Primary and secondary structural analyses of bovine and human thrombin together with data from chemical modification studies have suggested specific hypotheses concerning the relation of the structure of thrombin to its function. These hypotheses can be tested using molecular genetic techniques if there is an expression system that allows the synthesis of functional thrombin from cloned DNA. We have developed such a system in E. coli. Using this novel oligonucleotide mutagenesis scheme for deleting specific regions of genes (such as hypothetical functional domains), we have constructed two expression vectors, one of which produces bovine thrombin and the other the B-chain of thrombin. Both synthetic proteins actively hydrolyze highly specific chromogenic substrates and specifically bind fibrinogen. We propose to study in depth the B-chain of thrombin whose biological function has only been partially characterized to date. We shall examine the function of the so-called """"""""B"""""""" and """"""""E"""""""" loops of thrombin by removing these domains with our mutagenesis technique, then test the mutated proteins for their ability to cleave specific substrates, activate platelets, and bind fibrinogen and heparin. The results of these studies will then allow us to conduct a more detailed analysis in which specific tyrosine, arginine and lysine residues are mutated.
