| The life-threatening coagulopathy associated with acute promyelocytic leukaemia (APL) has been the defining clinical characteristic of this disease. Since the introduction of all-trans retinoic acid (ATRA) and its derivants into the therapy for patients with APL, disease-free survival and overall survival have improved dramatically, such that the disease is now highly curable. However, haemorrhage especially life-threatening coagulopathy in the important organ such as central nervous system still accounts for the majority of early deaths. Pathogenesis of the coagulopathy is complex and includes disseminated intravascular coagulation (DIC), primary fibrinolysis and proteolysis. Studing the mechanism of primary fibrinolysis will benefit the management of hemorragic events in APL.AnnexinⅡ(AnnⅡ), a phospholipid-binding protein, which serves as a co-receptor for plasminogen and tissue plasminogen activator (tPA), has been found expressed as a peripheral membrane protein on endothelial cells and some cancer cells in a wide variety of tissues and organs. It has been reported that APL cells expressed high levels of AnnⅡand stimulate cell surface tPA-dependent plasmin generation more efficiently than other cells with lower AnnⅡexpression in vitro, leading to an enhanced fibrinolysis and then abnormal bleeding in patient with APL. But whether the expression of AnnⅡis correlated with the fibrinolytic activity in APL has not been defined.The management of APL with either ATRA or arsenic trioxide (ATO) can cause terminal differentiation or apoptosis of leukemic promyelocytcs and induce complete remission (CR) in the majority of patients with APL through different ways. Remission induction with ATRA and ATO has been accompanied by rapid amelioration of the bleeding syndrome. The mechanisms by which ATRA and ATO leads to the rapid resolution of coagulopathy have not been completely defined. The previous data suggest that both ATRA and ATO can reduce tissue factor (TF) and procoagulant activity (PCA) of APL blast cells in vitro and in vivo, and in vitro studies aslo show ATRA and ATO can down regulate the expression of AnnⅡin APL blast cells, thus reduce the generation of plasmin in APL cells. But whether the remission induction therapy with ATRA and ATO could bring about the same effect in vivo has not been reported.In this study, the relationship between AnnⅡexpression and fibrinolytic activity was prospectively studied in 36 newly diagnosed APL patients and 64 patients with other types of leukemias. Bone marrow cells were collected before treatment and the expression of AnnⅡprotein in leukemic blasts was analyzed using flow cytometry and western blotting, and AnnⅡmRNA using real time-PCR. Initial rates of in vitro plasmin generation by tPA in the presence of APL cells or other leukemia cells were examined using chromogenic study. The highest expression of AnnⅡwas seen on APL and M5 cells, with the mean fluorescence intensity (MFI) was 9.93±2.23(n=12) and 9.79±2.44(n=12), respectively. The AML-M1 (3.19±0.56, n=10), M2 (3.38±0.76, n=11) and M4 (3.05±0.68, n=10) blasts showed relatively low amount of cell surface expression. The expression of AnnⅡon ALL-B (1.12±0.65, n=10) and ALL-T (0.5±0.26, n=11) blasts was the least with no significant difference when compared with normal controls (0.79±0.41, n=10). The expression of AnnⅡin APL and M5 blasts was further evaluated by western blotting of whole cell extract. With the use of a specific monoclonal IgG antibody against AnnⅡ, abundant AnnⅡwas detected . The most marked plasminogen activation was seen in the presence of APL (K=0.011±0.001) and M5 (K=0.010±0.002) cells. For M1, M2 and M4 cells, the rates of plasmin generation were 34.4%, 40.6% and 53.1% of the value obtained with APL cells. More over, a monoclonal anti-AnnⅡantibody reduced plasmin generation by APL cells to 64 percent (P<0.05) of that observed when APL cells were incubated with control IgG, suggesting AnnⅡmay play an important role in the fibrinolytic activity in APL cells.To further confirm the role of AnnⅡin the fibrinolytic activity in APL cells, an expression vector pcDNA3.1(-)-AnnⅡwas constructed. With the use of total RNA extracted from HUVEC cells, AnnⅡcDNA were amplified by PCR. The amplified cDNAs were cloned into pMD?19-T Simple Vector and subjected to nucleotide sequence analysis and then cloned into the expression vector pcDNA3.1(-). Then HL-60 cells, which have originally little AnnⅡexpression, were transiently transfected with pcDNA3.1(-)-AnnⅡusing electroporation techniques.On the other hand, NB4 cells were transfected with siRNA specific to AnnⅡ. After transfection, we found HL-60 transfectants stimulated plasmin production 5.1 times as effectively as cells transfected with the empty vector (P<0.01), and the enhancement of tPA dependent plasmin generation was decreased by 58.4% (P<0.05) in the AnnⅡ-depleted NB4 cells.Plasma fibrinolysis and coagulation markers in 18 APL patients and 12 other leukemias with overt bleeding symptoms were analysed using ELISA or chromogenic study. All the AL patients had evidence of fibrinolysis activation such as high levels of FDP and D-dimer. while the levels of plasma fibrinogen were relatively low in the majority of AL patients.The most marked hyperfibrinolysis was seen in APL patients, as indicated by highest levels of FDP, D-dimer, while the levels of fibrinogen were very low. The levels of tPA antigen and tPA activity at the onset of AL were comparable with normal controls, while PAI-1 antigen were elevated in 10 patients with DIC syndrome. Routine coagulation tests in 11 APLs without DIC syndrome suggested a limited coagulopathy and hyperfibrinolysis, with a marked increment of FDP and decrement of fibrinogen, while APTT (36.4±3.4 sec) and PT (15.2±2.1 sec) in the majority of patients were normal or only slightly prolonged, and the level of AT was normal. These results show a dissociation between the two syndromes-DIC and fibrinogenolysis, and high expression of AnnⅡin APL cells may contribute to the primary hyperfibrinolysis. But no significant correlation was observed in the 11 APLs without DIC between AnnⅡexpression and FDP, fibrinogen.These results indicate that AnnⅡexpression is properly not the sole determinant of fibrinolysis in APL. The alterations of AnnⅡexpression and fibrinolytic markers in APL during the inductive treatment with ATRA(n=9) or ATRA+ATO(n=9) were analysed. The abnormal fibrinolytic parameters, including FDP, D-dimer and fibrinogen, gradually returned to normal in 2-3 weeks after treatment with either ATRA or ATRA plus ATO. Elevated PAI-1 antigen in 7 APLs with DIC at the onset of disease restored when DIC syndrome was corrected. But the expression of AnnⅡin APL bone marrow cells(MFI=4.15±2.14) was still higher than normal controls (MFI=0.79±0.41)even when hematologic CR(HCR) was achieved. The abnormal expression of AnnⅡdecreased to normal range till molecular CR(MCR) was achieved, suggesting the PML/RARαfusion gene play a role in regulating the AnnⅡexpression in APL cells.Given the results described above, we can conclude that abnormally high levels of AnnⅡexpression in APL cells promote the generation of plasmin in vitro. However, the expression of AnnⅡis properly not the sole determinant of fibrinolysis in APL. Thus, several factors, in addition to AnnⅡ, cooperatively contribute to hyperfibirnolytic condition in APL. A rational antifibrinolytic management would be an effective way to decrease the incidence of severe hemorragic events in APL. |
PDF Full Text Request