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Prepublished online as a Blood First Edition Paper on April 30, 2002; DOI 10.1182/blood-2002-01-0243.
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Blood, 15 August 2002, Vol. 100, No. 4, pp. 1347-1353
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Molecular and functional characterization of a natural homozygous
Arg67His 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
From the Angelo Bianchi Bonomi Hemophilia and
Thrombosis Center, and Fondazione Luigi Villa, IRCCS Maggiore Hospital
University of Milan, Milan, Italy, and the Hemostasis Research Centre,
Catholic University School of Medicine, Rome, Italy.
In a patient who presented with a severe coagulation deficiency in
plasma contrasting with a very mild hemorrhagic diathesis a homozygous
Arg67His mutation was identified in the prothrombin gene.
Wild-type (factor IIa [FIIa]-WT) and mutant Arg67His 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 2.1 × 107
M 1s1 and 9 × 105
M1s1. Decreased activation of protein C
(PC) correlated with the 33-fold decreased binding affinity for
thrombomodulin (TM; Kd = 65.3 nM vs 2.1 nM, in FIIa-MT67
and in FIIa-WT, respectively). In contrast, hydrolysis of PC in the
absence of TM was normal. The Arg67His mutation had a dramatic effect
on the cleavage of protease-activated G protein-coupled receptor 1 (PAR-1) 38-60 peptide
(kcat/Km = 4 × 107
M1s1 to 1.2 × 106
M1s1). 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 sulfate, was found for the FIIa-MT67 compared with FIIa-WT,
suggesting a severe impairment of thrombin inhibition by HCII in vivo.
Finally, the Arg67His mutation was associated with 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 Arg67His 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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