Antiphospholipid syndrome is a systemic autoimmune disease mediated by antiphospholipid antibodies and characterized by venous and/or arterial thrombosis. The heterogeneous group of antiphospholipid antibodies comprises antibodies directed against various plasma proteins which can be determined in the serum either by specific immune tests or by coagulation tests. In our diploma thesis we focused on antiprothrombin antibodies, which are one of the subgroup of antiphospholipid antibodies and aimed to determine their binding site on prothrombin.
For epitope determination we have used phage display libraries containing different dodecapeptides. Specifically bound bacteriophages expressing peptides that interacted with antiprothrombin antibodies were afterwards isolated in serial selections. Using the ELISA test we determined peptides with highest avidity for antiprothrombin antibodies, then isolated their DNA and determined the amino acid sequence. Next, we tried to localize the revealed epitopes with the alignment to prothrombin tertiary structure. We determined four different peptide motives, with one observed in previous studies and came to the conclusion that prothrombin conformation has a big impact on alignment of antibodies. Using PepSurf algorithm we preformed the alignment to both closed (4O03) and open conformations of prothrombin (6BJR and 4NZQ). The first alignment to closed conformation revealed epitopes for antiphospholipid antibodies on proteolytic domain and »kringle«-2 domain of prothrombin, however when we performed the alignment of the same peptides on open conformation of prothrombin (6BJR and 4NZQ), the epitopes where on different domains.
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