Platelet factor XI

血小板因子XI

• 批准号: 6570521
• 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: 2002
• 资助国家: 美国
• 起止时间: 2002-02-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6570521
• 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).

申请人的实验室以及其他研究人员的研究重点是一种新的但鲜为人知的独特蛋白质--血小板FXI，它是目前申请的重点。申请人实验室最近利用逆转录聚合酶链式反应(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，或者是在巨核细胞中唯一表达的单独基因的产物。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(PAGE)，血小板FXI的表观MR约为220,000，还原后约为55,000，而血浆FXI的表观MR约为160,000，亚基MR约为80,000。血小板FXI凝血活性和抗原存在于洗净的血小板悬液中，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大约有300个血小板FXI分子。本研究的目的是在基因组和蛋白质水平上完成对血小板FXI的结构和功能的研究，确定血浆FXI缺乏症患者血小板FXI与血小板膜结合的分子基础，确定血小板FXI与血小板膜结合的机制，并确定其活化机制和酶活性的表达。这项建议的具体目标如下：1)完成对血小板FXI基因、mRNA和蛋白的结构表征。次目标1)确定血小板FXI和FXI基因的mRNA之间的关系，以解决血浆FXI基因的选择性剪接或单独基因的产物的问题。亚目的1b)通过体外翻译来确定血小板FXI mRNA和蛋白的关系。子目标1)通过纯化和生化鉴定血小板FXI蛋白来完成对其结构的表征。2)探讨血浆FXI缺乏症患者血小板FXI的分子基础、功能相关性及是否存在。次目标2a)检验II型FXI缺乏症患者(以外显子V的终止密码子为特征)能够正常表达血小板FXI的假设，因为血小板FXI中缺少外显子V。次目标2b)验证血小板FXI可替代血浆FXI止血的假说。3)通过探索一个血小板FXI(MR约55,000)与血小板膜糖蛋白Ib(MR约165,000)形成二硫键连接的复合体(MR约220,000)的假说，确定血小板FXI与血小板膜结合的机制。4)确定凝血酶、FXIa、FXIIa或其他酶激活血小板FXI的机制，并确定其表达或酶活性及其正常大分子底物(即FXI或FIX)的机制。