Cross-talk Between Coagulation, Inflammation, and Immunity

描述(申请人提供)：基于对pCPB KO小鼠的研究，我们已获得令人信服的数据来支持我们的一般假设，即凝血酶可激活的羧基肽酶B(CPB或TAFIA)在体内具有广泛的抗炎作用；CPB的靶标包括缓激肽、C5a、纤溶酶和凝血酶裂解的骨桥蛋白(OPN-Arg或OPN-R)。在类风湿关节炎患者的滑液样本中，我们发现OPN-R及其CPB裂解产物OPN-Leu(OPN-L)的水平显著升高，这些裂解的OPN形式影响中性粒细胞和成纤维细胞样滑膜细胞。此外，我们还发现，原趋化蛋白是一种新的趋化物质，其活性依赖于丝氨酸蛋白酶(S)C端的蛋白水解性切割，它可以被纤溶酶和体外循环连续切割而激活，从而为体外循环建立了一个新的底物，并在止血/血栓形成和组织炎症和免疫之间提供了新的分子联系。目的1.检测骨桥蛋白-R和骨桥蛋白-L在人脑胶质母细胞瘤中的生物学意义。我们发现基底膜患者脑脊液中骨桥蛋白受体和骨桥蛋白L水平显著升高。我们将用免疫组织化学方法检测OPN-FL、OPN-R和OPN-L在基底膜中的分布，并从细胞黏附、细胞增殖、细胞凋亡、蛋白磷酸化、基因表达和细胞因子产生等方面比较OPN-R和OPN-FL和OPN-L在细胞信号转导中的作用。我们将测试无论是静息状态还是刺激状态，GBM细胞是否会表达和释放OPN，导致局部OPN被切割。目的2.利用凝血酶不能裂解的OPN R153A敲击小鼠，确定凝血酶裂解OPN在炎症、造血干细胞动员、主动脉瘤和癌症模型中的体内重要性。我们已经获得了存活的纯合子OPN R153A敲门(KI)小鼠，并表明重组OPN R153A对凝血酶切割具有抵抗力。根据我们的初步数据，我们预测纯合子OPN Ki小鼠将对ConA诱导的肝炎产生抵抗力。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抵抗力。由于OPN与癌症之间有密切的联系，KI小鼠将在化学致癌物(ENU)诱导的癌症模型中进行测试，以确定取消OPN-R的产生是否会改变癌症的易感性。目的3.丝氨酸/半胱氨酸蛋白酶和CPB激活前趋化蛋白的生物化学和生物学研究。活性趋化蛋白存在于GBM脑脊液样本(初步结果)和RA滑液中。为了确定体内活性趋化蛋白的相关形式，我们制备了针对原趋化蛋白和纤溶酶裂解的多克隆抗体以及纤溶酶/CPB双裂解的趋化蛋白。这些将被用来开发特定的ELISA来测量它们在临床样本中的水平；我们将确定不同丝氨酸和半胱氨酸蛋白酶对前趋化蛋白的蛋白分解切割效率。初步研究表明，GBM U87细胞同时表达趋化蛋白及其受体，趋化蛋白通过STAT3和S6的磷酸化向GBM U87细胞发出信号。我们将研究GBM细胞是否促进纤溶酶的产生和促进局部活性趋化蛋白的产生。我们将对U87细胞的趋化蛋白信号进行表征，并测试来自GBM细胞的趋化蛋白或培养后的培养液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。 与公共卫生相关：凝血酶是主要的血液凝固酶，负责在血管损伤部位形成血栓。然而，除此之外，凝血酶还可以激活许多不同类型的细胞，并在引起组织炎症中发挥关键作用。作为正常控制机制的一部分，凝血酶与血管表面一种名为血栓调节蛋白的蛋白质结合，在这种情况下，凝血酶的功能受到“调节”，并将激活另外两种蛋白质，蛋白C和前羧基肽酶B(PCPB)。激活的蛋白C现在被确定为一种蛋白质，它将抑制血液凝固，并对血管起到保护作用，从而作为一种反馈机制，下调凝血酶的凝血和炎症功能。我们和其他人发表的数据表明，激活的CPB可能在这一过程中发挥类似的作用。这项应用的目的是研究CPB如何影响一些特定的炎症蛋白，并测量这种相互作用的产物，以证明这些相互作用实际上发生在我们的身体中。我们认为，凝血、炎症和免疫之间的这种串扰对于我们理解包括癌症、炎症性疾病和异常凝血在内的许多疾病是重要的和高度相关的。

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.