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, Pier M. Mannucci

Research output: Contribution to journalArticle

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Abstract

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 lla [Flla]-WT) and mutant Arg67His thrombin (Flla-MT67) had similar amidolytlc activity. By contrast, the kcat/Km value of fibrinopeptide A hydrolysis by Flla-WT and Flla-MT67 was equal to 2.1 × 107 M-1s-1 and 9 × 105 M-1s-1. 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 Flla-MT67 and In Flla-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 M-1s-1 to 1.2 x 106 M-1s-1). Flla-MT67 showed a weaker platelet activating capacity, attributed to a defective PAR-1 interaction, whereas the interaction with glycoprotein lb 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 Flla-MT67 compared with Flla-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.

Original languageEnglish
Pages (from-to)1347-1353
Number of pages7
JournalBlood
Volume100
Issue number4
DOIs
Publication statusPublished - Aug 15 2002

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Heparin Cofactor II
Prothrombin
Factor Va
Thrombin
Genes
Protein C
Phenotype
Defects
Mutation
Hydrolysis
Chemical activation
Fibrinopeptide A
Hemorrhagic Disorders
Thrombomodulin
Dermatan Sulfate
Factor Xa
G-Protein-Coupled Receptors
Platelets
Coagulation
Anticoagulants

ASJC Scopus subject areas

  • Hematology

Cite this

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. / Akhavan, Sepideh; De Cristofaro, Raimondo; Peyvandi, Flora; Lavoretano, Silvia; Landolfi, Raffaele; Mannucci, Pier M.

In: Blood, Vol. 100, No. 4, 15.08.2002, p. 1347-1353.

Research output: Contribution to journalArticle

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abstract = "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 lla [Flla]-WT) and mutant Arg67His thrombin (Flla-MT67) had similar amidolytlc activity. By contrast, the kcat/Km value of fibrinopeptide A hydrolysis by Flla-WT and Flla-MT67 was equal to 2.1 × 107 M-1s-1 and 9 × 105 M-1s-1. 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 Flla-MT67 and In Flla-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 M-1s-1 to 1.2 x 106 M-1s-1). Flla-MT67 showed a weaker platelet activating capacity, attributed to a defective PAR-1 interaction, whereas the interaction with glycoprotein lb 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 Flla-MT67 compared with Flla-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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T1 - 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

AU - Akhavan, Sepideh

AU - De Cristofaro, Raimondo

AU - Peyvandi, Flora

AU - Lavoretano, Silvia

AU - Landolfi, Raffaele

AU - Mannucci, Pier M.

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