Project 2: GeorgiaCenter for the Investigation of Factor VIII Inhibitors and Glycosylation

总结 先天性血友病A（HA）是一种X连锁出血性疾病，由于凝血FVIII缺乏或功能障碍 （FVIII）。每5，000名活产男婴中有近1名被诊断患有HA，其中40%涉及散发病例 是由新生突变引起的血浆源性凝血因子VIII（pdFVII）和重组凝血因子VIII（rFVII）是两种主要的凝血因子 HA治疗的静脉替代治疗药物。出现FVIII中和抗体或抑制剂， 在25-30%接受蛋白质替代治疗的严重HA患者中。这些FVIII抑制剂基本上消除了 凝血剂治疗的有效性，导致发病率和医疗保健成本的显著增加。 尽管rFVIII和pdFVIII都具有相似的相同的一级氨基酸，但在rFVIII和pdFVIII中FVIII抗体的风险是相同的。 据报道，rFVII治疗后既往未接受治疗的患者（PUP）的比例更高。作为糖蛋白， pdFVIII和rFVIII在糖基化方面不同，糖基化是一种翻译后修饰（PTM），其能够改变蛋白质的结构。 蛋白质免疫原性 因此，在本项目中，我们将开发一套针对FVIII抑制剂的新型糖分析工具集。为了 为了研究宿主糖环境对FVIII的影响，我们建议使用与FVIII密切相关的VWF 和FV作为替代标志物，以显示对发生FVIII的HA患者中FVIII糖基化的潜在影响 抑制剂的此外，基于我们开发的基于MS的方法，总免疫球蛋白的糖基化模式， 特异性FVIII抑制剂、FV、VWF和宿主细胞环境的总体糖组变化将被彻底 特点是大量的HA患者样本，密切关注发生免疫耐受的患者 诱导（ITI）。从这个项目中获得的总体结果将对理解基本的 FVIII免疫原性机制。

Summary Congenital Hemophilia A (HA) is an X-linked bleeding disorder due to a lack or dysfunction of coagulation FVIII (FVIII). Nearly 1 in 5,000 live male births are diagnosed with HA with 40% of whom involving of sporadic cases caused by de novo mutations. Plasma-derived FVIII (pdFVIII) and recombinant FVIII (rFVIII) are two major intravenous replacement therapy medicines for HA treatment. FVIII neutralizing antibodies, or inhibitors, occur in 25-30% of severe HA patients receiving protein replacement. These FVIII inhibitors essentially abolish the efficacy of the coagulant treatment, resulting in a considerable increase in the morbidity and healthcare cost. Although both rFVIII and pdFVIII share similar to identical primary amino acids, the risk of FVIII antibodies in previously untreated patients (PUPs) was reported to be higher following rFVIII treatment. As a glycoprotein, pdFVIII and rFVIII are different in glycosylation, a post-translation modification (PTM) that is capable of alter the immunogenicity of protein Therefore, in this project, we will develop a set of novel glycoanalysis toolset for FVIII inhibitors. In order to investigate the effect of the host glyco environment on FVIII, we propose to use FVIII closely associated VWF and FV as surrogate marker to show to potential effect on FVIII glycosylation in HA patients who developed FVIII inhibitors. Also, based on our developed MS-based approach, glycosylation pattern of total immunoglobulins, specific FVIII inhibitors, FV, VWF, and overall glycome changes of the host cell environment will be thoroughly characterized in a large number of HA patients’ samples, close attention to those undergone immune tolerance induction (ITI). The overall results obtained from this project will be significant for understanding the fundamental mechanism of FVIII immunogenicity.