Insight into the mechanism of asparaginase-induced depletion of antithrombin III in treatment of childhood acute lymphoblastic leukemia

Bushman, Jennifer E; Palmieri, Diane; Whinna, Herbert C; Church, Frank C

« Previous Next »Leukemia Research
Volume 24, Issue 7 , Pages 559-565, July 2000

Insight into the mechanism of asparaginase-induced depletion of antithrombin III in treatment of childhood acute lymphoblastic leukemia

Jennifer E Bushman
- Department of Chemistry, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3290, USA
Diane Palmieri
- Department of Pathology and Laboratory, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7525, USA
Herbert C Whinna
- Department of Pathology and Laboratory, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7525, USA
- Center for Thrombosis and Hemostasis, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7015, USA
Frank C Church
- Department of Pathology and Laboratory, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7525, USA
- Department of Pharmacology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365, USA
- Department of Medicine, Division of Hematology-Oncology, 932 Mary Ellen Jones Bldg., The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7035, USA
- Center for Thrombosis and Hemostasis, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7015, USA
- Corresponding author. At Division of Hematology-Oncology, Department of Medicine, 932 Mary Ellen Jones Bldg., The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7015, USA. Tel.: +1-919-9663311; fax: +1-919-9667639

Received 20 September 1999; accepted 21 January 2000.

Abstract

Asparaginase (ASNase) is a widely used and successful agent against childhood acute lymphoblastic leukemia (ALL). Asparaginase cleaves asparagine (Asn) to give aspartic acid and ammonia, thereby depleting free Asn in the blood. However, treatment with ASNase has been implicated in significant reduction of plasma levels of the coagulation serine protease inhibitor (serpin) antithrombin III (AT3), predisposing patients to thromboembolic complications. Our investigation was designed to delineate the biochemical mechanism of AT3 depletion that can occur in the plasma of ALL patients undergoing ASNase therapy. SDS-PAGE showed no cleavage of purified AT3 following treatment with ASNase. Furthermore, purified AT3 treated with ASNase demonstrated no decrease in inhibitory activity. Human plasma and whole blood treated with approximate therapeutic concentrations of ASNase showed no loss of AT3 activity as detected by a plasma-based factor Xa inhibition assay. Treatment of a confluent monolayer of HepG2 (hepatocarcinoma) cells with ASNase showed no gross loss in AT3 message levels detected by rtPCR. However, a decrease of cell viability was observed in cultures treated with ASNase. Interestingly, medium from HepG2 cells treated with ASNase showed a marked decrease in secretion of AT3 and another serpin, heparin cofactor II. Collectively, these data show that ASNase has no direct effect on AT3 in blood or plasma, but that ASNase may affect plasma levels of AT3 by interfering with translation and/or secretion of the protein in liver cells.

Keywords: l-asparaginase, Antithrombin III, Childhood acute lymphoblastic leukemia, Liver protein synthesis, Thrombosis

Abbreviations: ALL, acute lymphoblastic leukemia, ASNase, asparaginase, AT3, antithrombin III, BSA, bovine serum albumin, FXa, plasma factor Xa, HC2, heparin cofactor II, HNP, 20 mM HEPES buffer, 150 mM NaCl, 0.1% polyethylene glycol, 0.05% sodium azide., MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra-zolium bromide, SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, serpin, serine protease inhibitor