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Platelet dysfunction in chemotherapy
platelets (Figure 5C). Transmission electron microscopic images were also recorded, and these did not reveal struc- tural abnormalities of the mitochondria (data not shown).
Chemotherapeutics like anthracycline analogues can cause (cardio)myopathy and neuropathy by inducing mitochondrial damage, a process mediated by oxidative stress.29,30 To determine whether a similar process is oper- ative in the platelet lineage, activation markers, mitochon- drial function (TMRE) and reactive oxygen species (ROS) levels were measured in platelets from seven patients prior to chemotherapy, and two days after said chemotherapy. Additional blood samples were analyzed when severe thrombocytopenia occurred (median ten days after last treatment; median count 11x109/L). Before the start of chemotherapy, platelet reactivity in these patients was comparable to that of healthy controls (Figure 6A,B). After two days of therapy, the platelet count was slighter low- ered (median decrease: 15x109/L, IQR: 12.5-24.5), but platelet reactivity was not significantly changed. In con- trast, reactivity in response to all agonists decreased markedly when the patients became thrombocytopenic. Similarly, TMRE fluorescence only decreased in the latter case (Figure 6C), which only then was accompanied by a higher ROS production (Figure 6D). The reduction in TMRE fluorescence correlated with the production of ROS (Spearman’s rho=-0.459, P=0.012). Treatment of con-
trol platelets in vitro with chemotherapeutics affected nei- ther the mitochondrial membrane potential nor the pro- duction of ROS (Online Supplementary Figure S3C,D). Together, these results strongly suggest that mitochondrial dysfunction is not caused by a direct effect of chemother- apeutics on platelets, but by affecting the platelet precur- sor cells, the megakaryocytes.
Discussion
In this paper, we provide novel evidence that the platelets from thrombocytopenic patients suffering from hematological malignancies and treated with myeloabla- tive chemotherapy are dysfunctional in multiple aspects. We found that key agonist-induced responses of the patients' platelets, such as integrin activation, secretion and Ca2+ fluxes are impaired, at a remarkably variable extent. Furthermore, the platelets from almost all patients showed agonist-independent exposure of PS upon stor- age, which was not linked to apoptotic caspase activity, in contrast to the platelets from healthy subjects which did not display PS exposure. In the patients' platelets, the defective activation could be linked to an impaired mito- chondrial membrane potential and a decreased mitochon- drial respiratory activity.
AB
CD
E
Figure 4. Increased PS exposure in platelets from patients receiving chemotherapy in the absence of apoptosis. Isolated platelets from patients and controls were incubated at 37°C for 90 min, and stained for PS exposure with FITC-annexin A5. (A) Percentages of PS-exposing platelets, (patients n=15, controls n=12). (B) PS exposure measured after indicated times with vehicle or 5 μM ABT-737 to induce apoptosis, (n=6-9). Platelets (10x109/L) from patients or controls were pretreated with caspase inhibitor Q-VD-OPh (10 μM), as indicated, and then stimulated with ABT-737 (5 μM) or vehicle. (C) Fractions of platelets with PS exposure, measured with FITC-annexin A5, (n=8). (D) Caspase-3 activity determined with a fluorometric assay, (n=4). (E) Absence of caspase-dependent kindlin-3 cleavage in western blots from patient platelets. Control platelets were stimulated with ABT-737 with(out) Q-VD-OPh pretreatment; patient platelets were analyzed during the decreasing and recovery phases of platelet count, (n=7). Overall platelet responsiveness of the patients was 30.5 – 48.4% (IQR). Medians with IQR, *P<0.05, **P<0.01 and ***P<0.001. plt: platelets.
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