The goal of this project is to increase our understanding of the fibrinogen to fibrin clot conversion with the hope of providing a better foundation for clinical intervention in pathological clot formation. Recent advances in genetic engineering provide a unique opportunity to approach this problem in a way where the importance of each amino acid residue can be analyzed. That is, by manipulating the DNA which codes for fibrinogen, specific amino acid alterations can be made. I have isolated a human cDNA clone which codes for the entire A alpha polypeptide chain including a 19 amino acid signal peptide. This clone will be used to construct plasmids to direct the synthesis of the A alpha polypeptide in transfected cells. The expression of the A alpha chain will be detected immunologically. In periments I have constructed a vector which codes for a hybrid protein containing amino acids 1 to 551 of the A alpha polypeptide. Immunoreactivity has been detected in E. Coli transfected with this vector. The functional activity of the A alpha peptide separated from native fibrinogen will be tested by measuring its susceptability to thrombin cleavage and its ability to inhibit fibrin polymerization. Site specific mutants will then be constructed in the cDNA in order to produce altered A alpha peptides. Since thrombin recognition of the A alpha polypeptide has been extensively described, it will be straightforward to direct mutations towards residues which are known to be important to fibrinopeptide A cleavage. Similarly, areas of the A alpha chain which are considered to be important to fibrin polymerization can be tested by site directed mutagenesis. The effects of these alterations on the functional activity of the A alpha peptide will provide new information relevant to thrombin specificity and fibrin polymerization.
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