Abstract
The coagulopathy in acute promyelocytic leukemia (APL) is unique among the leukemias and accounts for the major complications of hemorrhage and thrombosis. The pathogenesis is in large part the result of increased expression by the APL promyelocytes of the procoagulant TF, and the fibrinolytic components of tPA , PAI-1 and Annexin A2. Recently, microparticles derived from plasma of APL patients and from culture medium of the human APL cell line NB4 have been shown to contain antigens of procoagulant tissue factor (TF), and the fibrinolytic components of tPA , Annexin A2 and PAI-1 (Kwaan HC, Rego EM. Role of microparticles in the hemostatic dysfunction in acute promyelocytic leukemia. Seminars in thrombosis and hemostasis 2010;36:917-24;Kwaan et al:Microparticles in acute promyelocytic leukemia. 53rdASH Annual Meeting; Blood 2011 Abstract No.3346). Results of functional activities of these components and thrombin generation in the microparticles are presented in the present study.
Blood samples were collected in citrate vacutainers from 23 patients at the time of diagnosis and after induction therapy with ATRA at one month when there was molecular remission, under the approval of the respective institutional review boards. Control samples were collected from 37 healthy subjects.
sodium citrated blood is centrifuged at 1500g for 20 minutes. The plasma portion is collected and centrifuged a second time at 3000g for 3 minutes to eliminate any extraneous platelets. 250µl of plasma is then centrifuged at 20,000g for 15 minutes at 4C. The resultant pellet contains the microparticles. [Standardization of platelet-derived microparticle enumeration by flow cytometry with calibrated beads: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop. Lacroix R. Robert S. Poncelet P. Kasthuri RS. Key NS. Dignat-George F. ISTH SSC Workshop. Journal of Thrombosis & Haemostasis. 8(11):2571-4, 2010 Nov.
| Microparticle samples: . | At Diagnosis . | At Molecular Remission . | Healthy Control . |
|---|---|---|---|
| Tissue factor activity* | 0-27 pg/ml | 0-55 pg/ml | 0 pg/ml |
| PAI-1 activity** | 4.9 IU | 4.2 IU | 4.6 IU |
| tPA activity** | 5.6 ng/ml | 5.5 ng/ml | 5.3 ng/ml |
| Microparticle samples: . | At Diagnosis . | At Molecular Remission . | Healthy Control . |
|---|---|---|---|
| Tissue factor activity* | 0-27 pg/ml | 0-55 pg/ml | 0 pg/ml |
| PAI-1 activity** | 4.9 IU | 4.2 IU | 4.6 IU |
| tPA activity** | 5.6 ng/ml | 5.5 ng/ml | 5.3 ng/ml |
Tissue factor actuvuty – method of Manley et al (Manly DA et al Increased microparticle tissue factor activity in cancer patients with Venous Thromboembolism. Thrombosis Research. 2010:125:511-2
tPA and PAI-1 – Spectrolyze chromogenic ELISA (Diagnostica Stago, NJ)
Thrombin Generation Assay*
| *Microparticle samples . | N= . | Lag phase (min) . | Peak thrombin (nM) . | Peak time (min) . |
|---|---|---|---|---|
| Diagnosis | 13 | 21 ±13 | 40 ± 44 | 28 ± 21 |
| Molecular remission | 6 | 12 ±22 | 13 ± 254 | 18± 31. |
| Healthy Control | 11 | 0 | 0 | 0 |
| *Microparticle samples . | N= . | Lag phase (min) . | Peak thrombin (nM) . | Peak time (min) . |
|---|---|---|---|---|
| Diagnosis | 13 | 21 ±13 | 40 ± 44 | 28 ± 21 |
| Molecular remission | 6 | 12 ±22 | 13 ± 254 | 18± 31. |
| Healthy Control | 11 | 0 | 0 | 0 |
Performed on microparticles by Technothrombin ™ TGA assay (Technoclone, Austria)
Microparticles bearing TF have been shown to play a thrombogenic role in cancer. We had previously shown that there is an increase of microparticles containing procoagulants and fibrinolytic factors in APL and that these microparticles are mostly derived from CD33 positive (myeloid) cells. Present findings show that procoagulant and fibrinolytic factors are functional. The TF activity, thrombin generation are increased in the APL microparticles. It is noteworthy that thrombin generation is increased in the APL microparticles. In contrast, there is a complete absence of thrombin generation in the control microparticles. This suggests that the TF in microparticles, which are components of cell membranes, are not activated in healthy subjects but in APL.
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
