A natural prothrombin mutant reveals an unexpected influence of A-chain structure on the activity of human α-thrombin

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 alpha-thrombin ( DeltaK9). 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 DeltaK9 of the synthetic substrate Phe-Pip- 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 DeltaK9 was found significantly attenuated compared with the WT form. DeltaK9 has a very weak platelet- activating capacity, attributed to a severely defective PAR1 interaction, whereas the binding to the platelet glycoprotein Ibalpha 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-alpha-[2-naphthylsulfonyl- glycyl]4- amidinophenylalanine- piperidide) thrombin inhibitors displayed a better binding to DeltaK9 than to the WT form, indicating a better accommodation of these inhibitors into the catalytic pocket of DeltaK9. A molecular dynamics simulation of the DeltaK9 thrombin in full explicit water solvent provided support to the role of the A-chain 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.