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Prepublished online as a Blood First Edition Paper on April 30, 2002; DOI 10.1182/blood-2002-01-0243.
Submitted January 28, 2002
Accepted April 9, 2002
Molecular and Functional Characterization of a Natural Homozygous Arg67 His Mutation in the Prothrombin Gene of a Patient with a Severe Procoagulant Defect Contrasting with a Mild Hemorrhagic Phenotype
Sepideh Akhavan*, Raimondo De Cristofaro, Flora Peyvandi, Silvia Lavoretano, Raffaele Landolfi, and Pier M Mannucci
Angelo Bianchi Bonomi, Hemophilia and Thrombosis Center, IRCCS Maggiore Hospital, University of Milan, Milan, Italy
Hemostasis Research Centre, Catholic University School of Medicine, Rome, Italy
* Corresponding author; email: sepidehakhavan{at}yahoo.it.
In a patient who presented a severe coagulation deficiency in plasma contrasting with a very mild hemorrhagic diathesis a homozygous R67H mutation was identified in the prothrombin gene. Wild type (FIIa-WT) and mutant R67H thrombin (FIIa-MT67) had similar amidolytic activity. By contrast, the kcat/Km value of fibrinopeptide A hydrolysis by FIIa-WT and FIIa-MT67 was equal to 2.1x107M-1sec-1 and 9x105M-1sec-1. Decreased activation of protein C (PC) correlated with the 33-fold decreased binding affinity for thrombomodulin (TM) (Kd=65.3nM vs 2.1nM, in FIIa-MT67 and in FIIa-WT). In contrast, hydrolysis of PC in the absence of TM was normal. The R67H mutation had a dramatic effect on the cleavage of PAR1 (protease-activated G protein-coupled receptor-1) 38-60 peptide (kcat/Km=4x107M-1sec-1 to 1.2x106M-1sec-1). FIIa-MT67 showed a weaker platelet activating capacity, attributed to a defective PAR-1 interaction, whereas the interaction with glycoprotein Ib was normal. A drastic decrease (up to 500-fold) of the second order rate constant pertaining to heparin cofactor II (HCII) interaction, especially in the presence of dermatan sulphate, was found for the FIIa-MT67 compared with FIIa-WT, suggesting a severe impairment of thrombin inhibition by HCII in vivo. Finally, the R67H mutation was associated to a 5-fold decrease of prothrombin activation by the factor Xa-factor Va complex, perhaps through impairment of the prothrombin-factor Va interaction. These experiments show that the R67H substitution affects drastically both the procoagulant and the anticoagulant functions of thrombin as well as its inhibition by HCII. The mild hemorrhagic phenotype might be explained by abnormalities that ultimately counterbalance each other.
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