Microparticles in Acute Promyelocytic Leukemia,
Authors: Kwaan Hau C. , Rego Eduardo M , McMahon Brandon , Weiss Ivy , Marvin James 
Published: October 2019

Abstract
Abstract 3346
Abnormal hemostatic function in acute promyelocytic leukemia (APL) contributes to the bleeding and thrombotic complications, which account for much of the morbidity and mortality. Increased procoagulants (tissue factor (TF) and cancer procoagulant) and increased fibrinolytic components (annexin A2 and tPA) have been found in the blood of APL patients and in APL cell line NB4. However, their role in the pathogenesis of these complications is not clear. In recent years, increased numbers of circulating microparticles (MP) containing tissue factor have been observed in many cancer patients. This abstract describes the first study of MP in APL.
Plasma samples were collected from 23 APL patients with 37 age-matched controls from healthy subjects. The APL patients were treated with a regimen of ATRA and anthracyclin according to the protocol of the International Consortium for APL (Sem. Thromb. Hemost. 2010, 369:17–924).The first sample was collected at diagnosis and second after induction therapy when molecular remission was obtained in all patients in this study. Platelet poor plasma (PPP) was obtained by centrifuging at 1,500 G for 20 minutes. 250 μL of PPP was centrifuged at 20,000 G for 10 minutes. The sediment containing MP was resuspended in 100μL of trisHCL buffer for fluorochrome-labelling with antibodies respectively against (1) human TF, (2) human annexin A2, (3) CD33, (4) human tPA, (5) human PAI-1 and (6) human annexin A5. Following incubation for 45 min, buffer was added to the suspension to a volume of 0.5 ml for FAC analysis using Coulter CyAn with Summit software. Electronic triggering was done on side-scatter, and samples were gated for size using enumeration beads (0.3 to 1.0 micron) and for annexin A5 positivity. Using MP sediment, the activities of TF, tPA and PAI-1 were respectively measured using chromogenic assays (Actichrome™ TF ELISA, American Diagnostica),(Spectrolyze™ tPA) and (Spectrolyze™ PAI-1).
At diagnosis, FAC analysis showed tissue factor, PAI-1 and tPA in the MPs were significantly higher than controls with respective P values of 0.008, 0.0001 and 0.002. MPs with annexin A2 and CD33 in APL samples were not different from controls. After achieving molecular remission, the levels of tissue factor, CD33, and annexin A2 were significantly reduced with respective P values of 0.0001, 0.0007 and 0.05, while those of PAI-1 and tPA were not. Of the MP bearing tissue factor, 83% were associated with MP with CD33. This fraction was high at diagnosis and significantly reduced at remission (P=0.016).
On the other hand, when assayed for activity, the TF was functional in all samples, but elevated in only one patient at diagnosis. Likewise, PAI-1 and tPA were functional but their respective activities were not elevated. Western blot showed formation of tPA/PAI-1 complex.
These results showed that the increased number of TF-bearing MPs in APL plasma must be viewed with caution. Although the TF was functional, but its activity was not higher than in the controls. The same is true with PAI-1 and tPA. We hypothesize that inhibitors of TF, such as tissue factor pathway inhibitor, are present in APL patients mitigating the hypercoagulability, and that PAI-1 is complexed with tPA, reducing both their activities. Further studies testing this hypothesis are in progress.

Disclosures:
No relevant conflicts of interest to declare.