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

Raimondo De Cristofaro, Sepideh Akhavan, Cosimo Altomare, Andrea Carotti, Flora Peyvandi, Pier Mannuccio Mannucci

Research output: Contribution to journalArticlepeer-review

Abstract

We have recently identified in two unrelated patients with bleeding tendency a homozygous mutation causing a deletion of one of the two contiguous Lys9/Lys10 residues in the A-chain of α-thrombin (ΔK9). We used in vitro expression analysis to clarify the role of the deletion of Lys9 or Lys10 in the thrombin function. The kcat/Km value of the hydrolysis by ΔK9 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 ΔK9 was found significantly attenuated compared with the WT form. ΔK9 has a very weak platelet-activating capacity, attributed to a severely defective PAR1 interaction, whereas the binding to the platelet glycoprotein Ibα was unaffected. Likewise, the interaction with protein C was severely impaired, whereas interaction with thrombomodulin had a normal Kd value. At variance with these findings, both low affinity (basic pancreatic trypsin inhibitor) and high affinity (N-α -[2-naphthylsulfonyl-glycyl]-4-amidinophenylalanine-piperidide) thrombin inhibitors displayed a better binding to ΔK9 than to the WT form, indicating a better accommodation of these inhibitors into the catalytic pocket of ΔK9. A molecular dynamics simulation of the ΔK9 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.

Original languageEnglish
Pages (from-to)13035-13043
Number of pages9
JournalJournal of Biological Chemistry
Volume279
Issue number13
DOIs
Publication statusPublished - Mar 26 2004

ASJC Scopus subject areas

  • Biochemistry

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