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. © 2018 Bosko M. Stojanovski et al., published by De Gruyter 2018.