Cross-talk Between Coagulation, Inflammation, and Immunity

Palo Alto Veterans Instit For Research, Palo Alto CA

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Abstract

DESCRIPTION (provided by applicant): Based on studies with the pCPB KO mice, we have obtained compelling data to support our general hypothesis that thrombin-activatable carboxypeptidase B (CPB or TAFIa) plays a broad anti-inflammatory role in vivo; the targets for CPB include bradykinin, C5a, the generation of plasmin and thrombin-cleaved osteopontin (OPN-Arg or OPN-R). We showed markedly elevated levels of OPN-R and its CPB-cleaved product, OPN-Leu (OPN-L), in synovial fluid samples from rheumatoid arthritis patients and these cleaved OPN forms affect neutrophils and fibroblast-like synoviocytes. Furthermore, we showed that prochemerin, a novel chemoattractant whose activity is dependent on C-terminal proteolytic cleavage by serine protease(s), can be activated by sequential cleavages by plasmin and CPB, thus establishing a new substrate for CPB and providing a novel molecular link between hemostasis/thrombosis and tissue inflammation and immunity. Aim 1. To determine the biology of OPN-R and OPN-L in human glioblastoma (GBM). We demonstrated significantly elevated levels of OPN-R and OPN-L in CSF samples of patients with GBM. We will examine the distribution of OPN-FL, OPN-R, and OPN-L in GBM by immunohistology, and characterize cell signaling by OPN-R in comparison to OPN-FL and OPN-L in terms of cell adhesion, cell proliferation, apoptosis, protein phosphorylation, gene expression and cytokine production. We will test whether GBM cells, either at resting or stimulated state, will express and release OPN, resulting in cleaved OPN locally. Aim 2. To determine the in vivo importance of thrombin cleavage of OPN using the thrombin non-cleavable OPN R153A knockin mice in inflammation, hemopoietic stem cell mobilization, aortic aneurysm, and cancer models. We have generated viable homozygous OPN R153A knockin (KI) mice and showed that recombinant OPN R153A is resistant to thrombin cleavage. Based on our preliminary data, we predicted that the homozygous OPN KI mice would be resistant to Con A-induced hepatitis. We will test whether the KI mice show enhanced hemopoietic stem cell mobilization and whether these animals are resistant to angiotensin II-induced abdominal aortic aneurysm formation in an ApoE null background. Since there is a close association between OPN and cancer, the KI mice will be tested in a chemical carcinogen (ENU)-induced cancer model to determine if abolition of OPN-R generation will alter the susceptibility to cancer development. Aim 3. Biochemistry and biology of activation of prochemerin by serine/cysteine proteases and CPB. Active chemerin is present in GBM CSF samples (preliminary result) and RA synovial fluid. To determine the relevant forms of active chemerin in vivo, we have generated polyclonal antibodies specific for prochemerin and plasmin-cleaved and plasmin/CPB-double cleaved chemerin. These will be used to develop specific ELISAs to measure their levels in the clinical samples; we will determine the proteolytic cleavage efficiencies of prochemerin by different serine and cysteine proteases. Preliminary studies showed that GBM U87 cells express both chemerin and its receptors and that chemerin signals GBM U87 cells by STAT3 and S6 phosphorylation. We will examine whether GBM cells enhance plasmin generation and promote local active chemerin production. We will characterize chemerin signaling of U87 cells and test whether chemerin or post-culture media from GBM cells will recruit plasmacytoid dendritic cells. Our long-term goal is to understand the roles of thrombin and carboxypeptidase B and their cleavage products in the cross-talk between coagulation, tissue inflammation, and immunity, which has direct relevance to many pathological processes including malignancy, host defenses, and inflammatory diseases.