A natural prothrombin mutant reveals an unexpected influence of A-chain structure on the activity of human alpha-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.
De Cristofaro
Raimondo
R
Hemostasis Research Centre, Institute of Internal Medicine and Geriatrics, Catholic University School of Medicine, Largo F. Vito 1, 00168 Rome, Italy. rdecristofaro@rm.unicatt.it
Akhavan
Sepideh
S
Altomare
Cosimo
C
Carotti
Andrea
A
Peyvandi
Flora
F
Mannucci
Pier Mannuccio
PM
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000737:chemistry; D001792:Blood Platelets / Q000378:metabolism; D019556:COS Cells; D020134:Catalytic Domain; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry; D005340:Fibrinogen / Q000737:chemistry; D005344:Fibrinopeptide A / Q000737:chemistry; D017353:Gene Deletion; D006801:Humans; D006868:Hydrolysis; D007020:Hypoprothrombinemias / Q000378:metabolism*; D007477:Ions; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011486:Protein C / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011516:Prothrombin / Q000235:genetics*; D012964:Sodium / Q000737:chemistry; D012965:Sodium Chloride / Q000494:pharmacology; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D007477:Ions; D011486:Protein C; D005344:Fibrinopeptide A; D012965:Sodium Chloride; C018833:arginine-4-nitroanilide; D011516:Prothrombin; D005340:Fibrinogen; D004247:DNA; D001120:Arginine; D012964:Sodium; D013917:Thrombin; D008239:Lysine