Structural studies of blood clotting proteins

描述(申请人提供)：本项目的总体目标是通过网络形成和Xllla因子催化的交联，确定纤维蛋白聚合和凝块机械稳定的具体相互作用。纤维蛋白聚合的研究已经到了一个关键的阶段，我们已经知道了一些基本的方面，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用传统的生物化学方法来研究，因为纤维蛋白是不溶的，每个分子上同时发生许多相互作用，而且有不同物种的混合物。在第一个具体目标中，我们将使用我们开发的基于激光镊子的力光谱新技术来研究单分子水平上的分子间相互作用，以便我们能够分离和量化这些不同的结合部位。除了纤维蛋白(原)片段外，还将使用具有特定受损结合部位的突变纤维蛋白原。对于第二个特定的目的，反卷积显微镜，它允许光学切片与低褪色的荧光，将被用来可视化聚合作为时间的函数。我们将描述血栓形成、分支网络的形成、侧向聚集和网络的机械稳定等鲜为人知的方面。在聚合的平台阶段，光漂白后的荧光恢复将被用来测量纤维蛋白的重塑。周转将受到多肽的调节，以与结合作用和超声波竞争。对于第三个特定目的，将通过测量XIII因子或X111a因子与纤维蛋白原或纤维蛋白以及变异和突变分子之间单个键的断裂力来研究XIII因子与纤维蛋白原(原)的特定相互作用。将确定特定交联物对凝块性能的影响。这些研究结果将有助于我们了解血栓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。

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.