Abstract
Meizothrombin is an active intermediate generated during the proteolytic activation of prothrombin to thrombin in the penultimate step of the coagulation cascade. Structurally, meizothrombin differs from thrombin because it retains the auxiliary Gla domain and two kringles. Functionally, meizothrombin shares with thrombin the ability to cleave procoagulant (fibrinogen), prothrombotic (PAR1) and anticoagulant (protein C) substrates, although its specificity toward fibrinogen and PAR1 is less pronounced. In this study we report information on the structural architecture of meizothrombin resolved by SAXS and single molecule FRET as an elongated arrangement of its individual domains. In addition, we show the properties of a meizothrombin construct analogous to the anticoagulant thrombin mutant W215A/E217A currently in Phase I for the treatment of thrombotic complications and stroke. The findings reveal new structural and functional aspects of meizothrombin that advance our understanding of a key intermediate of the prothrombin activation pathway.
Highlights
Binding of prothrombin to prothrombinase in Dedicated to Paul Placeholder the closed conformation exposes R320 for cleavage to Meizothrombin (Mz) is an important physiological generate meizothrombin, which assumes an open intermediate generated during the proteolytic conversion conformation promoting cleavage at R271, shedding of the of of pth1roethScrootamugbudilnaiteiionsntoactanhsrcdoamdIebninv[1-e7in]s. ttThihegepaeptnriouotlhtnirmosmatbeinsatespe auxiliary domains and generation of thrombin [16]
We report new details on the structure and function of Mz
We use small angle X-ray scattering (SAXS) and single molecule Förster resonance energy transfer to unravel features of the conformation of this intermediate that can be compared to those recently reported for prothrombin [12, 13, 15, 16]
Summary
Receptor thrombomodulin more efficiently than thrombin, due to the accelerating effect that phospholipids have on its anticoagulant function [18-20, 22]. The aforementioned catalytic properties implicate that Mz may function as a better anticoagulant enzyme than thrombin due to reduced activity toward procoagulant (fibrinogen) and prothrombotic (PAR1) substrates, linked to a slightly more pronounced activity toward the anticoagulant protein C in the presence of phospholipids [18, 19]. We report new details on the structure and function of Mz. We use small angle X-ray scattering (SAXS) and single molecule Förster resonance energy transfer (smFRET) to unravel features of the conformation of this intermediate that can be compared to those recently reported for prothrombin [12, 13, 15, 16].
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