The coagulation cascade is often the first responder at the site of a challenge. Thus, when the innate or adaptive immune systems respond, they do so in an environment that has been modified by activated factors of the coagulation system. Our long-term goal is to understand how the coagulation cascade modulates the immune response. Thrombomodulin (TM) is a cell surface glycoprotein that binds thrombin, the key effector enzyme of the coagulation cascade, converting it from a pro-coagulant and pro-inflammatory enzyme to an anti-coagulant, anti-fibrinolytic and anti-inflammatory enzyme by activation of Protein C (PC) and procarboxypeptidase2 (pCPB2 aka Thrombin Activatable Fibrinolysis Inhibitor). In addition, its lectin domain can interact directly with cells as well as antagonize signaling at the Receptor for Advanced Glycation End products (RAGE) by binding high mobility group protein B1 (HMGB1). The lectin domain has been suggested to bind to a carbohydrate, Lewisy, present on some endothelial cells. TM also catalyzes the inactivation of complement factor C3b. We have shown that TM treatment of dendritic cells (DCs) changes their properties from causing asthma (immunogenic) to ameliorating asthma (tolerogenic) in a murine bronchial asthma model. DCs treated with TM express increased levels of TM on their cell surface. After sorting TM treated DCs into TM+ and TM- DCs we found that the TM+ DCs were tolerogenic and TM- DCs were immunogenic. Only the lectin domain is necessary for modulation of DCs through an unknown receptor. In preliminary data, the lectin domain from TM was shown to bind in a specific and calcium dependent manner to DCs, CD4+ T cells and a CD4+ T cell leukemia line, Jurkat. Lysates of Jurkat cells contain bands that interact with the lectin domain on a Western. We hypothesize that TM binds to a glycoprotein on DCs and CD4+ T cells that functions as its own signaling receptor triggering cellular responses. This project will identify te TM receptor.
Aim 1. Identify the TM specific signaling receptor on immune cells. We will use the TM lectin domain-Fc fusion protein to isolate that receptor by affinity chromatography from Jurkat cells and identify it by proteomics.
Aim 2 : Characterization of TM binding protein(s) on DCs and other immune cells. The TM lectin receptor isolated in Aim 1 will be produced by expression in mammalian cells and characterized for its ability to interact with the TM lectin domain by surface plasmon resonance. We will also demonstrate that it is a functional antagonist of TM signaling in DCs and other immune cells, including CD4+ T cells. Identification of the TM receptor could suggest novel approaches to treatment of inflammatory diseases. In addition, delineating the mechanism by which TM modulates DCs and other immune cells would allow identification of alternative targets and assays by which drugs for inflammatory diseases could be discovered.
Our long-term goal is to understand how the blood clotting cascade modulates the immune response. Recombinant soluble thrombomodulin (TM), an established regulator of blood clotting, is approved for use in sepsis in Japan and is currently in Phase 2 testing in the US for prevention of deep vein thrombosis. Our studies showed that TM plays a surprising role in protecting against asthma by converting dendritic cells to an immune tolerant phenotype. In this proposal, we will study in detail how TM modulates the immune properties of dendritic cells. These observations could lead to novel therapy in asthma patients. In addition, delineating the mechanism by which TM modulates dendritic cells would allow identification of alternative targets and assays by which drugs for bronchial asthma and other immune disorders could be discovered.
|Schultz, Geoffrey; Tedesco, Maureen M; Sho, Eiketsu et al. (2010) Enhanced abdominal aortic aneurysm formation in thrombin-activatable procarboxypeptidase B-deficient mice. Arterioscler Thromb Vasc Biol 30:1363-70|