Annie Bezeaud, INSERM U-698, Faculté Xavier Bichat, 16, rue Henri Huchard, BP416, 75870, Paris Cedex 18, France. E-mail: email@example.com
A new prothrombin variant, with a point mutation at nucleotide 20 029 resulting in Asp 552 to Glu substitution (prothrombin numbering), has been identified in a male newborn. Plasma prothrombin level was <3%, 16% and 60% when measured by clotting, chromogenic and immunological assays respectively. The substitution did not affect the rate of prothrombin conversion to thrombin but altered thrombin activity. Amino acid 552 has been reported to be involved in the allosteric transition, which is induced by sodium binding to thrombin. This is the first known amino acid substitution at this site to result in dysprothrombinaemia.
Thrombin, a multifunctional serine protease generated from prothrombin, interacts with a number of natural substrates to regulate blood clotting and other important biological functions. The high specificity of thrombin is controlled by residues within the active site, and by separate recognition sites located on the surface of the enzyme. In addition, thrombin activity is regulated in an allosteric fashion by the binding of Na+, which optimises thrombin for its procoagulant and signalling functions (Dang et al, 1995; Pineda et al, 2004).
Prothrombin deficiency is a rare autosomal recessive bleeding disorder resulting either from hypoprothrombinaemia with concomitantly low levels of coagulant activity and antigen, or from dysprothrombinaemia with low activity and borderline or normal antigen level. To date, 41 different mutations have been identified in patients with hypo- or dys-prothrombinaemia. We report here a new mutation in the prothrombin gene that results in the substitution of an amino acid located within the Na+ binding site.
Materials and methods
Plasma prothrombin assays
Plasma prothrombin assays included: (i) the classical one stage assay; (ii) a prothrombin assay with Echis carinatus venom (Latoxan, Rosans, France) as activator and either fibrinogen (Sigma Aldrich Srl, St Louis, MO, USA) or H-d-phenylalanyl-l-pipecolyl-l-arginine-pNA (S-2238; Chromogenix, Montpellier, France) as thrombin substrates; (iii) a quantitative Laurell electroimmunoassay using a rabbit antiserum against human prothrombin (Assera II; Diagnostica Stago, Asnieres, France).
Prothrombin conversion to thrombin
Plasma vitamin K-dependent proteins were adsorbed onto barium citrate, eluted and incubated with 5 μmol/l phospholipids vesicles, 20 pmol/l bovine factor Xa (Enzyme Research Laboratory, South bend, IN, USA), 20 pmol/l human factor Va (Diagnostica Stago, Asnieres, France) and 10 mmol/l CaCl2. At timed intervals, aliquots were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting (Akhavan et al, 2005).
Genomic DNA was isolated from the blood leucocytes. The coding region, intron/exon boundaries and the 5′ and 3′ untranslated region (UTR) of the prothrombin gene were amplified by polymerase chain reaction using 32 oligonucleotide primers, purified and sequenced on an ABI PRISM® 3100 DNA Sequencer [Applied Biosystems, Courtaboeuf Cedex, France].
The proband was a male newborn referred to us at birth because of fetal pulmonary hypertension and left ventricular failure, which resolved spontaneously. Systematic blood coagulation evaluation revealed an isolated and severe prothrombin deficiency. The defect persisted at the age of 15 months, characterised (Table I) by a dramatic decrease in prothrombin activity when measured by clotting assays with thromboplastin (rabbit or human) or E. carinatus venom as the activator, combined with a mild decrease in prothrombin activity measured by chromogenic assay and a subnormal level of prothrombin antigen. The father and the mother, who are first cousins, had a slight decrease in prothrombin activity and normal levels of prothrombin antigen. No abnormal bleeding was observed in the proband at birth or in the first 30 months of his life, and both parents were asymptomatic.
Table I. Plasma prothrombin levels in the proband and his parents
Prothrombin antigen (%)
Prothrombin activity (%)
Clotting assay with thromboplastin
Clotting assay with ECV
Chromogenic assay with ECV
ECV, Echis carinatus venom.
DNA sequence analysis of all 14 exons identified a missense mutation in exon 14, where a G replaced a C at nucleotide 20 029, resulting in the substitution of Asp at position 552(221) by Glu in the prothrombin molecule [nucleotides and residues numbering according to the prothrombin sequence (Degen & Davie, 1987) with the chymotrypsinogen numbering of thrombin residues (Bode et al, 1989) in parentheses]. The proband was homozygous and both parents heterozygous for the mutation. The prothrombin variant characterised by the D552(221)E substitution was designated prothrombin Saint-Denis.
Proteolytic activation of prothrombin by the prothrombinase complex
Upon incubation with prothrombinase, prothrombin is cleaved by factor Xa at R320 and R271 to generate thrombin and fragment 1.2, while an autocatalytic cleavage catalysed by thrombin at R155 yields prethrombin 1, fragment 1 and fragment 2. The pattern of prothrombin Saint-Denis proteolysis (Fig. 1) indicated that the Xa-catalysed cleavages at R320 and R271 were not impaired by the D552(221)E substitution. In contrast, formation of prethrombin 1 and fragment 1 was significantly reduced, indicating a decrease in the rate of the autocatalytic cleavage at position R155 by the mutant thrombin.
The present study showed that the prothrombinase complex rapidly converts prothrombin Saint-Denis to thrombin molecules with a severe defect in clotting activity and poor hydrolytic activity toward prothrombin itself or the small chromogenic substrate S-2238.
The mutated residue D552(221) is absolutely conserved in thrombins from hagfish to human. Selective mutagenesis has recently shown that D552(221)A substitution almost abrogated the Na+-induced enhancement of formyl peptide receptor (FPR) hydrolysis although it had no effect on Na+ binding per se (Pineda et al, 2004). The allosteric transition, which is flipped through Na+ binding (Huntington & Esmon, 2003; Pineda et al, 2004) and optimises the cleavage of fibrinogen and small substrates, might be impaired in prothrombin Saint-Denis. In addition, the mild decrease in plasma prothrombin antigen that is associated with the severe clotting defect suggests that the mutation could result in a more global destabilisation and relative instability of the whole prothrombin molecule.
The assessment of the in vivo physiological importance of Na+-induced allosteric regulation of thrombin is a difficult challenge. The natural prothrombin variants characterised by a mutation in the Na+-binding site or its immediate surrounding, are poorly informative. The same homozygous mutation E466(146)A was associated with a severe prothrombin deficiency in two unrelated families (Miyata et al, 1992; Degen et al, 1995), with significant bleeding in one family (Degen et al, 1995) and not in the other (Bezeaud et al, 1988). Two other variants with a defect in the Na+ binding site (Henriksen et al, 1998; Sun et al, 2001) are not associated with bleeding but have been detected only in heterozygotes. The proband described here is homozygous for the D552(221)E mutation, with a severe defect in fibrinogen clotting but no bleeding. The absence of bleeding is intriguing and warrants a careful follow up of the proband in years to come. It has been suggested that, in some variants with a mild or no bleeding phenotype, abnormalities in the procoagulant functions of thrombin might be counterbalanced by abnormalities in the anticoagulant functions (i.e. defective thrombomodulin-driven activation of the protein C anticoagulant pathway) and/or decrease in the rate of inhibition (i.e. slow thrombin–antithrombin interaction) (Akhavan et al, 2002). The extent to which this hypothesis applies to the proband presented here remains to be explored.
We would like to thank Odile Bournier, Patrick Marie and William Davillé (Centre d'Investigations Biologiques PhenGen, AP-HP, Paris) for expert technical assistance.