Cross-talk Between Coagulation, Inflammation, and Immunity

描述（由申请方提供）：基于对pCP B 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，即凝血酶活化羧肽酶B（CP B或TAFIa）在体内发挥广泛的抗炎作用; CP B的靶点包括缓激肽、C5 a、纤溶酶和凝血酶裂解骨桥蛋白（OPN-Arg或OPN-R）的生成。我们在类风湿性关节炎患者的滑液样本中发现OPN-R及其CPB裂解产物OPN-Leu（OPN-L）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，我们表明，prochemerin，一种新的化学引诱剂，其活性依赖于丝氨酸蛋白酶的C-末端蛋白水解切割，可以通过纤溶酶和CPB的连续切割来激活，从而建立CPB的新底物，并提供止血/血栓形成与组织炎症和免疫之间的新分子联系。目标1.目的：研究人胶质母细胞瘤（GBM）中OPN-R和OPN-L的生物学特性。我们证明了GBM患者CSF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、凋亡、蛋白磷酸化、基因表达和细胞因子产生方面与OPN-FL和OPN-L相比表征OPN-R的细胞信号传导。我们将测试GBM细胞在静息或刺激状态下是否会表达和释放OPN，导致局部切割的OPN。目标2.在炎症、造血干细胞动员、主动脉瘤和癌症模型中，使用凝血酶不可切割的OPN R153 A敲入小鼠确定OPN的凝血酶切割的体内重要性。我们已经产生了活的纯合子OPN R153 A敲入（KI）小鼠，并显示重组OPN R153 A对凝血酶切割具有抗性。基于我们的初步数据，我们预测纯合子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，以及这些动物在ApoE空背景下是否对血管紧张素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关联，因此将在化学致癌物（ENU）诱导的癌症模型中测试KI小鼠，以确定OPN-R生成的消除是否会改变对癌症发展的易感性。目标3.丝氨酸/半胱氨酸蛋白酶和CPB激活prochemerin的生物化学和生物学。GBM CSF样本（初步结果）和RA滑液中存在活性chemerin。为了确定体内活性趋化蛋白的相关形式，我们已经产生了对前趋化蛋白和纤溶酶切割的和纤溶酶/CPB双切割的趋化蛋白特异的多克隆抗体。这些将用于开发特异性ELISA以测量其在临床样品中的水平;我们将确定不同丝氨酸和半胱氨酸蛋白酶对prochemerin的蛋白水解切割效率。初步研究表明，GBM U87细胞表达chemerin及其受体，chemerin通过STAT 3和S6磷酸化向GBM U87细胞发出信号。我们将研究GBM细胞是否能增强纤溶酶的产生并促进局部活性趋化蛋白的产生。我们将表征U87细胞的chemerin信号传导，并测试chemerin或来自GBM细胞的后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中的作用，这与许多病理过程包括恶性肿瘤、宿主防御和炎性疾病直接相关。 公共卫生相关性：凝血酶是主要的凝血酶，负责在血管损伤部位形成血凝块。然而，除此之外，凝血酶还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，凝血酶与血管表面上称为血栓调节蛋白的蛋白质结合，在这种情况下，凝血酶的功能被“调节”，并将激活另外两种蛋白质，蛋白质C和羧肽酶原B（pCP B）。活化蛋白C现在被确定为一种抑制血液凝固并对血管发挥保护作用的蛋白质，从而作为一种反馈机制来下调凝血酶的凝固和炎症功能。我们和其他人已经发表的数据表明，激活的CPB可能在这个过程中发挥类似的作用。本申请的目的是研究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相互作用是重要的，并且与我们对许多疾病的理解高度相关，包括癌症、炎症性疾病和异常凝血。

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.