Thromboembolic disease due to thermolabile conformational changes of antithrombin Rouen-VI (187 Asn-->Asp)

Abstract

A new variant of antithrombin (Rouen-VI, 187 Asn-+Asp) with increased heparin affinity was shown to have normal inhibitory activity which decreased slowly at 4C and rap- idly at 41C. On electrophoresis the freshly isolated variant had an anodal shift relative to native antithrombin due to the mutation. A further anodal transition occurred after either prolonged storage at 4C or incubation at 41C due to the formation of a new inactive uncleaved component with properties characteristic of L-form (latent) antithrom- bin. At the same time, polymerization also occurred with a predominance of di-, tri-, and tetra-mers. These findings fit with the observed mutation of the conserved asparagine (187) in the F-helix destabilizing the underlying A-sheet of the molecule. Evidence of A-sheet perturbation is provided by the increased rate of peptide insertion into the A-sheet and by the decreased vulnerability of the reactive loop to proteolysis. The spontaneous formation of both L-anti- thrombin and polymers is consistent with our crystal struc- ture of intact antithrombin where L-form and active anti- thrombin are linked together as dimers. The nature of this linkage favors a mechanism of polymerization whereby the opening of the A-sheet, to give incorporation of the reactive center loop, is accompanied by the bonding of the loop of one molecule to the C-sheet of the next. The accelerated lability of antithrombin Rouen-VI at 41 versus 37C pro- vides an explanation for the clinical observation that episodes of thrombosis were preceded by unrelated pyrexias.