Background. Natural mutations in the prothrombin gene, involving the substitution of Arg596 with polar/apolar amino acids, represent an important risk factor for thrombosis. Notably, Arg596 in prothrombin corresponds to position 221a in the Na+ -binding loop of mature -thrombin (T). Patients carrying these mutations (Arg221a→Gln/Leu/Trp) share a common clinical phenotype, characterized by a moderately reduced procoagulant function in coagulation tests and resistance of T variants to inactivation by antithrombin (ATIII). However, the biochemical mechanisms linking these mutations to the increased thrombotic risk remain elusive. Aims. To provide quantitative data on the effect that the recombinant Arg221aTrp T mutant (T-PD2) has on the activation of procoagulant and anticoagulant pathways and elucidate the structural the basis of the observed functional changes. Methods. A multi-pronged approach was used, integrating recombinant DNA techniques, clotting/fibrinolytic and enzymatic assays, microscopy, proteomics, spectroscopic, mass spectrometry, and molecular modelling techniques. Results. Arg221aTrp mutation abrogates physiological Na+ binding and reduces the proportion of the fully active T conformation. T-PD2 affects procoagulant pathways by less efficiently generating fibrin (7- fold) and activating platelets (10-fold). The mutation potently affects anticoagulant pathways, as T-PD2 is less efficiently inhibited by ATIII (3-fold), binds heparin less tightly (10-fold), and much less efficiently (41-fold) activates protein C. Conclusions. Our data indicate that the procoagulant phenotype, observed in Arg221aTrp mutation carriers, may result from opposing inhibitory effects on both procoagulant and anticoagulant pathways, with the impact on anticoagulant pathways being predominant and shifting the natural anticoagulant↔procoagulant equilibrium in vivo towards thrombosis.
The Natural Mutation Arg221aTrp in Human α-Thrombin Abrogates Physiological Na+ Binding and Preferentially Hinders the Protease Anticoagulant Functions
Ciancetta, Antonella;
2026
Abstract
Background. Natural mutations in the prothrombin gene, involving the substitution of Arg596 with polar/apolar amino acids, represent an important risk factor for thrombosis. Notably, Arg596 in prothrombin corresponds to position 221a in the Na+ -binding loop of mature -thrombin (T). Patients carrying these mutations (Arg221a→Gln/Leu/Trp) share a common clinical phenotype, characterized by a moderately reduced procoagulant function in coagulation tests and resistance of T variants to inactivation by antithrombin (ATIII). However, the biochemical mechanisms linking these mutations to the increased thrombotic risk remain elusive. Aims. To provide quantitative data on the effect that the recombinant Arg221aTrp T mutant (T-PD2) has on the activation of procoagulant and anticoagulant pathways and elucidate the structural the basis of the observed functional changes. Methods. A multi-pronged approach was used, integrating recombinant DNA techniques, clotting/fibrinolytic and enzymatic assays, microscopy, proteomics, spectroscopic, mass spectrometry, and molecular modelling techniques. Results. Arg221aTrp mutation abrogates physiological Na+ binding and reduces the proportion of the fully active T conformation. T-PD2 affects procoagulant pathways by less efficiently generating fibrin (7- fold) and activating platelets (10-fold). The mutation potently affects anticoagulant pathways, as T-PD2 is less efficiently inhibited by ATIII (3-fold), binds heparin less tightly (10-fold), and much less efficiently (41-fold) activates protein C. Conclusions. Our data indicate that the procoagulant phenotype, observed in Arg221aTrp mutation carriers, may result from opposing inhibitory effects on both procoagulant and anticoagulant pathways, with the impact on anticoagulant pathways being predominant and shifting the natural anticoagulant↔procoagulant equilibrium in vivo towards thrombosis.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
