PATHOBIOLOGY OF ANTITHROMBOTIC MECHANISMS

描述(摘自《研究人员摘要》)：抗凝血酶 (AT)-抗凝剂硫酸乙酰肝素蛋白多糖(HSPGact)相互作用 作为血管壁的主要抗凝机制的基础。 然而，内皮细胞HSPGact产生的调节和 AT-HSPGact在不同器官中相互作用的重要性尚不清楚 已定义。研究人员克隆了3-0-磺基转移酶(3-0-ST)和 提供了这种酶控制HSPGact产生的证据。这 提案将使用生化/免疫学相结合的方法来确定 内皮细胞中是否存在单一的3-0-ST形式，以及 内皮细胞HSPGact前体含量过高。会的 用表达3-0-ST的逆转录病毒载体感染内皮细胞，文献 酶浓度的升高，并定量测定在 HSPGact生成。突变的3-0STs也将产生大量 生物活性降低和以动力学分析为特征的缺陷。 其他研究将确定HSPGact前体和 HSPGinact前体，在3-0-硫酸基化时产生AT结合 位点或变异的非活性序列。这是为了及早描绘出 热休克蛋白生成中的生化事件。将采用基因打靶技术 创造出3-0-ST基因敲除的小鼠。如果没有存活的纯合子小鼠 通过产生嵌合小鼠，出生、胚胎死亡将被绕过 3-0-ST在不同血管床中的局限性缺失，导致小鼠 生物活性下降幅度最大的突变酶 与生存相适应，并创造出具有纯合内皮细胞的小鼠 用Cre/Lox系统对3-0-ST基因进行细胞特异性敲除。 转基因小鼠将在环境状态下接受检查，经过 血栓形成挑战，并在纤溶抑制的背景下 活动。这项研究将包括3-0-ST和HSPGact的测量， 活化肽/酶对凝血酶生成和抑制作用的定量 免疫捕获试验，以及通过检测 交联性纤维蛋白沉积、组织学检查以及评估 寿命的长短。这些研究将确定AT-HSPGact的重要性 不同抗凝机制与其他天然抗凝机制的相互作用 血管床。

DESCRIPTION (Adapted from Investigator's Abstract): The antithrombin (AT)-anticoagulant heparan sulfate proteoglycan (HSPGact) interaction serves as the basis for a major anticoagulant mechanism of the blood vessel wall. However, the regulation of endothelial cell HSPGact generation and the importance of AT-HSPGact interactions in different organs remain to be defined. The investigator has cloned 3-0-sulfotransferase (3-0-ST) and provided evidence that this enzyme controls HSPGact generation. This proposal will use a combined biochemical/immunologic approach to determine whether a single 3-0-ST form exists in endothelial cells and whether endothelial cell HSPGact precursor is present in excess amounts. It will infect endothelial cells with retroviral vectors expressing 3-0-ST, document the elevated concentrations of enzyme, and quantitate the augmentation in HSPGact generation. Mutated 3-0STs will also be produced with greatly reduced biologic activity and the defects characterized by kinetic analyses. Other studies will determine the structures of HSPGact precursor and HSPGinact precursor which produce upon 3-0-sulfation either the AT binding site or a variant inactive sequence. This is designed to delineate early biochemical events in HSPGact generation. Gene targeting will be employed to create mice with 3-0-ST gene knockouts. If no viable homozygous mice are born, embryonic lethality will be bypassed by generating chimeric mice with localized absence of 3-0-ST in different vascular beds, producing mice with mutated enzyme possessing the greatest reduction in biologic activity compatible with survival, and creating mice with a homozygous endothelial cell specific knockout of the 3-0-ST gene with the Cre/Lox system. Genetically engineered mice will be examined in the ambient state, after a thrombogenic challenge, and in backgrounds with suppressed fibrinolytic activity. The study will include measurement of 3-0-ST and HSPGact, quantitation of thrombin generation, inhibition by activation peptide/enzyme immunocapture assays, and determination of thrombotic events by detection of crosslinked fibrin deposition, histologic examination, as well as evaluation of life span. These studies will establish the importance of AT - HSPGact interactions vis a vis other natural anticoagulant mechanisms in different vascular beds.