PATHOBIOLOGY OF ANTITHROMBOTIC MECHANISMS

描述（改编自研究者摘要）：抗凝血酶 （AT）-抗凝剂硫酸乙酰肝素蛋白聚糖（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生成的调节和细胞内HSPGact表达的调节是不稳定的。 AT-HSPGact相互作用在不同器官中的重要性仍有待进一步研究。 定义了 研究者克隆了3-0-磺基转移酶（3-0-ST）， 提供了这种酶控制HSPGact生成的证据。 这 该提案将使用生物化学/免疫学方法来确定 内皮细胞中是否存在单一的3-0-ST形式， 内皮细胞HSP Gact前体过量存在。 它将 用表达3-0-ST逆转录病毒载体感染内皮细胞，文献 酶的浓度升高，并定量增加， HSP Gact生成。 突变的3- 0 ST也将产生， 降低的生物活性和通过动力学分析表征的缺陷。 其他研究将确定HSP Gact前体的结构， HSPGinact前体在3-0-硫酸化后产生AT结合 位点或变体无活性序列。 这是为了尽早描绘 HSPGact生成中的生化事件。 将采用基因靶向技术 来制造3-0-ST基因敲除的小鼠。 如果没有存活的纯合子小鼠， 出生时，胚胎致死性将通过产生嵌合小鼠来绕过， 在不同的血管床中局部缺乏3-0-ST，产生具有 生物活性降低最大的突变酶 与生存相容，并产生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。 基因工程小鼠将在环境状态下进行检查， 血栓形成挑战，并在背景下抑制纤维蛋白溶解 活动 该研究将包括测量3-0-ST和HSP Gact， 凝血酶生成的定量，通过活化肽/酶的抑制 免疫捕获测定，以及通过检测 交联纤维蛋白沉积，组织学检查，以及评价 生命的延续。 这些研究将确定AT-HSP Gact的重要性， 维斯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.