Limiting prothrombin activation to meizothrombin is compatible with survival but significantly alters hemostasis in mice

Thrombin-mediated proteolysis is central to hemostatic function but also plays a prominent role in multiple disease processes. The proteolytic conversion of fII to α-thrombin (fIIa) by the prothrombinase complex occurs through two parallel pathways: i) the inactive intermediate, prethrombin, or ii) the proteolytically active intermediate, meizothrombin (fIIa^MZ). FIIa^MZ has distinct catalytic properties relative to fIIa, including diminished fibrinogen cleavage and increased protein C activation. Thus, fII activation may differentially influence hemostasis and disease depending on the pathway of activation. To determine the in vivo physiologic and pathologic consequences of restricting thrombin generation to fIIa^MZ, mutations were introduced into the endogenous fII gene resulting in expression of prothrombin carrying three amino acid substitutions (R157A, R268A, and K281A) to limit activation events to yield only fIIa^MZ. Homozygous fIIMZ mice are viable, express fII levels comparable to fII^WT mice, and have reproductive success. Although in vitro studies revealed delayed generation of fIIa^MZ enzyme activity, platelet aggregation by fII^MZ is similar to fII^WT. Consistent with prior analyses of human fIIa^MZ, significant prolongation of clotting times was observed for fII^MZ plasma. Adult fII^MZ animals displayed significantly compromised hemostasis in tail bleeding assays, but did not demonstrate overt bleeding. More notably, fII^MZ mice had two significant phenotypic advantages over fII^WT animals: protection from occlusive thrombosis after arterial injury and markedly diminished metastatic potential in a setting of experimental tumor metastasis to the lung. Thus, these novel animals will provide a valuable tool to assess the role of both fIIa and fIIa^MZ in vivo.