Antithrombins Wibble and Wobble (T85M/K): archetypal conformational diseases with in vivo latent-transition, thrombosis, and heparin activation.
The inherent variability of conformational diseases is demonstrated by two families with different mutations of the same conserved aminoacid in antithrombin. Threonine 85 underlies the opening of the main beta-sheet of the molecule and its replacement, by the polar lysine, in antithrombin Wobble, resulted in a plasma deficiency of antithrombin with an uncharacteristically severe onset of thrombosis at 10 years of age, whereas the replacement of the same residue by a nonpolar methionine, antithrombin Wibble, gave near-normal levels of plasma antithrombin and more typical adult thromboembolic disease. Isolated antithrombin Wibble had a decreased thermal stability (Tm 56.2, normal 57.6 degreesC) but was fully stabilized by the heparin pentasaccharide (Tm 71.8, normal 71.0 degreesC), indicating that the prime abnormality is a laxity in the transition of the main sheet of the molecule from the 5- to 6-stranded form, as was confirmed by the ready conversion of antithrombin Wibble to the 6-stranded latent form on incubation. That this transition can occur in vivo was shown by the finding of nearly 10% of the proband's plasma antithrombin in the latent form and also, surprisingly, of small but definitive amounts of latent antithrombin in normal plasma. The latent transition will be predictably accelerated not only by gross mutations, as with antithrombin Wobble, to give severe episodic thrombosis, but also by milder mutations, as with antithrombin Wibble, to trigger thrombosis in the presence of other predisposing factors, including the conformational stress imposed by the raised body temperatures of fevers. Both antithrombin variants had an exceptional (25-fold) increase in heparin affinity and this, together with an increased inhibitory activity against factor Xa, provides evidence of the direct linkage of A-sheet opening to the conformational basis of heparin binding and activation.
Division of Molecular and Genetic Medicine, University of Sheffield, Royal Hallamshire Hospital, Sheffield, UK.
N J
NJ
Beauchamp
R N
RN
Pike
M
M
Daly
L
L
Butler
M
M
Makris
T R
TR
Dafforn
A
A
Zhou
H L
HL
Fitton
F E
FE
Preston
I R
IR
Peake
R W
RW
Carrell
D019943:Amino Acid Substitution; D020152:Antithrombin III Deficiency / Q000097:blood / Q000235:genetics*; D058833:Antithrombin Proteins; D000991:Antithrombins / Q000737:chemistry / Q000235:genetics*; D002648:Child; D018076:DNA, Complementary / Q000378:metabolism; D005165:Factor V / Q000235:genetics; D005260:Female; D006493:Heparin / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D008297:Male; D008875:Middle Aged; D020125:Mutation, Missense; D009844:Oligosaccharides / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D013329:Structure-Activity Relationship; D013912:Threonine
D058833:Antithrombin Proteins; D000991:Antithrombins; D018076:DNA, Complementary; D009844:Oligosaccharides; C115211:antithrombin Wibble; C115210:antithrombin Wobble; C095381:factor V Leiden; D013912:Threonine; C049714:IC 831423; D005165:Factor V; D006493:Heparin