STRUCTURAL STUDIES OF BLOOD CLOTTING PROTEINS

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The objective of this research project is to obtain structural information about fibrin and fibrinogen and their interactions in order to elucidate aspects of the molecular mechanism of clotting. Major goals are the investigation of the structure of the fibrin clot and the mechanism of its assembly and the determination of the structure of he fibrinogen molecule at higher resolution. Unstained, frozen, hydrated fibrin fibers will be examined by high resolution, low dose electron microscopy. The specific calcium to fibers will be measured by electron microprobe. The nature of the lateral aggregation of fibers will be explored by electron microscopy. The influence of calcium concentration, carbohydrate content and several platelet proteins on aggregation will be determined. Fibers formed by precipitation of fibrin monomer will be examined by electron microscopy. Quantitative measurements of the twisting of fibers will be made to evaluate a possible mechanism for diameter limitation. Electron microscopy will be used to study the branching of fibers, the dissolution of the clot by plasmin, and the extent of order in the arrangement of fibers in a clot. The nature of the alpha oc domain, missing from the modified fibrinogens, will be investigated by electron microscopy of individual fibrinogen and fibrin molecules. Our recent three-dimensional model for the shape of the molecule will be used to obtain initial phases for the X-ray crystallographic data that has been collected; these phases will be refined by use of non-crystallographic two-fold symmetry averaging and solvent flattening. Recently grown crystals of human fibrinogen and fragment D will be characterized. 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.