2-Antiplasmin Gene Deficiency in Mice Is Associated With Enhanced Fibrinolytic Potential Without Overt Bleeding

Abstract

2-antiplasmin (2-AP) is the main physiologic plasmin inhibitor in mammalian plasma. Inactivation of the murine 2-AP gene was achieved by replacing, through homologous recombination in embryonic stem cells, a 7-kb genomic sequence encoding the entire murine protein (exon 2 through part of exon 10, including the stop codon) with theneomycin resistance expression cassette. Germline transmission of the mutated allele was confirmed by Southern blot analysis. Mendelian inheritance of the inactivated 2-AP allele was observed, and homozygous deficient (2-AP−/−) mice displayed normal fertility, viability, and development. Reverse transcription-polymerase chain reaction confirmed the absence of 2-AP mRNA in kidney and liver from 2-AP−/− mice, in contrast to wild-type (2-AP+/+) mice. Immunologic and functional 2-AP levels were undetectable in plasma of 2-AP−/− mice, and were about half of wild-type in heterozygous littermates (2-AP+/−). Other hemostasis parameters, including plasminogen activator inhibitor-1, plasminogen, fibrinogen, hemoglobin, hematocrit, and blood cell counts were comparable for 2-AP+/+, 2-AP+/−, and 2-AP−/− mice. After amputation of tail or toe tips, bleeding stopped spontaneously in 2-AP+/+, as well as in 2-AP+/− and 2-AP−/− mice. Spontaneous lysis after 4 hours of intravenously injected 125I-fibrin–labeled plasma clots was significantly higher in 2-AP−/− than in 2-AP+/+ mice when injecting clots prepared from 2-AP+/+ plasma (78% ± 5% v 46% ± 9%; mean ± SEM, n = 6 to 7; P = .02) or from 2-AP−/−plasma (81% ± 5% v 46% ± 5%; mean ± SEM, n = 5; P = .008). Four to 8 hours after endotoxin injection, fibrin deposition in the kidneys was significantly reduced in 2-AP−/− mice, as compared with 2-AP+/+ mice (P ≤ .005). Thus, 2-AP−/− mice develop and reproduce normally; they have an enhanced endogenous fibrinolytic capacity without overt bleeding.

Thrombin-activatable fibrinolysis inhibitor (TAFI) deficiency is compatible with murine life

“…Following LPS injection (either 30 mg/kg or 40mg/kg), the survival rates were similar between TAFI-deficient and the wild-type mice (Figure 4). Because of reports on transient fibrin deposition in the kidney of mice injected with LPS (34,45), in the separate experiments kidneys from animals injected with either a sublethal dose (2 mg/kg) or a lethal dose (30 mg/kg) of LPS 3 hours earlier were stained for fibrin. While there was some specific fibrin staining within the glomeruli of both the wild-type and TAFI-deficient littermates following LPS injection, there was no significant difference in the extent of staining between the two groups (data not shown).…”

“…On the other hand, the effectiveness of an enhanced endogenous fibrinolytic system against a lethal dose of LPS has not been reported. However, reduced renal fibrin deposition in response to a nonlethal dose of endotoxin has been demonstrated in α 2 -antiplasmin-deficient mice (34,64), but not in PAI-1-deficient mice (64). In the current study, we did not detect any difference in the extent of fibrin deposition in the kidney of wild-type and their TAFI-deficient littermates in response to LPS.…”

“…Mouse models deficient in each component of the fibrinolytic system have been generated to examine their role in development, thrombosis, thrombolysis, reproduction, wound repair, and cell migration (30)(31)(32)(33)(34). Unlike deficiency in coagulation factors such as tissue factor or factor V, which are embryonic lethal, deficiency in a component of the fibrinolytic system results in animals that develop normally with no obvious phenotypic abnormalities.…”

“…However, plasminogendeficient mice suffered spontaneous thrombosis, and wound healing was grossly impaired (32,33). Furthermore, plasminogen activator-deficient mice had impaired lysis of microthrombi in the lung and increased fibrin deposition in response to endotoxin challenge (31), while mice deficient in either PAI-1 or α2-antiplasmin had increased thrombolysis and were more resistant to endotoxin-induced thrombosis (30,34). To examine the consequence of TAFI deficiency on endogenous fibrinolysis, as well as other prothrombotic challenges, we generated homozygous TAFI-deficient mice by targeted gene disruption.…”

α2-Antiplasmin: New Insights and Opportunities for Ischemic Stroke

“…34 Mice with genetic deficiency of a2AP (a2AP À/À ) induced by gene targeting have normal fertility, viability, and coagulation. 38 There was no increased bleeding or rebleeding after surgical procedures, including tail bleeding. 38 In a similar fashion, selective a2AP inactivation in vivo by monoclonal antibodies has not been associated with bleeding or consumption of fibrinogen or plasminogen in preclinical studies (see below).…”