OC 75.5 - Prothrombin-Wauwatosa, a natural prothrombin variant with increased anticoagulant function and increased bleeding

Background: A mucocutaneous bleeding predisposition, consistent with an autosomal dominant disorder, was identified in multiple related individuals. PT, PTT, and thrombin time were within normal limits, as were clinical tests of von Willebrand factor, platelet function, and rare bleeding disorders. Genetic sequencing of hematologic proteins identified a heterozygous prothrombin variant (Tyr242Cys; Prothrombin-Wauwatosa), which linked with the bleeding phenotype. Plasma prothrombin concentration was normal, but PT-based prothrombin activity was ~50%.

Aims: Determine the mechanism of Prothrombin-Wauwatosa-associated bleeding.

Methods: This study was approved by the Institutional Review Board. Biochemical analyses were performed on samples from affected family members and unaffected controls (2 female, 1 male each).

Results: Reduced thrombin generation was observed in platelet-poor and platelet-rich plasma in all patient samples following tissue factor or contact pathway initiation. By western blot, prothrombin cleavage was normal, but minimal thrombin was detectable. Exogenous prothrombin corrected thrombin generation, suggesting the prothrombin variant as causative. Tyr242 is in fragment 2 of prothrombin, a region removed upon thrombin activation, and therefore cannot directly impact thrombin activity. Given the proximity of the variant to the Arg271 activation site, we hypothesized impaired release of the activation fragments results in a meizothrombin-like product. Meizothrombin is an activation intermediate of thrombin that efficiently activates protein C (PC), even in the absence of thrombomodulin. Thrombin generation was corrected by mixing Prothrombin-Wauwatosa plasma with plasma depleted of either PC or its cofactor protein S. In control samples, addition of thrombomodulin reduced thrombin generation by >90%, but only by < 50% in samples containing Prothrombin-Wauwatosa, consistent with reduced anticoagulant dependence on thrombomodulin.

Conclusion(s): Prothrombin-Wauwatosa (Tyr242Cys) is an autosomal dominant variant, which causes a moderate bleeding phenotype and is dependent on PC but relatively independent of thrombomodulin. Our data support a novel mechanism in which Prothrombin-Wauwatosa activation results in significant accumulation of a meizothrombin-like product that expresses significant anticoagulant activity.