The purpose of the proposed research is to investigate the mechanisms of initiation, recognition and regulation of alternative pathway activation. Although the overall scheme has been defined, there are major gaps in our understanding of this pathway. First, the currently accepted description of how activation is initiated, which the author proposed in 1981, may be in error. Recent experiments suggest that the initiating molecule does not function as proposed. This labile molecule has now been trapped at low temperature, isolated by HPLC and its properties have begun to be studied. Alternative initiation mechanisms will be explored. Second, the important question of how the pathway recognizes pathogenic organisms and other activators is unanswered. It is not presently known which protein recognizes the activator nor is it known what markers are recognized. Vesicle-bound C3b and amphophilic derivatives of C3b will be utilized to investigate these interactions. The experiments described will help to quantitatively define the roles of the attachment site of C3b, of neighboring carbo hydrates and of sialic acid. An assay for quantitatively determining the activating potential of different carbohydrates is described. The lack of such an assay is the primary reason that the specificity of the alternative pathway has not yet been clarified. Third, the functional roles of the various molecular forms of properdin present in normal human plasma (dimer, trimer, etc.) will be examined. The role of each form in alternative pathway activation and C5 convertase formation will be determined. Fourth, the three dimensional topology of C3b will be defined by the approach used to reveal antibody structure in the electron microscope. Symmetrical C3b dimers linked at their thioester sites will be used to define the relative positions of binding sites for B, H, P, C5 and various monoclonal antibodies. Finally, a major goal of this proposal is to continue work toward the development of a model of alternative pathway activation. A computer simulated model is sought which accurately describes the activation process under physiological conditions, which has predictive value and which is experimentally verified. The model would be useful in predicting effects of complement depletion and deficiencies, in simulating anaphylatoxin release, in modeling changes in acute phase proteins and in the design and evaluation of drugs which affect complement.
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