L-asparaginase-induced antithrombin type I deficiency: implications for conformational diseases - PubMed (original) (raw)

L-asparaginase-induced antithrombin type I deficiency: implications for conformational diseases

David Hernández-Espinosa et al. Am J Pathol. 2006 Jul.

Abstract

Serpinopathies, a group of diseases caused by mutations that disrupt the structurally sensitive serpins, have no known acquired cause. Interestingly, l-asparaginase treatment of acute lymphoblastic leukemia patients causes severe deficiency in the serpin antithrombin. We studied the consequences of this drug on antithrombin levels, activity, conformation, and immunohistological and ultrastructural features in plasma from acute lymphoblastic leukemia patients, HepG2 cells, and plasma and livers from mice treated with this drug. Additionally, we evaluated intracellular deposition of alpha1-antitrypsin. l-Asparaginase did not affect functional or conformational parameters of mature antithrombin; however, patients and mice displayed severe type I deficiency with no abnormal conformations of circulating antithrombin. Moreover, l-asparaginase impaired secretion of antithrombin by HepG2 cells. These effects were explained by the intracellular retention of antithrombin, forming aggregates within dilated endoplasmic reticulum cisterns. Similar effects were observed for alpha1-antitrypsin in plasma, cells, and livers, and intracellular aggregates of additional proteins were observed in frontal cortex and pancreas. This is the first report of a conformational drug-associated effect on serpins without genetic factors involved. l-Asparaginase treatment induces severe, acquired, and transient type I deficiency of antithrombin (and alpha1-antitrypsin) with intracellular accumulation of the nascent molecule, increasing the risk of thrombosis.

PubMed Disclaimer

Figures

Figure 1

Effects of ASNase treatment on plasma antithrombin during the induction procedure of ALL patients and mice. A: Evolution of the anti-FXa anticoagulant activity in ASNase-treated ALL patients (n = 19). Arrows indicate the start of every single block of treatment with ASNase. B: Highest loss of antithrombin antigen levels and anti-FXa activity compared with the basal value of each patient (100%). C: Reduction of the anti-FXa activity in plasma of mice treated with 10,000 IU/m2 of ASNase for 2 consecutive days.

Figure 2

Effect of ASNase incubation of HepG2 cells after 24 hours. A: Antithrombin secreted to the medium as determined by enzyme-linked immunosorbent assay. B: Intracellular antithrombin of HepG2 lysates as evaluated by SDS-PAGE under reducing conditions and immunoblotting. C: Intracellular distribution of antithrombin in HepG2 as revealed by immunofluorescence. Arrowheads mark antithrombin aggregates. D: Antithrombin immunoblot of HepG2 immunoprecipitates after two-dimensional electrophoresis. Original magnifications, ×100 (C).

Figure 3

Consequences of ASNase treatment on mouse (2 days with 10,000 IU/m2) livers and hepatocyte morphology as revealed by H&E (A), Congo Red (B), and PAS (C) staining under light microscopy, and by electronic microscopy (EM) (D). Arrows indicate intracellular aggregates of proteins. We point out the Mallory body-looking inclusion within the ER observed by EM (bold arrow). Original magnifications: ×100 (A–C); ×40,000 (D).

Figure 4

Intracellular retention of antithrombin aggregates in mice treated with ASNase (2 days with 10,000 IU/m2). A: Intracellular antithrombin evaluated by SDS-PAGE under reducing conditions and immunoblotting of supernatant and pellets from liver lysates. B: Intracellular distribution of antithrombin and cellular morphology and ultrastructure of hepatocytes as revealed by immunohistochemistry (IH) under light microscopy and immunogold labeling electron microscopy (IG-EM). Arrows indicate antithrombin accumuli. Original magnifications: ×100 (B, left); ×40,000 (B, right).

Figure 5

Effects of ASNase on α1-antitrypsin secretion and retention. A: Effects of ASNase treatment on the levels of α1-antitrypsin in plasma during the induction procedure of ALL patients. B: Intracellular retention and aggregation of α1-antitrypsin in HepG2 cells as revealed by immunofluorescence (IF) after 24 hours of incubation with ASNase. C and D: Intracellular α1-antitrypsin in livers of ASNase-treated mice (2 days with 10,000 IU/m2) and their control littermates, as revealed by immunoblotting of supernatants from liver lysates (C) and by immunohistochemistry (IH) under light microscopy or immunogold-labeling and electron microscopy (IG-EM) (D). Arrows indicate α1-antitrypsin aggregates. Original magnifications: ×100 [B, D (top)]; ×40,000 (D, bottom).

Figure 6

Intracellular accumulation of proteins in pancreatic acinar cells (A) and frontal cortex white matter (B) from mice treated with ASNase (2 days with 10,000 IU/m2), as revealed by Congo Red and PAS staining under light microscopy. Arrowheads indicate β-amyloid (A, B) and glycoprotein (B) deposits. Original magnifications, ×100.

References

1. Ishiguro K, Kojima T, Kadomatsu K, Nakayama Y, Takagi A, Suzuki M, Takeda N, Ito M, Yamamoto K, Matsushita T, Kusugami K, Muramatsu T, Saito H. Complete antithrombin deficiency in mice results in embryonic lethality. J Clin Invest. 2000;106:873–878. - PMC - PubMed
1. Carrell RW, Huntington JA. How serpins change their fold for better and for worse. Biochem Soc Symp. 2003;70:163–178. - PubMed
1. Carrell RW, Huntington JA, Mushunje A, Zhou A. The conformational basis of thrombosis. Thromb Haemost. 2001;86:14–22. - PubMed
1. Corral J, Vicente V, Carrell RW. Thrombosis as a conformational disease. Haematologica. 2005;90:238–246. - PubMed
1. Wriston JC, Jr, Yellin TO. L-Asparaginase: a review. Adv Enzymol Relat Areas Mol Biol. 1973;39:185–248. - PubMed

L-asparaginase-induced antithrombin type I deficiency: implications for conformational diseases - PubMed (original) (raw)