IMPORTANCE OF THE RELEASE OF STRAND 1C TO THE POLYMERIZATION MECHANISM OF INHIBITORY SERPINS

Serpin polymerization is the underlying cause of several diseases, inc luding thromboembolism, emphysema, liver cirrhosis, and angioedema. Un derstanding the structure of the polymers and the mechanism of polymer ization is necessary to support rational design of therapeutic agents. Here we show that polymerization of antithrombin is sensitive to the addition of synthetic peptides that interact with the structure. A 12- mer peptide (homologous to P-14-P-3 of antithrombin reactive loop), re presenting the entire length of s4A, prevented polymerization totally. A 6-mer peptide (homologous to P-14-P-9 of antithrombin) not only all owed polymerization to occur, but induced it. This effect could be blo cked by the addition of a 5-mer peptide with the s1C sequence of antit hrombin or by an unrelated peptide representing residues 26-31 of chol ecystokinin. The s1C or cholecystokinin peptide alone was unable to fo rm a complex with native antithrombin. Moreover, an active antitrypsin double mutant, Pro 361 --> Cys, Set 283 --> Cys, was engineered for t he purpose of forming a disulfide bond between s1C and s2C to prevent movement of s1C. This mutant was resistant to polymerization if the di sulfide bridge was intact, but, under reducing conditions, it regained the potential to polymerize. We have also modeled long-chain serpin p olymers with acceptable stereochemistry using two previously proposed loop-A-sheet and loop-C-sheet polymerization mechanisms and have shown both to be sterically feasible, as are ''mixed'' linear polymers. We therefore conclude that the release of strand 1C must be an element of the mechanism of serpin polymerization.