Serum Levels of Soluble Annexin II in Patients with APL

Acute promyelocytic leukaemia (APL, M3) is associated with both a characteristic t(15;17) and severe bleeding diathesis caused by disseminated intravascular coagulation (DIC) and/or hyperfibrinolysis. It has been suggested that annexin II, a coreceptor for tissue plasminogen activator (t-PA) and plasminogen (PLG), is overexpressed on the surface of promyelocytes, leading to an increased fibrinolytic potential. However, little is known about the serum levels of annexin II in patients with APL. Therefore, we measured concentrations of annexin II in sera from patients with APL (n=10). The serum annexin II levels were determined by following methods (sandwich ELISA).

1. The EIA plates coated for 48h at 4· with 2ug human annexin II antibodies (monoclonal) in carbonate buffer pH 9.4 respectively.

2. The plates were then washed once with PBS and were done for non-specific blocking with 200ul/well PBS containing 1% BSA (PBSA) for 2h at room temperature (RT).

3. After blocking, the plates were washed once with PBS. Subsequently, 100ul of samples diluted by ten times were added to the wells.

4. After 2h incubation at RT, the plates were washed twice with PBS containing 0.05% Tween20 (PBST) and human annexin II rabbit antibodies (polyclonal) diluted 1:250 with PBSA were added to the wells.

5. After 2h incubation at RT, the plates were washed twice with PBST and then incubated for one hour at RT with 100ul/well HRP-conjugated goat anti-rabbit Ig diluted 1:250 in PBSA.

6. The plates were washed twice with PBST and reaction was developed by adding 100ul/well of substrate solution (0.04% O-phenylenediamine in phosphate/citrate buffer pH 5, with 0.04% H2O2).

7. The reaction was stopped after 10 min by adding 50ul/well 2.5M H2SO4, the absorbance was read at 492nm using a spectrophotometer In the patients with APL, we found significant elevations in serum annexin II levels as compared with normal controls.

In other words, serum levels of annexin II were 0.109 (mean OD values, n=10) from patients with APL.

On the other hand, concentrations of annexin II in sera from normal controls were 0.092 (mean OD values, n=10). There was correlation between the FDP and annexin II levels in the APL patients with DIC The present work demonstrates significant elevations in serum annexin II levels in patients with APL. Therefore, we suggest that elevation of annexin II may be associated with the development of an increased fibrinolytic potential.