Cross-talk Between Coagulation, Inflammation, and Immunity

描述（由申请人提供）：基于对pCPB KO小鼠的研究，我们获得了令人信服的数据来支持我们的一般假设，即凝血酶激活羧肽酶B （CPB或TAFIa）在体内发挥广泛的抗炎作用；CPB的靶点包括缓激肽、C5a、产生纤溶酶和溶栓性骨桥蛋白（OPN-Arg或OPN-R）。我们发现，在类风湿性关节炎患者的滑膜液样本中，OPN- r及其cpb切割产物OPN- leu （OPN- l）水平显著升高，这些被切割的OPN形式影响中性粒细胞和成纤维细胞样滑膜细胞。此外，我们发现prochemerin是一种新的化学吸引剂，其活性依赖于丝氨酸蛋白酶的c端蛋白水解裂解，可以被纤溶蛋白和CPB的顺序裂解激活，从而建立了CPB的新底物，并在止血/血栓形成与组织炎症和免疫之间提供了新的分子联系。目的1。探讨OPN-R和OPN-L在人胶质母细胞瘤（GBM）中的生物学意义。我们证实了GBM患者脑脊液样本中OPN-R和OPN-L水平显著升高。我们将通过免疫组织学检查OPN-FL、OPN-R和OPN-L在GBM中的分布，并在细胞粘附、细胞增殖、细胞凋亡、蛋白磷酸化、基因表达和细胞因子产生方面，将OPN-R与OPN-FL和OPN-L进行比较，表征OPN-R的细胞信号传导。我们将测试在静止或刺激状态下，GBM细胞是否会表达和释放OPN，导致OPN局部断裂。目标2。利用凝血酶不可切割的OPN R153A敲入小鼠，在炎症、造血干细胞动员、主动脉瘤和癌症模型中确定凝血酶切割OPN在体内的重要性。我们培育了活的纯合子OPN R153A敲除蛋白（KI）小鼠，并证明重组OPN R153A对凝血酶切割具有抗性。根据我们的初步数据，我们预测纯合的OPN KI小鼠将对Con - a诱导的肝炎具有抗性。我们将测试KI小鼠是否表现出增强的造血干细胞动员，以及在ApoE缺失的背景下，这些动物是否对血管紧张素ii诱导的腹主动脉瘤形成具有抗性。由于OPN与癌症之间存在密切联系，KI小鼠将在化学致癌物（ENU）诱导的癌症模型中进行测试，以确定消除OPN- r的产生是否会改变癌症发展的易感性。目标3。丝氨酸/半胱氨酸蛋白酶和CPB活化前趋化素的生物化学和生物学研究。活性趋化素存在于GBM CSF样本（初步结果）和RA滑液中。为了确定体内活性趋化素的相关形式，我们制备了针对前趋化素和纤溶蛋白切割和纤溶蛋白/ cpb双切割趋化素的多克隆抗体。这些将用于开发特异性elisa来测量它们在临床样品中的水平；我们将测定不同的丝氨酸和半胱氨酸蛋白酶对前趋化素的蛋白水解裂解效率。初步研究表明，GBM U87细胞同时表达趋化素及其受体，趋化素通过STAT3和S6磷酸化向GBM U87细胞传递信号。我们将研究GBM细胞是否会增强纤溶蛋白的产生并促进局部活性趋化素的产生。我们将对U87细胞的趋化素信号传导进行表征，并测试趋化素或GBM细胞培养后的培养基是否会招募浆细胞样树突状细胞。我们的长期目标是了解凝血酶和羧肽酶B及其裂解产物在凝血、组织炎症和免疫之间的相互作用，这与许多病理过程直接相关，包括恶性肿瘤、宿主防御和炎症性疾病。

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. PUBLIC HEALTH RELEVANCE: Thrombin is the main blood-clotting enzyme responsible for making a blood clot at the site of blood vessel injury. However, in addition to that, thrombin also activates many different types of cells and plays a key role in causing tissue inflammation. As part of a normal control mechanism, thrombin binds to a protein on the blood vessel surface called thrombomodulin and in that context, thrombin's function is "modulated' and will activate two other proteins, protein C and procarboxypeptidase B (pCPB). Activated protein C is now established as a protein that will dampen blood clotting, and exert a protective effect on blood vessels, thus serving as a feedback mechanism to down modulate thrombin's clotting and inflammatory functions. We and others have published data indicating that activated CPB may play a similar role in this process. The objective of this application is to study how CPB affects a number of specific inflammatory proteins and measure the products of this interaction to prove that these interactions actually occur in our body. We believe that this crosstalk between blood clotting, inflammation and immunity is important and highly relevant to our understanding of many diseases including cancer, inflammatory diseases, and abnormal blood clotting.