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都集中在一种新颖但知之甚少的独特蛋白质Platelet FXI上，这是当前应用的重点。来自申请人实验室利用逆转录酶聚合酶链反应（RT-PCR）和来自巨型杂质细胞（CHRF-288细胞）cDNA库的分子克隆的最新实验表明，血小板FXI是血小板FXI是缺乏等离子体FXI Gene Gene Exon V or Serveriatienty Gene的产品的替代性固定产物的可能性。血小板FXI通过SDS-聚丙烯酰胺凝胶电泳（PAGE）迁移，明显的MR约为220,000，减少后约55,000，而血浆FXI则具有约160,000的MR和大约80,000的亚基MR。血小板FXI凝血活性和抗原存在于正常血浆中大约0.5％的FXI活性的精心洗净的血小板悬浮液中，可以从中计算出大约300个小血小板分子的血小板FXI。该建议的目标是在基因组和蛋白质水平上完成血小板FXI的结构和功能表征，以确定血浆FXI缺乏患者血小板FXI与血小板 - 质膜膜的存在的分子基础，以确定与血小板FXI与血小板及时的衰落及时及时及时及时及时及时及时的膜片及时，并构成毫无用处的膜片，并构成肿瘤的机制，并构成象征的效果，并构成象征的效果，并构成了象征的效果，并构成象征的效果，并构成了象征的效果，并确定象征的效果和造成的效果，并构成了象征性的效果，并确定象征性的效果及其与血小板的机制相关性。酶活性。该建议的具体目的如下：1）完成血小板FXI基因，mRNA和蛋白质的结构表征。 sub-aim 1a）确定血小板FXI和FXI基因的mRNA之间的关系，以解决该问题是血浆FXI基因的替代剪接或单独基因的乘积。 Sub-aim 1b）通过体外翻译来确定血小板FXI mRNA和蛋白质之间的关系。 sub-aim 1c）通过纯化和血小板FXI蛋白的生化表征来完成血小板FXI的结构表征。 2）确定血浆FXI缺乏患者血小板中血小板FXI的分子基础和功能相关性。 Sub-aim 2a）为了测试以下假设：II型FXI缺乏症患者（以外显子V中的终止密码子为特征）能够正常表达血小板FXI，因为血小板FXI中没有外显子V。 Sub-aim 2b）测试了血小板FXI可以在止血中代替血浆FXI的假设。 3）通过探索一个假设，即一个血小板FXI（MR约55,000）形成二硫键连接的复合物（MR约220,000）与血小板膜膜Glycoprotein IB（GPIB（GPIB）（GPIB）（MR大约165,000））（MR大约165,000）来确定血小板FXI与血小板质膜的关联机制。 4）确定凝血酶，FXIA，FXIIA或其他蛋白酶激活血小板FXI的机理，并确定表达或其酶活性的机理及其正常的大分子分子底物（即FXI或FIX）。