The natural mutation Arg221aTrp in human α-thrombin abrogates physiological Na+ binding and preferentially hinders the protease anticoagulant functions

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 α-thrombin (αT). 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 αT variants to inactivation by antithrombin III (ATIII). However, the biochemical mechanisms linking these mutations to the increased thrombotic risk remain elusive. Objectives To provide quantitative data on the effect that the recombinant Arg221aTrp αT mutant (αT-PD2) has on the activation of procoagulant and anticoagulant pathways and elucidate the structural basis of the observed functional changes. Methods A multipronged approach was used, integrating recombinant DNA techniques, clotting/fibrinolytic and enzymatic assays, microscopy, proteomics, spectroscopic, mass spectrometry, and molecular modeling techniques. Results Arg221aTrp mutation abrogates physiological Na+ binding and reduces the proportion of the fully active αT conformation. αT-PD2 affects procoagulant pathways by less efficiently generating fibrin (7-fold) and activating platelets (10-fold). The mutation potently affects anticoagulant pathways, as αT-PD2 is less efficiently inhibited by ATIII (3-fold), binds heparin less tightly (10-fold), and much less efficiently (41-fold) activates protein C. Conclusion 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 toward thrombosis.