Structural studies of blood clotting proteins

凝血蛋白的结构研究

• 批准号: 7277654
• 负责人: JOHN W WEISEL
• 金额: $ 33.81万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7277654
• 关键词: 3-Dimensional; Amino Acids; Binding; Binding Sites; Biochemical; Biochemistry; Blood Clot; Blood coagulation; Chemicals; Clinical; Coagulation Process; Complex; Condition; Disease; Electron Microscopy; Factor XIII; Fiber; Fibrin; Fibrinogen; Fluorescence; Fluorescence Recovery After Photobleaching; Gel; Goals; Hemostatic Agents; Individual; Label; Lasers; Lateral; Measurement; Measures; Mechanics; Methods; Microscopy; Modeling; Molecular; Morphologic artifacts; Mutation; Nature; Object Attachment; Optics; Peptides; Pharmacia brand of estropipate; Phase; Physiological; Plasma; Polymers; Preparation; Prevention; Process; Property; Proteins; Rate; Recombinants; Relative (related person); Research; Research Personnel; Rupture; Scanning Electron Microscopy; Shadowing (Histology); Site; Specificity; Specimen; Spectrum Analysis; Staging; Structure; Surface; Techniques; Thermodynamics; Thinking; Time; Touch sensation; Transmission Electron Microscopy; Ultrasonography; Uncertainty; Variant; base; crosslink; fibrinopeptide; in vivo; inhibitor/antagonist; intermolecular interaction; light scattering; monomer; mutant; novel; physical process; physical property; polymerization; programs; reconstruction; research study; single molecule

DESCRIPTION (provided by applicant): The overall goal of this project is to determine specific interactions in the polymerization of fibrin and mechanical stabilization of the clot through network formation and Factor Xllla-catalyzed crosslinking. Research on fibrin polymerization has reached a critical stage at which we know some basic aspects, but fundamental molecular mechanisms remain a mystery. These interactions are difficult or impossible to study by conventional biochemical methods because fibrin is insoluble, many interactions are occurring simultaneously on each molecule, and there is a heterogeneous mixture of species. In the first specific aim, novel techniques we have developed using laser tweezers-based force spectroscopy will be used to study the intermolecular interactions at the single molecule level, so we can separate out and quantify these different binding sites. Mutant fibrinogens with specific impaired binding sites will be used, in addition to fibrin(ogen) fragments. For the second specific aim, deconvolution microscopy, which allows optical sectioning with low fading of fluorescence, will be used to visualize polymerization as a function of time. We will characterize little-known aspects of clot formation, the formation of a branched network, lateral aggregation, and the mechanical stabilization of the network. During the plateau phase of polymerization, fluorescence recovery after photobleaching will be used to measure the remodeling of fibrin. Turnover will be modulated by peptides to compete with the binding interactions and ultrasound. For the third specific aim, the specific interactions of Factor XIII with fibrin(ogen) will be studied by measuring the rupture forces of the individual bonds between Factor XIII or Xllla and fibrinogen or fibrin, as well as variant and mutant molecules. The effects of specific crosslinks on clot properties will be determined. The results of these studies will help us to understand molecular mechanisms of clot formation and stabilization, which may have clinical implications for the treatment and prevention of thrombotic and hemostatic disorders.

描述（由申请方提供）：本项目的总体目标是确定纤维蛋白聚合中的特定相互作用以及通过网络形成和因子XIIIa催化的交联实现凝块的机械稳定。纤维蛋白聚合的研究已经达到了一个关键阶段，我们知道一些基本方面，但基本的分子机制仍然是一个谜。这些相互作用很难或不可能通过常规的生物化学方法进行研究，因为纤维蛋白是不溶性的，许多相互作用同时发生在每个分子上，并且存在物种的异质混合物。在第一个具体目标中，我们使用基于激光镊子的力谱开发的新技术将用于研究单分子水平上的分子间相互作用，因此我们可以分离和量化这些不同的结合位点。除纤维蛋白（原）片段外，还将使用具有特异性受损结合位点的突变纤维蛋白原。对于第二个具体目标，去卷积显微镜，它允许与低褪色的荧光光学切片，将被用来可视化聚合作为时间的函数。我们将描述血凝块形成、分支网络的形成、侧向聚集和网络的机械稳定性的鲜为人知的方面。在聚合的平台期，光漂白后的荧光恢复将用于测量纤维蛋白的重塑。周转将通过肽调节以与结合相互作用和超声竞争。对于第三个具体目的，将通过测量因子XIII或XIIIa与纤维蛋白原或纤维蛋白以及变体和突变分子之间的各个键的断裂力来研究因子XIII与纤维蛋白（原）的特异性相互作用。将确定特定交联对血凝块特性的影响。这些研究的结果将有助于我们了解凝块形成和稳定的分子机制，这可能对血栓和止血疾病的治疗和预防具有临床意义。