The Experts below are selected from a list of 276 Experts worldwide ranked by ideXlab platform
Desire Collen - One of the best experts on this subject based on the ideXlab platform.
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Plasminogen Binding Properties of Macrophage Inflammatory Protein (MIP)-2α
Thrombosis and Haemostasis, 2017Co-Authors: Begoña Arza, Priscilla Fabregas, Jordi Félez, Yves Laroche, Desire Collen, H. Roger LijnenAbstract:The chemokine macrophage inflammatory protein (MIP)-2α was identified as a Plasminogen binding protein by phage display analysis. MIP-2and a truncated form lacking 5 lysine residues in the COOH-terminal region (mut-MIP-2α) were expressed in E. coli and purified to apparent homogeneity. Purified MIP-2α but not mut-MIP-2bound specifically to Plasminogen, with KA of 3.7105 M-1 for the interaction of Plasminogen with surface-bound MIP-2α. 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-2α. Activation of Plasminogen bound to surface-associated MIP-2α by two-chain urokinase-type Plasminogen activator (tcu-PA) was about 2.5-fold more efficient than in solution (catalytic efficiency kcat /KM of 0.1 µM-1s-1 , as compared to 0.04 M-1s-1 ). In contrast, binding of Plasminogen to MIP-2α in solution was very weak, as evidenced by the absence of competition of MIP-2α with lysine-Sepharose or with human THP-1 cells for binding of Plasminogen. In agreement with this finding, addition of excess MIP-2α did not affect the main functional properties of plasmin(ogen) in solution, as indicated by unaltered activation rates of Plasminogen by tcu-PA or tissuetype Plasminogen activator (t-PA), t-PA-mediated fibrinolysis, and inhibition rate of plasmin by α2-antiplasmin. Thus, association of MIP-2α with surfaces exposes its COOH-terminal Plasminogen-binding site, and may result in enhanced local plasmin generation.
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Plasminogen and Plasminogen activators protect against renal injury in crescentic glomerulonephritis.
Journal of Experimental Medicine, 1997Co-Authors: A. Richard Kitching, Desire Collen, Edward F Plow, Victoria A. Ploplis, Stephen R. Holdsworth, Peter Carmeliet, Peter G. TippingAbstract:The Plasminogen/plasmin system has the potential to affect the outcome of inflammatory diseases by regulating accumulation of fibrin and other matrix proteins. In human and experimental crescentic glomerulonephritis (GN), fibrin is an important mediator of glomerular injury and renal impairment. Glomerular deposition of matrix proteins is a feature of progressive disease. To study the role of Plasminogen and Plasminogen activators in the development of inflammatory glomerular injury, GN was induced in mice in which the genes for these proteins had been disrupted by homologous recombination. Deficiency of Plasminogen or combined deficiency of tissue type Plasminogen activator (tPA) and urokinase type Plasminogen activator (uPA) was associated with severe functional and histological exacerbation of glomerular injury. Deficiency of tPA, the predominant Plasminogen activator expressed in glomeruli, also exacerbated disease. uPA deficiency reduced glomerular macrophage infiltration and did not significantly exacerbate disease. uPA receptor deficiency did not effect the expression of GN. These studies demonstrate that Plasminogen plays an important role in protecting the glomerulus from acute inflammatory injury and that tPA is the major protective Plasminogen activator.
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staphylokinase requires nh2 terminal proteolysis for Plasminogen activation
Journal of Biological Chemistry, 1997Co-Authors: Bernhard Schlott, Manfred Hartmann, Karlheinz Guhrs, Anja Rocker, Desire CollenAbstract:Abstract Staphylokinase (Sak), a single-chain protein comprising 136 amino acids with NH2-terminal sequence, forms a complex with plasmin, that is endowed with Plasminogen activating properties. Plasmin is presumed to process mature (high molecular weight, HMW) Sak to low molecular weight derivatives (LMW-Sak), primarily by hydrolyzing the Lys10-Lys11 peptide bond, but the kinetics of Plasminogen activation by HMW-Sak and LMW-Sak are very similar. Here, the requirement of NH2-terminal proteolysis of Sak for the induction of Plasminogen activating potential was studied by mutagenesis of Lys10 and Lys11 in combination with NH2-terminal microsequence analysis of equimolar mixtures of Sak and Plasminogen and determination of kinetic parameters of Plasminogen activation by catalytic amounts of Sak. Substitution of Lys10 with Arg did not affect processing of the Arg10-Lys11 site nor Plasminogen activation, whereas substitution with His resulted in cleavage of the Lys11-Gly12 peptide bond and abolished Plasminogen activation. Substitution of Lys11 with Arg did not affect Lys10-Arg11 processing or Plasminogen activation, whereas replacement with His did not prevent Lys10-His11 hydrolysis but abolished Plasminogen activation. Substitution of Lys11 with Cys yielded an inactive processed derivative which was fully activated by aminoethylation. Deletion of the 10 NH2-terminal amino acids did not affect Plasminogen activation, but additional deletion of Lys11 eliminated Plasminogen activation. Thus generation of Plasminogen activator potential in Sak proceeds via plasmin-mediated removal of the 10 NH2-terminal amino acids with exposure of Lys11 as the new NH2 terminus. This provides a structural basis for the hypothesis, derived from kinetic measurements, that Plasminogen activation by Sak needs to be primed by plasmin and a mechanism for the high fibrin selectivity of Sak in a plasma milieu.
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Restoration of thrombolytic potential in Plasminogen-deficient mice by bolus administration of Plasminogen
Blood, 1996Co-Authors: H.r. Lijnen, P Carmeliet, A Bouche, L Moons, Va Ploplis, Ef Plow, Desire CollenAbstract:Abstract Homozygous Plasminogen-deficient (Plg-/-) mice had a significantly reduced thrombolytic capacity toward intravenously injected 125I-fibrin labeled plasma clots prepared from Plg-/- murine plasma (9% +/- 3% lysis after 8 hours; (mean +/- SEM, n = 6), as compared with 82% +/- 8% in wild-type mice; P < .0001). Bolus injection of 1 mg purified murine Plasminogen in 10- to 17-week-old Plg-/- mice increased the Plasminogen antigen and activity levels at 8 hours to normal levels (130 +/- 5 micrograms/mL). Plasminogen administration was associated with significant restoration of thrombolytic potential (64% +/- 7% spontaneous clot lysis; P < .0001 versus lysis without Plasminogen injection). Bolus injection of 1 mg Plasminogen in homozygous tissue- type Plasminogen activator-deficient (t-PA-/-) mice doubled the Plasminogen antigen and activity levels after 8 hours and increased 125I-fibrin clot lysis at 8 hours from 13% +/- 3% to 34% +/- 5% (P = .008). Fibrinogen, t-PA antigen and alpha 2-antiplasmin activity levels after 8 hours were not significantly different in the groups with or without Plasminogen injection. Injection of Plasminogen induced a variable increase (on average 7- to 10-fold) of PAI-1, but no correlation with the extent of spontaneous clot lysis was observed. Histopathologic examination at the end of the experiments revealed that fibrin deposition in the liver of Plg-/- mice was slightly reduced 8 hours after bolus Plasminogen injection (P = .007) and markedly reduced after 24 hours (P < .0001). Plasminogen antigen levels in liver extracts were comparable with those found in wild-type mice at 8 hours (130 +/- 20 versus 110 +/- 15 ng/mg protein) and decreased to 25 +/- 3.2 ng/mg protein at 24 hours. Thus, restoration of normal Plasminogen levels in Plg-/- mice normalized the thrombolytic potential toward experimentally induced pulmonary emboli, and resulted in removal of endogenous fibrin deposits within 24 hours.
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structure function relationships in staphylokinase as revealed by clustered charge to alanine mutagenesis
Journal of Biological Chemistry, 1995Co-Authors: K Silence, Desire Collen, Bernhard Schlott, Manfred Hartmann, Karlheinz Guhrs, Ariane Gase, Roger H LijnenAbstract:Abstract Eighteen mutants of recombinant staphylokinase (SakSTAR) in which clusters of two or three charged residues were converted to alanine (“clustered charge-to-alanine scan”) were characterized. Fifteen of these mutants had specific Plasminogen-activating activities of >20% of that of wild-type SakSTAR, whereas three mutants, SakSTAR K11A D13A D14A (SakSTAR13), SakSTAR E46A K50A (SakSTAR48), and SakSTAR E65A D69A (SakSTAR67) had specific activities of ≤3%. SakSTAR13 had an intact affinity for Plasminogen and a normal rate of active site exposure in equimolar mixtures with Plasminogen. The plasmin-SakSTAR13 complex had a 14-fold reduced catalytic efficiency for Plasminogen activation but was 5-fold more efficient for conversion of Plasminogen-SakSTAR13 to plasmin-SakSTAR13. SakSTAR48 and SakSTAR67 had a 10-20-fold reduced affinity for Plasminogen and a markedly reduced active site exposure; their complexes with plasmin had a more than 20-fold reduced catalytic efficiency toward Plasminogen. Thus, Plasminogen activation by catalytic amounts of SakSTAR is dependent on complex formation between plasmin(ogen) and SakSTAR, which is deficient with SakSTAR48 and SakSTAR67, but also on the induction of a functional active site configuration in the plasmin-SakSTAR complex, which is deficient with all three mutants. These findings support a mechanism for the activation of Plasminogen by SakSTAR involving formation of an equimolar complex of SakSTAR with traces of plasmin, which converts Plasminogen to plasmin and, more rapidly, inactive Plasminogen-SakSTAR to plasmin-SakSTAR.
Roger H Lijnen - One of the best experts on this subject based on the ideXlab platform.
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structure function relationships in staphylokinase as revealed by clustered charge to alanine mutagenesis
Journal of Biological Chemistry, 1995Co-Authors: K Silence, Desire Collen, Bernhard Schlott, Manfred Hartmann, Karlheinz Guhrs, Ariane Gase, Roger H LijnenAbstract:Abstract Eighteen mutants of recombinant staphylokinase (SakSTAR) in which clusters of two or three charged residues were converted to alanine (“clustered charge-to-alanine scan”) were characterized. Fifteen of these mutants had specific Plasminogen-activating activities of >20% of that of wild-type SakSTAR, whereas three mutants, SakSTAR K11A D13A D14A (SakSTAR13), SakSTAR E46A K50A (SakSTAR48), and SakSTAR E65A D69A (SakSTAR67) had specific activities of ≤3%. SakSTAR13 had an intact affinity for Plasminogen and a normal rate of active site exposure in equimolar mixtures with Plasminogen. The plasmin-SakSTAR13 complex had a 14-fold reduced catalytic efficiency for Plasminogen activation but was 5-fold more efficient for conversion of Plasminogen-SakSTAR13 to plasmin-SakSTAR13. SakSTAR48 and SakSTAR67 had a 10-20-fold reduced affinity for Plasminogen and a markedly reduced active site exposure; their complexes with plasmin had a more than 20-fold reduced catalytic efficiency toward Plasminogen. Thus, Plasminogen activation by catalytic amounts of SakSTAR is dependent on complex formation between plasmin(ogen) and SakSTAR, which is deficient with SakSTAR48 and SakSTAR67, but also on the induction of a functional active site configuration in the plasmin-SakSTAR complex, which is deficient with all three mutants. These findings support a mechanism for the activation of Plasminogen by SakSTAR involving formation of an equimolar complex of SakSTAR with traces of plasmin, which converts Plasminogen to plasmin and, more rapidly, inactive Plasminogen-SakSTAR to plasmin-SakSTAR.
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mechanisms of activation of mammalian plasma fibrinolytic systems with streptokinase and with recombinant staphylokinase
FEBS Journal, 1993Co-Authors: Desire Collen, Berthe Van Hoef, Bernhard Schlott, Manfred Hartmann, Karlheinz Guhrs, Roger H LijnenAbstract:The molecular basis of the marked interspecies variability in the response of plasma fibrinolytic systems to activation by streptokinase (SK) or recombinant staphylokinase (STAR) was studied using highly purified Plasminogens and a,-antiplasmins from five representative species (man, baboon, rabbit, dog and cow). Human Plasminogen reacted rapidly and stoichiometrically with both SK and STAR to yield potent Plasminogen activators (catalytic efficiencies, k,,,/K,, of 1 .O pM-' . s-' and 0.3 pM-' . sK', respectively). The complex with SK was insensitive to a,-antiplasmin, which, however, rapidly inhibited the complex with STAR (second-order rate constant, k,.,,, of 8 X 10" M-' . s-'). In a system composed of a 0.06-ml 'z51-fibrin-labeled plasma clot submerged in 0.30 ml plasma, both SK and STAR had potent fibrinolytic properties, causing 50% clot lysis in 2 h (EC,,), with 120 nM and 13 nM, respectively. Clot lysis with SK was non-fibrin specific (residual fibrinogen < lo%), whereas lysis with STAR was highly fibrin specific (residual fibrinogen 76%). Canine Plasminogen reacted avidly with SK, but SK was rapidly degraded: it reacted rapidly and quantitatively with STAR to form a potent Plasminogen-activating complex (kcd,/Km of 0.4 pM-' SK') which was sensitive to neutralization by a,-antiplasmin (k,,a,, of 6 X 10' M-' . s-'). In a canine plasma milieu, SK was relatively potent (EC,, 200 nM) and fibrin specific, whereas STAR was very potent (EC,, 1.3 nM) but poorly fibrin specific. Baboon and rabbit Plasminogen did not form stable stoichiometric complexes with SK, but reacted stoichiometrically and quantitatively with STAR. The complexes with STAR, however, had low catalytic efficiencies for the activation of their autologous Plasminogens (k,,JK,, 0.02 pM-' . s-') and reacted more slowly with a,-antiplasmin (k,,,,, 5-10 X 10' M-' . s-'). Bovine Plasminogen was virtually unreactive towards both SK and STAR as well as to their complexes with human Plasminogen, as monitored by measurement of the initial activation rates. The resistance to fibrinogen degradation with STAR observed in the human system could be transferred to the canine system by reconstituting canine plasma, depleted of Plasminogen and a,-antiplasmin, with the human proteins. Conversely, the sensitivity to fibrinogen degradation of the canine system could be transferred to the human system by reconstituting depleted plasma with canine Plasminogen and a,-antiplasmin. It is concluded that the variability in the response of mammalian plasma fibrinolytic systems to activation with SK or STAR is determined mainly by the extent of complex formation of these compounds with Plasminogen, by the catalytic efficiencies of the complexes for the activation of autologous Plasminogen and by the rate of inhibition of these complexes by a,-antiplasmin.
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interaction of staphylokinase with different molecular forms of Plasminogen
FEBS Journal, 1993Co-Authors: Roger H Lijnen, Berthe Van Hoef, Desire CollenAbstract:In order to obtain more information on the mechanism of Plasminogen activation by staphylokinase (STA), we have studied the interaction between recombinant STA (STAR) and different molecular forms of human Plasminogen, including Glu-Plasminogen (native moiety), Lys-Plasminogen (partially degraded moiety) and low-molecular-mass (LMM) Plasminogen (moiety lacking kringles 1–4). Addition of 2 μM STAR to 0.4 μM Glu-Plasminogen, Lys-Plasminogen or LMM Plasminogen resulted in the generation of proteolytic activity towards the chromogenic substrate D-Val-Leu-Lys-NH-PhNO2 (S-2251) corresponding to the exposure of 1 active center/Plasminogen molecule. Complex formation was associated with conversion of the one-chain Plasminogen moieties to two-chain plasmin, and with quantitative conversion of Glu-Plasminogen to Lys-plasmin. The stoichiometry of the Plasminogen-STAR complex, determined by binding of the complex to Lys-Sepharose and measurement of residual STAR, was found to be equimolar. The Plasminogen-STAR complexes were inhibited by α2-antiplasmin with second-order rate constants of 2.4 ± 0.17 × 106 M−1 s−1 for Glu-Plasminogen, 2.4 ± 0.21 × 106 M−1 s−1 for Lys-Plasminogen and 9.4 ± 1.5 × 104 M−1 s−1 for LMM Plasminogen. Glu-plasmin-STAR, Lys-plasmin-STAR and LMM plasmin-STAR had comparable catalytic efficiencies (kcat/Km) for the activation of Glu-Plasminogen (0.24–0.29 μM−1 s−1), Lys-Plasminogen (0.57–0.79 μM−1 s−1) or LMM Plasminogen (0.11–0.16 μM−1 s−1). In a human plasma milieu in vitro STAR, Glu-plasmin-STAR, Lys-plasmin-STAR and LMM-plasmin-STAR were equally effective for the lysis of 125I-fibrin-labeled human plasma clots [50% clot lysis in 2 h (EC50) with 11–13 nM test compound] and equally fibrin-selective (residual fibrinogen levels of 72–84% after 2 h at EC50). Our results thus confirm that Plasminogen and STAR form a 1:1 stoichiometric complex in which Plasminogen is converted to plasmin and Glu-Plasminogen to Lys-plasmin. The lysine-binding sites in kringles 1–4 of Plasminogen are not required for the complex formation with STAR, nor for the enzyme activity of the complex with STAR in purified systems and in a human plasma milieu. The lysine-binding sites are, however, important for the rate of the inhibition of the complexes by α2-antiplasmin.
Lindsey A. Miles - One of the best experts on this subject based on the ideXlab platform.
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Exposure of Plasminogen and the novel Plasminogen receptor, Plg-RKT, on activated human and murine platelets.
Blood, 2020Co-Authors: Claire S Whyte, Lindsey A. Miles, Gael B Morrow, Nagyung Baik, Nuala A Booth, Mohammed M Jalal, Robert J Parmer, Nicola J MutchAbstract:Plasminogen activation rates are enhanced by cell surface binding. We have previously demonstrated that exogenous Plasminogen binds to phosphatidylserine-exposing and spread platelets. Platelets contain Plasminogen in their α-granules but secretion of Plasminogen from platelets has not been studied. Recently, a novel transmembrane lysine-dependent Plasminogen receptor, Plg-RKT, has been described on macrophages. Here, we analyzed the pool of Plasminogen in platelets and examined whether platelets express Plg-RKT. Plasminogen content of the supernatant of resting and collagen/thrombin-stimulated platelets was similar. Pre-treatment with the lysine analogue, εACA, significantly increased platelet-derived Plasminogen (0.33 nmol/108 plts vs. 0.08 nmol/108 plts) in the stimulated supernatant, indicating a lysine-dependent mechanism of membrane retention. Lysine-dependent, platelet-derived Plasminogen retention on thrombin and convulxin activated human platelets was confirmed by flow cytometry. Platelets initiated fibrinolytic activity in fluorescently labelled Plasminogen-deficient clots and in turbidimetric clot lysis assays. A 17 kDa band, consistent with Plg-RKT, was detected in the platelet membrane fraction by Western blotting. Confocal microscopy of stimulated platelets revealed Plg-RKT co-localized with platelet-derived Plasminogen on the activated platelet membrane. Plasminogen exposure was significantly attenuated in thrombin and convulxin stimulated platelets from Plg-RKT-/- mice compared to Plg-RKT+/+ littermates. Membrane exposure of Plg-RKT was not dependent on Plasminogen, as similar levels of the receptor were detected in Plasminogen-/- platelets. These data highlight Plg-RKT as a novel Plasminogen receptor in human and murine platelets. We show for the first time that platelet-derived Plasminogen is retained on the activated platelet membrane and drives local fibrinolysis, by enhancing cell-surface mediated Plasminogen activation.
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proteomics based discovery of a novel structurally unique and developmentally regulated Plasminogen receptor plg rkt a major regulator of cell surface Plasminogen activation
Blood, 2010Co-Authors: Nicholas M. Andronicos, Nagyung Baik, Robert J Parmer, Emily I Chen, Caitlin M Parmer, William B Kiosses, Mark P Kamps, John R Yates, Lindsey A. MilesAbstract:Activation of Plasminogen, the zymogen of the primary thrombolytic enzyme, plasmin, is markedly promoted when Plasminogen is bound to cell surfaces, arming cells with the broad spectrum proteolytic activity of plasmin. In addition to its role in thrombolysis, cell surface plasmin facilitates a wide array of physiologic and pathologic processes. Carboxypeptidase B-sensitive Plasminogen binding sites promote Plasminogen activation on eukaryotic cells. However, no integral membrane Plasminogen receptors exposing carboxyl terminal basic residues on cell surfaces have been identified. Here we use the exquisite sensitivity of multidimensional protein identification technology and an inducible progenitor cell line to identify a novel differentiation-induced integral membrane Plasminogen receptor that exposes a C-terminal lysine on the cell surface, Plg-RKT (C9orf46 homolog). Plg-RKT was highly colocalized on the cell surface with the urokinase receptor, uPAR. Our data suggest that Plg-RKT also interacts directly with tissue Plasminogen activator. Furthermore, Plg-RKT markedly promoted cell surface Plasminogen activation. Database searching revealed that Plg-RKT mRNA is broadly expressed by migratory cell types, including leukocytes, and breast cancer, leukemic, and neuronal cells. This structurally unique Plasminogen receptor represents a novel control point for regulating cell surface proteolysis.
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Plasminogen receptors: the sine qua non of cell surface Plasminogen activation.
Frontiers in Bioscience, 2005Co-Authors: Lindsey A. Miles, Francis J. Castellino, Nagyung Baik, Stephen B. Hawley, Nicholas M. Andronicos, Robert J ParmerAbstract:: Localization of Plasminogen and Plasminogen activators on cell surfaces promotes Plasminogen activation and serves to arm cells with the broad spectrum proteolytic activity of plasmin. Cell surface proteolysis by plasmin is an essential feature of physiological and pathological processes requiring extracellular matrix degradation for cell migration including macrophage recruitment during the inflammatory response, tissue remodeling, wound healing, tumor cell invasion and metastasis and skeletal myogenesis. Cell associated plasmin on platelets and endothelial cells is optimally localized for promotion of clot lysis. In more recently recognized functions that are likely to be independent of matrix degradation, cell surface-bound plasmin participates in prohormone processing as well as stimulation of intracellular signaling. This issue of Frontiers in Bioscience on Plasminogen Receptors encompasses chapters focusing on the kinetics of cell surface Plasminogen activation and the regulation of Plasminogen receptor activity as well as the contribution of Plasminogen receptors to the physiological and pathophysiological processes of myogenesis, muscle regeneration and cancer. The molecular identity of Plasminogen receptors is cell-type specific, with distinct molecular entities providing Plasminogen receptor function on different cells. This issue includes chapters on the well studied Plasminogen receptor functions.
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Endogenous plasmin converts Glu‐Plasminogen to Lys‐Plasminogen on the monocytoid cell surface
Journal of Thrombosis and Haemostasis, 2003Co-Authors: Li Zhang, Francis J. Castellino, Davida K Grella, Yun Gong, Lindsey A. MilesAbstract:Summary. Recently, we showed that localization of Glu-Plasminogen on cell surfaces enhances its conversion to Lys-Plasminogen by exogenous plasmin. This leads to stimulation of Plasminogen activation because Lys-Plasminogen is the preferred substrate on cell surfaces. Here, we show that Glu-Plasminogen was converted to Lys-Plasminogen on monocytoid cells in the absence of exogenous plasmin. Culture of cells under serum-free conditions did not affect this conversion, suggesting that the enzymatic activity was cell-derived. Therefore, we tested whether endogenous monocytoid Plasminogen could provide a source of plasmin to convert cell-associated Glu-Plasminogen to Lys-Plasminogen because plasmin is the only enzyme known to effect this reaction. We used a recombinant human Plasminogen mutant, [D(646)E]Pg, which can be cleaved by Plasminogen activators, but cannot catalyze the generation of Lys-Plasminogen. Upon incubation with either THP-1 or U937 monocytoid cells, 35 and 38%, respectively, of the cell-bound ligand was converted to Lys-[D(646)E]Pg. Trasylol, α2-antiplasmin, and an anticatalytic antiPlasminogen monoclonal antibody decreased Lys-[D(646)E]Pg formation to < 5% on monocytoid cells, consistent with a plasmin-dependent mechanism. Plasminogen was detected in these cells by Northern blotting and RT-PCR. Our results suggest that plasmin converts cell-bound Glu-Plasminogen to Lys-Plasminogen and that this enzyme is produced by activation of monocytoid Plasminogen by endogenous monocytoid Plasminogen activators to enhance Plasminogen activation on the monocytoid cell surface.
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Critical role for conversion of glu-Plasminogen to Lys-Plasminogen for optimal stimulation of Plasminogen activation on cell surfaces.
Trends in Cardiovascular Medicine, 2003Co-Authors: Lindsey A. Miles, Francis J. Castellino, Yun GongAbstract:Abstract When Glu-Plasminogen, the native circulating form of the zymogen, is bound to cell surfaces, its activation is markedly enhanced compared with the reaction in solution. This results in localization of the broad-spectrum proteolytic activity of plasmin on cell surfaces. The cell-associated plasmin plays a key role in fibrinolysis, cell migration, and prohormone processing. It is well established that the localization of Plasminogen and Plasminogen activators on cell surfaces promotes the enhanced Plasminogen activation on the cell surface. The focus of this article is to review recent studies demonstrating that the conversion of Glu-Plasminogen to the more readily activated Lys-Plasminogen derivative is necessary for optimal stimulation of Plasminogen activation on the cell surface, and that the interaction of Glu-Plasminogen with cells serves to increase processing of Glu-Plasminogen to Lys-Plasminogen, thereby enhancing Plasminogen activation on the cell surface.
Robert J Parmer - One of the best experts on this subject based on the ideXlab platform.
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Exposure of Plasminogen and the novel Plasminogen receptor, Plg-RKT, on activated human and murine platelets.
Blood, 2020Co-Authors: Claire S Whyte, Lindsey A. Miles, Gael B Morrow, Nagyung Baik, Nuala A Booth, Mohammed M Jalal, Robert J Parmer, Nicola J MutchAbstract:Plasminogen activation rates are enhanced by cell surface binding. We have previously demonstrated that exogenous Plasminogen binds to phosphatidylserine-exposing and spread platelets. Platelets contain Plasminogen in their α-granules but secretion of Plasminogen from platelets has not been studied. Recently, a novel transmembrane lysine-dependent Plasminogen receptor, Plg-RKT, has been described on macrophages. Here, we analyzed the pool of Plasminogen in platelets and examined whether platelets express Plg-RKT. Plasminogen content of the supernatant of resting and collagen/thrombin-stimulated platelets was similar. Pre-treatment with the lysine analogue, εACA, significantly increased platelet-derived Plasminogen (0.33 nmol/108 plts vs. 0.08 nmol/108 plts) in the stimulated supernatant, indicating a lysine-dependent mechanism of membrane retention. Lysine-dependent, platelet-derived Plasminogen retention on thrombin and convulxin activated human platelets was confirmed by flow cytometry. Platelets initiated fibrinolytic activity in fluorescently labelled Plasminogen-deficient clots and in turbidimetric clot lysis assays. A 17 kDa band, consistent with Plg-RKT, was detected in the platelet membrane fraction by Western blotting. Confocal microscopy of stimulated platelets revealed Plg-RKT co-localized with platelet-derived Plasminogen on the activated platelet membrane. Plasminogen exposure was significantly attenuated in thrombin and convulxin stimulated platelets from Plg-RKT-/- mice compared to Plg-RKT+/+ littermates. Membrane exposure of Plg-RKT was not dependent on Plasminogen, as similar levels of the receptor were detected in Plasminogen-/- platelets. These data highlight Plg-RKT as a novel Plasminogen receptor in human and murine platelets. We show for the first time that platelet-derived Plasminogen is retained on the activated platelet membrane and drives local fibrinolysis, by enhancing cell-surface mediated Plasminogen activation.
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proteomics based discovery of a novel structurally unique and developmentally regulated Plasminogen receptor plg rkt a major regulator of cell surface Plasminogen activation
Blood, 2010Co-Authors: Nicholas M. Andronicos, Nagyung Baik, Robert J Parmer, Emily I Chen, Caitlin M Parmer, William B Kiosses, Mark P Kamps, John R Yates, Lindsey A. MilesAbstract:Activation of Plasminogen, the zymogen of the primary thrombolytic enzyme, plasmin, is markedly promoted when Plasminogen is bound to cell surfaces, arming cells with the broad spectrum proteolytic activity of plasmin. In addition to its role in thrombolysis, cell surface plasmin facilitates a wide array of physiologic and pathologic processes. Carboxypeptidase B-sensitive Plasminogen binding sites promote Plasminogen activation on eukaryotic cells. However, no integral membrane Plasminogen receptors exposing carboxyl terminal basic residues on cell surfaces have been identified. Here we use the exquisite sensitivity of multidimensional protein identification technology and an inducible progenitor cell line to identify a novel differentiation-induced integral membrane Plasminogen receptor that exposes a C-terminal lysine on the cell surface, Plg-RKT (C9orf46 homolog). Plg-RKT was highly colocalized on the cell surface with the urokinase receptor, uPAR. Our data suggest that Plg-RKT also interacts directly with tissue Plasminogen activator. Furthermore, Plg-RKT markedly promoted cell surface Plasminogen activation. Database searching revealed that Plg-RKT mRNA is broadly expressed by migratory cell types, including leukocytes, and breast cancer, leukemic, and neuronal cells. This structurally unique Plasminogen receptor represents a novel control point for regulating cell surface proteolysis.
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Plasminogen receptors: the sine qua non of cell surface Plasminogen activation.
Frontiers in Bioscience, 2005Co-Authors: Lindsey A. Miles, Francis J. Castellino, Nagyung Baik, Stephen B. Hawley, Nicholas M. Andronicos, Robert J ParmerAbstract:: Localization of Plasminogen and Plasminogen activators on cell surfaces promotes Plasminogen activation and serves to arm cells with the broad spectrum proteolytic activity of plasmin. Cell surface proteolysis by plasmin is an essential feature of physiological and pathological processes requiring extracellular matrix degradation for cell migration including macrophage recruitment during the inflammatory response, tissue remodeling, wound healing, tumor cell invasion and metastasis and skeletal myogenesis. Cell associated plasmin on platelets and endothelial cells is optimally localized for promotion of clot lysis. In more recently recognized functions that are likely to be independent of matrix degradation, cell surface-bound plasmin participates in prohormone processing as well as stimulation of intracellular signaling. This issue of Frontiers in Bioscience on Plasminogen Receptors encompasses chapters focusing on the kinetics of cell surface Plasminogen activation and the regulation of Plasminogen receptor activity as well as the contribution of Plasminogen receptors to the physiological and pathophysiological processes of myogenesis, muscle regeneration and cancer. The molecular identity of Plasminogen receptors is cell-type specific, with distinct molecular entities providing Plasminogen receptor function on different cells. This issue includes chapters on the well studied Plasminogen receptor functions.
Bernhard Schlott - One of the best experts on this subject based on the ideXlab platform.
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staphylokinase requires nh2 terminal proteolysis for Plasminogen activation
Journal of Biological Chemistry, 1997Co-Authors: Bernhard Schlott, Manfred Hartmann, Karlheinz Guhrs, Anja Rocker, Desire CollenAbstract:Abstract Staphylokinase (Sak), a single-chain protein comprising 136 amino acids with NH2-terminal sequence, forms a complex with plasmin, that is endowed with Plasminogen activating properties. Plasmin is presumed to process mature (high molecular weight, HMW) Sak to low molecular weight derivatives (LMW-Sak), primarily by hydrolyzing the Lys10-Lys11 peptide bond, but the kinetics of Plasminogen activation by HMW-Sak and LMW-Sak are very similar. Here, the requirement of NH2-terminal proteolysis of Sak for the induction of Plasminogen activating potential was studied by mutagenesis of Lys10 and Lys11 in combination with NH2-terminal microsequence analysis of equimolar mixtures of Sak and Plasminogen and determination of kinetic parameters of Plasminogen activation by catalytic amounts of Sak. Substitution of Lys10 with Arg did not affect processing of the Arg10-Lys11 site nor Plasminogen activation, whereas substitution with His resulted in cleavage of the Lys11-Gly12 peptide bond and abolished Plasminogen activation. Substitution of Lys11 with Arg did not affect Lys10-Arg11 processing or Plasminogen activation, whereas replacement with His did not prevent Lys10-His11 hydrolysis but abolished Plasminogen activation. Substitution of Lys11 with Cys yielded an inactive processed derivative which was fully activated by aminoethylation. Deletion of the 10 NH2-terminal amino acids did not affect Plasminogen activation, but additional deletion of Lys11 eliminated Plasminogen activation. Thus generation of Plasminogen activator potential in Sak proceeds via plasmin-mediated removal of the 10 NH2-terminal amino acids with exposure of Lys11 as the new NH2 terminus. This provides a structural basis for the hypothesis, derived from kinetic measurements, that Plasminogen activation by Sak needs to be primed by plasmin and a mechanism for the high fibrin selectivity of Sak in a plasma milieu.
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structure function relationships in staphylokinase as revealed by clustered charge to alanine mutagenesis
Journal of Biological Chemistry, 1995Co-Authors: K Silence, Desire Collen, Bernhard Schlott, Manfred Hartmann, Karlheinz Guhrs, Ariane Gase, Roger H LijnenAbstract:Abstract Eighteen mutants of recombinant staphylokinase (SakSTAR) in which clusters of two or three charged residues were converted to alanine (“clustered charge-to-alanine scan”) were characterized. Fifteen of these mutants had specific Plasminogen-activating activities of >20% of that of wild-type SakSTAR, whereas three mutants, SakSTAR K11A D13A D14A (SakSTAR13), SakSTAR E46A K50A (SakSTAR48), and SakSTAR E65A D69A (SakSTAR67) had specific activities of ≤3%. SakSTAR13 had an intact affinity for Plasminogen and a normal rate of active site exposure in equimolar mixtures with Plasminogen. The plasmin-SakSTAR13 complex had a 14-fold reduced catalytic efficiency for Plasminogen activation but was 5-fold more efficient for conversion of Plasminogen-SakSTAR13 to plasmin-SakSTAR13. SakSTAR48 and SakSTAR67 had a 10-20-fold reduced affinity for Plasminogen and a markedly reduced active site exposure; their complexes with plasmin had a more than 20-fold reduced catalytic efficiency toward Plasminogen. Thus, Plasminogen activation by catalytic amounts of SakSTAR is dependent on complex formation between plasmin(ogen) and SakSTAR, which is deficient with SakSTAR48 and SakSTAR67, but also on the induction of a functional active site configuration in the plasmin-SakSTAR complex, which is deficient with all three mutants. These findings support a mechanism for the activation of Plasminogen by SakSTAR involving formation of an equimolar complex of SakSTAR with traces of plasmin, which converts Plasminogen to plasmin and, more rapidly, inactive Plasminogen-SakSTAR to plasmin-SakSTAR.
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mechanisms of activation of mammalian plasma fibrinolytic systems with streptokinase and with recombinant staphylokinase
FEBS Journal, 1993Co-Authors: Desire Collen, Berthe Van Hoef, Bernhard Schlott, Manfred Hartmann, Karlheinz Guhrs, Roger H LijnenAbstract:The molecular basis of the marked interspecies variability in the response of plasma fibrinolytic systems to activation by streptokinase (SK) or recombinant staphylokinase (STAR) was studied using highly purified Plasminogens and a,-antiplasmins from five representative species (man, baboon, rabbit, dog and cow). Human Plasminogen reacted rapidly and stoichiometrically with both SK and STAR to yield potent Plasminogen activators (catalytic efficiencies, k,,,/K,, of 1 .O pM-' . s-' and 0.3 pM-' . sK', respectively). The complex with SK was insensitive to a,-antiplasmin, which, however, rapidly inhibited the complex with STAR (second-order rate constant, k,.,,, of 8 X 10" M-' . s-'). In a system composed of a 0.06-ml 'z51-fibrin-labeled plasma clot submerged in 0.30 ml plasma, both SK and STAR had potent fibrinolytic properties, causing 50% clot lysis in 2 h (EC,,), with 120 nM and 13 nM, respectively. Clot lysis with SK was non-fibrin specific (residual fibrinogen < lo%), whereas lysis with STAR was highly fibrin specific (residual fibrinogen 76%). Canine Plasminogen reacted avidly with SK, but SK was rapidly degraded: it reacted rapidly and quantitatively with STAR to form a potent Plasminogen-activating complex (kcd,/Km of 0.4 pM-' SK') which was sensitive to neutralization by a,-antiplasmin (k,,a,, of 6 X 10' M-' . s-'). In a canine plasma milieu, SK was relatively potent (EC,, 200 nM) and fibrin specific, whereas STAR was very potent (EC,, 1.3 nM) but poorly fibrin specific. Baboon and rabbit Plasminogen did not form stable stoichiometric complexes with SK, but reacted stoichiometrically and quantitatively with STAR. The complexes with STAR, however, had low catalytic efficiencies for the activation of their autologous Plasminogens (k,,JK,, 0.02 pM-' . s-') and reacted more slowly with a,-antiplasmin (k,,,,, 5-10 X 10' M-' . s-'). Bovine Plasminogen was virtually unreactive towards both SK and STAR as well as to their complexes with human Plasminogen, as monitored by measurement of the initial activation rates. The resistance to fibrinogen degradation with STAR observed in the human system could be transferred to the canine system by reconstituting canine plasma, depleted of Plasminogen and a,-antiplasmin, with the human proteins. Conversely, the sensitivity to fibrinogen degradation of the canine system could be transferred to the human system by reconstituting depleted plasma with canine Plasminogen and a,-antiplasmin. It is concluded that the variability in the response of mammalian plasma fibrinolytic systems to activation with SK or STAR is determined mainly by the extent of complex formation of these compounds with Plasminogen, by the catalytic efficiencies of the complexes for the activation of autologous Plasminogen and by the rate of inhibition of these complexes by a,-antiplasmin.
