A Natural Prothrombin Mutant Reveals an Unexpected Influence of A-chain Structure on the Activity of Human α-Thrombin

Abstract

We have recently identified in two unrelated patients with bleeding tendency a homozygous mutation causing a deletion of one of the two contiguous Lys 9 /Lys 10 residues in the A-chain of ␣-thrombin (⌬K9). We used in vitro expression analysis to clarify the role of the deletion of Lys 9 or Lys 10 in the thrombin function. The k cat /K m value of the hydrolysis by ⌬K9 of the synthetic substrate PhePip-Arg-p-nitroanilide (where Pip represents L-pipecolyl) and fibrinopeptide A was 18-and 60-fold lower, respectively, compared with wild type (WT). Interaction with antithrombin was also reduced in the mutant, the association rate being about 20-fold lower than in the WT thrombin. The sensitivity to sodium ion of ⌬K9 was found significantly attenuated compared with the WT form. ⌬K9 has a very weak platelet-activating capacity, attributed to a severely defective PAR1 interaction, whereas the binding to the platelet glycoprotein Ib␣ was unaffected. Likewise, the interaction with protein C was severely impaired, whereas interaction with thrombomodulin had a normal K d value. At variance with these findings, both low affinity (basic pancreatic trypsin inhibitor) and high affinity (N-␣-[2-naphthylsulfonyl-glycyl]-4-amidinophenylalanine-piperidide) thrombin inhibitors displayed a better binding to ⌬K9 than to the WT form, indicating a better accommodation of these inhibitors into the catalytic pocket of ⌬K9. A molecular dynamics simulation of the ⌬K9 thrombin in full explicit water solvent provided support to the role of the Achain in affecting conformation and catalytic properties of the B-chain, especially in some insertion loops of the enzyme, such as the 60-loop, as well as in the geometry of the catalytic triad residues.Recently, a homozygous deletion mutation of one of the two contiguous Lys 9 /Lys 10 residues 1 in the A-chain of ␣-thrombin was identified in two unrelated Iranian patients with severe prothrombin deficiency and hemorrhagic diathesis (1). The level of prothrombin antigen measured in plasma was 15%, whereas the coagulant activity ranged from less than 1% to about 2.5% (1). Prothrombin deficiency is an autosomal recessive bleeding disorder characterized by two phenotypes: hypoprothrombinemia, with concomitantly low levels of coagulant activity and antigen (type I), and dysprothrombinemia, with very low activity but subnormal or normal antigen levels (type II). These disorders are rare, and there is always residual prothrombin procoagulant activity measurable in patients, the phenotype found in prothrombin-deficient mice indicating that complete prothrombin deficiency may be lethal in humans (2, 3). To date, 38 defects in the prothrombin gene have been identified in patients with dysprothrombinemia or hypoprothrombinemia (1, 4 -7). Among these natural dysprothrombins, amino acid residues of the thrombin A-chain were found to be rarely involved (1, 4 -7).The A-chain in human thrombin is composed of 36 amino acid residues, which are connected to the catalytic B-chain by a single disulfide bond (8). This chain...

Role of the A chain in thrombin function

“…The results have been interpreted in terms of molecular dynamics calculations as a gross perturbation of the active site moiety propagating long-range from the site of deletion in the A chain [9]. Notably, the perturbed structure produced by modeling contains features documented in the X-ray crystal structure of the inactive form of thrombin E* [25].…”

“…Functional investigation of the ΔK9 mutant has revealed significant impairment of FpA release (64-fold), PAR1 activation (116-fold), protein C activation in the presence of thrombomodulin (34-fold) and hydrolysis of chromogenic substrate in both the E (6-fold) and E:Na + (18-fold) forms [9]. The results have been interpreted in terms of molecular dynamics calculations as a gross perturbation of the active site moiety propagating long-range from the site of deletion in the A chain [9].…”

“…Deletion of Lys9 in the A chain is associated with a bleeding phenotype [8]. The thrombin mutant ΔK9 shows defects in the activation of its zymogen form [9], but the mature enzyme features impaired Na + binding and significantly reduced activity toward a chromogenic substrate, fibrinogen, PAR1 and protein C [9] due to long-range perturbation of the active site moiety and catalytic triad [9,10]. These findings underscore the importance of the A chain in thrombin function and warrant a thorough analysis in terms of mutagenesis.…”

Thrombin

“…Other naturally mutations, like deletion of K9 or K10 (Akhavan et al, 2000), are also associated with severe bleeding. Interestingly, the defect causes impaired fibrinogen and PAR1 cleavage, reduced response to Na + activation (De Cristofaro et al, 2004, and long-range perturbation of active site residues (De Cristofaro et al, 2006). The A chain is rich in charged residues that make polar interactions with partners of the B chain.…”

Recessively inherited coagulation disorders

“…4,31 Several cases of dysprothrombinemia, characterized by normal levels of a dysfunctional protein, were reported and the relevant mutant proteins expressed and characterized. 18,32 FV deficiency FV deficiency is almost invariably expressed phenotypically as type I deficiency. 3 Only one genetic defect (FV New Brunswick, Ala221Val) causing type II deficiency was recently demonstrated to interfere with the stability of activated FV.…”