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 ...

Background Resolvins are lipid mediators generated by leukocytes during the resolution phase of inflammation. They have been shown to regulate the transition from inflammation to tissue repair; however, it is unknown whether resolvins play a role in tissue revascularization following ischemia. Methods We used a murine model of hind limb ischemia (HLI), coupled with laser Doppler perfusion imaging, micro computed tomography (microCT) and targeted mass spectrometry, to assess the role of resolvins in revascularization and inflammation-resolution. Results In mice undergoing HLI, we identified resolvin D2 (RvD2) in bone marrow and skeletal muscle by mass spectrometry (n=4-7 per group). We also identified RvD2 in skeletal muscle biopsies from humans with peripheral artery disease. Monocytes were recruited to skeletal muscle during HLI and isolated monocytes produced RvD2 in a lipoxygenase-dependent manner. Exogenous RvD2 enhanced perfusion recovery in HLI and microCT of limb vasculature revealed greater volume, with evidence of tortuous arterioles indicative of arteriogenesis (n=6-8 per group). Unlike other treatment strategies for therapeutic revascularization that exacerbate inflammation, RvD2 did not increase vascular permeability, but reduced neutrophil accumulation and the plasma levels of TNF-α and GM-CSF. In mice treated with RvD2, histopathological analysis of skeletal muscle of ischemic limbs showed more regenerating myocytes with centrally located nuclei. RvD2 enhanced endothelial cell migration in a Rac-dependent manner, via its receptor, GPR18, and Gpr18-deficient mice had an endogenous defect in perfusion recovery following HLI. Importantly, RvD2 rescued defective revascularization in diabetic mice. Conclusions RvD2 stimulates arteriogenic revascularization during HLI suggesting that resolvins may be a novel class of mediators that both resolve inflammation and promote arteriogenesis.

Plasminogen is the zymogen of plasmin, the major enzyme that degrades fibrin clots. In addition to its binding and activation on fibrin clots, plasminogen also specifically interacts with cell surfaces where it is more efficiently activated by plasminogen activators, compared with the reaction in solution. This results in association of the broad spectrum proteolytic activity of plasmin with cell surfaces that functions to promote cell migration. Here we review emerging data establishing a role for plasminogen, plasminogen receptors and the newly discovered plasminogen receptor, Plg-RKT, in macrophage recruitment in the inflammatory response and we address mechanisms by which the interplay between plasminogen and its receptors regulates inflammation.