Investigation into the mechanisms of LRP1-mediated factor VIII clearance

 描述（由申请人提供）：血友病A是一种众所周知的遗传性血液疾病，每5,000名男性中就有1名患者，由凝血因子VIII（fVIII）（一种关键凝血蛋白）缺乏引起。除了血友病A的高经济成本外，这种疾病对整体健康有重大影响。随着时间的推移，反复出血导致血友病性关节病和永久性关节损伤。此外，每次血友病患者经历出血事件时，血压偏离正常，许多严重的血友病患者在以后的生活中发展为高血压。目前血友病患者的治疗是通过常规注射重组FVIII来阻止出血发作的替代疗法。替代治疗包括终生注射FVIII以应对出血或每周约三次以预防出血发生。虽然有效，但由于注射的成本和频率，目前的血友病A治疗难以维持许多患者，导致需要更好的预防性治疗。在循环中，凝血因子VIII与一种称为血管性血友病因子（vWF）的蛋白质紧密结合，通过阻止凝血因子VIII与肝受体结合来保护其免受快速清除。研究表明，fVIII的肝脏清除由低密度脂蛋白受体相关蛋白（LRP 1）介导，LDL受体也有一定的作用。在脉管系统内损伤时，fVIII被凝血酶激活，导致其从vF解离。活化的fVIII与酶活性因子IXa结合以触发凝血级联的最后步骤。虽然已知FVIII如何发挥作用以及什么受体将其从循环中清除，但其清除的分子细节尚未完全了解。特别感兴趣的是揭示FVIII催化剂的分子机制，因为可以在循环中停留更长时间的更稳定的FVIII将提高替代疗法的效率。本项目的目的是确定有效的LRP 1介导的肝脏清除是否需要激活fVIII，并确定延迟fVIII清除的机制。在本项目中，将结合体外技术（如结合试验）和小鼠体内清除试验阐明FVIII清除的机制。该项目的成功完成将为FVIII研究界提供有关FVIII如何从循环中清除的宝贵信息。此外，可以通过将结合位点定位到FVIII上的LRP 1来延迟FVIII从循环中的清除，以开发在循环中具有更长半衰期的重组FVIII，和/或将可溶性LRP 1片段设计为阻断FVIII与LRP 1的结合。这些策略中的任何一种都可能代表血友病治疗的改进策略。

 DESCRIPTION (provided by applicant): Hemophilia A is a well-known inherited blood disorder that affects 1 in 5,000 males caused by a deficiency in factor VIII (fVIII), a critical blood coagulation protein. In addition to the high economic cost of Hemophilia A, this disease has a significant impact on overall health. Over time recurrent bleeds result in hemophilic arthropathy and permanent joint damage. Further, each time a hemophiliac experiences a bleeding episode, blood pressure deviates from normal and many severe hemophiliacs develop hypertension later in life. Current treatment for hemophiliacs is replacement therapy by routine injection of recombinant fVIII to arrest bleeding episodes. Replacement therapy involves lifetime injection of fVIII in response to a bleed or approximately three times a week to prevent bleeds from occurring. Although effective, current Hemophilia A treatments are difficult to maintain for many patients due to cost and frequency of injections, leading to the need for a better prophylactic treatment. In the circulation, fVIII binds tightly to a protein known as von Willebrand Factor (vWF), which protects it from rapid clearance by preventing fVIII from binding to hepatic receptors. Studies have shown that the hepatic clearance of fVIII is mediated by the low-density lipoprotein receptor-related protein (LRP1), with some contribution by the LDL receptor. Upon injury within the vasculature, fVIII is activated by thrombin, resulting in its dissociation from vF. Activated fVIII binds to enzymatically active factor IXa to trigger the final steps of the coagulaton cascade. While it is known how fVIII functions and what receptor clears it from circulation, the molecular details of its clearance are not well understood. It is particularly of interest to revea molecular mechanisms of fVIII catabolism as a more stable fVIII that can stay in circulation for extended time would improve efficiency of replacement therapy. The objectives for the current project are to define if activation of fVIII is required for efficient LRP1-mediated hepatic clearance and identify mechanisms to delay fVIII clearance. In this project, the mechanisms of fVIII clearance will be elucidated with a combination of in vitro techniques, such as binding assays, and in vivo clearance assays in mice. Successful completion of this project will provide the fVIII research community with invaluable information regarding how fVIII is cleared from the circulation. Further, fVIII clearance from circulation can be delayed by mapping bindings sites to LRP1 on fVIII to develop a recombinant fVIII that will have a longer half-life in circulation and/o soluble LRP1 fragments will be designed to block fVIII binding to LRP1. Either of these strategies may represent an improved strategy for hemophilia therapy.