title: Limiting prothrombin activation to meizothrombin is compatible with survival but significantly alters hemostasis in mice.
abstract: 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 2 parallel pathways: (1) the inactive intermediate, prethrombin; or (2) 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 3 amino acid substitutions (R157A, R268A, and K281A) to limit activation events to yield only fIIa(MZ) Homozygous fII(MZ) mice are viable, express fII levels comparable with 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 2 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.
authors.affiliation: Division of Hematology and.
authors.forename: Maureen A
authors.initials: MA
authors.lastname: Shaw
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Cancer and Blood Diseases Institute, Cincinnati Children's Research Foundation, Cincinnati, OH;
authors.forename: Keith W
authors.initials: KW
authors.lastname: Kombrinck
authors.affiliation: Division of Hematology and.
authors.forename: Kathryn E
authors.initials: KE
authors.lastname: McElhinney
authors.affiliation: Division of Hematology and.
authors.forename: David R
authors.initials: DR
authors.lastname: Sweet
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Cancer and Blood Diseases Institute, Cincinnati Children's Research Foundation, Cincinnati, OH;
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Flick
authors.affiliation: Division of Hematology and.
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Palumbo
authors.affiliation: Coagulation Biology Laboratory, Oklahoma Medical Research Foundation, Oklahoma City, OK;
authors.forename: Mei
authors.initials: M
authors.lastname: Cheng
authors.affiliation: Coagulation Biology Laboratory, Oklahoma Medical Research Foundation, Oklahoma City, OK;
authors.forename: Naomi L
authors.initials: NL
authors.lastname: Esmon
authors.affiliation: Coagulation Biology Laboratory, Oklahoma Medical Research Foundation, Oklahoma City, OK;
authors.forename: Charles T
authors.initials: CT
authors.lastname: Esmon
authors.affiliation: Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom; and Program in Cellular and Molecular Medicine and Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Brill
authors.affiliation: Program in Cellular and Molecular Medicine and Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA.
authors.forename: Denisa D
authors.identifier: 0000-0002-4494-413X
authors.initials: DD
authors.lastname: Wagner
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Cancer and Blood Diseases Institute, Cincinnati Children's Research Foundation, Cincinnati, OH;
authors.forename: Jay L
authors.initials: JL
authors.lastname: Degen
authors.affiliation: Division of Hematology and.
authors.forename: Eric S
authors.identifier: 0000-0002-1544-9068
authors.initials: ES
authors.lastname: Mullins
mesh_terms: D000483:Alleles; D000818:Animals; D001777:Blood Coagulation; D003018:Clot Retraction; D003435:Crotalid Venoms; D004622:Embryo, Mammalian / Q000378:metabolism; D004792:Enzyme Precursors / Q000378:metabolism*; D005355:Fibrosis; D006487:Hemostasis; D008666:Metalloendopeptidases; D008810:Mice, Inbred C57BL; D009206:Myocardium / Q000473:pathology; D009362:Neoplasm Metastasis; D010974:Platelet Aggregation; D011516:Prothrombin / Q000378:metabolism*; D016019:Survival Analysis; D013917:Thrombin / Q000378:metabolism*; D013927:Thrombosis / Q000378:metabolism / Q000473:pathology
chemical_list: D003435:Crotalid Venoms; D004792:Enzyme Precursors; D011516:Prothrombin; D013917:Thrombin; C026812:meizothrombin; C478420:FIIa; D008666:Metalloendopeptidases