Plg Receptor Research Articles

Mycobacterium tuberculosis H37Rv enolase (Rv1023)- expression, characterization and effect of host dependent modifications on protein functionality

Mycobacterium tuberculosis enolase is an essential glycolytic enzyme that catalyzes the conversion of 2, phosphoglycerate (PGA) to phosphoenol pyruvate (PEP). It is also a crucial link between glycolysis and the tricarboxylic acid (TCA) pathway. The depletion of PEP has recently been associated with the emergence of non-replicating drug resistant bacteria. Enolase is also known to exhibit multiple alternate functions, such as promoting tissue invasion via its role as a plasminogen (Plg) receptor. In addition, proteomic studies have identified the presence of enolase in the Mtb degradosome and in biofilms. However, the precise role in these processes has not been elaborated. The enzyme was recently identified as a target for 2-amino thiazoles - a novel class of anti-mycobacterials. In vitro assays and characterization of this enzyme were unsuccessful due to the inability to obtain functional recombinant protein. In the present study, we report the expression and characterization of enolase using Mtb H37Ra as a host strain. Our study demonstrates that the enzyme activity and alternate functions of this protein are significantly impacted by the choice of expression host (Mtb H37Ra or E. coli). Detailed analysis of the protein from each source revealed subtle differences in the post-translational modifications. Lastly, our study confirms the role of enolase in Mtb biofilm formation and describes the potential for inhibiting this process.

Abstract 3613: Effect of thrombomodulin and plasminogen interaction on melanoma spheroid invasion

Abstract Background: Thrombomodulin (TM), a transmembrane glycoprotein that acts as a plasminogen (Plg) receptor, has been found as a potential molecular and prognostic marker that impacts the spread of various cancers, including malignant melanoma. A previous study showed that TM plays a vital function in angiogenesis activity [1] but its role in the malignant melanoma invasion is unclear. Here, we investigate the role of TM-Plg by using melanoma spheroid (MS) embedded in an extracellular matrix (ECM) modeled to understand how TM-Plg affects the progression of MS invasion. Methods: In this work, A2058 (A2058-GFP and A2058-TMGFP) and MeWo (siC and siTM) MS with different TM expressions were formed, harvested, and embedded in type I collagen gel for invasion assay. TM and Plg expression were determined by western blot. The surface of A2058 MS was observed by SEM imaging. Human Plg, MMPs, and Plg inhibitors, tranexamic acid (TXA) and epsilon-aminocaproic acid (ε-ACA), were utilized to investigate the effect of TM on the plasmin system. Immunofluorescence of TM and F-actin were validated. Results: When the two cell lines expressed with TM, the formed MS invaded to the surrounding matrix more distinctively than that of the TM-silencing ones, with a 2.5-fold difference in the ratio invasive area change. Upon placing the two A2058-TMGFP MS in the vicinity of each other within the ECM, they showed a strong interaction with an observed distance between the two MS from 50 μm to 280 μm. Immunofluorescence of TMGFP assessed the role of TM in MS invasion and interaction in ECM. Microvilli structures were found on the surface of A2058-TMGFP cells that may help the cell have more cellular processes such as adhesion, signal transduction, and metabolic activities. Furthermore, TXA, ε-ACA, and MMP inhibitors inhibited the invasion of A2058-TMGFP MSs in a dose-dependent manner. Human Plg stimulated invasion in TM-expressing MSs while no effect on non-TM MSs. The results suggested that the human Plg only significantly increased the MS invasion with the presence of TM. Conclusion: We concluded that with the presence of TM, the MS invasion and interaction become stronger than MS without TM expression. Moreover, TM enhances MS invasion in the 3D model via interacting Plg. This study provides evidence for the development of TM antagonists in the future to analyze the drug's potential as an anti-metastasis treatment.[1] P. K. Chen et al., “Thrombomodulin functions as a plasminogen receptor to modulate angiogenesis,” FASEB J., vol. 27, no. 11, pp. 4520-4531, 2013, doi: 10.1096/fj.13-227561. Citation Format: Thi Kim Ngan Ngo, Hua-Lin Wu, Cheng-Hsiang Kuo, Ting-Yuan Tu. Effect of thrombomodulin and plasminogen interaction on melanoma spheroid invasion. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3613.

Plasminogen Binding and Activation at the Mesothelial Cell Surface Promotes Invasion through a Collagen Matrix.

Plasminogen (Plg) activation to the serine protease plasmin (Pla) plays a key role in regulating wound healing and fibrotic responses, particularly when bound to cell surface receptors. Our previous work suggested that mesothelial cells bind Plg at the cell surface, though no Plg receptors were described for these cells. Since mesothelial cells contribute to injury responses, including cellular differentiation to a mesenchymal-like phenotype and extracellular matrix remodeling, we hypothesized that Plg binding would promote these responses. Here, we confirm that Plg binds to both pleural and peritoneal mesothelial cells via the lysine-binding domain present in Plg, and we demonstrate the presence of three Plg receptors on the mesothelial cell surface: α-Enolase, Annexin A2, and Plg-RKT. We further show that bound-Plg is activated to Pla on the cell surface and that activation is blocked by an inhibitor of urokinase plasminogen activator or by the presence of animal-derived FBS. Lastly, we demonstrate that Plg promotes mesothelial cell invasion through a type I collagen matrix but does not promote cellular differentiation or proliferation. These data demonstrate for the first time that mesothelial cells bind and activate Plg at the cell surface and that active Pla is involved in mesothelial cell invasion without cell differentiation.

Essential role of DNA-PKcs and plasminogen for the development of doxorubicin-induced glomerular injury in mice.

ABSTRACTSusceptibility to doxorubicin-induced nephropathy (DIN), a toxic model for the induction of proteinuria in mice, is related to the single-nucleotide polymorphism (SNP) C6418T of the Prkdc gene encoding for the DNA-repair enzyme DNA-PKcs. In addition, plasminogen (Plg) has been reported to play a role in glomerular damage. Here, we investigated the interdependence of both factors for the development of DIN. Genotyping confirmed the SNP of the Prkdc gene in C57BL/6 (PrkdcC6418/C6418) and 129S1/SvImJ (PrkdcT6418/T6418) mice. Intercross of heterozygous 129SB6F1 mice led to 129SB6F2 hybrids with Mendelian inheritance of the SNP. After doxorubicin injection, only homozygous F2 mice with PrkdcT6418/T6418 developed proteinuria. Genetic deficiency of Plg (Plg−/−) in otherwise susceptible 129S1/SvImJ mice led to resistance to DIN. Immunohistochemistry revealed glomerular binding of Plg in Plg+/+ mice after doxorubicin injection involving histone H2B as Plg receptor. In doxorubicin-resistant C57BL/6 mice, Plg binding was absent. In conclusion, susceptibility to DIN in 129S1/SvImJ mice is determined by a hierarchical two-hit process requiring the C6418T SNP in the Prkdc gene and subsequent glomerular binding of Plg. This article has an associated First Person interview with the first author of the paper.

Plasminogen and the Plasminogen Receptor, Plg-RKT, Regulate Macrophage Phenotypic, and Functional Changes.

Inflammation resolution is an active process that functions to restore tissue homeostasis. Clearance of apoptotic leukocytes by efferocytosis at inflammatory sites plays an important role in inflammation resolution and induces remarkable macrophage phenotypic and functional changes. Here, we investigated the effects of deletion of either plasminogen (Plg) or the Plg receptor, Plg-RKT, on the resolution of inflammation. In a murine model of pleurisy, the numbers of total mononuclear cells recruited to the pleural cavity were significantly decreased in both Plg−/− and Plg-RKT−/− mice, a response associated with decreased levels of the chemokine CCL2 in pleural exudates. Increased percentages of M1-like macrophages were determined in pleural lavages of Plg−/− and Plg-RKT−/− mice without significant changes in M2-like macrophage percentages. In vitro, Plg and plasmin (Pla) increased CD206/Arginase-1 expression and the levels of IL-10/TGF-β (M2 markers) while decreasing IFN/LPS-induced M1 markers in murine bone-marrow-derived macrophages (BMDMs) and human macrophages. Furthermore, IL4-induced M2-like polarization was defective in BMDMs from both Plg−/− and Plg-RKT−/− mice. Mechanistically, Plg and Pla induced transient STAT3 phosphorylation, which was decreased in Plg−/− and Plg-RKT−/− BMDMs after IL-4 or IL-10 stimulation. The extents of expression of CD206 and Annexin A1 (important for clearance of apoptotic cells) were reduced in Plg−/− and Plg-RKT−/− macrophage populations, which exhibited decreased phagocytosis of apoptotic neutrophils (efferocytosis) in vivo and in vitro. Taken together, these results suggest that Plg and its receptor, Plg-RKT, regulate macrophage polarization and efferocytosis, as key contributors to the resolution of inflammation.

Plasminogen and the Plasminogen receptor, Plg‐RKT, regulate efferocytosis and macrophage reprogramming

Inflammation resolution is an active process that functions to restore tissue homeostasis. Phagocytic clearance of apoptotic leukocytes (efferocytosis) at sites of inflammation plays an important role in inflammation resolution by inducing remarkable macrophage phenotypic plasticity. The M1/M2 nomenclature provides a basic classification system to distinguish proinflammatory (M1) from anti‐inflammatory/resolving macrophages (M2/Mres). Plasminogen (Plg) is synthesized by the liver and is the proenzyme of the broad‐spectrum serine protease, plasmin (Pla), which is the major enzyme responsible for fibrinolysis. The participation of the Plg system in the productive phase of inflammation is well known, but the involvement of the Plg system in the resolving phase of inflammation is just emerging. Here we investigated the effects of deletion of Plg and the Plg receptor, Plg‐RKT, separately, in the context of resolution of inflammation in vivo. Plg−/− and Plg‐RKT −/− mice and their respective wild type littermates were challenged by intrapleural injection of LPS (250ng/mouse, i.pl.) or PBS and cells present in the pleural cavity were harvested at 4, 8, 24 and 48 hr and processed for total and differential leukocyte counts and flow cytometric analyses. In this model of pleurisy there is a time‐dependent influx of leukocytes into the pleural cavity. The number of neutrophils and M1 macrophages peaks at 8 hr, and decreases at 48 hr. There is a significant increase in mononuclear cell recruitment into the pleural cavity that coincides with the resolution phase of inflammation and with prevalence of M2 and Mres macrophages at 48hr. In the current study, in the setting of LPS‐induced inflammation, increased numbers of M1 macrophages were present in Plg−/− mice 8h after LPS challenge without changes in number of M2 or Mres macrophages, as compared to Plg+/+ littermates [Plg+/+ = 155 ± 39 and Plg−/− = 301 ± 57 × 103 M1 macrophages/cavity (P < 0.05, n=6)]. There were no genotype‐dependent effects on the number of neutrophils recruited to the pleural cavity at all time points evaluated. At 48 hr the total number of mononuclear cells recruited to the pleural cavity was significantly decreased in both Plg−/− and Plg‐RKT −/− mice, relative to their respective wild type littermate controls [Plg+/+ = 3,432 ± 335 and Plg−/− = 2,377 ± 242 (P < 0.01, n=5); Plg‐RKT +/+ = 2,780 ± 341 and Plg‐RKT −/− = 1,725 ± 95 × 103 mononuclear cells/cavity (P < 0.05, n=6)]. Additionally, the frequency of the M2 marker, CD206, and Annexin A1 (both important for clearance of apoptotic cells) was decreased in Plg−/− macrophages 48 hr after LPS injection. To explore the roles of Plg and Plg‐RKT in efferocytosis, mice were injected with zymosan intraperitoneally, followed by an intraperitoneal injection of apoptotic human neutrophils labeled with carboxyfluorescein succinimidyl ester (CFSE). Significantly decreased efferocytosis of apoptotic neutrophils by Plg−/− macrophages and by Plg‐RKT−/− macrophages, compared with their wild type littermate controls was observed [Plg+/+ = 20.16 ± 3.57 and Plg−/− = 9.04 ± 3.50 (P < 0.05, n=5); Plg‐RKT +/+ = 18.11 ± 4.57 and Plg‐RKT−/− = 5.80 ± 2.90 frequency of total cells/cavity (P < 0.05, n=6)]. These results suggest that Plg regulates macrophage polarization and efferocytosis of apoptotic cells, thereby contributing to the resolution of inflammation and that these effects are mediated by the Plg receptor, Plg‐RKT.Support or Funding InformationCAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil), National Institutes of Health (grant HL 081046 to L.A.M.) and by Merit Review Award #5I01BX002026 from the U.S. Department of Veterans Affairs (to R.J.P.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification and characterization of Taenia solium enolase as a plasminogen-binding protein

The larval stage of Taenia solium (cysticerci) is the causal agent of human and swine cysticercosis. When ingested by the host, T. solium eggs are activated and hatch in the intestine, releasing oncospheres that migrate to various tissues and evolve into cysticerci. Plasminogen (Plg) receptor proteins have been reported to play a role in migration processes for several pathogens. This work is aimed to identify Plg-binding proteins in T. solium cysticerci and determine whether T. solium recombinant enolase (rTsEnoA) is capable of specifically binding and activating human Plg. To identify Plg-binding proteins, a 2D-SDS-PAGE ligand blotting was performed, and recognized spots were identified by MS/MS. Seven proteins from T. solium cysticerci were found capable of binding Plg: fascicilin-1, fasciclin-2, enolase, MAPK, annexin, actin, and cytosolic malate dehydrogenase. To determine whether rTsEnoA binds human Plg, a ligand blotting was performed and the results were confirmed by ELISA both in the presence and absence of εACA, a competitive Plg inhibitor. Finally, rTsEnoA-bound Plg was activated to plasmin in the presence of tPA. To better understand the evolution of enolase isoforms in T. solium, a phylogenetic inference analysis including 75 enolase amino acid sequences was conducted. The origin of flatworm enolase isoforms, except for Eno4, is independent of their vertebrate counterparts. Therefore, herein we propose to designate tapeworm protein isoforms as A, B, C, and 4. In conclusion, recombinant enolase showed a strong plasminogen binding and activating activity in vitro. T. solium enolase could play a role in parasite invasion along with other plasminogen-binding proteins.

Moonlighting glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH): an evolutionarily conserved plasminogen receptor on mammalian cells.

Prokaryotic pathogens establish infection in mammals by capturing the proteolytic enzyme plasminogen (Plg) onto their surface to digest host extracellular matrix (ECM). One of the bacterial surface Plg receptors is the multifunctional glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In a defensive response, the host mounts an inflammatory response, which involves infiltration of leukocytes to sites of inflammation. This requires macrophage exit from the blood and migration across basement membranes, a phenomenon dependent on proteolytic remodeling of the ECM utilizing Plg. The ability of Plg to facilitate inflammatory cell recruitment critically depends on receptors on the surface of phagocyte cells. Utilizing a combination of biochemical, cellular, knockdown, and in vivo approaches, we demonstrated that upon inflammation, macrophages recruit GAPDH onto their surface to carry out the same task of capturing Plg to digest ECM to aid rapid phagocyte migration and combat the invading pathogens. We propose that GAPDH is an ancient, evolutionarily conserved receptor that plays a key role in the Plg-dependent regulation of macrophage recruitment in the inflammatory response to microbial aggression, thus pitting prokaryotic GAPDH against mammalian GAPDH, with both involved in a conserved role of Plg activation on the surface of their respective cells, to conflicting ends.-Chauhan, A. S., Kumar, M., Chaudhary, S., Patidar, A., Dhiman, A., Sheokand, N., Malhotra, H., Raje, C. I., Raje, M. Moonlighting glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH): an evolutionarily conserved plasminogen receptor on mammalian cells.

Host-derived extracellular RNA promotes adhesion of Streptococcus pneumoniae to endothelial and epithelial cells.

Streptococcus pneumoniae is the most frequent cause of community-acquired pneumonia. The infection process involves bacterial cell surface receptors, which interact with host extracellular matrix components to facilitate colonization and dissemination of bacteria. Here, we investigated the role of host-derived extracellular RNA (eRNA) in the process of pneumococcal alveolar epithelial cell infection. Our study demonstrates that eRNA dose-dependently increased S. pneumoniae invasion of alveolar epithelial cells. Extracellular enolase (Eno), a plasminogen (Plg) receptor, was identified as a novel eRNA-binding protein on S. pneumoniae surface, and six Eno eRNA-binding sites including a C-terminal 15 amino acid motif containing lysine residue 434 were characterized. Although the substitution of lysine 434 for glycine (K434G) markedly diminished the binding of eRNA to Eno, the adherence to and internalization into alveolar epithelial cells of S. pneumoniae strain carrying the C-terminal lysine deletion and the mutation of internal Plg-binding motif were only marginally impaired. Accordingly, using a mass spectrometric approach, we identified seven novel eRNA-binding proteins in pneumococcal cell wall. Given the high number of eRNA-interacting proteins on pneumococci, treatment with RNase1 completely inhibited eRNA-mediated pneumococcal alveolar epithelial cell infection. Our data support further efforts to employ RNAse1 as an antimicrobial agent to combat pneumococcal infectious diseases.

Podocyte injury: the role of proteinuria, urinary plasminogen, and oxidative stress.

Podocytes are the key target for injury in proteinuric glomerular diseases that result in podocyte loss, progressive focal segmental glomerular sclerosis (FSGS), and renal failure. Current evidence suggests that the initiation of podocyte injury and associated proteinuria can be separated from factors that drive and maintain these pathogenic processes leading to FSGS. In nephrotic urine aberrant glomerular filtration of plasminogen (Plg) is activated to the biologically active serine protease plasmin by urokinase-type plasminogen activator (uPA). In vivo inhibition of uPA mitigates Plg activation and development of FSGS in several proteinuric models of renal disease including 5/6 nephrectomy. Here, we show that Plg is markedly increased in the urine in two murine models of proteinuric kidney disease associated with podocyte injury: Tg26 HIV-associated nephropathy and the Cd2ap-/- model of FSGS. We show that human podocytes express uPA and three Plg receptors: uPAR, tPA, and Plg-RKT. We demonstrate that Plg treatment of podocytes specifically upregulates NADPH oxidase isoforms NOX2/NOX4 and increases production of mitochondrial-dependent superoxide anion (O2-) that promotes endothelin-1 synthesis. Plg via O2- also promotes expression of the B scavenger receptor CD36 and subsequent increased intracellular cholesterol uptake resulting in podocyte apoptosis. Taken together, our findings suggest that following disruption of the glomerular filtration barrier at the onset of proteinuric disease, podocytes are exposed to Plg resulting in further injury mediated by oxidative stress. We suggest that chronic exposure to Plg could serve as a "second hit" in glomerular disease and that Plg is potentially an attractive target for therapeutic intervention.

Lipoprotein(a) Binds to Recombinant Nontypeable <i>Haemophilus influenzae</i> Aspartase

The respiratory pathogen nontypeable <i>Haemophilus influenza</i> (NTHi) can recruit plasminogen (Plg) on the cell surface by its Plg receptor aspartase (ASP) and utilize host Plg and fibrinolytic system to achieve its adherence and immune invasion. Lipoprotein(a) [Lp(a)] consists of one molecule low-density lipoprotein (LDL) and one molecule apolipoprotein(a) [Apo(a)]. Apo(a) shares a high degree of homology with the human Plg, and both of them contain lysine-binding sites (LBS), which enables them to interact with various cell-surface receptors or fibrin(ogen). However, the definite physiological function of Lp(a) remains vague. Here, we present evidence that Lp(a) via its Apo(a) may bind to thePlg receptor ASP. Recombinant aspartase (rASP) and C-terminal lysine-deleted variant of ASP (rASPΔK) were used in the current study. The rASP specifically bound to Lp(a), but rASPΔK did not, indicating that C-terminal lysine residue of rASP was responsible for the interaction. In addition, rASP interacted with Lp(a), but not with LDL, revealing that LBS of Apo(a) was involved in the binding. Our results also showed that Lp(a) could inhibit the binding of Plg to rASP. Plasma Lp(a) might play a role in anti-NTHi infection by binding to its Plg receptor ASP.

The Interaction Between Recombinant Protein F Derived from Nontypeable &amp;lt;i&amp;gt;Haemophilus influenza&amp;lt;/i&amp;gt; and Lipoprotein(a)

Protein F (PF) is a surface plasminogen (Plg) receptor on the nontypeable Haemophilus influenza (NTHi). Plg via its lysine binding sites (LBS) can bind to PF. Apolipoprotein(a) [Apo(a)] is one component of Lipoprotein(a) [Lp(a)]. It has Kringle (K) domains, which contain LBS and has a high homology with Plg. Therefore, we speculated that Lp(a) might bind to Plg receptor on the surface of NTHi, subsequently competitively inhibiting the interaction of NTHi with Plg. In this study, recombinant PF (rPF) and its C-terminal lysine residue-deleted variant (rPFΔK) were expressed in E. coli BL21. The interactions of rPF with Plg and Lp(a) were tested by ELISA. The results showed that rPF could bind to Plg and Lp(a). The binding capacity of rPF was significantly higher than that of rPFΔK. The interactions of rPF with Plg and Lp(a) could be inhibited by EACA. 2 mmol/L of EACA significantly inhibited the binding of rPF to Plg, while 0.2 mmol/L of EACA could significantly reduce the binding of rPF to Lp(a). 50 ng/100 μL Lp(a) could significantly inhibit the interaction of rPF with Plg. In addition, affinity chromatography assay followed by Western biotting was also used to study the interaction. In overall, C-terminal lysine residue of rPF and the lysine binding sites (LBS) of Plg and Lp(a) should be responsible for these specifically bindings. Lp(a) could combine with rPF consequently inhibiting the interaction of Plg with rPF. This revealed that Lp(a) might play a role in anti-NTHi infection.

Lipoprotein(a) Binds to C-terminal Lysine Residues of Recombinant Enolase Derived from Group A &amp;lt;i&amp;gt;Streptococcus&amp;lt;/i&amp;gt;

The biological function of lipoprotein(a) [Lp(a)] remains elusive although it was identified in 1963. We previously hypothesized that Lp(a) might inhibit pathogens from hijacking host plasminogen (Plg) since apolipoprotein(a) [Apo(a)], a unique protein in Lp(a), shares a high homology with Plg. We demonstrate that Lp(a) bound to recombinant Streptococcal α-enolase (rSEN), which is a surface Plg receptor on group A Streptococcus (GAS). However, recombinant C-terminal lysines-deleted variant of enolase (rSENΔ434-435) did not bind to Lp(a). Moreover, epsilon-aminocaproic acid (EACA), a lysine analog, significantly inhibited the binding of rSEN to Lp(a). Collectively, Lp(a) via its LBS bound to the C-terminal lysines of rSEN. In addition, Lp(a) only competitively blocked the Plg-rSEN interaction but not Plg-rSENΔ434-435 interaction since Plg could also bind to the internal lysine residue of α-enolase. The preliminary study indicated that Lp(a) also interacted with GAS, consequently competitively inhibiting the Plg-GAS binding to some extent. Therefore, Lp(a) might play a limited role in preventing GAS infection since it only partially inhibited the pathogen from recruiting host Plg.

Plasminogen promotes macrophage phagocytosis in mice

The phagocytic function of macrophages plays a pivotal role in eliminating apoptotic cells and invading pathogens. Evidence implicating plasminogen (Plg), the zymogen of plasmin, in phagocytosis is extremely limited with the most recent in vitro study showing that plasmin acts on prey cells rather than on macrophages. Here, we use apoptotic thymocytes and immunoglobulin opsonized bodies to show that Plg exerts a profound effect on macrophage-mediated phagocytosis in vitro and in vivo. Plg enhanced the uptake of these prey by J774A.1 macrophage-like cells by 3.5- to fivefold Plg receptors and plasmin proteolytic activity were required for phagocytosis of both preys. Compared with Plg(+/+) mice, Plg(-/-) mice exhibited a 60% delay in clearance of apoptotic thymocytes by spleen and an 85% reduction in uptake by peritoneal macrophages. Phagocytosis of antibody-mediated erythrocyte clearance by liver Kupffer cells was reduced by 90% in Plg(-/-) mice compared with Plg(+/+) mice. A gene array of splenic and hepatic tissues from Plg(-/-) and Plg(+/+) mice showed downregulation of numerous genes in Plg(-/-) mice involved in phagocytosis and regulation of phagocytic gene expression was confirmed in macrophage-like cells. Thus, Plg may play an important role in innate immunity by changing expression of genes that contribute to phagocytosis.

Thrombomodulin functions as a plasminogen receptor to modulate angiogenesis

Urokinase-type plasminogen activator (uPA) activates plasminogen (Plg) through a major pericellular proteolytic system involved in cell migration and angiogenesis; however, the Plg receptor that participates in uPA-mediated Plg activation has not yet been identified. In this study, we demonstrated that thrombomodulin (TM), a type I transmembrane glycoprotein, is a novel Plg receptor that plays a role in pericellular proteolysis and cell migration. Plg activation at the cell surface and the extent of its cell migration- and invasion-promoting effect are cellular TM expression dependent. Direct binding of Plg and the recombinant TM extracellular domain, with a KD of 0.1-0.3 μM, was determined through surface plasmon resonance analysis. Colocalization of TM, Plg, and the uPA receptor within plasma membrane lipid rafts, at the leading edge of migrating endothelial cells, was demonstrated and was also shown to overlap with areas of major pericellular proteolysis. Moreover, the roles of TM and Plg in neoangiogenesis were demonstrated in vivo through the skin wound-healing model. In conclusion, we propose that TM is a novel Plg receptor that regulates uPA/uPA receptor-mediated Plg activation and pericellular proteolysis within lipid rafts at the leading edge of migrating cells during angiogenesis.

Phosphatidylserine as an anchor for plasminogen and its plasminogen receptor, Histone H2B, to the macrophage surface

Plasminogen (Plg) binding to cell surface Plg receptors (Plg-Rs) on the surface of macrophages facilitates Plg activation and migration of these cells. Histone H2B (H2B) acts as a Plg-R and its cell surface expression is up-regulated when monocytes are differentiated to macrophages via a pathway dependent on L-type Ca(2+) channels and intracellular Ca(2+). We sought to investigate the mechanism by which H2B, a protein without a transmembrane domain, is retained on the macrophage surface. THP-1 monocytoid cells were induced to differentiate with interferon gamma + Vitamin D3 or to undergo apoptosis by treatment with camptothecin. Flow cytometry and cell surface biotinylation followed by Western blotting were used to measure the interrelationship between Plg binding, cell surface expression of H2B and outer membrane exposure of phosphatidylserine (PS). H2B interacted directly with PS via an electrostatic interaction. Anti-PS or PS binding proteins, annexin V and protein S, diminished H2B interaction with PS on the surface of differentiated or apoptotic cells and these same reagents inhibited Plg binding to these cells. L-type Ca(2+) channels played a significant role in PS exposure, H2B surface expression and Plg binding induced either by differentiation or apoptosis. These data suggest that H2B tethers to the surface of cells by interacting with PS on differentiated or apoptotic monocytoid cells. L-type Ca(2+) channels regulate PS exposure on the surface of these cells. The exposed PS interacts directly with H2B and hence provides sites for Plg to bind to.

Colocalization of the novel plasminogen receptor, Plg‐RKT, with the epithelial sodium channel (ENaC)

Binding of plasminogen (Plg) to cells via specific receptors markedly enhances Plg activation, and localizes the proteolytic activity of plasmin (P) on cell surfaces. Exposure of kidney tubular cells to P results in cleavage and activation of ENaC leading to increased Na+ uptake, and Na+ retention in nephrotic syndrome in which Plg is present in urine. We recently isolated a novel transmembrane Plg receptor, Plg‐RKT, from macrophages. Here, we examined kidney cells for the presence and subcellular localization of Plg‐RKT as a potential component of the mechanism in which P processes ENaC. Human kidney sections were stained with specific monoclonal anti‐Plg‐RKT. Strong staining was observed in distal tubular epithelium (intensity=200–256) &gt; proximal tubular cells (intensity=140–199) ≫ glomeruli (intensity=80–139). In western blotting of MDCK cells and M1 mouse collecting duct cells Plg‐RKT was highly expressed in membrane fractions, but was not detected in cytoplasm. Using confocal microscopy, Plg‐RKT was immunodetected on the apical surface, an orientation allowing for interaction with urinary Plg. Furthermore, in merged images, Plg‐RKT colocalized with both ENaC and the urokinase receptor (uPAR). The extent of colocalization of Plg‐RKT with ENaC was 98±1% and 89±3% in MDCK and M1 cells, respectively, and with uPAR was 96±1% in both cell types. Thus, Plg‐RKT, uPAR and ENaC are present in very close proximity on the kidney tubular cell surface in an orientation to promote Plg activation and ENaC processing.

L-Type Calcium Channel Blockers Exert an Antiinflammatory Effect by Suppressing Expression of Plasminogen Receptors on Macrophages

L-type Ca(2+) channel (LTCC) blockers, represented by amlodipine and verapamil, are widely used antihypertensive drugs that also have antiinflammatory activities. Plasminogen (Plg) is an important mediator of macrophage recruitment, and this role depends on its interaction with Plg receptors (Plg-Rs). Plg-Rs include histone 2B, alpha-enolase, annexin 2, and p11, all proteins which lack signal sequences for cell surface export. When human or murine monocytoid cells were induced to differentiate into macrophages, their Plg binding and Plg-R expression increased by 4-fold. These changes were suppressed by pretreatment with verapamil and amlodipine. Expression of the Ca(v)1.2 LTCC pore subunit was induced in differentiated macrophages, and siRNA against this subunit suppressed the upregulation of Plg binding and Plg-Rs. In vivo, amlodipine and verapamil suppressed peritoneal macrophage recruitment in response to thioglycollate by >60% at doses that did not affect blood pressure. In drug-treated animals, macrophages migrated into but not through the peritoneal membrane tissue and showed reduced surface expression of Plg-Rs. These findings demonstrate that Plg-R expression on macrophages is dependent on Ca(v)1.2 LTCC subunit expression. Suppression of Plg-Rs may contribute to the antiinflammatory effects of the widely used LTCC blockers.

Receptor recognition specificity of plasminogen for the novel plasminogen receptor, Plg‐RKT

Plasminogen (Plg) receptors promote Plg activation to arm cells with plasmin to promote both thrombolysis and cell migration. Plg‐RKT is a new type II integral membrane Plg receptor on leukocytes and other tissues. Our objective was to define the recognition specificity of Plg for Plg‐RKT. The C‐terminal peptide of Plg‐RKT was coupled to microtiter wells. Biotinylated Plg was incubated with the immobilized peptide and detected with streptavidin‐HRP. Plg bound to the peptide in a concentration‐dependent manner (IC50=5 nM). In specificity controls, Plg binding was competed by unlabeled Plg and no binding to the reverse peptide was detected. Plg binding was inhibited in a dose‐dependent manner by the soluble peptide, but was not affected by a mutant peptide with the C‐terminal K replaced with A. A peptide with the proximal upstream R replaced with K was a more effective competitor than the wild type peptide. Human Plg also recognized the C‐terminal peptides of murine, rat, and bovine Plg‐RKT's that contain the same R6K substitution. Plg domains containing disulfide‐bonded kringles (K) inhibited the interaction with the following order of effectiveness: Plg&gt;K1‐3&gt;K5&gt;K4. In conclusion, the interaction of Plg‐RKT with Plg requires the presence of a C‐terminal lysine and can be modulated by a proximal upstream basic residue. Furthermore, kringle domains within the Plg molecule interact with the C‐terminus of Plg‐RKT.

Histone H2B as a functionally important plasminogen receptor on macrophages

AbstractPlasminogen (Plg) facilitates inflammatory cell recruitment, a function that depends upon its binding to Plg receptors (Plg-Rs). However, the Plg-Rs that are critical for cell migration are not well defined. Three previously characterized Plg-Rs (α-enolase, annexin 2, and p11) and a recently identified Plg-R (histone H2B [H2B]) were assessed for their contribution to Plg binding and function on macrophages. Two murine macrophage cell lines (RAW 264.7 and J774A.1) and mouse peritoneal macrophages induced by thioglycollate were analyzed. All 4 Plg-Rs were present on the surface of these cells and showed enhanced expression on the thioglycollate-induced macrophages compared with peripheral blood monocytes. Using blocking Fab fragments to each Plg-R, H2B supported approximately 50% of the Plg binding capacity, whereas the other Plg-Rs contributed less than 25%. Anti-H2B Fab also demonstrated a major role of this Plg-R in plasmin generation and matrix invasion. When mice were treated intravenously with anti-H2B Fab, peritoneal macrophage recruitment in response to thioglycollate was reduced by approximately 45% at 24, 48, and 72 hours, with no effect on blood monocyte levels. Taken together, these data suggest that multiple Plg-Rs do contribute to Plg binding to macrophages, and among these, H2B plays a very prominent and functionally important role.