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.
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