Background: Thrombin (αT) is the final effector protease in the coagulation cascade, converting fibrinogen into fibrin clots and inducing platelets agglutination via PAR-1 cleavage. αT is a Na⁺-activated enzyme and is irreversibly inhibited by antithrombin-III (AT-III). Simioni and colleagues have recently identified the mutation p.Arg596Trp in the prothrombin gene (Padua-2) of heterozygous members from two families, with strong pro-coagulant phenotype [Bulato et al., JTH (2016)]. The plasma of these patients showed significant prolongation of clotting time in thrombin generation assays, compared to healthy subjects, and almost complete resistance to AT-III.

Aims: Provide the structural basis for the pro-coagulant phenotype of patients with Padua-2 prothrombin mutation.

Methods: rPre2-PD2 was cloned and expressed in E. coli and, after disulfide renaturation and purification, activated with ecarin to yield rαT-PD2; the purity, chemical identity, disulfide bonds topology of rαT-PD2 was established by SDS‑PAGE, RP‑HPLC and MS; fibrin clotting parameters, the kinetic constants (k_cat, K_m) for the cleavage of the chromogenic substrate S2238, release of fibrinopeptides A and B (FpA, FpB), and inhibition by AT-III were determined as described [Pontarollo et al., JBC (2017)].

Results: For S2238 hydrolysis, the specificity constant (k_cat/K_m) determined with rαT-PD2 was 6-fold lower than that estimated for rαT. Likewise, rαT-PD2 releases FpA and FpB by 25- and 7-fold much less efficiently, respectively. Fibrin clotting time (t_c) on isolated was estimated as 22.7min for rαT-PD2 and 0.5min for rαT. Notably, rαT-PD2 was inhibited by AT-III (K_a = 2.6±0.2·10⁶ M⁻¹ min⁻¹) less efficiently than rαT (K_a = 7.3±0.2·10⁶ M⁻¹ min⁻¹).

Conclusions: Modelling studies indicate that Arg221a-Trp mutation in the Na+-binding loop disrupts the stabilizing salt bridge with Glu146 in the γ-loop, which may collapse onto the active site and part of exosite-I impairing substrates (fibrinogen) and inhibitors (AT-III) binding. This structural model is fully consistent with biochemical data reported above and previous clinical data.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/structural-basis-of-the-pro-coagulant-phenotype-of-prothrombin-variant-arg553trp-p-arg596trp-padua-2/