Evaluation of activation and activity of fII^MZ. (A) Schematic of prothrombin cleavage sites. An antibody was used that targets the N-terminus of prothrombin, and the immunoblot was performed under reducing conditions. Therefore, an activation event resulting in meizothrombin would result in a product including fragment 1.2 (F1.2) and the A chain (F1.2.A). Upon further cleavage to α-thrombin, fragments F1 and F2 are released from the A chain. (B-C) Activation of prothrombin was determined in both fII^WT and fII^MZ animals. Hemostatic factor activation was initiated with thromboplastin in plasma from fII^WT (B) and fII^MZ (C) animals. Aliquots were taken at specified time points and assayed via immunoblot for fII and products of fII activation. Although no remaining detectable fII or fragment 1.2.A is present in the fII^WT samples beyond 2 minutes, prothrombin from the fII^MZ animals remains at F1.2.A (representing meizothrombin) throughout the time period assayed. No further conversion/degradation was detected.