免疫性血小板减少症(ITP)最重要的病理特征之一是产生抗血小板自身抗体，但这些抗体产生的原因尚不明确。VH替换是免疫球蛋白重链基因的二次重排，能够向抗体互补决定区3（CDR3）中提供多余的正电荷氨基酸。我们的前期工作提示ITP患者存在中枢免疫耐受缺陷，并且大部分识别血小板抗原的B细胞受体（BCR）或抗血小板抗体由VH替换产物翻译。因此我们推断：VH替换能促进抗体与富集负电荷的血小板糖蛋白（如GPIIbIIIa）结合，是抗血小板抗体产生的主要来源。本项目中我们将利用单细胞PCR制作抗体、BCR高通量测序、L-PCR技术、生物信息学分析、抗体基因修饰等方法，进一步证明ITP患者存在中枢免疫耐受缺陷且VH替换发生率升高，并证明VH替换对于抗血小板抗体生成有决定性作用。这一假说的成功验证将为ITP诊断提供新的标志物，并为ITP治疗提供新的靶点。

One of the hallmark of primary immune thrombocytopenia (ITP) is the production of anti-platelet antibody; however,the underlying mechnism is unclear. VH replacement is the secondary rearrangement of Immunologbulin heavy chain (IgH) gene, which provides exata positively charged amino acids into the CDR3 regions of IgH. In the preliminary data, we found the B cell receptor (BCR) of ITP Naive B cells preferencially reacted with platelet antigens, indicating a defective central immune tolerance for B cells to platelets. Interestingly, most of these anti-platelet BCRs or previously studied anti-platelet antibodies were translated from VH replacement products. Based on these, we hypothesize that excessive VH replacements contribute to production of antibodies against the negative charge-rich platelet glycoproteins, such as GPIIbIIIa. We will investigate this hypothesis with comprehesive methods including single cell PCR for cloning antibody, BCR high through-put sequencing, ligation PCR, bioinformatic analysis, and immunoglobulin gene modification. We expect that the central tolerance for B cells in ITP patients is defective, and excessive VH replacements contribute to the generation of anti-platelet antibodies in ITP patients. Understanding the mechanisms responsible for the generation of anti-platelet antibodies will allow us to design specific approaches for ITP diagnosis and new targets for ITP treatment.