The scientific goal is to clarify the molecular basis of the interaction between fibrinogen and its receptor on human platelets, glycoprotein llb- lla. The binding of fibrinogen to the glycoprotein llb-lla complex is required for platelets to aggregate and form a hemostatic plug. Although the primary amino acid sequences of both fibrinogen and glycoprotein llb- lla have already been established, the precise binding domains on either of these molecules are not known. We will use a murine monoclonal antibody PAC1, whose epitope appears to be identical to or overlapping with a fibrinogen binding site on GP llb-lla. A cDNA expression system will be developed in which the variable regions of PAC1 are expressed, recovered, and used in direct binding studies with activated platelets. Such an expression system will help in understanding what structural features of PAC1 (and hence fibrinogen) determine its specificity for GP llb-lla. In addition, since anti-idiotypic antibodies raised against PAC1 may contain variable regions that are the """"""""internal image"""""""" of the fibrinogen receptor on GP llb-lla, we will develop and sequence PAC1 anti-idiotypic antibodies. He will then compare the primary sequences of their variable regions with the sequences of GP llb-lla in order to find area(s) of sequence similarity or identity. We will modify the variable regions of PAC1 by site-directed mutagenesis to determine the structural features of the molecule that confer its specificity for GP llb-lla. The binding of these antibody mutants to GP llb-lla and their ability to inhibit fibrinogen binding will be examined. In addition using the information obtained in from sequencing anti-idiotypic antibodies that contain an internal image of the fibrinogen receptor, we will attempt to identify putative binding domains for fibrinogen on GP llb-lla, synthesize peptides derived from these regions, and examine the inhibitory effects of these peptides on fibrinogen binding to platelets. Knowledge gained from these studies may be useful in the development of genetically-engineered antibodies or antibody fragments capable of serving as diagnostic or therapeutic reagents in thrombotic disorders of man.
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