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C.C.F.M.J. Baaten et al.
topenia, i.e., a platelet count of ≤50x109/L, develops in vir- tually all treated patients.2 These patients are at high risk of bleeding, with up to 43% experiencing clinically signif- icant bleeding (World Health Organization [WHO] grade 2 or higher), and 1% experiencing life-threatening bleed- ing.3 Prophylactic transfusion with platelet concentrates for preventing bleeding is given as standard care once the count drops below 10x109/L, or in case of active bleed- ing.2,4 Randomized clinical trials have indicated that the bleeding risk in this patient group is reduced by platelet transfusion, although it does not completely eliminate hemorrhagic events.3,5 Since bleeding is relatively infre- quent in non-malignant thrombocytopenia,6,7 it can be considered that a low platelet count is not the sole risk fac- tor for bleeding in chemotherapy-treated patients.
Earlier studies on patients with acute myeloid leukemia, of whom none received chemotherapy, have provided indications for impaired platelet function due to disease, as apparent from low platelet aggregation, reduced gran- ule secretion and weak thromboxane B2 production.8-10 It was proposed that low expression of the a-granule glyco- protein, P-selectin, can be used as a prognostic marker for hemorrhage.11 However, bleeding in combination with thrombocytopenia is more frequently observed in cancer patients treated with chemotherapy.12 The literature thus far only indicates that the anthracycline daunorubicin inhibits integrin aIIbβ3 activation, aggregation and secre- tion of platelets upon agonist stimulation.13,14 Daunorubicin and its analogue idarubicin were found to induce integrin activation and secretion in resting platelets.15 However, to what extent and by which mech- anism myelosuppressive chemotherapy in general affects platelet function has remained largely unclear.
In this study, we evaluated the platelet activation processes and coagulant activity in 77 patients with hema- tological malignancies treated with chemotherapy. Our results point to multiple functional defects in the patients' platelets which are related to impaired mitochondrial activity, independent of classical apoptosis. In the majority of patients, low platelet activity could be improved by platelet transfusion.
Methods
Materials and methods
See Online Supplementary Material.
Patients and control subjects
The study was approved by the local ethics committee (METC- 11-4-097). All participating patients and healthy volunteers gave written informed consent according to the Helsinki declaration. Patients, reporting at the hospital, fulfilling the inclusion criteria and providing informed consent, were consecutively included in the period between November 2014 and April 2018. Eligible patients were ≥18 years of age, received chemotherapy for treat- ment of a confirmed hematologic malignancy (acute myeloid leukemia, acute lymphocytic leukemia, multiple myeloma or malignant lymphoma), and had, or were expected to have, throm- bocytopenia (platelet count ≤50x109/L). Morning platelet counts were monitored daily as part of routine clinical care. According to standard practice, when the morning platelet count was <10x109/L, patients received prophylactic transfusion with one batch of platelet concentrate (leukocyte-depleted pooled buffy coat from five donors, median storage time: six days, median
platelet count: 357x109/L). Patient exclusion criteria were: sepsis, splenomegaly, signs of active bleeding at the time of blood with- drawal, previous platelet transfusion within three days (excluding the presence of donor platelets), and/or use of antithrombotic medication during the previous 14 days.
For clinical care, blood samples were collected before and dur- ing chemotherapeutic treatment at multiple time points: 1) before the start of chemotherapy, 2) before myelosuppression, 3) during myelosuppression (platelet count ≤50x109/L), 4) during myelosup- pression: before (platelet count ≤10x109/L) and one hour after platelet transfusion, and 5) during bone marrow recovery (platelet count ≤50x109/L). Patient blood samples were obtained via a cen- tral venous catheter, rinsed with 100 mL saline to remove residual traces of heparin (verified by measurement of thrombin time). Blood samples from healthy control subjects were obtained via venipuncture of the antecubital vein using a Vacutainer 21-gauge needle (Becton-Dickinson Bioscience, NJ, USA). Blood collection was always into 3.2% (w/v) trisodium citrate (Greiner Bio-One Vacuette, Alphen a/d Rijn, The Netherlands). For clinical care (hematological parameters), separate samples from patients were drawn into vacuette tubes containing K2-ethylenediaminete- traacetic acid (EDTA; Becton-Dickinson Bioscience, NJ, USA).
Experimental setup
Within the limitations of medical ethical permission, a total of 52 blood samples from patients (platelet count ≤50x109/L) could be obtained during myelosuppression (study A). In all these samples, platelet responsiveness was assessed using flow cytometry. Due to the limited blood volume and the low platelet counts, a restricted number of additional analyses was carried out per sample. When there remained sufficient sample volume, platelet function was further characterized by measuring the following platelet respons- es: platelet spreading, intracellular calcium signaling and phos- phatidylserine (PS) exposure. To gain a deeper understanding of the underlying mechanisms of platelet dysfunction, subsequent blood samples could be obtained from 25 additional patients (platelet count ≤50x109/L) during the myelosuppression phase (study B). The samples were used to investigate apoptotic signal- ing (caspase activity; western blotting for caspase-mediated pro- tein cleavage), mitochondrial respiration and structure (high-reso- lution respirometry, citrate synthase activity, transmission electron microscopy) or reactive oxygen species (ROS). The maximum of care was taken that for all measurements patients from the major treatment classes were represented (see Figure Legends).
For 36 of the patients in study A, blood samples could also be obtained at one hour after transfusion with platelet concentrate. Again, platelet responsiveness was determined by flow cytometry.
Statistical analysis
Data are represented as medians with interquartile ranges. Paired data were compared using the Wilcoxon signed-rank test, otherwise the Mann-Whitney U test was used. When comparing more than two groups, the Kruskal Wallis H test was used. P-values <0.05 were considered significant. Graphs were made using GraphPad Prism v6 (San Diego, CA, USA). Statistical analy- sis was performed using the SPSS Statistics 23 package (IBM, Armonk, NY, USA).
Results
Variable impairment of platelet activation in cancer patients with thrombocytopenia after chemotherapy
Blood samples were obtained from a total of 77 patients, who were diagnosed with acute myeloid leukemia or
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haematologica | 2018; 103(9)