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Platelet dysfunction in chemotherapy
ondary loss of function.33 Given that in the present patient group P-selectin expression and integrin activation were low without stimulation, there is no evidence for in vivo platelet activation linked to chemotherapy treatment. On the other hand, the patients’ platelets showed a tendency to expose PS, which is compatible with an apoptotic process, as apoptotic platelets are known to be defective in aggregation and secretion.22 However, ongoing apoptot- ic signaling could be excluded, since: (i) treatment with the pancaspase inhibitor Q-VD-OPh did not prevent PS expo- sure, (ii) measurable caspase-3 activity was absent, and (iii) caspase-dependent cleavage of kindlin-3 could not be detected.
Platelets rely on mitochondrial ATP production, in par- ticular upon activation when their energy demand increas- es.24 While the mitochondrial content and ultrastructure appeared normal in the patients’ platelets, we noticed a marked reduction of the platelet mitochondrial membrane potential and the mitochondrial oxidative phosphoryla- tion. Other authors have shown that anti-tumor antibi- otics (anthracyclines), an important class of chemothera- peutic agents used to treat hematological malignancies, induce cardiotoxicity and muscle weakness due to the impairment of mitochondrial function via an increased production of ROS.29,34,35 In cardiac cells, the accumulation of iron inside the mitochondria may contribute to the pro-
duction of ROS.36 Furthermore, the mitochondrial activity in myocardial and hepatic cells is known to be impaired by the chemotherapeutics cyclophosphamide and carmus- tine (BCNU).37-39 Our results suggest that a similar mecha- nism of ROS-linked mitochondrial dysfunction is opera- tive in the platelet precursor cells, as deduced from the strong correlation (at >2 days after treatment) between mitochondrial dysfunction and elevated ROS levels. The fact that platelet activation induced by strong agonists (CRP-XL, thrombin) was more affected than platelet acti- vation by ADP suggests a relatively larger role of mito- chondrial ATP production upon stimulation with stronger agonists.40 The slight decrease in GPVI (and GPIba) recep- tor levels might contribute to the lower responsiveness of platelets, although this can also be the consequence of receptor shedding induced by ROS and mitochondrial stress.41
Taken together, our findings suggest that ROS-induced dysfunction in the mitochondria (before the production of platelets) impairs platelet activity and induces PS expo- sure, thus leading to a shortened platelet lifetime. This conclusion is supported by a recent study in mice, devel- oping thrombocytopenia after 5-fluororacil treatment. In these animals, low-level laser therapy was found to increase the mitochondrial activity of megakaryocytes, resulting in a normalization of hemostasis.42 Another pos-
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Figure 6. Decreased responsiveness of patient platelets is accompanied by mitochondrial membrane depolarization and ROS production. Platelets (10x109/L) were isolated from healthy controls (healthy ctrl) and from patients at three time points; namely 1: directly before the start of chemotherapy, 2: at two days of chemotherapy and, 3: upon severe thrombocytopenia (count ≤50x109/L). Washed platelets were activated with thrombin (4 nM), CRP-XL (10 μg/mL) or 2MeS-ADP (1 μM) in the presence of 2 mM CaCl2. After 15 min activation, integrin aIIbβ3 activation (A) and P-selectin expression (B) were measured by flow cytometry using labeled PAC-1 and anti-P-selectin antibody, respectively. Depicted is mean platelet responsiveness to thrombin, CRP-XL and ADP. Platelet samples were loaded with TMRE (C) to assess mitochondrial membrane potential, indicative of mitochondrial function, or with H2DCFDA (D) to measure ROS levels. Platelets from healthy controls were treated with CCCP as a positive control (h.c. CCCP). Medians with interquartile ranges (IQR); n=7-10 (patients) and n=7 (healthy controls), *P<0.05,**P<0.01.
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