Human SH-SY5Y neuroblastoma cells treated with retinoic acid, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or nerve growth factor differentiated morphologically to neuronlike cells with increased amounts of neurofilament protein and mRNA. All three effectors induced an increase in the amount of relative molecular weight (Mr) 70,000 tissue-type plasminogen activator (t-PA) and its mRNA, as determined by immunocapture, enzyme activity, and Northern blotting analyses. About 90% of the t-PA activity was secreted to the culture medium. In contrast, of the three effectors studied, only TPA induced transcription of the proto-oncogene c-fos, studied as a control gene responsive to various stimuli, and induced a rapid increase in urokinase-type PA (u-PA). Most of the u-PA activity induced by TPA remained cell-associated. Because induction of differentiation correlated closely with induction of t-PA, and not u-PA, the authors propose that t-PA may have a functional role in the morphological differentiation of neuronal cells.

Cell Differentiation↗
PubMed1989

Activation of pro-urokinase and plasminogen on human sarcoma cells: a proteolytic system with surface-bound reactants.

Human HT-1080 fibrosarcoma cells produce urokinase-type plasminogen activator (u-PA) and type 1 plasminogen activator inhibitor (PAI-1). We found that after incubation of monolayer cultures with purified native human plasminogen in serum-containing medium, bound plasmin activity could be eluted from the cells with tranexamic acid, an analogue of lysine. The bound plasmin was the result of plasminogen activation on the cell surface; plasmin activity was not taken up onto cells after deliberate addition of plasmin to the serum-containing medium. The cell surface plasmin formation was inhibited by an anticatalytic monoclonal antibody to u-PA, indicating that this enzyme was responsible for the activation. Preincubation of the cells with diisopropyl fluorophosphate-inhibited u-PA led to a decrease in surface-bound plasmin, indicating that a large part, if not all, of the cell surface plasminogen activation was catalyzed by surface-bound u-PA. In the absence of plasminogen, most of the cell surface u-PA was present in its single-chain proenzyme form, while addition of plasminogen led to formation of cell-bound two-chain u-PA. The latter reaction was catalyzed by cell-bound plasmin. Cell-bound u-PA was accessible to inhibition by endogenous PAI-1 and by added PAI-2, while the cell-bound plasmin was inaccessible to serum inhibitors, but accessible to added aprotinin and an anticatalytic monoclonal antibody. A model for cell surface plasminogen activation is proposed in which plasminogen binding to cells from serum medium is followed by plasminogen activation by trace amounts of bound active u-PA, to form bound plasmin, which in turn serves to produce more active u-PA from bound pro-u-PA. This exponential process is subject to regulation by endogenous PAI-1 and limited to the pericellular space.

Cell Line↗
PubMed1989

Tissue-type plasminogen activator in subretinal fluid.

The plasminogen activation system is quantitatively the major mechanism of extracellular proteolysis. To evaluate its role in retinal detachment, plasmin and plasminogen activators were measured in subretinal fluid (SRF) from 12 eyes of twelve patients. Plasmin was detected in 5 eyes (mean 6.26 micrograms/ml, SD = 3.7 micrograms/ml). Tissue-type plasminogen activator was present in 5 eyes (mean activity 0.33 IU/ml, SD = 0.24 IU/ml) but the activity did not associate with the plasmin activity in all of the SRF samples. Urokinase-type plasminogen activator was not detected in SRF. We conclude that the plasmin system has been activated in SRF in some eyes with retinal detachment with tissue-type plasminogen activator as the predominant activator. Plasmin in SRF may enhance dispersion of pigment epithelial cells into the subretinal space and the vitreous, a phenomenon seen frequently in eyes with retinal detachment.

Adult↗
PubMed1988

Urokinase-type plasminogen activator in colorectal carcinomas and adenomatous polyps: quantitative expression of active and proenzyme.

Total urokinase-type plasminogen activator (u-PA) content (proenzyme plus active enzyme) was significantly higher in 20 colorectal carcinomas and in 27 adenomatous polyps than in metaplastic polyps and autologous normal mucosa. u-PA content was also markedly increased in adenomatous polyps and autologous colonic mucosa removed from familial polyposis coli patients. Using a new monoclonal antibody technique to distinguish the proenzyme of u-PA from the active enzyme, we found that 70% of the u-PA in polyp and cancer tissue was present in the proenzyme form compared to 47% in normal colonic mucosa. For colon cancers, there was a significant correlation between their stage of invasiveness and the levels of proenzyme. No correlation was observed between the u-PA content of adenomatous polyps and their size or degree of dysplasia. Study of the u-PA content of the colonic mucosa may offer a useful biochemical correlate of epithelial cell transformation in the colon.

Adenoma↗
PubMed1988

Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A)+ RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the lambda PL promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated Mr of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 (3 amino acid differences) and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). Like ovalbumin, mPAI-2 appears to have no typical amino-terminal signal sequence. The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators.

Amino Acid Sequence↗
PubMed1988

Detection and partial characterization of a specific plasminogen activator inhibitor in human chondrocyte cultures.

Serum-free culture medium collected from primary monolayer cultures of human articular chondrocytes was found to inhibit human urokinase [EC 3.4.21.31] activity. Although chondrocyte culture medium contained a small amount of endothelial-type plasminogen activator inhibitor which could be demonstrated by reverse fibrin autography, most of the urokinase inhibitory activity of chondrocyte culture medium was shown to be due to a different molecule from endothelial-type inhibitor, since it did not react with a specific antibody to this type of inhibitor. The dominant urokinase inhibitor in chondrocyte culture medium was partially purified by concanavalin A-Sepharose affinity chromatography. The partially purified inhibitor inhibited high-Mr urokinase more effectively than low-Mr urokinase, but no obvious inhibition was detected against tissue-type plasminogen activator, plasmin, trypsin, and thrombin. The inhibitor had an apparent Mr of 43,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and it was unstable to sodium dodecyl sulfate, acid, and heat treatments. Inhibition of urokinase by the inhibitor was accompanied with the formation of a sodium dodecyl sulfate-stable high-Mr complex between them. Inhibition and complex formation required the active site of urokinase. The partially purified inhibitor was thought to be immunologically different from the known classes of plasminogen activator inhibitors, including endothelial-type inhibitor, macrophage/monocyte inhibitor, and protease nexin, since it did not react with specific antibodies to these inhibitors.

Antibodies↗
PubMed1988

Human articular cartilage contains an inhibitor of plasminogen activator.

The presence of plasminogen activator (PA) inhibitor in human articular cartilage extracts was shown using a microtiter plate assay using immunofixed urokinase. Cartilage urokinase inhibitor had a molecular weight of 66,000 on gel chromatography. Cartilage extracts also contained alpha 1-proteinase inhibitor; however, the urokinase inhibitor was distinguishable from such serum inhibitors immunologically. In sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin overlay, inhibition of urokinase was observed accompanying higher molecular weight complex formation. The cartilage urokinase inhibitor was unstable with acid, heat and SDS treatment, and required the active site of urokinase for inhibition.

Antibodies↗