Life-Threatening Thrombosis in Mice With Targeted Arg48-to-Cys Mutation of the Heparin-Binding Domain of Antithrombin

Abstract

Abstract-Antithrombin (AT) inhibits thrombin and some other coagulation factors in a reaction that is dramatically accelerated by binding of a pentasaccharide sequence present in heparin/heparan-sulfate to a heparin-binding site on AT.Based on the involvement of R47 in the heparin/AT interaction and the frequent occurrence of R47 mutations in AT deficiency patients, targeted knock-in of the corresponding R48C substitution in AT in mice was performed to generate a murine model of spontaneous thrombosis. The mutation efficiently abolished the effect of heparin-like molecules on coagulation inhibition in vitro and in vivo. Mice homozygous for the mutation (AT m/m mice) developed spontaneous, life-threatening thrombosis, occurring as early as the day of birth. Only 60% of the AT m/m offspring reached weaning age, with further loss at different ages. Thrombotic events in adult homozygotes were most prominent in the heart, liver, and in ocular, placental, and penile vessels. In the neonate, spontaneous death invariably was associated with major thrombosis in the heart. This severe thrombotic phenotype underlines a critical function of the heparin-binding site of antithrombin and its interaction with heparin/heparan-sulfate moieties in health, reproduction, and survival, and represents an in vivo model for comparative analysis of heparin-derived and other antithrombotic molecules.

Targeted mutagenesis of zebrafish antithrombin III triggers disseminated intravascular coagulation and thrombosis, revealing insight into function

“…Concordant with our data, embryonic At3 2/2 mutant mice display a consumptive coagulopathy with secondary hemorrhage, although this causes in utero thrombosis and subsequent demise. 5 It is notable that in both the complete mouse knockout and a heparin-binding mutant knockin (Arg48Cys), 55 the heart is one of the most prominent sites of spontaneous thrombosis. The structure of the adult zebrafish heart has been described in detail, 56 and overall cardiovascular development is highly comparable to mammalian and avian species.…”

Crystal Structure and Mutational Analysis of Heparan Sulfate 3-O-Sulfotransferase Isoform 1

“…A severe thrombosis phenotype is observed in mice carrying an AT mutant defective in heparin binding, suggesting a key role for HS-AT interaction in balancing procoagulant and anticoagulant activities in vivo (7). Additionally, patients with AT mutants defective in heparin and HS binding suffer from thrombosis (8 -10).…”

Normal levels of anticoagulant heparan sulfate are not essential for normal hemostasis

“…Heterozygotes remain protected, which further supports that only a subfraction of normal AT is required for HS act function. Finally, a critical role for HS act is suggested by knockin mice that exclusively express a type II heparin binding site mutant of AT and spontaneously die throughout the first year of life, apparently from gross thrombosis (11).…”