The major goals of this research project are the elucidation of aspects of the molecular mechanisms of assembly of the fibrin clot, as well as interactions of fibrinogen with GPIIb/IIIa and of high molecular weight kininogen with several proteins. Various methods of structural molecular biology, including transmission, scanning and intermediate voltage electron microscopy and computer image processing will be employed in these studies. Most of the required specimen preparation procedures and applications of the appropriate methods have already been developed in this laboratory. Aspects of the mechanism of assembly of the fibrin clot will be studied through analysis of differences in the structures formed by highly selected variant fibrinogens that have specific molecular defects. Electron microscopy of unstained, frozen-hydrated microcrystals of fibrinogen and fibrin will be used to obtain structural data and define intermolecular interactions in fibrin fibers. We will determine whether the Factor XIIIa-induced intermolecular cross-linking between gamma chains occurs between fibrin molecules that are interacting in an end-to-end or half- staggered manner. The alphaC domains of fibrin in protofibrils will be localized by electron microscopy of protofibrils with antibody bound to this region, and their interactions visualized in polymers of alphaC fragments and in complexes of alphaC fragment and fibrin. The functions of this part of the molecule will be further studied by analysis of oligomers made from desA fibrin monomer missing the alphaC domains. Protofibrils formed by cleavage of fibrinopeptide B with venzyme will be examined by electron microscopy. Structural studies of whole clots are necessary for observation and analysis of fiber branch points to understand the mechanisms involved in branching. Specifically designed software will be used to reconstruct and analyze quantitatively networks from stereo images of clots obtained with an intermediate voltage electron microscope. The nature of the inter- actions between GPIIb/IIIa and fibrinogen will be further investigated, using electron microscopy of several complexes as one approach to developing a more detailed molecular model for adhesive interactions between various fibrinogen domains and GPIIb/IIIa. The shape of high molecular weight kininogen will be determined and its structural and functional domains will be localized, providing insight into its mechanisms of action. One of the long-term goals of this research is to relate the results of these structural studies to biochemical, physiological and clinical work in the same area. The complex clotting/fibrinolytic system is unbalanced in many pathological conditions; to develop more effective and specific methods to control these processes, it is necessary to understand the molecular structures and interactions of the proteins involved.
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