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

Abstract

Key Points Juvenile zebrafish tolerate widespread coagulopathy due to complete ablation of antithrombin III, but develop lethal thrombosis as adults. In vivo structure/function analysis of antithrombin III in zebrafish reveals limited roles for heparin-binding and anti-IXa/Xa activity.

Conformational Activation of Antithrombin by Heparin Involves an Altered Exosite Interaction with Protease

“…3 Moreover, the observed 3-8-fold decreases in heparin affinity caused by the Tyr 253 /His 319 mutations (Table 6) suggest that the activated fraction in these mutants is considerably reduced from wild-type. 4 The observed 1 to 2 order of magnitude losses in wild-type antithrombin reactivity produced by Tyr 253 and His 319 mutations in the absence of heparin thus imply that the mutations are almost exclusively affecting an intrinsic native state reactivity and not just the reactivity of a minor equilibrium fraction of activated serpin. 253 and His 319 for exosite function is supported by the ability to create a factor Xa/IXaspecific exosite in a P1 Arg variant of the related serpin, ␣ 1 -protease inhibitor, in which His 319 is conserved, by substituting Tyr in the homologous strand 3C 253 position (29).…”

“…If Tyr 253 /His 319 mutations only affected the 0.1% activated fraction, then the observed reactivities of these exosite mutant antithrombins in the absence of heparin should not be less than ϳ2500 M Ϫ1 s Ϫ1 and ϳ150 M Ϫ1 s Ϫ1 . 4 Assuming the changes in heparin affinity of the exosite mutants reflect changes in the conformational equilibrium between native and activated states of antithrombin in the unactivated serpin and given the observation that the observed wild-type heparin affinity significantly exceeds that of the native state (28), the fold-change in the fraction of activated antithrombin caused by the exosite mutations can be approximated by the ratio, critical Tyr 253 and His 319 residues were present and otherwise enhanced reactivity (Fig. 7).…”

“…The physiologic importance of this anticoagulant protein is indicated from the increased risk of thrombotic diseases associated with acquired or inherited deficiencies of the protein in humans (2) and the lethality and coagulopathy associated with complete antithrombin deficiency in mice (3) and zebrafish (4). Antithrombin circulates in the blood in a repressed state capable of slow inhibition of its target coagulation proteases and becomes activated at sites of injury by extravascular heparin and heparan sulfate glycosaminoglycans to localize and prevent the dissemination of blood clots (5,6).…”

Animal Models of Thrombosis From Zebrafish to Nonhuman Primates

“…157,162 The recent introduction of the 146 Gregory et al, 147 Hughes et al, 154 Day et al, 155 Khandekar and Jagadeeswaran, 156 Tournoij et al, 158 Williams et al, 159 and O'Connor et al 161 …”

“…146,156 Recently, the zinc finger nuclease knockout method was used to mutate the antithrombin III gene, which modeled disseminated intravascular coagulation in zebrafish. 157 With all these available tools, it should be possible to discover more novel factors that participate in thrombosis. To date, the laser injury model was used in conjunction with knockdown methods to analyze the role of prothrombin, factor VII, factor VIIi, hepsin, FSAP, Mlck1a, protein kinase c α, protein kinase c β, G6fl, and fibrinogen in hemostasis.…”