Published online before print October 30, 2003, doi: 10.1161/01.RES.0000103634.69868.4F
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© 2003 American Heart Association, Inc.
Integrative Physiology |
Life-Threatening Thrombosis in Mice With Targeted Arg48-to-Cys Mutation of the Heparin-Binding Domain of Antithrombin
From the Center for Transgene Technology and Gene Therapy (M.D., G.W., L. Moons, D.C., P.C.), VIB, KULeuven Campus Gasthuisberg O&N, Leuven, Belgium; Cardiovascular/Thrombosis Research Department (J.-P.H., M.P., P.S., L. Millet, J.-M.H.), Sanofi-Synthélabo, Toulouse Cedex, France; Hamilton Civic Hospitals Research Centre (J.I.W.), Hamilton, Ontario, Canada.
Correspondence to Mieke Dewerchin, PhD, Center for Transgene Technology and Gene Therapy, VIB, KULeuven Campus Gasthuisberg O&N, Herestraat 49, B-3000 Leuven, Belgium. E-mail mieke.dewerchin{at}med.kuleuven.ac.be
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Abstract |
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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 (ATm/m mice) developed spontaneous, life-threatening thrombosis, occurring as early as the day of birth. Only 60% of the ATm/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.
Key Words: coagulation • gene targeting • knock-in • heparin • antithrombin deficiency
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Introduction |
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Antithrombin (AT) is a major physiological coagulation inhibitor, primarily inhibiting thrombin and factor Xa.1 In the presence of heparin, a potent anticoagulant and antithrombotic agent, the moderate inactivation rate by AT is dramatically accelerated. Heparan-sulfate proteoglycans intercalated in the endothelial cell membrane are thought to have a similar effect in vivo.2 The acceleration depends on a specific pentasaccharide sequence within the heparin glycosaminoglycan chain, binding of which induces a conformational change in the AT molecule.3,4 The interaction with the pentasaccharide appears sufficient to enhance the inhibition of factor Xa. However, full enhancement of thrombin or factor IX inhibition requires heparin species with longer polysaccharide chains.5–8 Biochemical and structural studies with wild type and variants of AT have shown that the heparin/pentasaccharide-binding domain of AT involves two clusters of basic amino acids (residues 41 to 49 and 107 to 156).1,5,8–12
The importance of AT in maintaining normal hemostasis is emphasized by the increased incidence of thromboembolism in individuals with inherited deficiency.1,13 Among these are several mutations affecting the heparin-binding site (type II HBS AT deficiency). Heterozygous type II HBS patients have a low incidence of thrombosis, whereas all homozygous patients display thrombotic disease that may include venous as well as arterial events.1 To date, 70 reports on 12 distinct HBS mutations have been published,1,13,14 35 of which affect R47 [substitution to cysteine (19 reports), histidine,15 or serine1; see also16].
Heparin-based pentasaccharides or mimetics, which act, at least in part, by binding to the heparin-binding site in AT, have potent anticoagulant and antithrombotic properties.4,15,17 To develop a murine model of thrombosis suitable for evaluation of such anticoagulants, mutant mice with targeted Arg48-to-Cys substitution (R48C; corresponding to the human "Toyama" R47C mutation) were generated.
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Materials and Methods |
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Generation of the AT Mutant Mice
A targeting vector, pPNT.ATneo, was constructed to replace the wild-type exon 2 with a mutated exon 2 encoding the R48C substitution and an additional silent mutation creating a diagnostic SacI site (for details, see the online data supplement and online Figure S1, available at http://www.circresaha.org). Electroporation of R1 embryonic stem cells, subsequent Cre-mediated excision of the loxP-flanked neo cassette, generation of mice, and genotyping and expression controls were performed as detailed in the online data supplement. All experiments were performed on littermates. Housing and procedures involving experimental animals were approved by the Institutional Animal Care and Research Advisory Committee of the KULeuven, Belgium.
Developmental, Histological, and Immunohistochemical Analysis
Animals and embryos were initially evaluated by visual inspection and stereomicroscopy. Organs of dead or anesthetized saline- and fixative-perfused mice or embryos were processed for immunohistochemical analysis as described.18 Fibrin(ogen) staining was as described.19 Histological staining for collagen (Sirius red) and reticulin (Gordon and Sweets silver staining) were performed using standard procedures.
Coagulation and Hematological Parameters
Whole blood was collected from anesthetized animals by cardiac puncture into 0.1 vol 3.2% trisodium citrate and centrifuged twice for 10 minutes at 3000 rpm to obtain plasma. All blood collections proceeded fast and with instantaneous mixing with the citrate solution. Measurements were performed immediately after sample preparation or after storage at -80°C without intermediate thawing. Antithrombin inhibitory activity was measured using the Coamatic amidolytic anti-Xa heparin cofactor assay (Chromogenix, Sweden). Progressive fXa inhibitory activity was measured analogously but using heparin-free buffer and longer reaction times. Coagulant activities were determined in one-stage clotting assays as described.19 A similar analysis was performed after preconditioning of the mice with lepirudin to temporarily neutralize a possible in vivo activation of the intrinsic coagulation factors (see online data supplement). Plasma fibrinogen was determined by a coagulation rate assay.20 Factor VIII plasma levels were additionally determined using the Coatest Factor VIII kit (Chromogenix, Sweden)21 and by ELISA.22 Plasma AT antigen levels were determined by rocket immunoelectrophoresis23 using a polyclonal rabbit anti-human AT antibody (Dako) that cross-reacts with murine AT. Blood cell counts were determined as described.18 Prothrombin time (PT) and activated partial thromboplastin time (aPTT) was measured using routine assays. Plasma TAT levels were determined using the Enzygnost TAT kit (Dade Behring). Blood was collected, processed, and assayed for FPA as described.24
Thrombin Generation in Plasma and Venous Thrombosis Model
Platelet poor plasma was pooled from at least 3 mice, defibrinated, and 20 µL-aliquots were used for continuous monitoring of tissue factor-induced thrombin generation25 (see online data supplement), with or without addition of the pentasaccharide fondaparinux (Arixtra),17,26 the heparin mimetic SanOrg123781A,27 heparin, or hirudin (Sanofi-Synthélabo) as indicated. Thrombus formation in vivo was measured in a thromboplastin-induced vena cava stasis model with or without administration of fondaparinux (see online data supplement).
Statistical Analysis
Data are mean±SD unless otherwise indicated. The statistical significance of differences between groups was determined by unpaired t test, unless mentioned otherwise. A value of P<0.05 was considered significant.
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Results |
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Targeted R48C Mutation of the AT Gene
Mice with an Arg48-to-Cys (R48C) substitution were generated by targeted mutation using homologous recombination in embryonic stem cells (see online Figure S1a). This mutation corresponds to the most prevalent of the type II mutations affecting heparin binding in patients with hereditary AT deficiency, which have an increased risk of thrombosis (Toyama type II HBS AT deficiency).14,28 RNA analysis (semiquantitative RT.PCR on liver mRNA; not shown), and plasma antigen determination showed normal expression of the mutant protein in heterozygous (AT+/m) and homozygous (ATm/m) mutant mice (plasma antigen levels by rocket immunoelectrophoresis were 100±8% in AT+/m and 110±10% in ATm/m mice, n=3 to 4, P=NS; expressed in percent of wild-type control values). DNA sequencing of the entire cDNA prepared from ATm/m liver samples confirmed the integrity and correct mutation of the expressed message (not shown).
Viability and Survival
Among 167 progeny of heterozygous parents, ATm/m offspring were somewhat underrepresented at birth (P0: 16% instead of the expected 25%) and suffered frequent neonatal death, often within 24 hours after birth, with only 60% of them reaching weaning age (Table 1). Spontaneous death of AT+/m neonates was occasionally observed. Beyond weaning age, spontaneous death among wild-type and heterozygous animals was rare, whereas survival of ATm/m mice remained compromised with death at various ages and only about 30% survival beyond 6 months. No underrepresentation of ATm/m offspring was found at embryonal ages up to embryonal day (E) 18.5 (Table 1), suggesting that the early loss of ATm/m pups occurred during or immediately after birth, likely with immediate cannibalism by the mother (occasionally witnessed) and therefore without recovery of these dead pups.
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Heparin Binding and Inhibitor Activity of the R48C AT Mutant Protein
ATm/m plasma samples displayed low heparin cofactor activity in the Coamatic fXa inhibition assay (96±4.3% residual fXa activity at 1.5 minutes versus 8.4±2% for wild type; mean±SD, n=6 to 8, P<0.001) but normal progressive fXa inhibitory activity (37±7.8% residual fXa activity at 30 minutes versus 42±10% for wild type; mean±SD, n=6 to 8, P=NS). Tissue factor-induced thrombin generation measured in defibrinated plasma from ATm/m animals was comparable to that in wild-type samples (71±10 versus 63±3 mOD for wild type; mean±SD, n=3, P=NS) (Figure 1A). However, the R48C mutation totally prevented the action of AT-mediated inhibitors added at concentrations up to 8-fold higher than those affecting wild-type AT (heparin, pentasaccharide, or the heparin mimetic SanOrg123781A, which comprises both an AT and a thrombin-binding domain27), whereas the effect of direct inhibitors (hirudin) was not affected (Figure 1A).
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In vivo, using a stasis-plus-tissue factor-induced thrombosis model in the caval vein, the pentasaccharide inhibited thrombus formation in wild-type mice (69% inhibition), but not in ATm/m mice (only 5% inhibition; P=NS) (Figure 1B). These results confirm effective abolition of heparin interaction by the R48C mutation.
Spontaneous Thrombosis in ATm/m Mice
Homozygosity for the R48C mutation was associated with spontaneous, often massive, thrombosis in the heart and, less frequently, lungs. Out of 16 adult ATm/m mice euthanized for histology at different ages (2 to 17 months), 6 showed massive thrombosis in the atria and/or ventricles, staining positive for fibrinogen/fibrin (Figures 2a and 2b), and often associated with leukocyte infiltration (Figure 2b). Out of 14 adult ATm/m mice, 3 showed fibrin deposition and/or vessel occlusion in the lungs (Figures 2c and 2d). In addition, although obstruction of hepatic blood flow was not directly observed, signs of portal hypertension were seen in the majority of adult ATm/m mice analyzed (12/15), characterized by nodular regenerative hyperplasia (Figures 2e and 2f), dilatation of the sinusoids and formation of shunt vessels (Figures 2g and 2h). Abnormalities at the cellular level included macrovesicular steatosis and the presence of neutrophil clusters in the sinusoids and of phagocytosing macrophages in the parenchyme indicating an inflammatory response (not shown). In 1-day-old ATm/m pups (2 of 4 analyzed), infarcted zones in the liver with coagulative necrosis were observed (Figures 3a through 3c), presumably illustrating acute impaired blood flow in the liver and contributing to the development of the liver pathology. Although liver/body weight ratios were normal in ATm/m animals, they showed enlarged spleens (see online data supplement), which is frequently seen in liver disease. Both males (8/31; 26%) and, more frequently, females (9/19; 47%) displayed severe degeneration of the eyes (Figures 2i and 2j), often with disruption of the retina and occasionally perforation of the cornea (not shown), likely due to ocular vein occlusion (Figure 2k). No obvious thrombosis was observed in other organs nor in the larger vessels (caval vein, femoral artery and vein, brachial vein; not shown). However, in animals used for breeding, severe thrombosis was observed in the placenta of pregnant females, and in the penile veins of sexually active males. Placental thrombosis in ATm/m females (Figures 2l and 2m) occurred irrespective of the genotype of the embryo (Table 1), and likely caused the decreased litter sizes observed for ATm/m mothers (3.9±2.8 pups per litter, n=7, versus 8.6±2.9 for AT+/m mothers, n=44; mean±SD, P<0.005). Fifty percent of all sexually active males developed irreversible priapism (9 out of 18 mated males) due to occlusion of the dorsal penile vein and impaired drainage and thrombosis of the corpora cavernosa (not shown).
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All ATm/m neonates [6 at postnatal day (P) 1 or P2, one at P7] recovered dead and analyzed histologically, showed massive thrombosis in the heart (Figures 3d and 3e). Three succumbed heterozygous AT+/m neonates (age P1) also revealed a small clot in the heart and an occluded lung vessel in one of them (not shown). Neonates euthanized immediately after birth (P0) revealed, apart from the liver infarction zones mentioned above, clots in the heart in 2 out of 4 ATm/m pups (not shown), whereas no abnormalities in the wild-type or AT+/m neonates were observed.
Histological analysis of ATm/m offspring at E18.5 revealed no abnormalities (7 embryos analyzed; not shown). However, although rare, thrombosis was observed at earlier embryonal age: in 1 out of 16 E14.5 ATm/m embryos, a thrombus was found in the atrium, and a second embryo showed bleeding and fibrin deposition in the myocardial tissue, whereas all of 18 AT+/m and 4 AT+/+ embryos appeared normal (not shown).
Hemostasis Parameters in Adult Wild-Type and Mutant Mice
Hematological parameters (blood cell counts, hematocrit, platelets; not shown), plasma TAT, and fibrinogen levels (Table 2) in AT+/m and ATm/m blood samples were similar to those in wild-type samples. When measured using one-stage clotting assays, which are sensitive to preactivation of the coagulation factors, intrinsic clotting factors were elevated (Table 2). However, at least for fVIII, this increase was not observed in a preactivation-insensitive two-stage activity assay (Table 2).21 Similarly, fVIII antigen levels by ELISA were comparable in AT+/+ and ATm/m mice (500±100 ng/mL for ATm/m versus 410±230 for AT+/+ mice; mean±SD, n=4 to 8, P=NS). These results suggested a low level continuous activation of the coagulation system in ATm/m mice, consistent with the observed thrombotic phenotype. This possibility was further verified by one-stage clotting assay measurements on samples from mice preconditioned with lepirudin to prevent in vivo activation. Levels of intrinsic factors in lepirudin-treated ATm/m mice were reduced to values close, although not entirely comparable, to those of wild-type mice (for details, see online data supplement). A similar increase in intrinsic factor levels is seen in mice with targeted truncation of tissue factor. Presumably, this mutation results in the generation of soluble tissue factor. These mice exhibit severe thrombosis, but have a normal antithrombin molecule (Melis E, Moons L, Arnout J, Collen D, Carmeliet P, Dewerchin M, unpublished data, 2003). Taken together, these data suggest that low level activation of coagulation may be a feature of thrombosis-prone mice. Plasma FPA levels were normal (12±3.8 nmol/L for ATm/m mice versus 13±4.2 nmol/L for wild-type control mice; mean±SD, n=4 to 5, P=NS). Prothrombin times (PT) and activated partial thromboplastin times (aPTT) also were normal in ATm/m mice (PT, 9.8±0.9 versus 9.8±1.9 seconds in AT+/+ mice, mean±SD, n=9 to 11, P=NS; aPTT, 28±3.8 versus 32±4 seconds in AT+/+ mice, mean±SD, n=6, P=NS), although aPTT values tended to be slightly reduced.
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Discussion |
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In this study, knock-in of the R48C mutation in the heparin-binding site (HBS) of murine AT was shown to result in life-threatening, spontaneous thrombosis during neonatal and adult life. The mutant AT was synthesized and present in the plasma at levels comparable to those of wild-type AT in wild-type mice. Similar to the corresponding human R47C mutation,29 plasma from the ATm/m mice showed low heparin cofactor activity but normal progressive fXa inhibitory activity. The R48C mutation abolished the action of AT-mediated inhibitors (heparin, heparin mimetic, or AT-binding pentasaccharide) in plasma, whereas the effect of direct thrombin inhibitors was not affected. The less pronounced inhibition by the pentasaccharide as compared with heparin (mimetic) in wild-type plasma (Figure 1A) shows that the effect of the latter molecules on coagulation via AT not only results from inhibition of factor Xa but also of other coagulation enzymes such as thrombin. However, the lack of inhibition of thrombin generation by the pentasaccharide and heparin mimetic in ATm/m plasma, indicates that the R48C mutation effectively abolishes the functional interaction that results in AT-mediated inhibition of factor Xa and/or thrombin by these molecules. The weak dose-dependent response observed with heparin may reflect an AT-independent effect, possibly related to heparin cofactor II activation. These in vitro and in vivo results, together with the spontaneous thrombotic phenotype of the ATm/m mice, underline the importance of heparin binding of AT for efficient inhibition of coagulation and show that normally functioning AT plays a key role with regard to other "natural" inhibitors such as heparin cofactor-II in particular.
Thrombosis in the adult ATm/m mice involved the heart, lung, liver, and eyes, and the reproductive organs in sexually active animals. Unlike in adult AT+/m animals, spontaneous death and thrombotic events did occur in AT+/m neonates, although less frequently and less severely than in ATm/m neonates. This might be due to a critical imbalance between pro- and anticoagulation in a fraction of the AT+/m neonates, likely due to the presence of the mutation in one allele on top of presumed lower neonatal plasma AT levels.30 Spontaneous thrombosis was more frequent and more severe in homozygous neonates, accounting for the frequent early loss of ATm/m offspring. As in humans, our R48C phenotype was less severe than the embryonic lethal phenotype of AT-null mice.31 However, we occasionally observed fibrin deposition in the myocardium in E14.5 ATm/m embryos, reminiscent of the AT-null findings,31 although not in older embryos (E18.5). Whether such affected E14.5 ATm/m embryos recover or do not survive remains unclear. Nevertheless, the normal genotype distribution at these embryonal ages, the absence of apparent abnormalities before birth (E18.5), and the obvious thrombotic problems from early neonatal life onwards, suggest that the trauma of birth represents a major trigger of thrombosis in the mutant mice.
Unlike patients with homozygous type II HBS AT deficiency,1,29 ATm/m mice displayed no obvious large vessel thrombosis, but showed massive thrombosis in the heart, severe thrombotic eye disease, liver pathology consistent with portal hypertension, placental thrombosis, and priapism. However, ocular vein occlusion, portal vein thrombosis, placental thrombosis, and although rarely, priapism are observed in patients with Factor V Leiden, prothrombin mutation, or decreased protein S, protein C, or AT levels.32–36 Thrombosis in the heart has been described in neonates with proved or suspected AT deficiency, in addition to thrombus formation in the large vessels and intracranial venous sinus (see review37).
The ATm/m thrombotic phenotype showed signs of inflammatory response (leukocyte infiltration in thrombi, neutrophil clusters, and phagocytosing macrophages in the liver). This response might contain a direct AT-dependent component, not only through impaired thrombin inhibition and increased PAR-mediated cytokine production, but also by impaired binding to cell surface heparan-sulfate proteoglycans (HSPG). Indeed, AT, via interaction with HSPG present on endothelial cells or on neutrophils, promotes the release of antiinflammatory prostacyclin and blocks the activation of the proinflammatory NF-
B, thereby decreasing platelet and neutrophil activation, chemotaxis, and interaction with the endothelium,38–41 effects that are lost after chemically blocking the heparin-binding domain of AT.39,40
No thrombotic phenotype was observed so far in mice with altered heparan-sulfate (HS) moieties or deficiencies in the HSPG core proteins. 3-O-sulfation of the pentasaccharide core sequence of heparin/heparan-sulfate is essential for interaction with AT.42 This modification is thought to be catalyzed mainly by heparan-sulfate-3-O-sulfotransferase-1 (3-OST-1), which consequently plays a major role in the synthesis of AT-binding anticoagulant HS.43 However, mice deficient in 3-OST-1 did not display a procoagulant phenotype, perhaps due to redundancy by other 3-OST isoforms.44 Of note is the phenotype of mice deficient in glycosaminyl N-deacetylase/N-sulfotransferase-2, which lack endogenous sulfated heparin but show no obvious signs of thrombosis, indicating that endogenous heparin is not critically involved in coagulation.45 On the other hand, mice deficient in the HSPG core protein syndecan-4 are healthy and fertile, but show impaired coagulation in fetal vessels in the placental labyrinth.46
Heterozygous AT knockout mice recently were reported to develop thrombosis only after challenge, largely analogous to heterozygous AT type I deficiency patients in which one AT allele is not expressed giving low functional and immunological AT.47 Mice deficient in heparin cofactor II displayed a shorter time to thrombotic occlusion of the carotid artery after endothelial denudation, but otherwise did not show spontaneous thrombosis nor other morphological abnormalities.48 In contrast, transgenic mice with inactivation or mutation of plasminogen system components49 or of coagulation inhibitors31,50,51 display spontaneous thrombotic phenotypes that, however, are either mild, or more severe with early, sometimes embryonal, lethality, or characterized by additional nonthrombotic abnormalities.
In conclusion, knock-in of an R48C substitution in the heparin-binding site of antithrombin in mice effectively abolished the effect of heparin or heparin derivatives on coagulation inhibition in vitro and in vivo. Homozygous mutant mice displayed life-threatening thrombosis at different sites, most prominently in the heart, liver, and in ocular, placental, and penile vessels, and represent an in vivo model for spontaneous thrombosis suitable for the analysis of heparin-like and other antithrombotic molecules.
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Acknowledgments |
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Acknowledgments
The authors gratefully acknowledge the excellent assistance of C. Gaich, S. Grailly, E. Gils, A. Hubert, L. Kieckens, R. Lavend’homme, T. Vancoetsem, A. Van Nuffelen, M. Vanrusselt, the assistance with artwork by A. Vandenhoeck and S. Jansen, and the help with statistics by N. Boussac-Marlière. We are grateful to P. Lollar (Emory University, Atlanta, Ga) for kindly supplying the rabbit anti-murine fVIII antibody, to I. Stalmans for help with the eye phenotype, to T. Roskams (University Hospital, Leuven) for help with the liver pathology, and to J. Arnout, E. Conway, and M. Jacquemin for helpful advice and critical discussion.
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Footnotes |
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Original received February 25, 2003; resubmission received July 23, 2003; revised resubmission received October 16, 2003; accepted October 16, 2003.
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