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-R KT (C9orf46 homolog). Plg-R KT was highly colocalized on the cell surface with the urokinase receptor, uPAR.Our data suggest that Plg-R KT also interacts directly with tissue plasminogen activator. Furthermore, Plg-R KT IntroductionLocalization of plasminogen on cell surfaces is a crucial control point for positive regulation of cell surface plasmin proteolytic activity that facilitates both physiologic and pathologic processes, 1,2 including macrophage recruitment during the inflammatory response, [3][4][5][6] tissue remodeling, 7 wound healing, 8,9 tumor cell invasion and metastasis, 10-12 skeletal myogenesis, 13 neuroendocrine prohormone processing, 14,15 and neurite outgrowth. 16,17 Cell surface plasminogen binding sites promote plasminogen activation by reducing the Km (11-to 60-fold) for plasminogen activation. [18][19][20][21][22][23][24] Active plasmin also associates with the cell surface, where its activity is protected from inhibitors. 25,26 Plasminogen binding sites are very broadly distributed on both eukaryotic and prokaryotic cells. 27 Of the many eukaryotic cells examined to date, only erythrocytes do not bind plasminogen. 28 The interactions of plasminogen with eukaryotic cells are mediated by lysine binding sites within the disulfide-bonded kringle domains of plasminogen. 18,29 Therefore, plasminogen binding to eukaryotic cells is blocked in the presence of lysine and lysine analogs, including ⑀-aminocaproic acid (EACA). 27 Because most cell types have a very high capacity for plasminogen, no single molecule can account for the entire plasminogen binding capacity of a given cell type. 27 However, a subset of plasminogen binding proteins exposing C-terminal basic residues on cell surfaces are predominantly responsible for the ability of eukaryotic cells to enhance plasminogen activation because carboxypeptidase B (CpB) treatment abrogates cell surface-dependent plasminogen activation. 24 Correspondingly, plasminogen-dependent macrophage recruitment in vivo is mediated by CpB-sensitive plasminogen receptors, and plasminogen binding to recruited macrophages is increased, compared with peripheral blood monocytes. 6,30 Therefore, we probed the monocyte ...

Localization of plasmin on macrophages and activation of pro-MMP-9 play key roles in macrophage recruitment in the inflammatory response. These functions are promoted by plasminogen receptors exposing C-terminal basic residues on the macrophage surface. Recently, we identified a novel transmembrane plasminogen receptor, Plg-R KT , which exposes a C-terminal lysine on the cell surface. In the present study, we investigated the role of Plg-R KT in macrophage invasion, chemotactic migration, and recruitment. Plg-R KT was prominently expressed in membranes of human peripheral blood monocytes and monocytoid cells. Plasminogen activation by urokinasetype plasminogen activator (uPA) was markedly inhibited (by 39%) by treatment with anti-Plg-R KT mAb. Treatment of monocytes with anti-Plg-R KT mAb substantially inhibited invasion through the representative matrix, Matrigel, in response to MCP-1 (by 54% compared with isotype control). Furthermore, chemotactic migration was also inhibited by treatment with anti-Plg-R KT mAb (by 64%). In a mouse model of thioglycollate-induced peritonitis, anti-Plg-R KT mAb markedly inhibited macrophage recruitment (by 58%), concomitant with a reduction in pro-MMP-9 activation in the inflamed peritoneum. Treatment with anti-Plg-R KT mAb did not further reduce the low level of macrophage recruitment in plasminogennull mice. We conclude that Plg-R KT IntroductionActivation of plasminogen, the zymogen of the primary thrombolytic enzyme plasmin, is markedly promoted when plasminogen is bound to cell surfaces (for review, see Miles et al 1 ) and cellassociated plasmin is protected from inactivation. 2,3 Therefore, cells become armed with the broad-spectrum proteolytic activity of plasmin. 4 This provides a mechanism to facilitate both physiologic and pathologic processes requiring cell migration. Plasminogendependent cell migration is involved in macrophage recruitment during the inflammatory response, 4-10 tissue remodeling, 11 wound healing, 12,13 tumor cell invasion and metastasis, 14,15 skeletal myogenesis, 16 neuroendocrine prohormone processing, 17,18 and neurite outgrowth. 19,20 Studies in plasminogen-deficient mice have demonstrated that plasminogen plays a key role in cell migration in a diverse array of physiologic and pathophysiologic settings, notably, macrophage recruitment in response to inflammatory stimuli in the thioglycollate-induced model of peritonitis. Plasmin-dependent cell migration is accomplished by direct degradation of extracellular matrix components by plasmin and also by activation of matrix metalloproteinases for further degradation of extracellular matrices. [4][5][6][7] Among the plasminogen-binding proteins, those exposing Cterminal basic residues on cell surfaces are predominantly responsible for the ability of eukaryotic cells to enhance plasminogen activation, because carboxypeptidase B (CpB) treatment abrogates cell surface-dependent plasminogen activation. 21 Furthermore, plasminogen-dependent macrophage recruitment is mediated by CpB-sensitive plasminoge...

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-R KT , 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-R KT −/− 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-R KT −/− 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-R KT −/− mice. Mechanistically, Plg and Pla induced transient STAT3 phosphorylation, which was decreased in Plg −/− and Plg-R KT −/− 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-R KT −/− 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-R KT , regulate macrophage polarization and efferocytosis, as key contributors to the resolution of inflammation.

An emerging area of research has documented a novel role for the plasminogen activation system in the regulation of neurotransmitter release. Prohormones, secreted by cells within the sympathoadrenal system, are processed by plasmin to bioactive peptides that feed back to inhibit secretagogue-stimulated release. Catecholaminergic cells of the sympathoadrenal system are prototypic prohormone-secreting cells. Processing of prohormones by plasmin is enhanced in the presence of catecholaminergic cells, and the enhancement requires binding of plasmin(ogen) to cellular receptors. Consequently, modulation of the local cellular fibrinolytic system of catecholaminergic cells results in substantial changes in catecholamine release. However, mechanisms for enhancing prohormone processing and cellsurface molecules mediating the enhancement on catecholaminergic cells have not been investigated. Here we show that plasminogen activation was enhanced Ͼ6.5-fold on catecholaminergic cells. Carboxypeptidase B treatment decreased cell-dependent plasminogen activation by ϳ90%, suggesting that the binding of plasminogen to proteins exposing C-terminal lysines on the cell surface is required to promote plasminogen activation. We identified catecholaminergic plasminogen receptors required for enhancing plasminogen activation, using a novel strategy combining targeted specific proteolysis using carboxypeptidase B with a proteomics approach using twodimensional gel electrophoresis, radioligand blotting, and tandem mass spectrometry. Two major plasminogen-binding proteins that exposed C-terminal lysines on the cell surface contained amino acid sequences corresponding to ␤/␥-actin. An anti-actin monoclonal antibody inhibited cell-dependent plasminogen activation and also enhanced nicotine-dependent catecholamine release. Our results suggest that cell-surface-expressed forms of actin bind plasminogen, thereby promoting plasminogen activation and increased prohormone processing leading to inhibition of neurotransmitter release.

Monocytes are major mediators of inflammation, and apoptosis provides a mechanism for regulating the inflammatory response by eliminating activated macrophages. Furthermore, as a consequence of apoptosis, plasminogen binding is markedly increased on monocytoid cells. Therefore, we investigated the ability of plasminogen to modulate monocyte apoptosis. Apoptosis of monocytoid cells (human monocytes and U937 cells) was induced with either TNF␣ or cycloheximide. When apoptosis was induced in the presence of increasing concentrations of plasminogen, apoptosis was inhibited in a dose-dependent manner with full inhibition achieved at 2 M plasminogen. Plasminogen treatment also markedly reduced internucleosomal DNA fragmentation and reduced levels of active caspase 3, caspase 8, and caspase 9 induced by TNF␣ or by cycloheximide. We examined the requirement for plasmin proteolytic activity in the cytoprotective function of plasminogen.