Platelet factor XI

血小板因子XI

• 批准号: 6323057
• 负责人: PETER Newton WALSH
• 金额: $ 20.93万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6323057
• 关键词: SDS polyacrylamide gel electrophoresis; coagulation factor XI; disulfide bond; enzyme activity; gene interaction; genetic translation; hemostasis; human subject; messenger RNA; molecular cloning; phlebotomy; platelet activation; protein protein interaction; protein structure function; structural genes; thrombin

Studies from the applicant's laboratory, as well as those from other investigators, have focused on a novel but poorly understood unique protein, platelet FXI, which is the focus of the current application. Recent experiments from the applicant's laboratory utilizing reverse transcriptase polymerase chain reaction (RT-PCR) and molecular cloning from a megakaryocyte (CHRF-288 cell) cDNA library suggest the possibilities that platelet FXI is either an alternative splicing product of the plasma FXI gene lacking exon V or the product of a separate gene expressed exclusively in megakaryocytes. Platelet FXI migrates by SDS- polyacrylamide gel electrophoresis (PAGE) with an apparent Mr of approximately 220,000, approximately 55,000 after reduction, compared with plasma FXI which has an Mr approximately 160,000 and a subunit Mr of approximately 80,000. Platelet FXI coagulant activity and antigen are present in well-washed platelet suspensions constituting approximately 0.5% of the FXI activity in normal plasma, from which it can be calculated that there are approximately 300 molecules of platelet FXI per platelet. The objectives of this proposal are to accomplish a structural and functional characterization of platelet FXI both at the genomic and protein levels, to determine the molecular basis for the presence of platelet FXI with the platelet-plasma membrane in patients with plasma FXI deficiency, to determine the mechanism of association of platelet FXI with the platelet-plasma membrane, and to ascertain the mechanisms of its activation and the expression of its enzymatic activity. The specific aims of this proposal are as follows: 1) To accomplish a structural characterization of the platelet FXI gene, mRNA and protein. Sub-aim 1a) To determine the relationship between the mRNA for platelet FXI and the FXI gene in order to resolve the issue where it is an alternative splicing of the plasma FXI gene or the product of a separate gene. Sub-aim 1b) To determine the relationship between platelet FXI mRNA and protein by in vitro translation. Sub-aim 1c) To accomplish a structural characterization of platelet FXI by purification and biochemical characterization of the platelet FXI protein. 2) To determine the molecular basis and functional relevance and the presence of platelet FXI in the platelets of patients with plasma FXI deficiency. Sub-aim 2a) To test the hypothesis that patients with type II FXI deficiency (characterized by a stop codon in exon V) are able to express platelet FXI normally because of the absence of exon V in platelet FXI. Sub-aim 2b) To test the hypothesis that platelet FXI can substitute for plasma FXI in hemostasis. 3) To determine the mechanism of association of platelet FXI with the platelet plasma membrane by exploring the hypothesis that one platelet FXI (Mr approximately 55,000) forms a disulfide-linked complex (Mr approximately 220,000) with platelet membrane glycoprotein Ib (GPIb) (Mr approximately 165,000). 4) To determine the mechanism of activation of platelet FXI by thrombin, by FXIa, by FXIIa, or by other proteases and to determine the mechanism of expression or its enzymatic activity and its normal macromolecular substrate (i.e., FXI or FIX).

来自申请人实验室以及其他研究人员的研究集中在一种新的但知之甚少的独特蛋白质，血小板FXI，这是当前应用的重点。申请人实验室最近利用逆转录聚合酶链反应（RT-PCR）和巨核细胞（CHRF-288细胞）cDNA文库的分子克隆进行的实验表明，血小板FXI可能是缺乏外显子V的血浆FXI基因的替代剪接产物，或者是巨核细胞中专门表达的单独基因的产物。血小板FXI通过SDS-聚丙烯酰胺凝胶电泳（PAGE）迁移，其表观Mr约为22万，还原后约为55000，而血浆FXI的Mr约为16万，亚基Mr约为8万。血小板FXI凝血剂活性和抗原存在于洗涤良好的血小板悬浮液中，约占正常血浆中FXI活性的0.5%，由此可以计算出每个血小板约有300个血小板FXI分子。本课题的目的是在基因组和蛋白水平上完成血小板FXI的结构和功能表征，确定血浆FXI缺乏患者血小板FXI与血小板-质膜存在的分子基础，确定血小板FXI与血小板-质膜的关联机制，确定其活化及其酶活性的表达机制。本课题的具体目的如下：1)完成血小板FXI基因、mRNA和蛋白的结构表征。子目标1a)确定血小板FXI mRNA与FXI基因之间的关系，以解决血浆FXI基因的替代剪接或单独基因的产物的问题。Sub-aim 1b)通过体外翻译确定血小板FXI mRNA与蛋白的关系。子目标1c)通过血小板FXI蛋白的纯化和生化表征完成血小板FXI的结构表征。2)确定血浆FXI缺乏症患者血小板中血小板FXI的存在及其分子基础和功能相关性。Sub-aim 2a)为了验证II型FXI缺陷患者（以外显子V中的停止密码子为特征）由于血小板FXI中外显子V的缺失而能够正常表达血小板FXI的假设。亚目的2b)验证血小板FXI可替代血浆FXI止血的假设。3)通过探索一个血小板FXI （Mr约为55,000）与血小板膜糖蛋白Ib (GPIb) （Mr约为165,000）形成二硫化物连接复合物（Mr约为220,000）的假设，确定血小板FXI与血小板质膜的关联机制。4)确定凝血酶、FXIa、FXIIa或其他蛋白酶活化血小板FXI的机制，确定其酶活性及其正常大分子底物（即FXI或FIX）的表达机制。