To the editor:

Leukemia cells express high levels of Bcl-21  and BH3 mimetics that antagonize the prosurvival function of Bcl-2 and related proteins, thereby inducing apoptosis, are useful treatments for patients with chemotherapy-refractory leukemia.2  BH3 mimetics such as ABT-737 and ABT-263 also inhibit Bcl-xL and trigger acute thrombocytopenia in dogs,3  mice,4  and humans.5  In preclinical studies, they induced a rapid thrombocytopathy associated with shedding of GPVI and GPIbα ectodomains, platelet-specific adhesion receptors. This results in a loss of platelet adhesive function after ABT-263 treatment of human platelets in vitro or mice in vivo.6  The pretreatment platelet count and bleeding risk are important clinical parameters when considering BH3 mimetics as treatment options in refractory chronic lymphocytic leukemia (CLL).5 

We evaluated platelet receptor levels in citrated platelet-rich plasma (PRP) samples from patients before and after receiving ABT-263 by flow cytometry using phycoerythrin-conjugated anti-GPIbα (AK2), anti-GPVI (1G5), anti-CD9, or anti-αIIbβ3 (CD41a) monoclonal antibodies. We compared data from 5 patients with lymphoproliferative diseases refractory to standard therapies who received ABT-263,5,7  with data obtained from 15 healthy donors or 7 patients with immunothrombocytopenia (ITP; chronic ITP > 6 months, 2 receiving steroid therapy) to assess the impact of reduced platelet count on our findings. All subjects provided informed consent and the study was approved by Monash University and Royal Melbourne Hospital Human ethics committees in agreement with the Declaration of Helsinki 1975 (revised 1983). Flow cytometry was performed in a FACSCalibur (BD Biosciences) using a platelet gate identified using the platelet markers GPVI, αIIbβ3, and GPIbα in healthy-donor PRP. Fluorescence geomeans were ascertained using CellQuest Version 3.3 software and soluble GPVI ectodomain (sGPVI) by sandwich ELISA.8 

Compared with healthy donors, circulating platelets from 5 patients with lymphoproliferative disorders demonstrated stable levels of αΙΙbβ3 and tetraspanin CD9, but significantly diminished surface levels of GPIbα and GPVI (Table 1). Loss of GPVI was not due to increased metalloproteolysis,9  because plasma sGPVI was within healthy donor ranges.10  Levels of GPIbα, GPVI, and CD9 were all diminished in PRP from ITP patients and sGPVI was significantly elevated, with a different mechanism underlying the thrombocytopenia. The progressive form of leukemias such as CLL are characterized by anemia, neutropenia, and thrombocytopenia; patients with progressive CLL may also develop secondary ITP and display a higher incidence of bleeding.11  Temporal analysis of platelet receptors in PRP from patients receiving ABT-263 (100-150 mg/d for 7 days) indicated that, despite a significant drug-associated thrombocytopenia, there was no discernible ABT-263–associated loss of platelet adhesion receptors. This apparent disparity between patient and experimental systems (using non-CLL platelets with normal base levels of adhesion receptors)6  may reflect differences in plasma drug concentrations or may mean that detectable drug-induced decreases in patients are masked by low pretreatment levels of these receptors.

Table 1

Surface levels of platelet receptors in healthy donors, ITP patients or patients with lymphoproliferative disorders before and during treatment with ABT-263

nPlatelet countGPIbα geomeanPCD9 geomeanPGPVI geomeanPαΙΙbβ3 geomeanPsGPVI ng/mlP
Lymphoproliferative patients 
    Presample 151 ± 43 271 (73) CI 165-491 *** 367 (188) CI 110-376 ns 90 (24) CI 66-290 ** 342 (175) CI 154-220 ns 23 (9) CI 5.9-15 ns 
    + ABT-263 Day 1 95 ± 22 350 (129) CI 77-421 ** 329 (205) CI 75-417 ns 105 (40) CI 50-276 ** 474 (271) CI 50-380 ns 28 (17) CI 10-12 ns 
    + ABT-263 Day 7 91 ± 30 *226 (137) CI 199-547 *** 291 (191) CI 34-452 ns 96 (38) CI 59-285 ** 346 (149) CI 144-218 ns 16 (8) CI 5.4-15 ns 
ITP patients 35 ± 13 366 (187) CI 8-398 ** 208 (104) CI 98-486 ** 101 (53) CI 70-264 ** 399 (257) CI 102-282 ns 43 (10) CI 13-32 *** 
Healthy donors 15 457 ± 114 599 (166)  500 (233)  268 (116)  309 (172)  21 (10)  
nPlatelet countGPIbα geomeanPCD9 geomeanPGPVI geomeanPαΙΙbβ3 geomeanPsGPVI ng/mlP
Lymphoproliferative patients 
    Presample 151 ± 43 271 (73) CI 165-491 *** 367 (188) CI 110-376 ns 90 (24) CI 66-290 ** 342 (175) CI 154-220 ns 23 (9) CI 5.9-15 ns 
    + ABT-263 Day 1 95 ± 22 350 (129) CI 77-421 ** 329 (205) CI 75-417 ns 105 (40) CI 50-276 ** 474 (271) CI 50-380 ns 28 (17) CI 10-12 ns 
    + ABT-263 Day 7 91 ± 30 *226 (137) CI 199-547 *** 291 (191) CI 34-452 ns 96 (38) CI 59-285 ** 346 (149) CI 144-218 ns 16 (8) CI 5.4-15 ns 
ITP patients 35 ± 13 366 (187) CI 8-398 ** 208 (104) CI 98-486 ** 101 (53) CI 70-264 ** 399 (257) CI 102-282 ns 43 (10) CI 13-32 *** 
Healthy donors 15 457 ± 114 599 (166)  500 (233)  268 (116)  309 (172)  21 (10)  

Citrated PRP samples were analyzed by flow cytometry in CLL patients prior to and after receipt of 100 to 150 mg ABT-263 daily. Values reported as platelet count × 103/μl, mean fluorescence intensity geomeans for the entire platelet population (standard deviation), and sGPVI levels in citrated plasma ng/ml (standard deviation). P values derived from a 2-tailed unpaired t test comparing data from healthy donors with each patient group; ns indicates no significant difference. **P < .01; ***P < .001; CI = 95% confidence intervals.

*

One day 7 GPIbα measurement was lost.

Seven clinically diagnosed ITP patients (6 chronic, 1 persistent) with platelet count below 50 × 103/μl on the day of blood collection. Two of the 7 were receiving 50 mg or 75 mg prednisolone, the others were not receiving medication for ITP; the autoantibody target was not determined.

This is the first report demonstrating that circulating platelets in patients with lymphoproliferative disorders display very low levels of platelet adhesion receptors. It seems probable that this is because of a platelet production defect, because receptor levels did not normalize even after the platelet count improved beyond day 7 of treatment (not shown). However, we cannot rule out peripheral mechanisms or other thrombocytopathy causing low levels of platelet receptors. How lymphoproliferative disorders at different stages affect platelet production and function is not understood, but quantitative assessment of platelet quality and receptor levels are not routinely performed clinically. Diminished GPIbα and GPVI receptor levels may contribute to the increased bleeding risk observed in these patients.11 

Authorship

Acknowledgments: This work was supported by the National Health and Medical Research Council of Australia, the Victorian Cancer Agency, Victorian State Government Operational Infrastructure Support, and the China Scholarship Council.

Contribution: J.Q. performed the research; J.Q., R.K.A., A.W.R., and E.E.G. analyzed the data and wrote the manuscript; and all authors designed the research.

Conflict-of-interest disclosure: A.W.R. is an investigator on 3 clinical trials funded by Abbott and Genentech and receives funding for laboratory research that is part of those trials. The work described herein was not funded by either Abbott or Genentech. The remaining authors declare no competing financial interests.

Correspondence: Dr Elizabeth Gardiner, Monash University, Australian Centre for Blood Diseases, AMREP, 89 Commercial Road, Level 6, Melbourne, VI, 3004 Australia; e-mail: elizabeth.gardiner@monash.edu.

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Author notes

*

A.W.R. and E.E.G. contributed equally to this work.