Annexin II and Bleeding in Acute Promyelocytic Leukemia

Reviewers: Kenneth Blank, MD
Source: The New England journal of Medicine April 1, 1999 Vol. 340 No 13 p994

Background

Acute promyelocytic leukemia (APL) is a unique variant of acute myeloid leukemia, both in its biology and response to chemotherapy. APL ischaracterized genetically by the fusion of two genes, the retinoic acid receptor gene (RAR) and a transcription factor gene called the promyelocyticleukemia gene (PML). The fusion of these two genes creates a unique gene called the PML/RAR gene that codes for a chimeric protein thatpresumably plays a role in the etiology of APL.

Scientists and physicians discovered a method to take advantage of APL's unique biology when it was discovered that all-trans-retinoic acid (ATRA)causes APL cells to differentiate and stop rapidly dividing. Today, the treatment of APL differs from all other acute myeloid leukemias byincorporating ATRA in the induction and consolidation phases of treatment. The use of ATRA has significantly improved treatment results overconventional chemotherapy.

Another unique aspect of APL is its association with bleeding disorders. Patients with APL frequently have hemorrhagic events, which may besevere enough to lead to the patient's death. The reason for this syndrome is not clear. Some investigators believe it is caused by disseminatedintravascular coagulation (DIC), a syndrome in which clotting occurs throughout the body causing a depletion of clotting factors and subsequentbleeding. Another theory attributes the bleeding diathesis to rapid clot breakdown. Specifically, clots, which are formed largely by fibrin, are rapidlydissolved in APL patients because of an overproduction of the enzyme plasmin that cleaves fibrin. Support for the latter theory appears in a paperpublished in the April 1, 1999 New England Journal of Medicine.

The authors of the report hypothesized that plasmin production in APL is enhanced because APL cells express great quantities of a cell surfaceprotein called annexin II, a protein that activates plasmin. To test this theory the authors studied APL cells for annexin II quantities and correlated thiswith plasmin levels.

Materials and Methods

Samples of peripheral blood or bone marrow from six patients with APL and eight patients with other forms of leukemia were studied for annexin IIlevels by immunofluorescence, flow cytometry and Western blot techniques. Generation of plasmin was examined in an APL cell line (termed NB4cells) as well as inpatient blood. Specifically, cells from patients with APL who lacked the PML/RAR translocation had the annexin II genetransfected into the cells. The effect of all-trans-retinoic acid on the synthesis of annexin II was examined using ribonuclease protection assays andnuclear run-on analyses.

Results

Five patients with APL who expressed the characteristic PML/RAR translocation strongly expressed annexin II compared to seven patients withother forms of acute leukemia and one APL patient without the characteristic translocation. Flow cytometric studies revealed that PML/RAR-positiveAPL patients had mean fluorescence intensities with anti-annexin II antibody of 6.9 versus 2.9 for other leukemias.

The role of annexin II in the production of plasmin was examined using advanced molecular techniques. For example, cells lacking the PML/RARtranslocation were transfected with a plasmid containing a full-length copy of the annexin II gene. Transfected cells were found to express annexin IItwice as effectively as un-transfected control cells, suggesting that the expression of annexin II directly correlates with the capacity of produceplasmin.

All-trans-retinoic acid significantly decreased the synthesis of annexin II. This finding related to both the cell surface expression of annexin II and themRNA levels of annexin II. Using nuclear run on analysis, the authors found that ATRA inhibition of annexin II expression is likely secondary to adecrease in the annexin II gene transcription, and not enhanced degradation of the protein.

Discussion and Conclusion

Patients with APL and the characteristic PML/RAR gene translocation express high levels of the cell surface protein annexin II. Annexin II plays animportant role in the formation of plasmin, an enzyme that dissolves clots. Therefore, bleeding disorders in APL patients may be partly explained bythe enhanced amounts of annexin II on the cell surface of APL leukemia cells. In addition, the treatment of APL with ATRA is known to reverse theclotting diathesis within one week. The authors provide a biologic explanation for this phenomenon showing that ATRA decreases transcription ofthe annexin II gene.