Thromboembolic disease due to thermolabile conformational changes of antithrombin Rouen-VI (187 Asn-->Asp)
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 4 degrees C and rapidly at 41 degrees C. 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 4 degrees C or incubation at 41 degrees C due to the formation of a new inactive uncleaved component with properties characteristic of L-form (latent) antithrombin. 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-antithrombin and polymers is consistent with our crystal structure of intact antithrombin where L-form and active antithrombin 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 37 degrees C provides an explanation for the clinical observation that episodes of thrombosis were preceded by unrelated pyrexias.
Bruce
D
D
Department of Haematology, University of Cambridge, United Kingdom.
Perry
D J
DJ
Borg
J Y
JY
Carrell
R W
RW
Wardell
M R
MR
D000328:Adult; D000595:Amino Acid Sequence; D058833:Antithrombin Proteins; D000991:Antithrombins / Q000737:chemistry / Q000235:genetics* / Q000648:ultrastructure; D005260:Female; D005334:Fever / Q000150:complications; D006358:Hot Temperature; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011249:Pregnancy Complications, Cardiovascular / Q000209:etiology; D017434:Protein Structure, Tertiary; D017422:Sequence Analysis, DNA; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism; D013924:Thrombophlebitis / Q000209:etiology / Q000235:genetics*
D058833:Antithrombin Proteins; D000991:Antithrombins; C090782:antithrombin Rouen-VI; D013917:Thrombin