2种蛋白C突变（Ile73Asn和Gly74Ser）导致抗凝功能缺陷的分子机制研究

Inherited Protein C (PC) deficiency resulted from PC mutations is one of the most important risk factors for venous thromboembolism in Asian population. It mainly consists of five domains, including γ-carboxyglutamic acid domain (Gla) and epidermal growth factor-like domains (EGF1) and et al. Mutations located on the EGF1 domain are associated with thromboembolism, but the pathogenetic mechanism is unclear. Normal binding between calcium and EGF1 is the prerequisite for maintaining the function of EGF1 and Gla domains. The ligation between calcium and consensus residues on EGF1 of PC is not yet elucidated，and it is reported that the key residue of calcium binding to EGF1 is the aspartic acid 71 (Asp71). We have identified two novel mutations Ile73Asn and Gly74Ser, located on EGF1, which are close to Asp71 in two unrelated patients with Based on current findings, we speculate that the two mutations might impair the calcium affinity for EGF1 by altering adjacent structure, thus influencing the normal function of EGF1. In this study, we will express and purify these two variants and wild-type recombinant PC to investigate: 1. the effects of the two mutant PC on the affinity for calcium; 2. the effects of the two mutant PC on the interaction between protein S (PS) and EGF1 by in vitro and in vivo detection; 3. the indirect impairment of the two mutant PC to the binding of Gla and endothelial protein C receptor (EPCR) or Gla and phospholipids. Therefore, the molecular pathogenesis of anticoagulation defects caused by two mutations will be explored through current project.

蛋白C（PC）缺陷症是亚洲人静脉血栓形成最重要危险因素之一。PC由r-羧基谷氨酸区（Gla）和表皮生长因子样区（EGF1）等五个结构区构成。EGF1区的突变与血栓发生相关,但其致病机制均不明确。EGF1区与Ca2+结合是维持EGF1及Gla区正常功能的保证，但PC的EGF1与Ca2+的结合方式尚不明确，有报道EGF1区的Asp71是Ca2+结合的关键位点。本项目组在两例Ⅱ型PC缺陷症伴血栓患者中分别发现了两种新突变Ile73Asn和Gly74Ser，均位于EGF1且与Asp71相邻。据此我们推测突变可能通过相邻结构的改变影响Ca2+的结合及EGF1功能。拟通过体外表达突变及野生型PC探讨：突变对EGF1区与Ca2+的结合的影响；突变对EGF1区与辅因子蛋白S结合的影响（体外和动物体内）；突变是否间接影响Gla区与内皮细胞PC受体及磷脂结合。从而阐明两种PC突变导致抗凝功能异常分子机制。

蛋白C（PC）缺陷症是亚洲人静脉血栓形成最重要危险因素之一。通过对先证者及家系成员的凝血相关功能检测，初步明确了II型蛋白C缺陷症的Ile73Asn和Gly74Ser两种突变与患者及家系相关成员静脉血栓发生密切相关。本项目组通过构建2种突变型(Ile73Asn和Gly74Ser)及野生型人PC的表达质粒以及稳转细胞株，对两种突变蛋白(Ile73Asn和Gly74Ser)的相关功能以及结构进行研究，证明了凝血酶-TM复合物可以正常活化两种突变PC，并且所得的两种突变PC（Ile73Asn和Gly74Ser）不影响活化PC催化小分子底物的能力；通过体外酶动力学研究发现两种突变PC（Ile73Asn和Gly74Ser）能够被正常活化且在无蛋白S的条件下对活化的FVIII和FV的灭活能力正常，但在PS存在，即体内生理条件下，该突变PC灭活FVIIIa和FVa的能力大大降低，证明突变的APC（Ile73Asn和Gly74Ser）与PS的结合异常。此外，对两种突变PC（Ile73Asn和Gly74Ser）的晶体结构分析显示，Ile73和Gly74位点两个新的氨基酸的替代会产生新的残基，阻碍APC EGF1结构域与PS的Gla-TSR-EGF1结构域的相互结合，减弱了APC对PS的亲和力，导致突变PC的抗凝活性降低。综上所述，本项目从功能检测及结构分析两方面共同阐明了两种突变PC（Ile73Asn和Gly74Ser）可能通过影响APC与PS的亲和力，导致突变PC的抗凝活性降低而导致静脉血栓形成的分子致病机制，为蛋白C突变导致的静脉血栓的预防和治疗提供依据。

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