Regulation of factor VIII Levels and Activity by Members of LDL Receptor Family

描述（由申请人提供）：因子VIII （fVIII）和因子IX （fIX）分别是出血性疾病血友病A和血友病B中缺乏的两种关键血浆蛋白。因子VIII作为复合体与其载体蛋白血管性血友病因子（vWf）一起循环。当在脉管系统内受到损伤时，该辅助因子通过有限的蛋白水解被激活为因子viii (fviii)，导致其与vWf分离，随后与酶活性形式的fIX （fIXa）组装到膜表面，形成大分子Xase复合物。这个复合物激活了凝血级联中的下一个前酶因子X。最近的研究表明，Xase复合物的功能活性可能受低密度脂蛋白受体相关蛋白（LRP-1或LRP）的调控，LRP是一种大的内吞受体，在几种配体的分解代谢中起重要作用，包括载脂蛋白丰富的脂蛋白、纤溶酶原激活剂和蛇形酶复合物。LRP与LDL受体（LDLR）在体内起调节肝脏清除fVIII的作用。阐明与fVIII分解代谢相关的分子机制是很重要的，因为fVIII用于血友病A的替代治疗，而重组fVIII分子的生物工程将在循环中停留更长时间，从而增强治疗效果。该应用的中心假设是LDL受体家族成员，特别是LRP，通过介导两个关键因子fVIII(a)和fIXa的细胞分解代谢，在调节凝血过程中发挥重要作用。需要验证的具体假设是：1)硫酸肝素蛋白聚糖（HSPG）调节fVIII从vWF的解离，导致LRP/LDLR对fVIII的摄取增加；2)我们可以设计一种具有延迟清除特性的fVIII分子，这种分子将有效地用于治疗；3) LDL受体家族成员（即LRP， LDLR和VLDLR）通过促进大分子Xase复合物的快速细胞内化来调节局部血栓形成。这些假设将在三个具体目标中得到检验。在第一个目标中，将探索fVIII分解代谢途径的初始步骤，并测试两种可能调节fVIII从vWf解离导致LRP/LDLR对fVIII摄取增强的机制-由HSPG介导的非蛋白水解机制和激活依赖机制。在第二个目标中，将产生具有最小体内LRP/ ldlr介导的清除特性的功能活性重组fVIII。这将通过fVIII分子上有助于LRP-， LDLR-和hspg结合的关键区域的定点突变来实现。在第三个目标中，我们将探索LDL受体家族成员调节内在Xase复合物活性的能力。Xase复合物在LRP基因缺陷巨噬细胞上的组装、功能和清除将被研究。总的来说，这些研究将深入了解LDL受体家族成员如何调节血液凝固过程，并将导致具有增强治疗用途的重组fVIII分子的开发。公共卫生相关性：出血性疾病血友病A与凝血因子VIII （fVIII）缺乏有关，可通过反复注射外源性fVIII治疗。替代疗法仍然很昂贵，估计每名成年患者每年高达20万美元。这个项目的目标是确定参与fVIII分解代谢的分子机制。该项目的完成将产生具有功能活性的重组fVIII，其在循环中的寿命将显著延长，这将提高血友病a替代治疗的疗效并降低成本。

DESCRIPTION (provided by applicant): Factor VIII (fVIII) and factor IX (fIX) are two key plasma proteins that are deficient in the bleeding disorders Hemophilia A and Hemophilia B, respectively. Factor VIII circulates as a complex with its carrier protein von Willebrand factor (vWf). Upon injury within the vasculature, this cofactor is activated to factor VIIIa (fVIIIa) by limited proteolysis resulting in its dissociation from vWf and subsequent assembly onto the membrane surface with an enzymatically active form of fIX (fIXa) to form a macromolecular Xase complex. This complex activates factor X, the next proenzyme in the coagulation cascade. Recent work has suggested that the functional activity of the Xase complex may be regulated by the low-density lipoprotein receptor-related protein (LRP-1 or LRP), a large endocytic receptor that plays an important role in the catabolism of several ligands including apoE-enriched lipoproteins, plasminogen activators, and serpin-enzyme complexes. LRP along with the LDL receptor (LDLR) functions in vivo to mediate the hepatic clearance of fVIII. Elucidation of molecular mechanisms associated with fVIII catabolism is important, as fVIII is used in replacement therapy for Hemophilia A, and bioengineering of a recombinant fVIII molecule which would stay in the circulation for longer time would enhance therapy. The central hypothesis of this application is that LDL receptor family members, especially LRP, play an important role in regulating the blood coagulation process by mediating the cellular catabolism of two key players, fVIII(a) and fIXa. The specific hypotheses to be tested are: 1) that heparan sulfate proteoglycans (HSPG) modulate the dissociation of fVIII from vWF leading to enhanced uptake of fVIII by LRP/LDLR; 2) that we can engineer a fVIII molecule with delayed clearance properties that would be effective for therapeutic use; and 3) that LDL receptor family members (i.e. LRP, LDLR and VLDLR) modulate localized thrombosis by promoting the rapid cellular internalization of the macromolecular Xase complex. These hypotheses will be tested in three specific aims. In the first aim, the initial step in the catabolic pathway of fVIII will be explored and two mechanisms which may modulate dissociation of fVIII from vWf leading to enhanced uptake of fVIII by LRP/LDLR will be tested - a non-proteolytic mechanism mediated by HSPG and an activation-dependent mechanism. In the second aim, functionally active recombinant fVIII with minimized in vivo LRP/LDLR-mediated clearance properties will be generated. This will be accomplished by site-directed mutagenesis of key regions on the fVIII molecule that contribute to LRP-, LDLR- and HSPG-binding. In the third aim, we will explore the ability of the LDL receptor family members to modulate activity of the intrinsic Xase complex. The assembly, function and clearance of the Xase complex on macrophages genetically deficient in LRP will be investigated. Overall, these studies will give insight into how the LDL receptor family members modulate the blood coagulation process, and should lead to the development of a recombinant fVIII molecule with enhanced properties for therapeutic use. PUBLIC HEALTH RELEVANCE: The bleeding disorder Hemophilia A is associated with deficiency of coagulation factor VIII (fVIII) and is treated by repeated injections of exogenous fVIII. Replacement therapy remains expensive and is estimated as up to $200,000/adult patient/year. The goals of this project are to define molecular mechanisms involved in the catabolism of fVIII. Accomplishment of this project will result in generation of functionally-active recombinant fVIII with a significantly prolonged lifetime in the circulation, which would improve efficacy and reduce the cost of replacement therapy of Hemophilia A.