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Platelet dysfunction in chemotherapy
rophenoxymethyl ketone (Q-VD-OPh) fully inhibited the PS exposure triggered by ABT-737. However, Q-VD-OPh failed to affect the storage-dependent PS exposure (Figure 4C). Furthermore, whereas ABT-737 stimulation resulted in high caspase-3 activity, no such activity could be detect- ed during storage (Figure 4D). Additional confirmation for the absence of apoptotic signaling was obtained by assess- ing the caspase-dependent cleavage of the integrin-bind- ing protein, kindlin-3.23 Western blot analysis indicated that, in platelets from control subjects, ABT-737 treatment induced full cleavage of kindlin-3, which was prevented by Q-VD-OPh (Figure 4E). In the patient platelets (with
confirmed functional impairment of integrin activation and P-selectin expression), however, no kindlin-3 cleavage could be detected in the absence of ABT-737.
Platelet activation is known to rely on mitochondrial activity for sufficient ATP production.24 Given that mito- chondrial impairment can lead to PS exposure,25,26 we assessed the activity of mitochondria in several ways. As part of the initial characterization of the patient platelets, the mitochondrial membrane potential was assessed by staining with TMRE. Whereas control platelets displayed high TMRE fluorescence, the patient platelets showed much less fluorescence intensity (Figure 5A). This suggest-
A
B
C
D
Figure 2. Impaired platelet responsiveness in relation to phase of treatment and/or recovery. Platelet integrin aIIbβ3 activation and P-selectin expression were measured (see Figure 1). Patients (n=52) were divided into two categories: (i) decreasing platelet count 50-11 x109/L (n=15) and (ii) decreasing platelet count ≤10 x109/L (n=37). Furthermore, from a subset of patients a sample could be collected when the platelet count increased independently of platelet transfusion (iii): 11- 50 x109/L (n=8). Data are expressed as % of platelets positive for PAC-1 or anti-P-selectin staining in the absence of stimulation (A), or after stimulation with thrombin (B), CRP-XL (C) or 2MeS-ADP (D). Medians with IQR for patients and healthy controls (n=27); **P<0.01 and ***P<0.001. plt: platelet.
haematologica | 2018; 103(9)
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