丝氨酸蛋白酶21作为血小板中凝血因子XI样活性蛋白的鉴定及凝血相关功能研究

Modern coagulation theory thinks that extrinsic activation is the main pathway for the initiation of blood coagulation cascade, the immediate suppression of FVIIa activity by tissue factor pathway inhibitor (TFPI) and impermeability of platelet plug formed upon injury site for activated factor X suggest that the initiating processes might also need the participation of intrinsic activation pathway. Factor XI (FXI) is commonly accepted as the initiator in the intrinsic coagulation pathway. Previous studies have shown that there is FXI like activity presenting on activated platelet surface, however, there is no idea which protein plays the role. Preliminary studies hinted that serine protease 21 (PRSS21) was expressed in platelets. Moreover, partially purity of recombinant PRSS21 expressed in pichia pastoris showed FIX cleavage activity, which put it as one candidate mediating platelet FXI activity. In the proposed studies, we are going to prove the existence of PRSS21 in platelet both transcriptionally and transnationally. With highly purified PRSS21 expressed from yeast system, we also plan to characterize its enzymatic properties and study its function related to the initiation of blood coagulation. Finally, we are going to establish PRSS21 knock out mouse model and probe its contribution to the development of thrombosis using inferior vena cava thrombosis and MCAO stroke model. Through this study, we expect to improve the blood coagulation theory and offer a new target for antithrombotic therapy.

现代凝血理论认为凝血启动过程主要由外源激活途径完成，但外源激活途径中的启动因子FVIIa活性很快会被抑制，且初级凝血块阻碍了其所活化的FX向外弥散。因此凝血启动过程尚需内源激活途径的共同完成。一般认为FXI是内源激活途径中参与凝血启动的因子，近来的研究发现血小板也存在FXI样活性。但尚不明确何种蛋白质与此相关。我们前期研究发现血小板中有丝氨酸蛋白酶21（PRSS21）表达，且体外表达的PRSS21具有FXI活性。故推测PRSS21可能就是我们希望寻找的血小板中具有FXI样活性参与凝血启动过程的蛋白。本项目拟在转录和翻译水平分析PRSS21在血小板中的表达特性；通过真菌表达的PRSS21酶学特性测定，探讨其在凝血启动过程中的作用；最终将利用在PRSS21基因敲除小鼠中建立的下腔静脉血栓和大脑中动脉中风模型，评估PRSS21在病理血栓形成中的作用。完善止凝血理论并为抗血栓治疗提供新的靶点。

丝氨酸蛋白酶21（PRSS21）主要表达于嗜酸性粒细胞和睾丸组织中，通过C端序列与细胞膜表面糖磷脂酰肌醇锚定，参与精子生成发育和生殖系统肿瘤调控过程。前期研究表明PRSS21 催化区与凝血因子XI高度同源，推测其为可能在机体止凝血中发挥作用。本研究中，我们利用逆转录PCR和Western Blot确定嗜酸性粒细胞表达PRSS21；利用真核细胞表达并纯化PRSS21蛋白，采用不同活化凝血因子进行激活，并对不同发色底物/生理性底物进行裂解，发现PRSS21不被凝血酶或者活化凝血因子X激活。活化鼠PRSS21可以裂解四种不同的凝血相关发色底物，但对人凝血因子IX和人凝血酶没有激活作用。后续将采用更多蛋白尝试对重组人PRSS21进行激活，以排除小鼠与人之间的种属差异；PRSS21基因敲除的小鼠具备正常生殖能力，血常规结果显示敲除小鼠白细胞总数升高，且主要来源于颗粒细胞比例增高；敲除小鼠血浆部分活化凝血活酶时间、凝血酶原时间、凝血酶时间和凝血酶生成与野生型小鼠均无明显差异，提示PRSS21基因敲除对血浆凝血因子含量或凝血活性无影响。鉴于该蛋白为膜锚定蛋白，主要在细胞膜表面发挥作用，因此需要采用更全面的凝血实验对其凝血功能进行评估。本研究成果提示PRSS21在嗜酸性粒细胞表面发挥重要生理功能，基因敲除小鼠能为研究嗜酸性粒细胞功能提供帮助。