Conformational Asn187Asp/Lys antithrombin variants and thrombosis

Summary

Antithrombin Rouen VI (N187D) is a rare conformational thermolabile variant.The unique symptomatic carrier reported in the literature developed 3 thrombotic events during pregnancy, in each case in a context of pyrexial infection. In fresh plasma, antithrombin activity and antigen level were normal but in vitro experiments demonstrated the presence of a thermolabile variant, suggesting that fever could be a trigger for thrombosis in N187D carriers.The RouenVI variant was further found in two asymptomatic brothers. In these subjects, it was associated with normal antigen level but reduced activity. In order to better delineate the functional and clinical consequences of the N187 variants,we have studied a series of seven subjects from two distinct families heterozygous for the Rouen VI mutation. Antithrombin levels were normal or borderline in these patients. Thermostability of plasma antithrombin was normal. We have also studied six subjects heterozygous for a new mutation, 6462C>G,which results in an asparagine to lysine substitution at residue 187. In these patients, the N187K mutation is associated with a clear type II deficiency and decreased thermostability of the plasma protein has been demonstrated. That the N187D mutation has milder consequences on plasma antithrombin activity than the N187K mutation is in agreement with structural predictions. About 50% of the N187 carriers studied have suffered venous thrombotic events, strongly suggesting that both mutations are risk factors for thrombosis, but none occurred during pyrexial infections.

Keywords

Antithrombin - conformational variant - thrombophilia - serpin

 

• References

• 1 Egeberg O. Inherited antithrombin deficiency causing thrombophilia. Thromb Diath Haemorrh 1965; 13: 516-30.
• 2 Heijboer H, Brandjes DP, Buller HR. et al Deficiencies of coagulation-inhibiting and fibrinolytic proteins in outpatients with deep-vein thrombosis. N Engl J Med 1990; 323: 1512-6.
• 3 Rosendaal FR. Risks factors for venous thrombotic disease. Thromb Haemost 1996; 82: 616-9.
• 4 Tait RC, Walker ID, Perry DJ. et al Prevalence of antithrombin deficiency in the healthy population. Br J Haematol 1994; 87: 106-12.
• 5 Lane DA, Bayston T, Olds RJ. et al Antithrombin mutation database: 2nd (1997) update. For the Plasma Coagulation Inhibitors Subcommittee of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis. Thromb Haemost 1997; 77: 197-211.
• 6 Perry DJ, Marshall C, Borg JY. et al Two novel antithrombin variants, Asn187Asp and Asn187Lys, indicate a functional role for asparagine 187. Blood Coagul Fibrinolysis 1995; 6: 51-4.
• 7 Bruce D, Perry DJ, Borg JY. et al Thromboembolic disease due to thermolabile conformational changes of antithrombin Rouen-VI (187 Asn → Asp). J Clin Invest 1994; 94: 2265-74.
• 8 Beauchamp NJ, Pike RN, Daly M. et al Antithrombins Wibble and Wobble (T85M/K): archetypal conformational diseases with in vivo latent-transition, thrombosis, and heparin activation. Blood 1998; 92: 2696-706.
• 9 Picard V, Dautzenberg MD, Villoutreix BO. et al Antithrombin Phe229Leu: a new homozygous variant leading to spontaneous antithrombin polymerization in vivo associated with severe childhood thrombosis. Blood 2003; 102: 919-25.
• 10 Creagh MD, Roberts IF, Clark DJ. et al Familial antithrombin III deficiency and Mycoplasma pneumoniae pneumonia. J Clin Pathol 1991; 44: 870-1.
• 11 Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988; 16: 1215.
• 12 Picard V, Bura A, Emmerich J. et al Molecular bases of antithrombin deficiency in French families: identification of seven novel mutations in the antithrombin gene. Br J Haematol 2000; 110: 731-4.
• 13 Sas G, Pepper DS, Cash JD. Plasma and serum antithrombin III: differentiation by crossed immunoelectrophoresis. Thromb Res 1975; 6: 87-91.
• 14 Picard V, Marque PE, Paolucci F. et al Topology of the stable serpin-protease complexes revealed by an autoantibody that fails to react with the monomeric conformers of antithrombin. J Biol Chem 1999; 274: 4586-93.
• 15 McKay EJ. A simple two-step procedure for the isolation of antithrombin III from biological fluids. Thromb Res 1981; 21: 375-82.
• 16 Wardell MR, Chang WS, Bruce D. et al Preparative induction and characterization of L-antithrombin: a structural homologue of latent plasminogen activator inhibitor-1. Biochemistry 1997; 36: 13133-42.
• 17 Carrell RW, Huntington JA, Mushunje A. et al The conformational basis of thrombosis. Thromb Haemost 2001; 86: 14-22.
• 18 Eldor A. Thrombophilia, thrombosis and pregnancy. Thromb Haemost 2001; 86: 104-11.
• 19 Martinelli I, Legnani C, Bucciarelli P. et al Risk of pregnancy-related venous thrombosis in carriers of severe inherited thrombophilia. Thromb Haemost 2001; 86: 800-3.
• 20 Conard J, Horellou MH, Van Dreden P. et al Thrombosis and pregnancy in congenital deficiencies in AT III, protein C or protein S: study of 78 women. Thromb Haemost 1990; 63: 319-20.
• 21 Pabinger I, Schneider B. Thrombotic risk in hereditary antithrombin III, protein C, or protein S deficiency. Arterioscler Thromb Vasc Biol 1996; 16: 742-8.
• 22 Walker ID. Thrombophilia in pregnancy. J Clin Pathol 2000; 53: 573-80.
• 23 Hellgren M. Hemostasis during normal pregnancy and the puerperium. Semin Thromb Hemost 2003; 2: 125-30.
• 24 Eichinger S, Weltermann A, Philipp K. et al Prospective evaluation of hemostatic system activation and thrombin potential in healthy pregnant women with and without factor V Leiden. Thromb Haemost 1999; 82: 1232-6.
• 25 Irving JA, Pike RN, Lesk AM. et al Phylogeny of the serpin superfamily: implications of patterns of amino acid conservation for structure and function. Genome Res 2000; 10: 1845-64.
• 26 Gettins PG. The F-helix of serpins plays an essential, active role in the proteinase inhibition mechanism. FEBS Lett 2002; 523: 2-6.
• 27 Schreuder HA, de Boer B, Dijkema R. et al The intact and cleaved human antithrombin III complex as a model for serpin-proteinase interactions. Nat Struct Biol 1994; 1: 48-54.